KR100842351B1 - Pharmaceutical composition for the prevention and treatment of cancers containing indole derivatives as an active ingredient - Google Patents

Abstract

A pharmaceutical composition containing indole derivatives is provided to inhibit activity of HIF-1(hypoxia inducible factor-1) as a transcription factor induced hypoxia, so that the composition is useful for the prevention and treatment of cancers including liver cancer, stomach cancer and breast cancer. A pharmaceutical composition for the prevention and treatment of cancers contains indole derivatives represented by the formula(1) or pharmaceutically acceptable salts thereof, wherein R is COORa, CONRbRc or C1-C4 alkyl group; Ra is H or C1-C4 alkyl; and Rb and Rc are each independently C1-C5 alkyl group substituted by C3-C5 heteroaryl group containing N, O or S or C3-C5 heterocycle group containing N, O or S, H or C1-C5 alkyl group. Further, the pharmaceutical composition is used as a pharmaceutical composition for treating diabetic retinopathy or rheumatoid arthritis.

Description

Pharmaceutical composition for the prevention and treatment of cancers containing indole derivatives as an active ingredient

1 illustrates the inhibition of HIF-1a accumulation under hypoxic conditions of a compound according to an embodiment of the present invention.

Figure 2 shows the degree of inhibition of VEGF expression in hypoxic conditions of the compound according to an embodiment of the present invention,

3 shows in vivo anticancer activity of nude mice of the compound according to one embodiment of the present invention.

The present invention relates to a pharmaceutical composition for preventing and treating cancer containing an indole derivative as an active ingredient. In particular, it can be used as a therapeutic agent for various cancer diseases such as liver cancer, gastric cancer and breast cancer by inhibiting HIF-1 activity, and is effective for treating diabetic retinopathy or arthritis, which is exacerbated by an increase in expression of VEGF by HIF-1 in hypoxia. It relates to a pharmaceutical composition for preventing and treating cancer containing an indole derivative which can be used as an active ingredient.

Cancer remains one of the incurable diseases, despite human endeavors over the past decades. Recently, anti-cancer drugs with new mechanism of action such as Gleevec have been developed along with remarkable advances in various fields such as cancer cell biology and medical chemistry, and new target molecules have emerged since the Human Genome Project. Hypoxia Inducible Factor-1 (HIF-1) is a transcription factor induced in hypoxia and consists of a HIF-1a subunit that is oxygen-dependently degraded and a heterodimer that is constantly expressed. ( Cancer Metastasis Rev. , 17, 187-195, 1998; Trends Mol . Med . , 7, 345-350, 2001).

Under normal oxygen concentration conditions, HIF-1a protein is oxygenated to proline moieties of 402 and 564 proline residues to bind ubiquitin and proteasome by binding to the cancer suppressor gene pVHL (Von Hippel-Lindau) protein. But in a hypoxic state, this series of reactions is inhibited and HIF-1a protein accumulates and binds to the existing HIF-1b protein and moves to the nucleus ( Science , 292, 468-472, 2001; Science ; , 292, 468-472, 2001). The stability of HIF-1a is influenced by factors involved in the oxygen sensing pathway in addition to the partial pressure of oxygen. These factors include transition metal ions, iron chelators or Antioxidants; and the like. In addition, HIF-1a protein may be expressed in epidermal growth factor, heregulin, insulin-like growth factor-I and insulin-like growth factor-II irrespective of oxygen concentration. It may also accumulate by the same growth factor or by activation of oncogenes such as ErbB2. When these growth factors bind to their respective receptors, PI3K-AKT and MAPK signaling pathways are activated to increase HIF-1a protein synthesis and accumulate HIF-1a protein.

HIF transferred to the nucleus binds to HRE (Hypoxia Responsive Element, 5'-ACGTG-3 ') on the promoter of the target gene to induce the expression of the gene. More than about 60 species are known to date, including vascular endothelial growth factor (VEGF) ( Nat . Rev. Cancer , 2, 38-47, 2002; J. Biol . Chem . , 278, 19575-19578, 2003; Nat , Med . , 9, 677-684, 2003; Biochem . Pharmacol . , 64, 993-998, 2002).

