A compound and its application in preparing anti-tumor drugs

Abstract

The present invention relates to the field of medicine, and in particular to a compound 7-bromo-6-hydroxy-N-methyl-1,2,3,4-tetrahydro-β-carboline used as a tyrosine kinase C-kit receptor inhibitor or a malignant tumor drug, the structural formula of the compound being: The compound described in the present invention is a new compound, which has a good inhibitory effect on the tyrosine kinase C-kit receptor and is suitable for preparing a drug for treating malignant tumors.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a brominated-β-carboline compound obtained from Ascidianus stygoscelis and its application as a tyrosine kinase C-kit receptor inhibitor and in the preparation of anti-tumor drugs. Background Art

[0002] Tyrosine kinase C-kit receptor (hereinafter referred to as: C-kit receptor), also known as CD117 or stem cell factor receptor (SCFR), has a relative molecular mass of 145kD. It is a type I transmembrane glycoprotein receptor encoded by the C-kit proto-oncogene. This protein belongs to a member of the receptor tyrosine kinase family, and its ligand is stem cell factor (SCF). The C-kit receptor is divided into three parts: the extracellular region, the transmembrane region, and the cytoplasmic region. Under normal physiological conditions, the C-kit receptor is normally expressed in hematopoietic stem cells, endothelial cells, and mast cells, but is rarely expressed or not expressed at all in acute lymphoblastic leukemia. However, in tumor populations, detection has found that the C-kit receptor is highly expressed on the surface of some malignant tumor cells. Therefore, the abnormal expression of the C-kit receptor is closely related to the occurrence and development of tumors.

[0003] Under normal circumstances, the activation of C-kit receptor requires the participation of ligand SCF. When C-kit receptor binds specifically to ligand SCF, it triggers C-kit receptor activation, i.e. homodimerization and phosphorylation of tyrosine residues in the cell membrane, thereby transmitting extracellular signals to the interior of the cell, triggering a series of physiological and biochemical reactions, and regulating metabolism and growth and development. Therefore, the C-kit / SCF system is an important link in the protein kinase / phosphatase signal transduction process.

[0004] When the C-kit gene mutates, it will lead to abnormal activation of the C-kit receptor, that is, spontaneous receptor dimerization independent of the ligand SCF, which will cause the continuous activation and high expression of the C-kit receptor, constantly stimulating excessive cell proliferation, leading to uncontrolled cell anti-apoptosis signals. It has been found that high expression of the C-kit receptor exists in many malignant tumors, including gastrointestinal stromal tumors, breast cancer, prostate cancer, small cell lung cancer and germ cell tumors, especially 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 to effectively treat tumors.

[0006] Compared with some compounds that inhibit DNA synthesis, C-kit receptor inhibitors can significantly increase the efficacy and reduce toxic side effects. The action site of compounds that inhibit DNA synthesis is targeted at cell division, especially tumor cells with rapid division characteristics, which have good toxicity. However, the metabolism of normal cells also requires the cell division process. Therefore, drugs that inhibit DNA synthesis are not only toxic to rapidly dividing tumor cells, but also have extensive toxicity to the cell division process of normal cells. It can be seen that for the treatment of tumors, drugs that inhibit DNA synthesis are not highly selective for tumor cells and have greater 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, and 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 being prone to drug resistance that is difficult to resolve. Therefore, the development of compounds with better selectivity or efficacy, or less toxicity and side effects as novel C-kit receptor inhibitors is still necessary for the field of tumor treatment.

[0009] The inventors obtained a novel compound from a marine organism, Ascidianus tunicate. Activity tests showed that the compound had significant inhibitory activity against C-kit receptors and could be used for the prevention and treatment of malignant tumors that highly expressed C-kit receptors. Summary of the invention

[0010] The present invention aims at the above-mentioned deficiencies in the prior art and provides a compound obtained from the marine organism Ascidianus stalkensis and having a novel structure and strong inhibitory activity on tyrosine kinase C-kit receptor.

