JP2016210687A - Benzofuropyrazole derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel material having HIF inhibitory activity.SOLUTION: The present invention provides a compound represented by the following general formula (I) [where R, R, R, and Reach represent a hydrogen atom, a methoxy group, an ethoxy group, a hydroxy group, an N-morpholylurea group, or an N-(4-aminophenyl) urea group, or adjacent ones of them bind together to form an alkylenedioxy group; R, R, R, R, and Reach represent a hydrogen atom, a methoxy group, a hydroxy group, a methoxy carbonyl group, a carboxyl group, an isopropyl group, or an isobutoxy group, or adjacent ones of them bind together to form an alkylenedioxy group].SELECTED DRAWING: None

Description

  The present invention relates to a novel benzofuropyrazole derivative, and an HIF inhibitor, pharmaceutical composition and anticancer agent using the same.

  HIF-1 (hypoxia inducible factor-1) is a transcription factor that regulates the expression of genes involved in angiogenesis, glucose metabolism, cell proliferation and survival, and invasion of solid tumors. It is considered to be a major factor controlling the adaptive response to oxygen status. HIF-1 is a heterodimer consisting of a hypoxia-inducible HIF-1α subunit and a constitutively expressed HIF-1β subunit.

  Under normal oxygen conditions, HIF-1α degradation is promoted by hydroxylation of proline residues by proline hydroxylase. Hydroxylated HIF-1α is degraded by the ubiquitin proteasome system via von Hippel-Lindau protein. On the other hand, in the hypoxic state, the activity of proline hydroxylase decreases, so that hydroxylation of HIF-1α is suppressed. HIF-1α that has escaped degradation is translocated into the nucleus and forms a dimer with HIF-1β. This dimer binds to a specific region called a hypoxia responsive region on DNA and activates the target gene. Such target genes include genes such as vascular endothelial growth factor (VEGF), erythropoietin, glucose transporter, and insulin-like growth factor.

  It is known that HIF-1α is detected at high levels in various types of human tumor cells (Talks, KL et al., Am. J. Pathol. 2000, 157, 411-421, Birner, P et al., Clin. Cancer Res. 2001, 7, 1661-1668, Birner, P. et al., Cancer 2001, 92, 165-171, Giatromanolaki, A. et al., Br. J. Cancer 2001, 85, 881-891, Zaggag, D et al., Cancer 2000, 88, 2606-2918, Birner, P. et al., Cancer Res. 2000, 60, 4693-4696). This fact indicates that inhibitors of HIF-1α can be potential candidates for anticancer agents. In fact, PX-478 (Koh, MY et al., Mol. Cancer Ther. 2008, 7, 90-100), PX-866 (Le Cras, TD et al., Am. J. Pathol. 2010, 176, 679 -686), EZN-2968 (Greenberger, LM et al., Mol. Cancer Ther. 2008, 7, 3598-3608) and other HIF-1α inhibitors are currently undergoing clinical research. Recently, the present inventor has reported that an indenopyrazole derivative is an inhibitor of HIF-1α (Non-patent Document 1).

Hidemitsu Minegishi, Shinji Fukashiro, Hyun Seung Ban, Hiroyuki Nakamura, ACS Med. Chem. Lett., 2013, 4 (2), pp 297-301

  Even today, with the development of medicine, cancer remains a highly fatal disease. Since HIF inhibitors can be effective therapeutic agents for cancer, development of new HIF inhibitors is desired. In view of such a background, an object of the present invention is to provide a novel substance having HIF inhibitory activity.

  As a result of intensive studies to solve the above problems, the present inventor has found that a compound having a benzofuropyrazole skeleton has a high HIF inhibitory activity, and based on this finding, has completed the present invention.

That is, the present invention provides the following (1) to (6).
(1) The following general formula (I)
(Wherein R ¹ , R ² , R ³ and R ⁴ each represent a hydrogen atom, a methoxy group, an ethoxy group, a hydroxy group, an N-morpholyl urea group, or an N- (4-aminophenyl) urea group, Or adjacent ones are bonded to form an alkylenedioxy group, and R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are each a hydrogen atom, a methoxy group, a hydroxy group, a methoxycarbonyl group, a carboxyl group, An isopropyl group, an isobutoxy group, or adjacent ones are bonded to form an alkylenedioxy group. ]
A compound represented by

(2) The compound according to (1), wherein R ⁶ and R ⁷ in the general formula (I) are bonded to form an alkylenedioxy group.

(3) R ⁶ and R ⁷ in the general formula (I) are bonded to form a methylenedioxy group, an ethylenedioxy group, or a 1,3-n-propylenedioxy group (1) Compound described in 1.

(4) A HIF inhibitor comprising the compound according to any one of (1) to (3).