Hypoxia in cancer, especially solid cancer, is a common phenomenon, and solid cancer cells undergo various genetic changes to adapt to these hypoxic conditions, making them more malignant and resistant to anticancer agents. It is known as a major inducer of malignancy in more than 70% of species ( Nature , 386, 403, 1997; Hockel M. and Vaupel P., Semin . Oncol . , 28, 36-41, 2001; Nature Med . , 6, 1335, 2000; Bos et al. Cancer , 97, 1573-1581, 2003). HIF-1 is known to be the most important molecule that controls the adaptation of cancer cells to this hypoxic state, and the amount of HIF-1a protein is known to have a close correlation with the prognosis of cancer patients. HIF-1 activated by cancer cells by hypoxic conditions or by stimulation of growth factors or oncogenes as mentioned above, or by inactivation of cancer suppressor genes such as pVHL, is known as hexokinase 2, Induces the expression of genes such as glucose transporter 1, erythropoietin, IGF-2, endoglin, VEGF, MMP-2, uPAR, MDR1 and so on apoptosis Tolerance, increase in angiogenesis, increase in cell proliferation, increase invasion (invasion), and the like to acquire traits and eventually cancer cells become malignant. Therefore, thus HIF cancer, and especially the growth of solid tumors, because it plays an important role in malignant proliferation and research to develop targeted cancer drugs in this proceeding very actively (Cancer Res . , 62, 4316, 2002; Nat . Rev. Drug . Discovery , 2, 1, 2003; Semenza et al. Nature Reviews Cancer , 3, 721-732, 2003). Recently, a large number of known anticancer agents such as taxol, rafamycin or 17-allylaminogeldanamycin (17-AAG) or the small molecule compound YC-1 which is a guanylaly cyclase activator (3- (5'-hydroxymethyl-2'-furyl) -1-benzylindazole, 3- (5'-hydroxymethyl-2'-furyl) -1-benzylindazole) are HIF-1 inhibitors that have several steps In clinical trials (Johnson et al. Nature Reviews Drug Discovery , 2, 1-9, 2003; Semenza et al. Nature Reviews Cancer , 3, 721-732, 2003; JNCI., 95, 516, 2003 ), reporter analysis in the one-cell stage utilizing HRE (cell based reporter assay), but with the progress also actively HIF-1 inhibitors, development of a new structure (Cancer Res . , 65, 4918, 2005; Cancer Cell . , 6, 33, 2004; Cancer Res . , 62, 4316, 2002), still at an early stage.

On the other hand, HIF-1 can be used as a target for the development of therapeutic agents for diseases in which not only cancer but also activation of angiogenesis is associated with worsening of the disease. Angiogenesis factors, such as VEGF, derived from HIF-1 that are activated in hypoxia, are associated with the development of diabetic retinopathy and arthritis as well as cancer. Thus, compounds that inhibit HIF-1, which is activated from hypoxia of diseased tissue, could be used as novel therapeutics for diseases such as diabetic retinopathy or rheumatoid arthritis (Eiji Ikeda, Pathology International , 55, 603-610, 2005). But this area is still in its infancy.

Therefore, the inventors of the present invention, while studying a new substance of indole derivatives, found that the indole derivative of the new structure can inhibit HIF-1, a transcription factor induced in hypoxia, and the indole derivatives of the present invention are liver cancer, stomach cancer, breast cancer. The present invention has been completed by discovering that it can be used as an active ingredient for treating various cancer diseases and diabetic retinopathy or arthritis.

An object of the present invention to provide a pharmaceutical composition for preventing and treating cancer containing an indole derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition containing an indole derivative represented by Formula 1 and their pharmacologically acceptable salts as an active ingredient.