[0011] The technical solution of the present invention to solve the above technical problems is as follows: a bromo- β -Carboline compound, the structure of the compound is shown in the following formula:

[0012]

[0013] The above compound can be named as: 7-bromo-6-hydroxy-N-methyl-1,2,3,4-tetrahydro -β -Carboline.

[0014] When preparing the above-mentioned compound, the powder of the stalked sea squirt is first alkalized with ammonia water, and then extracted with chloroform at room temperature to remove lipid substances until the chloroform extract is colorless. The sheath residue after chloroform extraction is soaked in 60-80% ethanol at 4-10 times its weight for 12-36 hours, then filtered and the filtrate is collected. In order to completely extract the compound, it is extracted with 60-90% ethanol for 3-6 times, the filtrates of 3-6 times are combined, and then the ethanol is recovered by a rotary evaporator under reduced pressure (pressure ≤ 0.1 atmosphere, temperature at 45-55 degrees Celsius) to obtain a concentrated extract.

[0015] Then, an appropriate amount of distilled water is added to the concentrated extract to fully suspend the concentrated extract in the water, and then chloroform and ethyl acetate are used to fully extract, the chloroform extract is removed, and the ethyl acetate extract is taken, and a silica gel column (first using 200-300 mesh coarse silica gel, and then using silica gel H for thin layer chromatography) is used for multiple chromatography, and chloroform-acetone and chloroform-methanol are used as eluents, and the chromatographic liquid is collected in sections, and the chromatographic liquid containing the same components is combined, and then the chromatographic liquid rich in alkaloid components is separated and purified by a preparative high performance liquid column to obtain the brominated-β-carboline compound of the present invention, and the structure of the compound is determined by spectral identification analysis.

[0016] The above compounds can be used as tyrosine kinase C-kit receptor inhibitors or applied to the preparation of drugs for treating malignant tumors.

[0017] The present invention has the following advantages: the compound described in the present invention is a new compound, and molecular biology experiments and pharmacological experiments show that the compound of the present invention can significantly inhibit the activity of tyrosine kinase C-kit receptor, and the compound can be used to prepare drugs for treating malignant tumors. DETAILED DESCRIPTION

[0018] The principles and features of the present invention are described in detail below. The examples given are only used to explain the present invention but not to limit the scope of the present invention.

[0019] Example 1 Extraction, purification and structural identification of the compound of the present invention

[0020] The stalked sea squirt was collected from the seashore of Zhifu Island, Zhifu District, Yantai City. After biological identification, the sample was a stalked sea squirt of the class Ascidian. Style clava). The collected stalked tunicate was rinsed with tap water at room temperature to remove attached impurities, remove attachments growing on the surface of the theca, peel off the theca and the theca, homogenize the theca with a high-speed tissue crusher, and then freeze-dry at low temperature to remove the water contained. The dried theca tissue was crushed with a high-speed universal grinder to obtain the theca powder. The stalked tunicate theca powder was alkalized with ammonia water, and first extracted with chloroform at room temperature to remove lipid substances until the last chloroform extract was colorless. The theca residue after the chloroform extraction to remove fat was then extracted and separated and purified.

