KR102152091B1 - Use of carbazol derivatives having apoptosis of brain cancer stem cells - Google Patents

Abstract

The present invention relates to a novel use of a carbazole derivative compound having apoptosis ability of brain tumor stem cells, specifically, the present invention is 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9- Regarding a pharmaceutical composition for killing brain tumor stem cells comprising a tetrahydro-1H-carbazole-1-amine hydrochloride compound as an active ingredient, a pharmaceutical composition for preventing or treating brain tumors, and a method for inducing apoptosis of brain tumor stem cells will be. In particular, the compound according to the present invention can inhibit the lipid metabolism of brain tumor stem cells and has an excellent effect of promoting apoptosis of brain tumor stem cells, so it can be used as a novel treatment for brain tumors targeting brain tumor stem cells. It can be used in various ways as an anticancer auxiliary medicine to increase the anticancer efficiency of the treatment.

Description

Use of carbazol derivatives having apoptosis of brain cancer stem cells {Use of carbazol derivatives having apoptosis of brain cancer stem cells}

The present invention relates to the use of a carbazole derivative compound having apoptosis ability of brain tumor stem cells, specifically 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol- It relates to a composition for killing brain tumor stem cells comprising a 1-amine hydrochloride compound as an active ingredient.

Brain tumor refers to a tumor in the brain tissue or the membrane tissue surrounding it, and refers to a primary brain tumor and a secondary brain tumor that has metastasized to the brain tissue or meninges from other areas. Brain tumors found in adults include glioma, glioblastoma, meningioma (meningioma), pituitary adenoma (adenoma), and schwannoma, among which glioblastoma starts from glial cells in brain tissue. It is a tumor that accounts for 12-15% of all brain tumors, and is known as the most common primary brain tumor in adults.

These brain tumors are representative refractory solid cancers with insignificant improvement in survival rate by treatment over the past 30 years. Despite the recent improved complex treatment technique, postoperative anticancer-radiation therapy, the average survival rate of brain tumor patients has developed to 12 15 months. There is no situation.

On the other hand, non-specific chemotherapy, which is a method of removing rapidly growing cells, has many side effects and has been presented with a problem that it cannot effectively remove cancer cells. Since then, Imatinib (Gleevec), an inhibitor of cancer-specific expression of BCR-ABL kinase, was developed and showed good effect on leukemia patients, and the concept of'targeted treatment' was introduced, and the target treatment was tumor cells. Since it'selectively' targets only the main factors involved in the characteristics of the drug, it is more effective in treatment with far fewer side effects than non-specific existing anticancer drugs.

Since Imatinib was developed, targeted treatments targeting cancer-activated phosphorylating enzymes or tumor-inducing signaling systems (RTK/RAS/PI3K), such as Gefitinib (Iressa), Sorafenib (Nexavar), Bevacizumab (Avastin), etc. It has been developed steadily. However, because the therapeutic effects of these target therapies are limited, patients are still forced to receive treatment using existing non-specific anticancer drugs such as TMZ. In addition, despite the use of these targeted treatments, a number of studies have been reported that eventually recurrence of cancer occurs. In particular, it has been reported that target therapies developed so far in brain tumors do not improve the prognosis of patients at all.

Meanwhile, since cancer stem cells were first isolated from leukemia, various cancer stem cells such as breast cancer stem cells, brain tumor stem cells, and colon cancer stem cells have been isolated and identified, and cancer stem cells are characteristic of normal stem cells. It has been reported to have chromosomal and genetic modification, tumorigenesis, generation of differentiated cancer cells, and high anticancer drug resistance, which are characteristic of cancer cells, along with the ability to self-renewal, pluripotency, and proliferate cells. Therefore, even if multiple signaling mechanisms related to cancer formation are suppressed in combination, if the cancer stem cells themselves are not targeted, cancer recurrence cannot be prevented. Therefore, conventional cancer treatment methods can derive the fundamental therapeutic effect of cancer. Can't.

Therefore, for a more effective and fundamental brain tumor treatment, the development of a new target therapy for brain tumor stem cells is urgently needed.