Where

R is any one selected from the group consisting of COOR _a , CONR _b R _c and C ₁ ~ C ₄ alkyl group, wherein R _a is any one selected from the group consisting of H and C ₁ ~ C ₄ alkyl group, wherein R _b and R _c are as any one or more are each independently selected from N, O or S, C ₃ ~ C ₅ selected from the heteroaryl group, and N, C ₃ ~ C ₅ heterocyclic group consisting of the cycle group containing O or S containing Substituted C ₁ -C ₅ alkyl group; H and any one selected from the group consisting of C ₁ to C ₅ alkyl group,

는 단일 또는 이중 결합이다.remind

Is a single or double bond.

The derivative of Formula 1 used as an active ingredient of the pharmaceutical composition for preventing and treating cancer according to the present invention

1) 3- (3-1 H -indol-3-yl-propionylamino) -benzamide;

2) 3- (3-1 H -indol-3-yl-acryloylamino) -benzamide;

3) 3- (3-1 H -indol-3-yl-propionylamino) -benzoic acid methyl ester;

4) 3- (3-1 H -indol-3-yl-acryloylamino) -benzoic acid methyl ester;

5) 3- (3-1 H -indol-3-yl-propionylamino) -benzoic acid;

6) 3- (3-1 H -indol-3-yl-acryloylamino) -benzoic acid;

7) 3- (3-1 H-indol-3-yl-amino-acryloyl) - N - (2- piperidin-1-yl-ethyl) -benzamide;

8) 3- (3-1 H-indol-3-yl-amino-acryloyl) - N - (3- morpholin-4-yl-propyl) -benzamide;

9) N -furan-2-ylmethyl-3- (3-1 H -indol-3-yl-acryloylamino) -benzamide.

Table 1 summarizes the structure and R of the derivative of Chemical Formula 1.

Chemical rescue R Coupling Form   One

CONH ₂   Single bond   2

CONH ₂   Double bond   3

COOCH ₃   Single bond   4

COOCH ₃   Double bond   5

  COOH   Single bond   6

  COOH   Double bond   7

  Double bond   8

  Double bond   9

  Double bond

In addition, the indole derivative represented by Formula 1 according to the present invention may be used in the form of a pharmaceutically acceptable salt. As pharmaceutically acceptable salts, addition salts formed with acceptable free acids are useful. Suitable free acids may be organic and inorganic acids, inorganic acids may be hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid, and organic acids may be citric acid, acetic acid, lactic acid, tartariac acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Etc. can be used.

Furthermore, the indole derivative represented by Formula 1 according to the present invention may include all salts, hydrates and solvates that can be prepared by conventional methods, as well as pharmaceutically acceptable salts.

Indole derivatives of Formula 1 used in the use according to the invention can be synthesized as shown in Scheme 1 below. This is outlined as follows.

(In the above scheme, R is as defined in Formula 1, wherein the compound of Formula 1a is included in the indole derivative of Formula 1 of the present invention.)

Specifically, 3- ( 1H -indol-3-yl) propionic acid (2) or 3- ( 1H -indol-3-yl) -acrylic acid (2 which is commercially available or readily prepared by known methods ) And amines (3), either commercially available or synthesized by known methods, are prepared using benzodthiazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), ethylenedichloride (EDC) and N Compound (1a) is reacted at room temperature in the presence of at least one condensation reagent selected from the group consisting of hydroxybenzotriazole (HOBt), dimethylformamide and diisopropylethylamine. It can manufacture.

When R is COOR _a in Scheme 1, Scheme 2 may be further performed. The following compounds 1b, 1c and 1d correspond to the derivatives of Chemical Formula 1.

(Wherein R _a , R _b and R _c are as defined in Formula 1, and derivatives of Formulas 1b to 1d are included in indole derivatives of Formula 1 of the present invention.)

Specifically describing the reaction step is as follows.

1) Purchasable with 3- ( 1H -indol-3-yl) propionic acid (2) or 3- (1H-indol-3-yl) -acrylic acid (2), commercially available or readily prepared by known methods The amine (4), which is possible or synthesized by known methods, is prepared by benzodthiazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP), ethylenedichloride (EDC) and N-hydroxy Compound (1b) may be prepared by reacting at least one condensation reagent selected from the group consisting of benzotriazole (HOBt) with dimethylformamide and diisopropylethylamine at room temperature.

