Indole compound and application thereof in preparation of antitumor drugs

Abstract

The invention relates to the field of medicines, in particular to a compound 5-hydroxy-6-bromo-12-methyl-10, 11, 12, 13-tetrahydro-1H-azepine ketone [5, 4, 3-cd] indole applied to a tyrosine kinase C-kit receptor inhibitor or a malignant tumor medicine, and the structural formula of the compound is shown in the specification. The compound disclosed by the invention has a good inhibition effect on a tyrosine kinase C-kit receptor and is suitable for preparing medicines for treating malignant tumors.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an indole compound obtained from the marine organism Stipes tunicate and its application as a tyrosine kinase C-kit receptor inhibitor and in the preparation of anti-tumor drugs. Background Art

[0002] The tyrosine kinase C-kit receptor (C-kit receptor), also known as CD117 or stem cell factor receptor (SCFR), has a relative molecular mass of 145 kD and is a type I transmembrane glycoprotein receptor encoded by the C-kit proto-oncogene. This protein belongs to the receptor tyrosine kinase family, and its ligand is stem cell factor (SCF). The C-kit receptor consists of three regions: the extracellular domain, the transmembrane domain, and the cytoplasmic domain. Under normal physiological conditions, the C-kit receptor is normally expressed in hematopoietic stem cells, endothelial cells, and mast cells, but is rarely or not expressed at all in acute lymphoblastic leukemia. However, in tumor populations, detection has revealed elevated expression of the C-kit receptor on the surface of certain malignant tumor cells. Therefore, abnormal expression of the C-kit receptor is closely associated with the development and progression of tumors.

[0003] Normally, C-kit receptor activation requires the participation of its ligand, SCF. When the C-kit receptor specifically binds to its ligand, SCF, it triggers C-kit receptor activation, namely homodimerization and phosphorylation of tyrosine residues within the cell membrane. This transmits extracellular signals to the cell interior, triggering a series of physiological and biochemical reactions that regulate metabolism and growth and development. Therefore, the C-kit / SCF system is a crucial link in the protein kinase / phosphatase signal transduction process.

[0004] Mutations in the C-kit gene lead to abnormal activation of the C-kit receptor, resulting in spontaneous receptor dimerization independent of its ligand, SCF. This leads to sustained activation and high expression of the C-kit receptor, continuously stimulating excessive cell proliferation and causing uncontrolled anti-apoptotic signaling. Overexpression of the C-kit receptor has been found in many malignant tumors, including gastrointestinal stromal tumors, breast cancer, prostate cancer, small cell lung cancer, and germ cell tumors, particularly in gastrointestinal stromal tumors.

[0005] Therefore, inhibiting the signal transduction pathway mediated by high expression of C-kit receptor in tumor cells is one of the important methods for effectively treating tumors.

[0006] Compared to certain compounds that inhibit DNA synthesis, C-kit receptor inhibitors can significantly enhance therapeutic efficacy and reduce toxic side effects. Compounds that inhibit DNA synthesis target cell division, and are particularly toxic to rapidly dividing tumor cells. However, normal cell metabolism also requires cell division. Therefore, drugs that inhibit DNA synthesis are not only toxic to rapidly dividing tumor cells, but also have a wide range of toxic effects on the cell division process of normal cells. Therefore, for tumor treatment, drugs that inhibit DNA synthesis are not highly selective for tumor cells and have significant side effects.

[0007] C-kit receptor inhibitors are different. They do not work by inhibiting DNA synthesis. Small molecule C-kit receptor inhibitors bind to the intracellular kinase functional region of C-kit, inhibit its catalytic activity, block the conduction of cell proliferation signals, and promote tumor cell apoptosis. Compared with traditional cytotoxic anti-tumor drugs, small molecule C-kit receptor inhibitors have the characteristics of significant efficacy, few adverse reactions, and easy preparation. At present, many tyrosine kinase C-kit receptor inhibitors on the market can be used as selective inhibitors of mammalian cancer cell growth. The earliest drug to come out was imatinib mesylate, which was approved for marketing by the US FDA in 2001. New drugs are also constantly emerging, such as dasatinib, nirofenib, sunitinib, etc., which can act on C-kit targets. Some are multi-target inhibitors with significant efficacy.