[0021] Take an appropriate amount of the above sheath powder, and extract it 4 times at room temperature with 80% ethanol of a volume concentration of 8 times its weight, each time for 24 hours, then combine the 4 ethanol extracts, recover the ethanol at a temperature of 45°C and a pressure of 0.1 atmosphere until the final recovered liquid has no alcohol taste, add an appropriate amount of distilled water to fully suspend it, first extract it with chloroform for multiple times until the last chloroform layer is colorless, and detect it by thin layer chromatography with an alkaloid detection reagent. The chloroform layer extract does not contain alkaloid components and is set aside. The residual sheath powder suspension was then extracted with ethyl acetate solvent for 5 times, and the ethyl acetate extracts were collected and combined. The ethyl acetate solvent was recovered by a rotary evaporator under reduced pressure (pressure ≤ 0.1 atmosphere) at a temperature of ≤ 45°C to obtain a brown ethyl acetate extract. The brown ethyl acetate extract was weighed, and a silica gel column (200-300 mesh) of 50 times its weight was taken to prepare a silica gel column. The ethyl acetate extract was loaded onto the silica gel column and eluted with chloroform:acetone in different volume ratios (100:0, 50:1, 20:1, 10:1, 5:1, 2:1, 0:100) as the eluent, and the extract was collected in sections. Each section of the eluent was developed by thin layer plate for detection (modified potassium bismuth iodide test solution was used as a color developer) and the sections with the same components were combined to obtain 6 crude fractions (1-6). Each crude fraction was further separated and purified by preparative high performance liquid chromatography (eluted with methanol and distilled water in different volume ratios) to obtain the compound of the present invention. After analysis, the compound was named: 7-bromo-6-hydroxy-N-methyl-1,2,3,4-tetrahydro -β -Carboline, the structure is shown below:

[0022] .

[0023] The compound has the following physical and chemical characteristics:

[0024] Molecular formula: C 12 H 13 Bn 2 O;

[0025] Appearance: white solid;

[0026] Melting point: mp.231.4-232.6℃;

[0027] Ultraviolet: UV max(MeOH): 218.2, 282.4 nm;

[0028] Infrared: IR(KBr): 3245, 2973, 1636, 1589, 1458, 1399, 1262, 1151, 967,927, 803 cm -1 ;

[0029] Proton spectrum: 1 H NMR and carbon spectroscopy 13 C NMR (DMSO- d 6 ) Data are shown in Table 1;

[0030] Table 1 Nuclear magnetic resonance hydrogen spectrum δ of the compounds of the present invention H and carbon spectrum δ C data

[0031] (NMR frequency: 400 MHz for 1 H and 100 MHz for 13 C, δ, ppm ( J , Hz), solvent: DMSO- d 6 )

[0032] Position <![CDATA[δ C (a lot)]]> <![CDATA[δ H (a lot)]]> 1 53.1 (t) 3.52 (s, 2H) 3 53.0 (t) 2.71 (d,= 4.9Hz, 2H) 4 20.9(t) 2.65 (d,= 4.9Hz, 2H) 4a 106.1 (s) 4b 126.7 (s) 5 103.1 (d) 6.66 (s, 1H) 6 149.6 (s) 7 109.0 (d) 8 113.8 (d) 6.86 (s, 1H) 8a 129.7 (s) 9a 132.5 (s) 9(NH) 10.44 (s, 1H) <![CDATA[N-CH 3 ]]> 2.29 (s, 3H) OH 9.69 (s, 1H)

[0033] Example 2 Activity test of the compounds of the present invention as tyrosine kinase C-kit receptor inhibitors:

[0034] Methods: Enzyme-linked immunosorbent assay (ELISA);

[0035] Receptor kinase: C-kit;

[0036] Test compound: Compound of the present invention

[0037] Positive control drug: Glivic.

[0038] Activity test steps: According to the instructions of the C-kit kit, firstly, an appropriate amount of the compound of the present invention was accurately weighed with an electronic balance, diluted to a concentration of 2 μg / ml, accurately measured with a pipette and added to a 96-well plate, and 100 μl of serum specimen was added to each well except the blank control well, and then 50 μl of the biotinylated antibody working night was added, mixed with a pipette and incubated at 25°C for 60 min. After the incubation, the plate was washed 4 times, and 100 μl of horseradish peroxidase-labeled avidin was added to each well (including the blank control well), and then incubated at 25°C for 30 min, and then 100 μl of substrate color development solution (OPD) was added to each well, and incubated at room temperature for 15 min under light-proof conditions, and then 100 μl of color reaction stop solution (1.8 mol / L sulfuric acid) was added to each well, and the absorbance value of the solution in each well at a wavelength of 490 nm was immediately measured in an ELISA instrument to detect the degree of substrate phosphorylation.