2) The corresponding carboxylic acid compound (1c) can be prepared by reacting the carboxy ester compound (1b) at room temperature under a mixed solution of an inorganic base such as lithium hydroxide, tetrahydrofuran, dioxane, methanol or ethanol and water. have.

3) Carboxylic acid compound (1c) was added to O- (benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate in the presence of dimethylformamide and diisopropylethylamine. HBTU) and O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (HATU) and a condensation reagent selected from the group consisting of Reaction with room temperature or reflux can produce the corresponding amide compound (1d).

Hereinafter, the use of the derivative of Formula 1 or a pharmaceutically acceptable salt thereof will be described in detail.

First, the present invention provides a pharmaceutical composition for preventing and treating cancer, which contains an indole derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The indole derivative represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof does not exhibit anticancer activity by non-selective cytotoxicity, but is a transcription factor HIF-1 that plays an important role in the growth and metastasis of cancer cells. It inhibits activity and shows anticancer activity.

The inhibition of HIF-1 activity includes all of a series of phenomena that inhibit transcriptional activity, inhibit HIF-1 protein expression, or inhibit accumulation of HIF-1 protein.

Indole derivatives of the present invention measured the effect of the derivatives of the present invention on HIF-1-mediated transcriptional activation induced by hypoxic conditions. You can see that it has. Therefore, the indole derivative of the present invention can be used as an active ingredient of an anticancer agent because it can inhibit the growth and metastasis of cancer by inhibiting the expression of genes associated with the malignancy of cancer expressed by HIF-1.

The derivative according to the present invention can inhibit the production of HIF-1a protein in a concentration dependent manner without affecting the production of topoisomerase-1 (TOPO-1) under hypoxic conditions (see Example 2). Therefore, the derivative of the present invention does not exhibit anticancer activity due to non-selective cytotoxicity, but can inhibit the growth and metastasis of cancer by concentration-dependently inhibiting accumulation of HIF-1a protein, and thus exhibit side effects while exhibiting anticancer activity. Can be minimized.

In addition, the indole derivative according to the present invention can inhibit the expression of VEGF in a concentration-dependent manner without affecting the expression of the control gene GAPDH in hypoxic conditions (see Example 3). Therefore, the compound of the present invention can be used as an active ingredient of an anticancer agent because it has a selective expression inhibitory effect on VEGF, which is a target gene of HIF-1, and can inhibit cancer growth and metastasis.

Therefore, the pharmaceutical composition containing the indole derivative of the formula (1) of the present invention, or a pharmaceutically acceptable salt thereof as an active ingredient inhibits HIF-1 activity, thereby inhibiting liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, tofu or Cervical cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulva carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, It can be used as a therapeutic agent for various cancer diseases such as ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor.

In addition, the present invention provides a pharmaceutical composition showing a therapeutic effect of diabetic retinopathy or arthritis through the inhibition of HIF-1 activity containing the indole derivative of Formula 1, or a pharmaceutically acceptable salt thereof as an active ingredient do.

The inhibition of HIF-1 activity includes all of a series of phenomena that inhibit transcriptional activity, inhibit HIF-1 protein expression, or inhibit accumulation of HIF-1 protein.

HIF-1 can be used as a target for the development of therapeutic agents for diseases in which activation of angiogenesis is associated with exacerbation of the disease. In particular, angiogenic factors such as VEGF induced by HIF-1 activated in the hypoxic state are associated with the development of arthritis such as diabetic retinopathy or rheumatoid arthritis. Diabetic retinopathy or arthritis may be exacerbated by increased expression of VEGF by HIF-1 in hypoxia. Therefore, the compound that inhibits HIF-1 activated from the hypoxic state of diseased tissue is utilized as a therapeutic agent for diabetic retinopathy or arthritis (Eiji Ikeda, Pathology International , 55, 603-610, 2005).