[0008] Although anticancer drugs such as those mentioned above have made significant contributions to the field of tumor treatment, they still have the disadvantage of easily generating drug resistance that is difficult to solve. Therefore, the development of compounds with better selectivity or efficacy, or less toxicity and side effects as novel C-kit receptor inhibitors is still very necessary for the field of tumor treatment.

[0009] The inventors obtained a novel indole compound from a marine organism, Ascidianus tunicate. Activity tests showed that it has significant inhibitory activity against the C-kit receptor and can be used to prevent and treat malignant tumors that overexpress the C-kit receptor. Summary of the Invention

[0010] The present invention addresses the deficiencies in the prior art and provides an indole compound with a novel structure obtained from the marine organism Stipes tunicate, which has a strong inhibitory activity on the tyrosine kinase C-kit receptor.

[0011] The technical solution of the present invention to solve the above technical problems is as follows: an indole compound, the structure of which is shown in the following formula: .

[0012] The compound can be named as 5-hydroxy-6-bromo-12-methyl-10,11,12,13-tetrahydro-1H-azepin-5-ol.

[0013] In the preparation of the compound, first, the Styela clava body powder is alkalized by ammonia water, and then the lipid material is removed by chloroform extraction at room temperature until the chloroform extract is colorless, which represents that the lipid material is completely removed. The body residue after chloroform extraction is soaked in 60-80% ethanol at 4-10 times of the weight of the residue for 12-36 hours, then filtered, and the filtrate is collected. In order to completely extract the compound, the filtrate is extracted by 60-90% ethanol for 3-6 times, and then all the filtrates are combined. The ethanol is recovered by rotary evaporation under reduced pressure (pressure ≤0.1 atm, temperature 45-55℃) to obtain a concentrated extract.

[0014] Then, the concentrated extract is fully suspended by using an appropriate amount of distilled water, and then fully extracted by chloroform and ethyl acetate in sequence. The chloroform extract is removed, and the ethyl acetate extract is retained, concentrated, and then extracted by ethyl acetate to obtain an ethyl acetate extract. Then, the ethyl acetate extract is separated by chromatography. First, a conventional silica gel column (200-300 mesh coarse silica gel) is used, and then the eluent containing the target component is subjected to multiple chromatography by thin layer chromatography using silica gel H. Different volume ratios of chloroform-acetone and chloroform-methanol are used as eluents in sequence, and the chromatography liquid is collected in sections. The chromatography liquids containing the same components are combined, and then the chromatography liquid rich in alkaloid components is separated and purified by a semi-preparative high-performance liquid chromatography column to obtain the indole compound of the present application. The structure of the compound is determined by spectral identification analysis.

[0015] The indole compound can be used for preparing a tyrosine kinase C-kit receptor inhibitor drug or for preparing a drug for treating malignant tumors.

[0016] The present application has the following advantages: the compound of the present application is a novel bromine-containing indole compound, and the molecular biology experiments and pharmacological experiments show that the compound of the present application can significantly inhibit the activity of tyrosine kinase C-kit receptor. DETAILED DESCRIPTION

[0017] The principles and characteristics of the present application are specifically described below, and the examples are only used to explain the present application, and are not used to limit the scope of the present application.

[0018] Example 1: Extraction, purification and structure identification of the compound of the present application The compound of the present application is derived from the marine organism Styela clava, which is collected from the sea of Zhifu Island in Zhifu District, Yantai City. The sample is Styela clava of the class Ascidiacea, which is identified by biology. Styela clava). The collected ascidian was washed with tap water at room temperature to remove the attached impurities, and the attached growth on the surface of the mantle sac was removed. The mantle sac and the body were peeled off, and the body was homogenized with a high-speed tissue grinder. Then, the body was frozen and dried at low temperature to remove the water content. The dried body tissue was crushed with a high-speed universal crusher to obtain a body powder. The body powder of the ascidian was alkalinized with ammonia water, and was first extracted with chloroform at room temperature to remove lipid substances until the last chloroform extract was colorless. The residue of the body after the removal of the fat was further extracted and purified.