(5) A pharmaceutical composition comprising the compound according to any one of (1) to (3).

(6) An anticancer agent comprising the compound according to any one of (1) to (3).

  Since the benzofuropiazole derivative of the present invention has HIF inhibitory activity, it is useful for the treatment of diseases such as cancer associated with HIF.

The figure showing the influence of compound 13 on HIF-1 (alpha) protein stabilization. The figure showing the influence on the intracellular localization of HIF-1 (alpha) protein by the compound 13. FIG. The figure in the left column (HIF-1α) shows the position of the HIF-1α protein, the figure in the middle column (DAPI) shows the position of the nucleus, and the figure in the right column (Merge) shows the figure in the left and middle columns. Are superimposed.

Hereinafter, the present invention will be described in detail.
In the present invention, the “alkylenedioxy group” is, for example, a linear alkylenedioxy group represented by the formula: —O— (CH ₂ ) _n —O—. N in the formula may be an integer of 1 or more, but is preferably an integer of 1 to 5. Specific examples of the alkylenedioxy group include methylenedioxy group, ethylenedioxy group, 1,3-n-propylenedioxy group, 1,4-n-butylenedioxy group, 1,5-n-pentylenediene An oxy group etc. can be mentioned.

  In the present invention, “inhibiting HIF” means inhibiting the function of HIF as a transcription factor. Whether a substance inhibits HIF can be determined, for example, by the luciferase assay described in Example 2. The HIF to be inhibited in the present invention is mainly HIF-1α, but HIF-1β and HIF other than HIF-1 are also included in the inhibition target.

The compound of the present invention is represented by the general formula (I).
In the general formula (I), R ¹ represents a hydrogen atom, a methoxy group, an ethoxy group, a hydroxy group, an N-morpholyl urea group, or an N- (4-aminophenyl) urea group, or is bonded to R ² to form an alkylene group. An oxy group may be formed, but when the compound represented by the general formula (I) is used as an HIF inhibitor, R ¹ is preferably a hydrogen atom.

In the general formula (I), R ² represents a hydrogen atom, a methoxy group, an ethoxy group, a hydroxy group, an N-morpholyl urea group, or an N- (4-aminophenyl) urea group, or bonded to R ¹ or R ^3. An alkylenedioxy group may be formed, but when the compound represented by the general formula (I) is used as an HIF inhibitor, R ² is preferably a hydrogen atom.

In the general formula (I), R ³ represents a hydrogen atom, a methoxy group, an ethoxy group, a hydroxy group, an N-morpholyl urea group, or an N- (4-aminophenyl) urea group, or bonded to R ² or R ^4. An alkylenedioxy group may be formed, but when the compound represented by the general formula (I) is used as an HIF inhibitor, R ³ is preferably a hydrogen atom.

In the general formula (I), R ⁴ represents a hydrogen atom, a methoxy group, an ethoxy group, a hydroxy group, an N-morpholyl urea group, or an N- (4-aminophenyl) urea group, or bonded to R ³ to form an alkylene group. An oxy group may be formed, but when the compound represented by the general formula (I) is used as an HIF inhibitor, R ⁴ is preferably a hydrogen atom.

In the general formula (I), R ⁵ represents a hydrogen atom, a methoxy group, a hydroxy group, a methoxycarbonyl group, a carboxyl group, an isopropyl group, or an isobutoxy group, or is bonded to R ⁶ to form an alkylenedioxy group. However, when the compound represented by the general formula (I) is used as an HIF inhibitor, R ⁵ is preferably a hydrogen atom.

In the general formula (I), R ⁶ represents a hydrogen atom, a methoxy group, a hydroxy group, a methoxycarbonyl group, a carboxyl group, an isopropyl group, or an isobutoxy group, or bonded to R ⁵ or R ⁷ to form an alkylenedioxy group. may be formed, when using the compound represented by the general formula (I) as HIF inhibitors, R ⁶ is preferably to form a alkylenedioxy group bonded to R ^7, combined with R ⁷ It is more preferable to form a methylenedioxy group, an ethylenedioxy group, or a 1,3-n-propylenedioxy group, and bond with R ⁷ to form a 1,3-n-propylenedioxy group. Is more preferable.

In the general formula (I), R ⁷ represents a hydrogen atom, a methoxy group, a hydroxy group, a methoxycarbonyl group, a carboxyl group, an isopropyl group, or an isobutoxy group, or bonded to R ⁶ or R ⁸ to form an alkylenedioxy group. may be formed, when using the compound represented by the general formula (I) as HIF inhibitors, R ⁷ is preferably formed alkylenedioxy group bonded to R ^6, bonded to R ⁶ It is more preferable to form a methylenedioxy group, an ethylenedioxy group, or a 1,3-n-propylenedioxy group, and bond with R ⁶ to form a 1,3-n-propylenedioxy group. Is more preferable.