As shown in Example 3, the compound of the present invention can inhibit the expression of VEGF in a concentration-dependent manner without affecting the expression of the control gene GAPDH in hypoxic conditions. Accordingly, the compound of the present invention has a selective expression inhibitory effect on VEGF, a target gene of HIF-1, and is an active ingredient of a therapeutic agent for diabetic retinopathy or arthritis, which is exacerbated by an increase in VEGF expression by HIF-1 in a hypoxic state. Can be used.

The pharmaceutical compositions of the invention can be formulated in a variety of oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations may contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose). Rose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally such as starch, agar, alginic acid or its sodium salt. Disintegrant or boiling mixtures and / or absorbents, colorants, flavors, and sweeteners.

The pharmaceutical composition comprising the indole derivative represented by Formula 1 according to the present invention as an active ingredient may be administered parenterally, and the parenteral administration may be by injection of subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. . At this time, in order to formulate into a formulation for parenteral administration, the derivative of Formula 1 or a pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or a buffer to prepare a solution or suspension, which is in ampule or vial unit dosage form. It can manufacture. The compositions may contain sterile and / or preservatives, stabilizers, hydrating or emulsifying accelerators, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating It may be formulated according to the formulation or coating method. As an active ingredient, the derivative of formula 1 may be used orally once a day or in divided doses in an amount of 0.1 to 500 mg / kg body weight, preferably 0.5 to 100 mg / kg body weight, for mammals including humans. Or by parenteral routes.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> HIF By Mediated  Transcription Inhibition Measurement Experiment

In order to measure the degree of inhibition of HIF-1 transcriptional activity of the indole derivative of Formula 1 of the present invention, the following experiment was performed.

A complex cloning (HRE (Hypoxia Responsive Element, 5'-ACGTG-3 ') present in the human VEGF gene is repeated six times in a pGL3-basic vector (promega) using luciferase as a reporter. pGL3-HRE-luciferase vector was prepared and used in the multi-cloning site.

Hep3B cells (ATCC, American Type Culture Collection), which are human liver cancer cell lines, were grown to about 70% in the cell culture vessel, and then internal control vectors (pRL) were prepared using Lipofectamine Plus Reagent (Invirogen). -CMV, Promega) and pGL3-HRE-luciferase vector were transfected together. At this time, the compound of Formula 1 was treated at a concentration of 10 μM. After 48 hours of incubation, each compound of Formula 1 was treated, and 16 hours of incubation under hypoxic conditions (1% oxygen, 94% nitrogen and 5% carbon dioxide), followed by a dual-luciferase reporter assay system (Dual-luciferase reporter assay). HIF-1 inhibitory activity of the compound of Formula 1 was measured by measuring the activity of luciferase induced under hypoxic conditions using the system, manufactured by Promoga. The activity of the luciferase was measured for 10 seconds using a luminometer (Microlumat Plus luminometer-EG & G Berthold). Meanwhile, renilla luciferase activity of the control vector pRL-CMV (promega) was measured and corrected. The measurement results are shown in Table 2 below.

The remaining percentage of HIF activity upon compound treatment was calculated by correction based on the activity of Renilla luciferase. YC-1 (AG Scientific Inc. San Diego, Calif.), Commercially available as a control compound, was tested in the same manner as above, and the results are shown together in Table 2 above.

% Remaining HIF activity upon compound (10 μM) treatment compound HIF% (10 μM) One 111.9 2 86.9 3 92.0 4 64.3 5 104.7 6 58.2 7 44.9 8 81.9 9 20.9 YC-1 84.8

As shown in Table 2 , as a result of measuring the effect of the derivatives of the present invention on HIF-1 mediated transcriptional activation induced in hypoxic conditions, the derivatives of the present invention inhibited HIF-1 transcriptional activity in cancer cell lines. We can see that we have a degree. Therefore, the derivative of the present invention can be used as an active ingredient of an anticancer agent because it can inhibit the growth and metastasis of cancer by inhibiting the expression of genes related to the malignancy of cancer expressed by HIF-1.