[0039] Table 2 Inhibition rate of test samples on tyrosine kinase C-kit activity (%)

[0040] Sample name Inhibition rate % (concentration) evaluate Compounds of the present invention 62.4 (2µg / ml) Has a significant inhibitory effect Positive Control Glivic 68.7 (10µg / ml) Has a significant inhibitory effect .

[0041] Experimental conclusion: The compound of the present invention has a good inhibitory effect on protein tyrosine kinase and can be used as a preventive and therapeutic drug for malignant tumors with high expression of C-kit receptor.

[0042] Example 3 Testing of the application of the compounds of the present invention in inhibiting the proliferation of malignant tumors.

[0043] 1. Experimental Materials

[0044] Tumor cell line: Mouse B16-F0 melanoma cell line, provided by the Institute of Materia Medica, Shandong First Medical University (Shandong Academy of Medical Sciences);

[0045] Experimental animals: C57BL / 6J mice, 30 in number, half male and half female, with an average weight of 12±1 g per mouse, SPF grade, provided by Qingdao Experimental Animals and Animal Experiment Center.

[0046] 2. Experimental methods (1) Tumor-bearing mouse model: The concentration of B16-F0 melanoma cell suspension was adjusted to 5.0×10 6 Each mouse was injected subcutaneously with 0.2 ml (1.0×10 6 cells); 24 hours after the injection of all mice, the mental state of the mice was observed, and the 30 mice were randomly divided into 3 groups, with half of the mice in each group being female and half being male, 10 mice in each group, and each group of mice was placed in one cage and fed separately.

[0047] (2) Grouping and dosing

[0048] Test group: Compounds of the present invention, take an appropriate amount of the compound, dissolve it in dimethyl sulfoxide with a volume concentration of 25%, and dilute it to the corresponding dosing concentration according to the dose of 100 mg / kg of mouse body weight;

[0049] Blank control group: 25% volume concentration of dimethyl sulfoxide;

[0050] Positive control group: 50 mg / kg dacarbazine

[0051] From the date of completion of inoculation, no drugs were given to the mice from the first day to the fourth day. Starting from the fifth day, drugs were administered every day for 14 consecutive days by intraperitoneal injection. The growth of 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.

[0052] (3) Detection indicators

[0053] 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).

[0054] The results are shown in Table 3. Statistical analysis showed that at the experimental dose, the average tumor weight of mice in the compound group of the present invention and the positive control group was significantly lower than that in the blank control group, and the difference was significant (P < 0.05).

[0055] Table 3 Tumor weight and tumor inhibition rate of mice (mean ± SD, n=10)

[0056] Group Tumor weight (g) Tumor inhibition rate (%) Blank control group 0.3020±0.0247 --- Compounds of the present invention 0.0426±0.0201* 85.9 Positive control group 0.0403±0.0194* 86.7

[0057] Note: * indicates P < 0.05 (compared with blank control group).

[0058] Example 4

[0059] Preparation of tablets of the compound of the present invention: accurately weigh 1000 mg of the raw material of the compound obtained in Example 1, add 29000 mg of medicinal starch and dextrin (weight ratio 3:1) as excipients, mix well, make granules, and press the whole granules into tablets to obtain 100 tablets, each weighing 0.30 g, with a compound content of 10 mg / tablet.

[0060] Example 5

[0061] Preparation of freeze-dried powder injection of the compound of the present invention: accurately weigh 10 g of the raw material of the compound obtained in Example 1 and 20 g of medicinal mannitol, dissolve them in 500 mL of water for injection, stir them 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 with a carbon rod, filter the filtrate through a 0.25 μm microporous filter membrane for sterilization, and pack them into 1000 vials, with a filling volume of 1.0 mL / vial, and freeze-dry to obtain, each vial contains 10 mg of the compound.

[0062] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound, characterized in that The name of the compound is: 7-bromo-6-hydroxy-N-methyl-1,2,3,4-tetrahydro-β-carboline, and the structural formula of the compound is shown below:

2. Use of the compound according to claim 1 in the preparation of tyrosine kinase C-kit receptor inhibitors.

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

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