Korean Patent Registration No. 10-1872950

Accordingly, the present inventors have shown that the carbazole derivative compound effectively inhibits SREBP1, which is known to be involved in lipid metabolism in undifferentiated brain tumor stem cells than brain tumor cells and plays an important role in tumor signaling, thereby inhibiting lipid metabolism of brain tumor stem cells. , The present invention was completed by finding out that the compound can be used as a therapeutic agent for brain tumors through the death of brain tumor stem cells since it has an activity to induce apoptosis of brain tumor stem cells.

Therefore, the object of the present invention is 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride (6-cyclohexyl-N-(1 -Phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride) compound or a pharmaceutically acceptable salt thereof, as an active ingredient, to provide a pharmaceutical composition for killing brain tumor stem cells .

Another object of the present invention is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound or a pharmaceutically acceptable salt thereof It is to provide a pharmaceutical composition for the prevention or treatment of brain tumors, comprising as an active ingredient.

Another object of the present invention is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound or a pharmaceutically acceptable It is to provide a brain tumor treatment adjuvant comprising a salt as an active ingredient.

Another object of the present invention is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound in brain tumor stem cells in vitro. It is to provide a method for inducing apoptosis of brain tumor stem cells, comprising the step of treating.

In order to achieve the object of the present invention as described above, the present invention is 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride Brain tumor stem containing a (6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride) compound or a pharmaceutically acceptable salt thereof as an active ingredient It provides a pharmaceutical composition for cell death.

In one embodiment of the present invention, the compound may reduce lipid metabolism by inhibiting the expression of SREBP1 in brain tumor stem cells.

In one embodiment of the present invention, the brain tumor is anaplastic astrocytomas, glioblastomas, meningiomas, pituitary tumors, schwannomas, central nervous system lymphoma (CNS lymphoma). ), oligodendrogliomas, ependymomas, low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma , Diffuse astrocytomas, pleomorphic xanthoastrocytomas, supependymal giant cell astrocytomas, anaplastic oligodendrogliomas, oligoastrocytomas, Anaplastic oligoastrocytomas, myxopapillary ependymomas, subependymomas, ependymomas, anaplastic ependymomas, astroblastoma astroblastomas), chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, connective tissue producing desmoplastic infantile astrocytomas, conjunctiva Desmoplastic infantile gangliogliomas, fetal hypoplasia neuroepithelial (dyse) mbryoplastic neuroepithelial tumors), central neurocytomas, cerebellar liponeurocytomas, paragangliomas, ependymoblastomas, supratentorial primitive neuroectodermal tumors, choroid Choroids plexus papilloma, pineocytomas, pineoblastomas, moderately differentiated pineal parenchymal tumors of intermediate differentiation, hemangiopericytomas, tumors in the saddle area ( tumors of the sellar region), craniopharyngioma, hemangioblastoma (capillary hemangioblastoma), and primary CNS lymphoma (primary CNS lymphoma).

In addition, the present invention is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound or a pharmaceutically acceptable salt thereof It provides a pharmaceutical composition for the prevention or treatment of brain tumors, including as an active ingredient.

In addition, the present invention is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound or a pharmaceutically acceptable salt thereof It provides an adjuvant for treating brain tumors, including as an active ingredient.

Further, the present invention is to treat the 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound to brain tumor stem cells in vitro. It provides a method for inducing apoptosis of brain tumor stem cells comprising the step.

The 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound of the present invention inhibits the expression of SREBP1 in brain tumor stem cells. It can effectively reduce lipid metabolism and has excellent activity to promote apoptosis of brain tumor stem cells, so it can be used as a novel brain tumor treatment targeting brain tumor stem cells, and at the same time, anticancer aid to increase the anticancer efficiency of brain tumor treatments. It can be used in various ways as well as pharmaceuticals.

1 is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound of the present invention in 5 types of brain tumor stem cell lines. After treatment by concentration, the degree of apoptosis in each cell line is shown in a linear graph.
Figure 2 shows the 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound of the present invention in 5 types of brain tumor stem cell lines. After treatment by concentration, the degree of apoptosis in each cell line is shown as a bar graph.
3 is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound of the present invention in 5 types of brain tumor stem cell lines. After treatment, the result of confirming the expression level of SREBP1 in each cell line by Western blot is shown.

In the present invention, 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound promotes apoptosis of brain tumor stem cells It is characterized by the fact that this is the first to be identified.

