KR20190143297A - A chroman compound having anti-cancer effect and pharmaceutical composition for treating or preventing cancer comprising the same - Google Patents

Abstract

The present invention relates to a chroman compound having anticancer activity and a pharmaceutical composition for preventing and treating cancer comprising the same as an active component. More specifically, the present invention relates to a chroman compound having anticancer activity represented by chemical formula (I), a pharmaceutical composition for preventing and treating cancer, and a pharmaceutical composition for anticancer adjuvant comprising the same.

Description

A chroman compound having anti-cancer effect and pharmaceutical composition for treating or preventing cancer comprising the same}

The present invention relates to a chromman compound having anticancer activity and an effective cancer prevention and treatment pharmaceutical composition comprising the same as an active ingredient.

In the rapidly changing modern society, many modern people are threatened with health by various lifestyle diseases such as cancer, hypertension, angina pectoris, diabetes, obesity, and myocardial infarction due to wrong lifestyles. According to the World Health Organization's statistics, cancer is one of the world's leading mortality rates, threatening the lives of modern people, and the mortality rate from cancer is expected to increase continuously.

In particular, modern people are increasing the incidence of various lifestyle diseases due to excessive stress, lack of exercise, and changes in meat-based eating habits. Among them, according to the National Statistical Office of 2017, colorectal cancer outperforms stomach cancer for the first time since the survey. The number of patients dying from colorectal cancer has surged to the third place. Colorectal cancer is often unrecognized because of its early symptoms, and when it is found, it is already advanced.

On the other hand, despite the development of modern medicine and the development of various medicines, anticancer drugs that have been used until now have limited side effects and resistance due to the formation of side effects and resistance. As an alternative to such synthetic drug side effects, there is increasing interest in the development of anti-cancer drugs derived from natural products based on the pharmacological effects of natural products. However, when natural products are used in high concentrations, not only normal cell toxicity is exhibited, but also various problems such as deterioration of natural biological materials, safety controversy such as detection of carcinogens, and the like have been raised.

Recently, research on the development of high value-added biological materials by applying the radiation technology to improve the extraction rate of the active ingredient and enhancing the physiological activity according to the structural transformation of the active ingredient is underway. Although studies have been reported to increase the activity, change the structure or content of a specific substance (Korean Patent No. 2009-0043706, Korean Patent No. 2008-0046311, etc.), the study on the structural conversion of various compounds due to irradiation There is not much research yet.

Therefore, if the technology to improve the intrinsic physiological activity of the compound by applying irradiation technology is expected to be usefully applied in the related fields of domestic natural biological material use and food / drug development.

Accordingly, one aspect of the present invention is to provide a compound having anticancer activity.

Another aspect of the present invention is to provide a pharmaceutical composition for preventing and treating cancer, comprising the compound as an active ingredient.

Another aspect of the invention to provide an anticancer auxiliary pharmaceutical composition comprising the compound as an active ingredient.

According to one aspect of the present invention, a Chroman compound is provided, which is represented by the following general formula (I) and has anticancer activity.

Formula (I)

(X is independently selected from the group consisting of hydroxy, hydrogen and C _{1- 6} alkyl, hydroxy, and at least one Y is phenyl and the other is independently selected from the group consisting of C _{1- 6} alkyl and hydrogen, n is Is an integer of 1 to 4, and m is an integer of 1 or 2.)

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating cancer, comprising the compound of formula (I).

According to another aspect of the present invention, there is provided a pharmaceutical composition for anticancer assistance, comprising the compound of formula (I).

According to the present invention can be obtained Chromman compound having anticancer activity, and furthermore, it is possible to obtain an effective cancer preventive and therapeutic pharmaceutical composition, and an anticancer auxiliary pharmaceutical composition comprising the compound according to the present invention as an active ingredient. Therefore, such a component may be usefully used as an anticancer composition or as an adjuvant of anticancer therapy during an anticancer treatment process.