< Example  2> Hypoxia  In the state HIF -1a accumulation inhibition measurement

In Example 1, the inhibition of HIF-1a accumulation was measured in AGS cells, gastric cancer cell lines, Hep3B cell lines, or HepG2 cell lines, which showed strong HIF transcriptional activity inhibition levels.

Specifically, inhibition of HIF-1a accumulation of the compound prepared in Compound 6 was measured in the following method in HepG2 cell line, a liver cancer cell line. The effect of compound 6 on inhibiting HIF-1a protein production induced by hypoxic conditions was measured using Western blot analysis. In the cell culture vessel, HepG2 cells (ATCC, American Type Culture Collection), which are human liver cancer cell lines, were grown to about 70%. Compound 6 was dissolved in dimethyl sulfoxide (DMSO) solvent and treated to liver cancer cell lines at various concentrations. In addition, only DMSO solvent was treated to liver cancer cell line without the addition of compounds in a control experiment (indicated by 'DMSO' in FIG. 1 ). Liver cancer cell lines were incubated for 12 hours under hypoxic conditions (1% oxygen and 94% nitrogen and 5% carbon dioxide, labeled '1% O ₂ ' in FIG. 1 ), followed by NE-PER reagent (NE-PER reagent, Pierce, Inc.). Product) to prepare a nuclear extract. About 30 μg of each sample of the nuclear extract was separated by SDS PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), transferred to a polyvinylidene fluoride (PVDF) membrane, and HIF-1a antibody (manufactured by R & D System Co., Ltd.). The amount of HIF-1a protein was detected using a secondary antibody (Amersham-Pharmacia) labeled with) and (horseradish peroxidase, HRP). To reconfirm that each PVDF membrane contains the same amount of nuclear extract, remove the HIF-1a antibody using a buffer solution containing 2-mercaptoethanol from the membrane used for detection of HIF-1a, and again The amount of topoisomerase-1 was detected using topoisomerase-1 (topoisomerase-1, indicated as 'TOPO-1' in FIG. 1) antibody (manufactured by Santa Cruz). The results are shown in Fig.

As shown in FIG. 1 , it can be seen that Compound 6 inhibits the production of HIF-1a protein in a concentration-dependent manner without affecting the production of topoisomerase-1 (TOPO-1) under hypoxic conditions. It can be seen that the inhibitory effect of HIF-1 transcriptional activity on hypoxic conditions of the derivative compounds of the present invention is due to the inhibition of HIF-1a protein production induced in hypoxic conditions. On the other hand, HIF-1a protein is one of the proteins constituting HIF-1 plays an important role in the expression of HIF-1 target genes. Therefore, the derivative of the present invention can be used as an active ingredient of an anticancer agent because it can inhibit the growth and metastasis of cancer by concentration-dependently inhibit the accumulation of HIF-1a protein important in cancer growth and metastasis.

< Example  3> Hypoxia  In the state HIF -1 target gene VEGF  Expression of the present invention Compound Impact measurement

In order to confirm the HIF-1 inhibitory activity of the derivatives according to the present invention, the extent to which the derivatives inhibit the expression of VEGF, a representative target gene of HIF-1, was measured. VEGF is one of the target genes of HIF-1 and is an important angiogenesis factor for cancer growth and cancer metastasis to other tissues. Specifically, the degree of inhibiting the expression of VEGF by the derivative of the present invention was measured in AGS cells, gastric cancer cell lines, Hep3B cells and HepG2 cells. Compound 6 was used as a derivative, and HepG2 cells, which are liver cancer cell lines, were measured as follows.