According to an embodiment of the present invention, after treatment with the compound of the present invention by concentration for five types of brain tumor stem cell lines, the degree of apoptosis of brain tumor stem cells was analyzed. As a result, the compound of the present invention was not treated. Compared to the untreated group, the degree of apoptosis in the brain tumor stem cell lines treated with the compounds of the present invention was significantly superior, and this apoptosis effect was shown in all five types of brain tumor stem cell lines.

Further, in another embodiment, after treatment with the compound of the present invention for five types of brain tumor stem cell lines, the degree of expression change of SREBP1, which is known to play an important role in lipid metabolism in brain tumor stem cells, and affects the signaling mechanism of cancer, is determined. Analyzed.

As a result, it was found that the expression of SREBP1 was effectively suppressed in all 5 types of brain tumor stem cells treated with the compound of the present invention compared to the group not treated with the compound.

On the other hand, according to the results of in silico analysis of the recently reported gene expression data of brain tumor stem cells, undifferentiated brain tumor stem cells are known to have more active lipid synthesis metabolism than differentiated brain tumor cells. In particular, SREBP1 (sterol regulatory Element-binding protein 1) is known as a regulatory factor for adipogenic metabolism, and recently, it is known to play an important role in cancer metabolism and tumor signaling.

In this respect, if the lipid synthesis metabolism of brain tumor stem cells can be effectively inhibited, the growth, proliferation and activity of brain tumor stem cells can be inhibited, and thus brain tumors can be ultimately prevented or treated by targeting brain tumor stem cells.

Accordingly, the present inventors analyzed whether the compound of the present invention has inhibitory activity against SREBP1 in brain tumor stem cells, and confirmed that it can effectively inhibit the expression of SREBP1, and thus inhibiting the lipid metabolism of cells by inhibiting the expression of SREBP1 It was found that it can reduce or inhibit the activity of brain tumor stem cells.

Therefore, the present invention is effective in a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound or a pharmaceutically acceptable salt thereof. It is possible to provide a pharmaceutical composition for killing brain tumor stem cells and a pharmaceutical composition for preventing or treating brain tumors, including as components.

6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound according to the present invention preferably has the structural formula of the following formula (1). It can be a compound.

<Formula 1>

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , Sebacate, fumarate, maleate, butin-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, me Toxoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol Rate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

In the present invention,'brain tumor' refers to all tumors occurring in the skull, and includes all tumors occurring in the brain and structures around the brain.

Specifically, the brain tumor is not limited thereto, but anaplastic astrocytomas, glioblastomas, meningiomas, pituitary tumors, schwannomas, central nervous system lymphoma (CNS lymphoma) , Oligodendrogliomas, ependymomas, low-grade astrocytomas, medulloblastomas, astrocytic tumors, pilocytic astrocytoma, Diffuse astrocytomas, pleomorphic xanthoastrocytomas, subependymal giant cell astrocytomas, anaplastic oligodendrogliomas, oligoastrocytomas, inverse Anaplastic oligoastrocytomas, myxopapillary ependymomas, subependymomas, ependymomas, anaplastic ependymomas, astroblastomas ), chordoid gliomas of the third ventricle, gliomatosis cerebris, glangliocytomas, desmoplastic infantile astrocytomas, connective tissue Desmoplastic infantile gangliogliomas, fetal hypoplasia neuroepithelial tumors (d ysembryoplastic neuroepithelial tumors), central neurocytomas, cerebellar liponeurocytomas, paragangliomas, ependymoblastomas, supratentorial primitive primitive neuroectodermal tumors, choroid Choroids plexus papilloma, pineocytomas, pineoblastomas, moderately differentiated pineal parenchymal tumors of intermediate differentiation, hemangiopericytomas, tumors in the saddle area ( tumors of the sellar region), craniopharyngioma, capillary hemangioblastoma, or primary CNS lymphoma.

In the present invention,'brain tumor stem cells', which are a kind of cancer stem cells, are important cells for the growth and metastasis of brain tumors, have the ability to maintain an undifferentiated state, self-renewal and differentiation capabilities, and can generate tumors. In addition, since it is differentiated from other groups and generates new tumors, it is present in a small amount in the cancer, but causes recurrence and metastasis of the cancer, so it can be said to be a key target for brain tumor treatment.