1 shows the HPLC chromatography results of irradiated chrysine (1 mg / mL).
Figure 2 shows an LC-MS chromatogram of the compound of Example 1.
3 (a) and 3 (b) show ¹³ C-NMR and ¹ H-NMR of the compound of Example 1, respectively.
4 shows the structure of a compound of Example 1 which is a novel irradiation product.
Figure 5 shows the results of confirming the toxicity of the compound of Example 1 on HT-29 cells by MTT assay.
Figure 6 shows the apoptosis effect of the compound of Example 1 on HT-29 cells. HT-29 cells were stained with Annexin V and PI and the ratio of positive cells in each quadrant was initiated.
FIG. 7 shows the effect of Example 1 compounds on HT-29 cell proliferation based on the identification of morphological changes in cells by Lumascope 620.
8 shows the apoptosis effect of the compound of Example 1 on HT-29 cells. Characteristic apoptotic morphology change was confirmed by fluorescence microscopy using Bis-Benzimide (Hoechst 33258) staining.
Figure 9 shows the apoptosis effect of the compound of Example 1 on HT-29 cells. The mitochondrial membrane potential of the compound of Example 1 was analyzed using JC-1 staining analysis using a fluorescence microscope.
10 shows the intracellular reactive oxygen species (ROS) generation of the compound of Example 1 in HT-29 cells by flow cytometry and fluorescence microscopy using dichloro fluorescein-diacetate (DCFH-DA).
Figure 11 shows the results confirmed by Western blot analysis of the compound of Example 1 in HT-29 cells.

Hereinafter, with reference to the accompanying drawings will be described a preferred embodiment of the present invention. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, a new compound having anticancer activity is provided, and in more detail, the compound of the present invention is a Chromman compound represented by the following formula (I) and having anticancer activity.

화학식(I)

Formula (I)

In the formula (I), X is independently selected from the group consisting of hydroxy, hydrogen and C _{1- 6} alkyl, hydroxy, and at least one Y is phenyl, and the remaining are independently from the group consisting of C _1-6 alkyl and hydrogen Is selected, n is an integer of 1 to 4, m is an integer of 1 or 2.

In this case, when X and / or Y is hydrogen, it means that the carbon position of the Chromman compound is not substituted with a substituent other than hydrogen and hydrogen is present.

Chromman compounds of the invention are preferably substituted phenylchroman compounds, for example substituted 2-phenylchroman compounds.

Exemplary preferred compounds of formula (I) are those wherein n is 3, m is 1 and Y is phenyl in formula (I).

Wherein n is 3. In a more preferred formula (I) compounds Formula (I), wherein to at least one of X is a C _{1- 4} alkyl, hydroxy, and the others are hydroxyl groups, wherein m is 1, Y is phenyl . At this time, a carbon position not specified as a substituent of X and Y means that hydrogen is present without being substituted with another substituent.

For example, the Croman compound represented by the formula (I) may be a compound represented by the following formula (Ia).

화학식 (Ia)

Formula (Ia)

The compound of formula (I) of the present invention may be synthesized by any synthetic process well known in the art.

For example, the Chroman compound represented by the formula (I), for example, the compound represented by the formula (Ia), may be obtained as a radiation product of chrysin by irradiation with Chrysin. .

In this case, the radiation that can be used in the present invention may be selected from the group consisting of gamma rays, electron beams and X-rays, preferably gamma rays.

In addition, the gamma rays may be emitted from any one radioisotope selected from the group consisting of cobalt (Co) -60, krypton (Kr) -85, strontium (Sr) -90, and cesium (Cs) -137, Preferably, the gamma ray may be cobalt (Co) -60, but is not limited thereto.

The radiation is irradiated below a dose at which the compound (s) exhibiting an improved anticancer effect due to a change in the structure of the chrysine and below the dose at which all the radiation products generated from the chrysin are degraded, e.g. 10 It is preferable to irradiate at a dose of 100 kGy or more, more preferably at a dose of 50 or more and 100 kGy, more preferably at a dose of about 50 kGy.

Chromane compound represented by the formula (I), for example, the compound represented by the formula (Ia) is a liquid chromatography, radiation (preparative-HPLC, LC-MS, NMR, etc.) after irradiation with Can be obtained separately through the process.

At this time, the step of irradiating the radiation may be performed at a temperature of room temperature.

The step of irradiating the radiation may be performed on the chrysine solution subsequent to dissolving the chrysine in an organic solvent to prepare the chrysine solution.

More specifically, a method for producing a radiation product of chrysine having anticancer activity, such as a Chroman compound represented by the above formula (I), for example, a compound represented by the above formula (Ia), is provided with Dissolving to prepare a crysine solution; And irradiating radiation to the chrysine solution.