In the general formula (I), R ⁸ represents a hydrogen atom, a methoxy group, a hydroxy group, a methoxycarbonyl group, a carboxyl group, an isopropyl group, or an isobutoxy group, or bonded to R ⁷ or R ⁹ to form an alkylenedioxy group. However, when the compound represented by formula (I) is used as an HIF inhibitor, R ⁸ is preferably a hydrogen atom.

In the general formula (I), R ⁹ represents a hydrogen atom, a methoxy group, a hydroxy group, a methoxycarbonyl group, a carboxyl group, an isopropyl group, or an isobutoxy group, or is bonded to R ⁸ to form an alkylenedioxy group. However, when the compound represented by the general formula (I) is used as an HIF inhibitor, R ⁹ is preferably a hydrogen atom.

In the general formula (I), R ¹ , R ² , R ³ , and R ⁴ may be the same group or different groups. Similarly, R ⁵ , R ⁶ , R ⁷ , R ⁸ , and R ⁹ may be the same group or different groups.

In addition to the inhibitory activity against HIF, the compound represented by the general formula (I) has an inhibitory activity against cancer cells, an inhibitory activity against cyclin-dependent kinase (CDK), platelet growth factor receptor tyrosine kinase (PDGFR-TK) It is considered to have biological activity such as inhibitory activity against EGF, epidermal growth factor receptor tyrosine kinase (EGFR-TK) and vascular endothelial growth factor receptor 2 tyrosine kinase (VEGFR-2-TK), and cytotoxicity. Therefore, the compound represented by the general formula (I) is not only a HIF inhibitor, but also a cell growth inhibitor, a CDK inhibitor, a PDGFR-TK inhibitor, an EGFR-TK and / or a VEGFR-2-TK inhibitor, a cell It can be used as a toxic drug. The general formula (I) compounds represented by, when used as cytostatic agents, R ² is methoxy, R ⁶ is methoxy ^{group, R 1, R 3 -R 5} , R 7 - R ⁹ is preferably a hydrogen atom, and when used as a CDK inhibitor, R ¹ is an N-morpholyl urea group, R ⁷ is a methoxy group, R ² -R ⁶ , R ⁸ -R ⁹ are hydrogen Preferably, it is an atom, and when used as a PDGFR-TK inhibitor, R ² and R ³ are methoxy groups, R ⁶ is a fluorine atom, R ¹ , R ⁴ -R ⁵ , R ⁷ -R ⁹ Is preferably a hydrogen atom, and when used as an EGFR-TK and / or VEGFR-2-TK inhibitor, R ¹ is an N- (4-aminophenyl) urea group, and R ⁶ and R ⁷ are bonded. Thus, a methylenedioxy group is formed, and R ² -R ⁵ and R ⁸ -R ⁹ are preferably hydrogen atoms. When used as a cytotoxic agent, R ² is preferably a methoxy group.

  The compound represented by general formula (I) may form a salt with an acid or a base. As such a salt, a pharmaceutically acceptable salt is preferable, for example, an alkali metal salt (sodium salt, potassium salt), an alkaline earth metal salt (calcium salt, magnesium salt), sulfate, hydrochloride, nitrate. And so on. Moreover, the compound represented by general formula (I) may form a hydrate or a solvate by mixing with water or an organic solvent. Such salts, hydrates and solvates of the compound represented by the general formula (I) are also included in the compound represented by the general formula (I).

  In addition, the compound represented by the general formula (I) may have an optical isomer. Such separated isomers or mixtures thereof are also included in the compound represented by the general formula (I).

Specific examples of the compound represented by the general formula (I) include, but are not limited to, the following (I-1) to (I-40). In the structural formula, “Me” represents a methyl group, and “Et” represents an ethyl group.

  Among these compounds, I-7 (compound 13) can be mentioned as a preferable compound when used as an HIF inhibitor, and I-16 is preferable as a compound when used as a cell growth inhibitor. Can do.

Since the compound represented by the general formula (I) has a structure similar to that of pyrazoloindole, indenopyrazole and the like, known production methods for these compounds (for example, Kumar, AS et al., Synthesis, 2011, 23, 3878) and can be produced according to a method in which alterations or modifications are made. For example, a compound represented by the following general formula (II) and a compound represented by the following general formula (III) are reacted to produce a compound represented by the following general formula (IV). The compound represented by the general formula (I) can be produced from the compound represented by the formula (IV).
In the above formula, R ^{1 to} R ⁹ have the same meaning as described above.

  The reaction of the compound represented by the general formula (II) and the compound represented by the general formula (III) can be performed as follows.