The composition of the present invention may contain a pharmaceutically acceptable additive, and pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, Mannitol, syrup, arabic rubber, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnaubanap, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar Etc. can be used.

The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

In the present invention, the composition may be administered in various oral or parenteral formulations at the time of actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. or It can be formulated using excipients, and suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently Mack Publishing Company, Easton PA. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. In addition, various excipients in addition to water and liquid paraffin, which are commonly used simple diluents, include suspensions, liquid solutions, emulsions, and syrups as liquid preparations for oral administration, such as wetting agents, sweeteners, fragrances, preservatives, etc. May be included.

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories.

As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used. The parenteral administration may be performed using external skin or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, type of infected virus, and Activity, sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field.

The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.

As used herein, the term'administering' means providing a given composition of the present invention to an individual in any suitable way.

In the present invention, the composition may be administered to an individual by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.

In the present invention, the composition may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods of using a biological response modifier to inhibit or kill brain tumor stem cells.

In the present invention,'prevention' means any action that suppresses or delays the onset of brain tumors by administration of the composition.

In the present invention,'improvement' means any action in which symptoms caused by brain tumors are improved or beneficially changed by administration of the composition.

In the present invention,'treatment' refers to all actions of healing a brain tumor by administration of the composition.

In the present invention,'metastasis' refers to a state that spreads from the brain to other tissues such as lungs or stomachs as a brain tumor, which is a malignant tumor, progresses.

In addition, the present invention is a 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound or a pharmaceutically acceptable salt thereof It is possible to provide an adjuvant for treating brain tumors, including as an active ingredient.

In the present invention, the term'brain tumor treatment adjuvant' refers to a material that can be auxiliaryly used to enhance the effect of brain tumor therapy generally used in the art, and by using the mixed extract according to the present invention, It can enhance the effect.

In the present invention, the types of brain tumor therapy that can be used together with the brain tumor adjuvant include all of brain surgery, radiation therapy, chemotherapy using anticancer agents, etc., and a combination of two or more of the above-described treatments Can be More specifically, the brain tumor therapy may be preferably concurrent chemoradiotherapy (CCRT) or a combination therapy of brain surgery and CCRT, but is not limited thereto.

In addition, the brain tumor therapy may be selected under general principles considered when selecting a brain tumor therapy such as the type of cancer cells, the rate of absorption of the anticancer agent (the treatment period and the route of administration of the anticancer agent), the location of the tumor, and the size of the tumor.

Furthermore, the present invention can provide a method for inducing apoptosis of brain tumor stem cells using the compound of the present invention, that is, 6-cyclohexyl-N-(1-phenylethyl)-2, in brain tumor stem cells in vitro. Apoptosis of the brain tumor stem cells may be induced through the step of treating the 3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound.

Therefore, the 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound of the present invention targets brain tumor stem cells to prevent brain tumors. It can be used as a new therapeutic agent for brain tumors, and at the same time, it can be used in various ways as an anticancer auxiliary medicine to increase the anticancer efficiency of brain tumor therapy.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

<Preparation example>

Sample and cell culture

The brain tumor stem cells used in the following examples were 528NS (glioblastoma, Ichiro Nakano, University of Alabama at Birmingham), MD13 (glioblastoma, University of Alabama at Birmingham), GSC11 (glioblastoma, Joshua), which is a brain tumor stem cell line derived from a brain tumor patient. M. Shulman, Texas Children's Hospital), GSC23 (glioblastoma, Joshua M. Shulman, Texas Children's Hospital), and GSC20 (glioblastoma, Joshua M. Shulman, Texas Children's Hospital) were used. Each cell is a brain tumor stem cell culture condition: 1% antimicrobial agent (apenicillin/streptomycin, Lonza, Basel, Switzerland), 0.04% B27 (Invitrogen, MA, USA), epidermal growth factor, 20 ng/mL; R&D Systems, MN, USA) and fibroblast growth factor (basic broblast growth factor, 20 ng/mL; R&D Systems) in DMEM/F12 media (Hyclone, GE healthcare life sciences, IL, USA) culture at 37°C, 5 It was used after incubation in% CO ₂ conditions.

In addition, 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (ChemBridge Corporation, San Diego, CA, USA) compound It was used after dissolving in DMSO to a final concentration of 10mM.