HepG2 cells (ATCC, American Type Culture Collection), which are human liver cancer cell lines, were grown to about 70% in the cell culture vessel, and then Compound 6 was treated at various concentrations (0 μM, 1 μM, 3 μM, and 10 μM), and hypoxic conditions ( After 12 hours of incubation under 1% oxygen, 94% nitrogen, 5% carbon dioxide, and '1% O ₂ ' in FIG. 2 , total RNA was isolated using an RNA mini kit (produced by Qiagen). Total RNA (2 μg) was isolated using the RT-PCR kit (Invitrogen) to make cDNA, VEGF specific primers were prepared to amplify VEGF using PCR, and isolated on agarose gel to express VEGF The degree was measured. At this time, GAPDH was simultaneously amplified by an internal control gene to measure selective expression inhibitory activity of VEGF of each compound. The measurement result is shown in Fig.

The base sequences of the primers of VEGF and GAPDH used in this example are as follows.

VEGF; 5'-GCTCTACCTCCACCATGCCAA-3 '(sense), 5'-TGGAAGATGTCCACCAGGGTC-3' (antisense)

GAPDH; 5'-ACCACAGTCCATGCCATCAC-3 '(sense), 5'-TCCACCACCCTGTTGCTGTA-3' (antisense).

As shown in FIG. 2 , Compound 6 had no effect on the expression of GAPDH, an internal control gene, in hypoxic conditions. However, it can be seen that Compound 6 inhibited the concentration-dependent expression of VEGF under hypoxic conditions.

Therefore, it can be seen that the derivative of the present invention has a selective expression inhibitory action against VEGF, which is a target gene of HIF-1. Therefore, the derivatives of the present invention can selectively inhibit the growth of cancer and the expression of VEGF, an angiogenesis factor that is important for cancer to metastasize to other tissues, thereby inhibiting the growth and metastasis of cancer, etc. Can be used.

In addition, since the compound of the present invention has a selective expression inhibitory effect on VEGF, which is a target gene of HIF-1, it is effective in treating diabetic retinopathy or arthritis, which is exacerbated by an increase in VEGF expression by HIF-1 in hypoxia. Can be used as a component.

< Example  4> on mouse in vivo  Anticancer activity measurement

In order to measure the iv vivo anticancer activity in the mouse of the derivative according to the present invention, the following experiment was performed.

Female nude mice (Crj: BALB / c nu / nu, Charles River), 5-6 weeks old, were bred in a sterile state maintained at constant temperature and humidity during the experiment. After anesthetizing nude mice, the skin of the chest is incised and transplanted with 106 cells / mouse metastatic breast cancer cell lines, MDA-MB-435 cells (DR. DR Welch, Univ. Alabama) into mammary fat pads. Afterwards, it was sealed with a surgical clip. Experimental and control groups consisted of six nude mice. Thereafter, the size of the cancer was measured by calipers, and when the size of the cancer grew to about 50 mm ³ after implanting the breast cancer cell line, Compound 6 of the derivative of Formula 1 of the present invention was administered at various concentrations. Specifically, in the experimental group, the compound was dissolved in a solvent composed of 94.5% of physiological saline, 0.5% of DMSO, and 5% of Tween 80 (hereinafter, referred to as 'solvent A'), and the concentration of the compound was 20 mg / kg or 50 mg / kg were administered once daily at a 100 μl dose each. As for the control group, 100 μl of solvent A alone without the compound was administered once daily. Thereafter, the size and weight of cancer cell lines were measured once a week. The measurement results are shown in FIG. 3 . The tumor volume was calculated by the following equation.

Size of cancer (mm ³ ) = (length of major axis of cancer cell, mm) X (length of axis of cancer cell, mm) ² × 0.5

As shown in Figure 3, in the case of compound 6 of the present invention, it can be seen that in the experimental group administered 50 mg / kg inhibited the growth of cancer cells by 64.6% and 58.6%, respectively, compared to the control group. In addition, when the compound 6 was administered at 20 mg / kg or 50 mg / kg, all six nude mice did not die and no symptoms were found in the weight gain and feed intake. Therefore, the derivative of the present invention can be used as an active ingredient of an anticancer agent because it has anti-cancer activity in mouse and has little toxicity.

< Example  5> Toxicity Test

In order to examine the acute toxicity test of the compound according to the present invention, the following acute toxicity test was performed using C57BL / 6 mouse (Samtaco Bio Korea Co., Ltd.) of a five-week-old specific hospital.