[0019] The body powder was extracted with 80% ethanol at room temperature for 4 times, 24 hours each time. The extract was filtered after each extraction, and then the filtrates were combined. The ethanol was recovered at 45°C and 0.1 atm until the last recovered liquid was alcohol-free. Then, the combined filtrate was fully suspended with distilled water. The suspension was extracted with chloroform for several times until the last chloroform layer was colorless. The chloroform extract was low in polarity and did not contain alkaloid components, and was discarded. The residual body powder suspension was further extracted with ethyl acetate solvent for 5 times. The ethyl acetate extract was collected, and the ethyl acetate solvent was recovered at a temperature of ≤45°C under reduced pressure (pressure ≤0.1 atm) using a rotary evaporator. A brown ethyl acetate extract was obtained, and its weight was measured. The weight of the ethyl acetate extract was 50 times the weight of the silica gel (200-300 mesh) to prepare a silica gel column. The ethyl acetate extract was loaded onto the silica gel column, and was eluted with chloroform:acetone eluent with different volume ratios (100:0, 50:1, 20:1, 10:1, 5:1, 2:1, and 0:100) to collect and detect each eluate by TLC thin layer plate development (improved bismuth potassium iodide solution as color developing agent). The eluates with the same components were combined, and were divided into 6 crude fractions (1-6). Each crude fraction was further separated and purified by semi-preparative high-performance liquid chromatography (eluted with different volume ratios of methanol and distilled water). The compound was obtained by separation and purification in the crude fraction 4. The compound was named as 5-hydroxy-6-bromo-12-methyl-10,11,12,13-tetrahydro-1H-azepin-3-one [5,4,3-cd] indole, and its structure is shown in the following formula: .

[0020] The compound has the following physicochemical characteristics: Molecular formula: C 12 H 13 BrN2O; Form: light yellow solid; UV spectral data: UV max (MeOH): 276, 220 nm; Infrared spectrum data: IR (KBr): 3431, 2927, 1579, 1439, 1390, 1251, 1078, 1007,798cm -1 ; Proton spectrum 1 H NMR and carbon spectroscopy 13 C NMR data are shown in Table 1: Table 1 H NMR and C NMR data of the compounds of the present invention ( 1 H NMR: 400 MHz and 13 C NMR: 100 MHz, solvent: D2O + DMSO- d 61:1) Position <![CDATA[ δ C ]]> δ H <!-- 3 -->]]> ​ <![CDATA[23456789101113N 12 -CH3]]> 125.0 (d)113.1 (s)108.0 (t)147.9 (s)109.9(s)114.8 (d)132.3 (s)127.7 (s)23.0 (t)75.1(t)71.2 (t)58.4 (q) 7.26 (s)7.01 (s)3.58 (dd,= 7.1, 4.5Hz)3.31 (m)4.12 (m, 2H)5.11d (=14.5)4.87d(=14.5)3.49 (s, 3H)

[0021] Example 2 Activity test of the compound of the present invention as a tyrosine kinase C-kit receptor inhibitor: Methods: Enzyme-linked immunosorbent assay (ELISA); Receptor kinase: C-kit; Test compound: Compound of the present invention Positive control drug: Glivic.

[0022] Activity test steps: The activity test experiment was performed according to the instructions of the C-kit kit. First, an appropriate amount of the compound of the present invention was accurately weighed using an electronic balance, diluted to a concentration of 10 μg / ml, and accurately measured with a pipette and added to a 96-well plate. 100 μl of serum sample was added to each well except the blank control well. Then, 50 μl of biotinylated antibody working night was added, mixed with a pipette, and incubated at 25°C for 60 minutes. After incubation, the plate was washed four times, and 100 μl of horseradish peroxidase-labeled avidin was added to each well (including the blank control well). Then, 100 μl of substrate color development solution (OPD) was added to each well. After incubation at room temperature in the dark for 15 minutes, 100 μl of color reaction stop solution (1.8 mol / L sulfuric acid) was added to each well, and the solution in each well was immediately measured at a wavelength of 490 nm using a microplate reader. The absorbance value was used to detect the degree of substrate phosphorylation.

[0023] Table 2 Inhibition rate of the compounds of the present invention on tyrosine kinase C-kit activity (%) Sample name Inhibition rate% (concentration) Evaluation of experimental results Compounds of the present invention 71.2 (10µg / ml) Has a significant inhibitory effect Positive control Glivic 70.6 (10µg / ml) Has a significant inhibitory effect

[0024] Experimental conclusion: The compound of the present invention has a good inhibitory effect on protein tyrosine kinase C-kit and can be used as a preventive and therapeutic drug for malignant tumors with high expression of C-kit receptors.