< Example 1>

Of the present invention Carbazole Apoptosis Effect of Derivative Compounds on Brain Tumor Stem Cells

The present inventors believe that 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound, a carbazole derivative compound, is used for brain tumor stem cells. The following experiment was performed to confirm whether there is apoptosis effect.

To this end, 528NS, MD13, GSC11, GSC23, GSC20 cells, which are brain tumor stem cell lines, were dispensed into a 96-well plate (SPL, Korea) at 5,000 cells/well, respectively, and after 24 hours, 6-cyclohexyl-N of the present invention -(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound by concentration (0, 0.05, 0.1, 0.5, 1, 5, 10 μM) After treatment, it was incubated for 3 days. Thereafter, 20 μL of the assay reagent of the Ez-Cytox kit (DOGEN, Seoul, Korea) was added per well and reacted for 4 hours, followed by a PowerWaveXS microplate-reader (Bio-Tek Instruments Inc., VT, USA) device at 450 nm wavelength. By measuring the absorbance using, the degree of apoptosis according to the compound treatment concentration for each cell line was measured. At this time, the measurement value was described as mean ± SEM.

As a result, as shown in FIGS. 1 and 2, the compound of the present invention was found to have apoptosis effect on all five types of brain tumor stem cells, and the survival rate of the cells was not treated with the compound of the present invention. Compared to the treatment of the compound of the present invention was found to be significantly reduced.

Therefore, through these results, the present inventors believe that the carbazole derivative compound 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazole-1-amine hydrochloride compound It was confirmed that brain tumor stem cells have apoptosis ability, and it was found that the compound can be used as a new brain tumor treatment targeting brain tumor stem cells.

< Example 2>

According to the invention Carbazole Derivative compounds in brain tumor stem cells SREBP1 Inhibitory effect

Next, the present inventors analyzed whether the compound of the present invention has inhibitory activity against SREBP1, which plays an important role in lipid metabolism and cancer signaling in brain tumor stem cells.

To this end, the brain tumor stem cell lines 528NS, MD13, GSC11, GSC23, GSC20 were dispensed at 2.0 × 10 ⁶ cells/9 mL, and after 24 hours, the compound of the present invention was diluted in 1 mL of the culture solution to reach a total of 10 mL. After treatment to reach 10 μM, the cells were collected after 72 hours, washed with PBS, and then precipitated at 3500 rpm, and RIPA solution was added to the precipitated cell pellet to lyse the cells, and then precipitated at 15000 rpm to collect only the supernatant and collect the protein concentration. Measured. Protein was transferred to the membrane by loading 15 μg of protein for each sample. Thereafter, the membrane was blocked using 5% skim milk powder, and the primary antibody was reacted at 4°C for 12 hours. Anti-SREBP1 (sc-8984, Santa Cruz Biotechnology, TX, USA), anti-β-actin (sc-47774, Santa Cruz Biotechnology) were used as antibodies. After washing 3 times for 5 minutes each using TBST, a secondary antibody (Vector Laboratories, CA, USA) conjugated to Horseradish peroxidase (HRP) was added and reacted for 1 hour at room temperature, and then 3 times for 5 minutes each using TBST. After washing, the ECL (EBP-1073, ELPIS biotech. Inc., Korea) solution was reacted for 1 minute and then developed by reacting with the film in a dark room.

As a result of the analysis, as shown in FIG. 3, it was confirmed that the protein expression of SREBP1 was suppressed in all five types of brain tumor stem cells treated with the compound of the present invention.

Through these results, the compound of the present invention can be used as a new brain tumor treatment targeting brain tumor stem cells by inhibiting the expression and activity of SREBP1 in brain tumor stem cells, reducing lipid metabolism of brain tumor stem cells, and ultimately inducing apoptosis. And it was found.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (6)

delete delete delete 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound or a pharmaceutically acceptable salt thereof as an active ingredient , A pharmaceutical composition for the prevention or treatment of brain tumors. 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound or a pharmaceutically acceptable salt thereof as an active ingredient , Brain tumor treatment adjuvant. In vitro treatment of brain tumor stem cells with 6-cyclohexyl-N-(1-phenylethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride compound. , A method of inducing apoptosis of brain tumor stem cells.

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