Compound 6 was suspended in a solvent consisting of 94.5% physiological saline, 0.5% DMSO, and 5% Tween 80, and orally administered to five mice at a dose of 300 mg / kg in 0.5 ml. After administration, the mortality, clinical symptoms, and weight changes of the mice were observed and necropsied.

As a result, no oral clinical symptoms were observed in all orally administered mice, no animals died, and no toxic changes were observed in weight changes and autopsy findings. Compounds 18 and 126 did not show toxic changes up to 300 mg / kg in mice and were determined to be safe substances with a minimum lethal dose (LD ₅₀ ) of 300 mg / kg or more.

According to the present invention, indole derivatives may inhibit the growth and metastasis of cancers by inhibiting the expression of genes related to the malignancy of cancers expressed by HIF-1, and also anticancer by non-selective cytotoxicity under hypoxic conditions. Rather than showing activity, the concentration-dependent inhibition of HIF-1a protein can inhibit cancer growth and metastasis. Therefore, the indole derivative of the present invention inhibits HIF-1 activity, thereby inhibiting liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, colon cancer, Treatment of various cancer diseases such as fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor Can be used as In addition, since the indole derivative of the present invention has a selective expression inhibitory effect on VEGF, which is a target gene of HIF-1, an agent for treating diabetic retinopathy or arthritis that is exacerbated by an increase in expression of VEGF by HIF-1 in hypoxia. Can be used as an active ingredient.

Claims (7)

A pharmaceutical composition for preventing and treating cancer, which comprises an indole derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. <Formula 1>

(Wherein R is any one selected from the group consisting of COOR _a , CONR _b R _c and C ₁ ~ C ₄ alkyl group, wherein R _a is any one selected from the group consisting of H and C ₁ ~ C ₄ alkyl group, wherein R _b and R _c are as any one or more are each independently selected from N, O or S, C ₃ ~ C ₅ selected from the heteroaryl group, and N, C ₃ ~ C ₅ heterocyclic group consisting of the cycle group containing O or S containing Substituted C ₁ -C ₅ alkyl group; H and any one selected from the group consisting of C ₁ to C ₅ alkyl group, remind

Is a single or double bond.) The method of claim 1, wherein the derivative of Formula 1, 1) 3- (3-1 H -indol-3-yl-propionylamino) -benzamide; 2) 3- (3-1 H -indol-3-yl-acryloylamino) -benzamide; 3) 3- (3-1 H -indol-3-yl-propionylamino) -benzoic acid methyl ester; 4) 3- (3-1 H -indol-3-yl-acryloylamino) -benzoic acid methyl ester; 5) 3- (3-1 H -indol-3-yl-propionylamino) -benzoic acid; 6) 3- (3-1 H -indol-3-yl-acryloylamino) -benzoic acid; 7) 3- (3-1 H-indol-3-yl-amino-acryloyl) - N - (2- piperidin-1-yl-ethyl) -benzamide; 8) 3- (3-1 H-indol-3-yl-amino-acryloyl) - N - (3- morpholin-4-yl-propyl) -benzamide; 9) N -furan-2-ylmethyl-3- (3-1 H -indol-3-yl-acryloylamino) -benzamide, any one selected from the group consisting of the prevention and treatment of cancer Pharmaceutical composition for. The method of claim 1 or 2, wherein the compound inhibits the activity of hypoxia inducible factor-1 (Hypoxia Inducible Factor-1, HIF-1), characterized in that the pharmaceutical for preventing and treating cancer Composition. A pharmaceutical composition for treating cancer, comprising the compound of claim 1 as an active ingredient. According to claim 4, wherein the cancer is liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma Cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor, characterized in that any one or more selected from the group consisting of Pharmaceutical compositions. A pharmaceutical composition for treating diabetic retinopathy containing the compound of any one of claims 1 or 2 as an active ingredient. A pharmaceutical composition for treating rheumatoid arthritis, comprising the compound of claim 1 as an active ingredient.

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