[0025] Example 3 Testing of the application of the compounds of the present invention in inhibiting the proliferation of malignant tumors.

[0026] 1. Experimental Materials Tumor cell line: Mouse B16-F0 melanoma cell line, provided by the Institute of Materia Medica, Shandong Academy of Medical Sciences; Experimental animals: 30 C57BL / 6J mice were randomly divided into three groups, with 10 mice in each group, half male and half female. The average weight of each mouse was 12.0 ± 1.0 g. They were provided by Qingdao Experimental Animal and Animal Experiment Center.

[0027] 2. Experimental methods (1) Tumor-bearing mouse model: a concentration of 5.0×10 6 The B16-F0 melanoma cell suspension with a concentration of 1.0×10 cells / ml was administered subcutaneously on the left side of the back of each mouse. Each mouse was injected with 0.2 ml of B16-F0 melanoma cell suspension (1.0×10 6 cells); within 24 hours after the injection of all mice, the mental state of the mice was observed. Each group of mice was placed in one cage and fed separately.

[0028] (2) Grouping and administration Test group: 50 mg / kg of the compound of the present invention was administered by dissolving an appropriate amount of the compound in 25% by volume dimethyl sulfoxide and diluting the solution to a dose of 50 mg / kg of the mouse body weight. The compound was administered by intraperitoneal injection. Blank control group: 25% dimethyl sulfoxide; Positive control group: 50 mg / kg dacarbazine From the day of inoculation, no drugs were given to the mice from the first to the fourth day. Starting from the fifth day, drugs were given daily for 14 consecutive days by intraperitoneal injection. The growth of the mice and tumors was observed. 24 hours after the last administration, the tumor mass of each mouse was removed, the attachments of the tumor were peeled off, the tumor was weighed, and the inhibition rate of tumor growth (tumor inhibition rate) was calculated.

[0029] (3) Detection indicators Tumor inhibition rate (%) = (average tumor weight of blank control group - average tumor weight of experimental group) / (average tumor weight of blank control group) × 100% (wherein: experimental group refers to the compound of the present invention group or positive control group).

[0030] The results are shown in Table 3. Statistical analysis showed that at the experimental dose, the average tumor weights of mice in the compound group and the positive control group were significantly lower than those in the blank control group, with significant differences (P < 0.05), indicating that the compound of the present invention has a significant tumor inhibitory effect.

[0031] Table 3 Tumor weight and tumor inhibition rate in mice (mean ± SD, n=10) Group Tumor weight (g) Tumor inhibition rate (%) Blank control group 0.2980±0.0312 --- Positive control group 0.0511±0.0207* 82.9 Compound group of the present invention 0.0495±0.0186* 83.4 Note: * indicates significant difference (P < 0.05) (compared with blank control group).

[0032] Example 4 Tablet preparation method for the compound of the present invention: 100 mg of the raw material of the compound obtained in Example 1 is mixed with 2900 mg of medicinal starch and dextrin (starch to dextrin mass ratio of 3:1) as excipients; after thorough mixing, granulation, whole granulation and tableting are carried out to finally prepare 10 tablets, each weighing 0.30 g and containing 10 mg of the compound.

[0033] Example 5 Preparation of lyophilized powder injection of the compound of the present invention: Weigh 10 g of the raw material compound obtained in Example 1 and 20 g of medicinal mannitol, dissolve them in 50 mL of water for injection, stir thoroughly to dissolve, add water for injection to 1000 mL, add 2.0 g of activated carbon for injection, heat to 60°C and stir for 30 minutes, filter through a carbon rod, and filter the filtrate through a 0.25 μm microporous membrane for sterilization. Dispense into 1000 vials, filling 1.0 mL / vial, and freeze-dry to obtain the product, each vial containing 10 mg of the compound.

[0034] 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 in the scope of protection of the present invention.

Claims

1. An indole compound, characterized in that The compound is named: 5-hydroxy-6-bromo-12-methyl-10,11,12,13-tetrahydro-1H-azepinone[5,4,3-cd]indole, and the structural formula of the compound is shown below: 。 2. Use of the indole compound according to claim 1 in the preparation of tyrosine kinase C-kit receptor inhibitor drugs.

3. Use of the indole compound according to claim 1 in the preparation of drugs for treating malignant tumors.

Images