The organic solvent that can be used at this time is preferably methanol, ethanol or a mixture thereof.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating cancer and a pharmaceutical composition for adjuvant anticancer, which comprises the Chroman compound of formula (I) of the present invention.

In the present invention, "administration" means providing the patient with the desired substance in any suitable way, and the route of administration of the composition of the present invention is oral or parenteral via all common routes as long as the target tissue can be reached. Oral administration.

"Patient" in the present invention means a human and all other animals with a disease that can improve symptoms by administering the composition of the present invention. The composition according to the invention can be applied to humans (treatment, inhibition or prevention) as well as to other commercially useful animals.

In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective amount level refers to the type, severity, and drug of the patient's disease. Activity, sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.

The pharmaceutical composition for preventing and treating cancer according to the present invention comprises a compound of formula (I), and may alternatively include a pharmaceutically acceptable salt thereof, and administering such pharmaceutical composition to a patient to Inhibitory, prophylactic and therapeutic effects of cancer metastasis can be obtained.

The pharmaceutical composition for preventing and treating cancer may be for the treatment of cancer selected from the group consisting of skin cancer, lung cancer, leukemia, breast cancer, gastric cancer, and colorectal cancer, and is preferably for treating colorectal cancer.

Furthermore, the composition of the present invention can be administered in parallel with other conventional cancer therapeutic agents, and from this aspect, the present invention provides an anticancer adjuvant pharmaceutical composition comprising the compound of formula (I) as an active ingredient.

The anticancer adjuvant pharmaceutical composition may have anticancer adjuvant activity against cancer selected from the group consisting of skin cancer, lung cancer, leukemia, breast cancer, stomach cancer and colorectal cancer, preferably the anticancer adjuvant pharmaceutical composition of the present invention is colorectal cancer It is an auxiliary pharmaceutical composition.

The pharmaceutical composition for preventing and treating cancer of the present invention and the pharmaceutical composition for supplementing cancer support are oral formulations of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It may be formulated and used in various forms, such as in the form of a sterile injectable solution, and may be administered orally or through various routes including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration.

Examples of suitable carriers, excipients and diluents that may be included in such cancer preventive and therapeutic pharmaceutical compositions and anticancer adjuvant pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. .

In addition, the pharmaceutical composition for preventing and treating cancer and the pharmaceutical composition for supplementing cancer may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which solid preparations include at least one excipient, for example, starch, in the pharmaceutical composition for preventing and treating cancer and the pharmaceutical composition for supplementing cancer. It can be formulated by mixing calcium carbonate, sucrose, lactose and gelatin. In addition to the simple excipients, lubricants such as magnesium stearate, talc can also be used.

Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Bases for injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

By administering such a composition of the present invention to a patient, cancer and cancer metastasis can be inhibited, prevented and treated, and the cancer preventive and therapeutic pharmaceutical composition and the anticancer pharmaceutical composition of the present invention are administered in a pharmaceutically effective amount. .

The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

In particular, the pharmaceutically effective amount of the compound according to the present invention may vary depending on the age, sex and weight of the patient, and generally 1 to 50 mg, preferably 1 to 10 mg per 1 kg body weight daily or every other day. It may be administered as or divided into 1 to 3 times a day.

However, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

Hereinafter, the present invention will be described in more detail with reference to specific examples. The following examples are merely examples to help understanding of the present invention, but the scope of the present invention is not limited thereto.

Example

Experimental Example  One: Croman  Preparation of the compound

After making a crysin solution by dissolving chrysin powder at a concentration of 1 mg / mL using methanol as a solvent, gamma-rays were irradiated to chrysine at room temperature using a gamma irradiation facility installed in the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute. Was irradiated with 50 kGy, and the comparative example 1 was made into the chrysine which was not irradiated with gamma rays (0 kGy).

 As a result of checking the HPLC peak of the compound with 50 kGy of gamma ray on the chrysine, as shown in FIG. 1, it was observed that the production of two radioactive peaks (Radiolytic peak) was increased. LC-MS was measured to determine the molecular weight of the compound (Example 1 compound) increased from the front, and found to be 299.08334 (FIG. 2). In this case, compounds other than the compound of Example 1 are referred to as Comparative Example 2 compound.

In addition, ¹ HNMR and ¹³ CNMR were performed to identify the exact structure of the compound of Example 1, and these results were shown as follows (FIGS. 3 (a) and 3 (b)).

¹ HNMR (500MHz, DMSO-d6): 11.8 (1H, s), 7.4 (2H, d), 7.4 (2H, d, J = 8Hz), 7.2 (2H, d, J = 8.7Hz), 5.9 ( 1H, s), 5.8 (1H, s), 5.1 (1H, Br), 4.6 (1H, Br), 3.7 (3H, q, J = 8.6 Hz), 3.3 (3H, m).

¹³ CNMR (125 MHz, DMSO-d6): 197.9, 166.9, 163.3, 160.8, 141.5, 139.8, 128.6, 127.8, 126.8, 101.4, 96.0, 86.4, 66.2, 60.0, 52.6.

Based on these results, the structure of the compound of Example 1 of the present invention as shown in FIG. 4 was identified.

The chemical formula of the compound is represented by C ₁₇ H ₁₆ O ₅ , the chemical name is referred to as 5,7-dihydroxy-3- (2-hydroxyethyl) -2-phenylchroman -4-one, the compound of Example 1 in the present specification Also called (Ia) compound.

Experimental Example  2: Example  Toxicity Assessment of Colorectal Cancer Cell (HT-29)

The compound of Example 1 obtained in Experimental Example 1 was fractionated into a gradient program as described below using a preparative HPLC 1260 infinity system (Agilent Technologies) to study anticancer activity. Proceeded: 0-5 min (20% B), 12 min (40%), 25 min (55%), 30 min (80%) 38 min (80%)

Treatment of HT-29 cells, which are colon cancer cell lines isolated from humans, with Example 1 compound and Comparative Example 1 compound at various concentrations (12.5, 25, 50, and 100 μg / mL), respectively, was carried out to determine cell viability by MTT method after 24 hours. saw.

As a result, as shown in FIG. 5, all cytotoxicity was increased in a concentration-dependent manner, and when the compound of Example 1 was treated, it was confirmed that the cytotoxicity was increased the most (FIG. 5). In FIG. 5, the control means a case in which nothing is treated in HT-29 cancer cells.

Furthermore, after treating Example 1 compound and Comparative Example 1 compound with various concentrations (50, 100 μg / ml) in HT-29 cancer cells, respectively, and using a flow cytometer (FACS) equipment to evaluate toxicity (Anext V / PI apoptosis detection was performed. As a result, as shown in FIG. 6, when HT-29 cells were treated with the compound of Example 1, it was confirmed that apoptosis was significantly induced as compared with the compound of Comparative Example 1.

In addition, as a result of observing the shape of the cells through a microscope, it was possible to visually observe that more cells died when the Example 1 compound was treated (FIG. 7).

In conclusion, the compound of Example 1 significantly increases the cytotoxicity against cancer cells, and this study is expected to be used as a basic data suggesting the possibility of irradiation technology in terms of new biomaterials and drug development. .

Experimental Example  3: Example  Colon cancer cell (HT-29) of 1 compound Apoptosis ( Apoptosis Induction effect

(One) Example  Colon Cancer Cells by Compound 1 (HT- 29)  Observe nuclear changes

In order to observe nuclear changes by the compound of Example 1 and Comparative Example 1, it was observed using the Hoechst 33258 reagent. In order to observe the morphological changes indicative of cell death when HT-29 cells were treated with Example 1 compound and Comparative Example 1 compound, 50 and 100 μg / ml of Example 1 compound and Comparative Example 1 compound were respectively added to HT-29 cells. 29 cells were treated. After culturing for 24 hours, the nuclei were stained with a Hoechst 33258 reagent that specifically binds to DNA, and the change of the nucleus was confirmed through confocal laser microscopy (FIG. 8).

As a result, as shown in FIG. 8, more damaged cell nuclei could be observed in the compound treated in Example 1, and cell death was confirmed to decrease the number of cells. Therefore, it can be confirmed morphologically that the compound of Example 1 is more effective in inducing apoptosis.

(2) Example  Mitochondria of HT-29 Cells Treated with Compound 1 Membrane potential  change

Mitochondria in cells synthesize ATP necessary for cells to nourish the cells and play an important role in maintaining the life of cells. Therefore, the role of mitochondria in cancer cell proliferation is very important, and various studies on mitochondria have been conducted in relation to cancer cell suicide.

In the present experiment, the Example 1 compound and the Comparative Example 1 compound were treated at 50 and 100 μg / ml, respectively, on the HT-29 colorectal cancer cells based on the above result that the compound of Example 1 inhibited the proliferation of HT-29 colorectal cancer cells. Membrane potential change of the mitochondria in the cell that plays an important role in cancer cell suicide was observed by fluorescence microscope through JC-1 fluorescence staining (Fig. 9).

As a result, it was confirmed that the cells treated with the compound of Example 1 showed more bright green fluorescence. This result can be analyzed that the membrane potential of the mitochondria of the cells treated with the compound of Example 1 was lowered in a concentration-dependent manner, and the suicide of the cells was progressing.

(3) Example  1 Treatment of Compounds in HT-29 Cells ROS Of Reactive Oxygen Species

Example 1 Compounds and Comparative Example 1 Compounds were stained with DCFH-DA (2 ', 7'-dichlorofluorescein-diacetate) reagent to evaluate ROS production in colorectal cancer cell lines HT-29 cells, and then measured using a fluorescence microscope. It was.

10 is a result of observing ROS expression of HT-29 cells in the form of an image through a fluorescence microscope and measuring the intensity of fluorescence. As a result, it was observed that the concentration of ROS fluorescence increased in the cells treated with the compound of Example 1. In conclusion, it can be seen that the compound of Example 1 has an effect on causing apoptosis by increasing ROS production of HT-29 cells.

Experimental Example  4: Example  1 colorectal cancer cell (HT-29) Apoptosis  Mechanism

Example 1 In order to investigate the mechanism of HT-29 cell death of the compound, the expression of proteins involved in various mechanisms of cell death was measured by Western blotting method and the results are shown in FIG. 11. As a result, as shown in FIG. 11, the expression of Bcl related to apoptosis in the mitochondria and the activation of Bax resulted in the apoptosis of HT-29 cells in association with the mitochondria, an intracellular organelle, in Example 1 compound. It is thought to promote this. Increasing cytochrome C released in the mitochondria activates caspase-9, an initiation factor for apoptosis, thereby increasing caspase-3, an early executive, leading to cleavage of PARP, the most important factor. It can be seen that the cells die. In conclusion, the signaling mechanism of anticancer activity of the compound of Example 1 was established.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and changes can be made without departing from the technical spirit of the present invention described in the claims. It will be obvious to those of ordinary skill in the field.

Claims (8)

Chromman compounds represented by the following formula (I) and having anticancer activity:

Formula (I)
In the above formula (I),
X is independently selected from the group consisting of hydroxy, hydrogen and C _{1- 6} alkyl, hydroxy,
Is at least one of Y is phenyl and the other is independently selected from the group consisting of C _{1- 6} alkyl and hydrogen,
n is an integer of 1-4, m is an integer of 1 or 2.
The chroman compound according to claim 1, wherein n is 3, m is 1, and Y is phenyl.
The method of claim 1 wherein n is 3, and wherein at least one X is a C _{1- 4} alkyl, hydroxy, and the others are hydroxyl groups,
M is 1, wherein Y is phenyl, Chromman compound having anticancer activity.
The Kroman compound having an anticancer activity according to claim 1, wherein the Kroman compound represented by the formula (I) is represented by the following formula (Ia).

Formula (Ia)
A pharmaceutical composition for preventing and treating cancer, comprising the Chroman compound of any one of claims 1 to 4.
The pharmaceutical composition for preventing and treating cancer of claim 5, wherein the cancer is a cancer selected from the group consisting of skin cancer, lung cancer, leukemia, breast cancer, stomach cancer, and colorectal cancer.
An anticancer adjuvant pharmaceutical composition comprising the Chroman compound of any one of claims 1 to 4.
The pharmaceutical composition of claim 7, wherein the anticancer adjuvant pharmaceutical composition has anticancer adjuvant activity against cancer selected from the group consisting of skin cancer, lung cancer, leukemia, breast cancer, gastric cancer, and colorectal cancer.

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