  This reaction is performed in the presence of sodium acetate or the like. Instead of sodium acetate, potassium acetate, calcium acetate, sodium carbonate, potassium carbonate and the like can be used.

  The amount of the compound represented by the general formula (III) used in this reaction is not particularly limited, but is usually 0.5 to 3 mol with respect to 1 mol of the compound represented by the general formula (II), preferably 1 to 1.2 mol. The amount of sodium acetate and the like is not particularly limited, but is usually 0.5 to 5 mol, preferably 1 to 1.2 mol, relative to 1 mol of the compound represented by the general formula (II).

  The solvent to be used is not particularly limited as long as it does not inhibit the reaction, and ethanol, methanol, propanol, isopropanol, butanol and the like can be used.

  Although reaction temperature is not specifically limited, Usually, it is room temperature-150 degreeC, Preferably it is 50-100 degreeC.

  The reaction time is not particularly limited, but is usually 1 to 60 minutes, and preferably 5 to 15 minutes.

  Purification of the compound represented by the general formula (VI) from the reaction product can be performed according to a conventional method using chromatography or the like.

  The reaction for producing the compound represented by the general formula (I) from the compound represented by the general formula (IV) can be performed as follows.

  This reaction is performed in the presence of copper (I) iodide, cyclohexane-1,2-diamine, potassium carbonate and the like. Instead of copper iodide (I), copper iodide (II), copper bromide (I), copper bromide (II), copper acetate (I), copper acetate (II), copper chloride (I), copper chloride (II), copper (I) cyanide, copper (II) cyanide, etc. can also be used, instead of cyclohexane-1,2-diamine, cyclopentane-1,2-diamine, cycloheptane-1,2- Diamine, cyclooctane-1,2-diamine, bipyridine and the like can also be used, and sodium carbonate, potassium hydrogen carbonate, sodium hydrogen carbonate and the like can be used instead of potassium carbonate.

  The amount of copper iodide (I) used in this reaction is not particularly limited, but is usually 0.05 to 1 mol, preferably 0.1 to 0.2 mol, relative to 1 mol of the compound represented by the general formula (IV). . The amount of cyclohexane-1,2-diamine or the like is not particularly limited, but is usually 0.05 to 1 mol, preferably 0.2 to 0.4 mol, relative to 1 mol of the compound represented by the general formula (IV). The amount of potassium carbonate and the like is not particularly limited, but is usually 0.5 to 5 mol, preferably 1.5 to 2 mol, relative to 1 mol of the compound represented by the general formula (IV).

  The solvent to be used is not particularly limited as long as it does not inhibit the reaction, and N-methyl-2-pyrrolidone, tetrahydrofuran, N, N-dimethylformamide, N, N-dimethylacetamide and the like can be used.

  Although reaction temperature is not specifically limited, Usually, it is room temperature-150 degreeC, Preferably it is 80-120 degreeC.

  The reaction time is not particularly limited, but is usually 1 to 60 minutes, and preferably 5 to 10 minutes.

  Purification of the compound represented by the general formula (I) from the reaction product can be performed according to a conventional method using chromatography or the like.

  The compound represented by the general formula (I) has HIF inhibitory activity. Since HIF is considered to be involved in diseases such as cancer, the compound represented by the general formula (I) can be used as a pharmaceutical composition, specifically as an anticancer agent.

When the compound represented by the general formula (I) is used as a pharmaceutical composition or an anticancer agent, the compound can be formulated by mixing with a pharmaceutically acceptable carrier or diluent according to a known method. . The dosage form is not particularly limited, and may be tablets, powders, granules, capsules, solutions, injections, suppositories, sustained release agents, and the like. The administration method is not particularly limited, and it can be administered orally or parenterally (topical, rectal, intravenous administration, etc.). The dose varies depending on the administration subject, administration method, and the like. For example, when administered orally to an adult as an anticancer agent, the compound represented by the general formula (I) is 10 to 100 mg per administration. It can be administered in 1 to several times a day.
Since the compound represented by the general formula (I) contains an oxygen atom in the ring, it is considered that the compound is more soluble in water than the indenopyrazole derivative. In addition, since the compound represented by the general formula (I) does not prevent the transfer of HIF-1α into the nucleus, it is less toxic than an HIF inhibitor such as LW6 that promotes the degradation of HIF-1α. Conceivable. From these properties, it is considered that the compound represented by the general formula (I) is more suitable for medicine than known HIF inhibitors.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these Examples.

Example 1 Production of 1-phenyl-1H-benzofuro [2,3-c] pyrazole derivative
The above compound having a 1-phenylbenzofuropyrazole skeleton was produced as follows.
(1) Synthesis of 2-chlorobenzofuran-3-carbaldehyde (compound 2)
Benzofuran-2 (3H) -one (compound 1) (1.063 g, 7.9 mmol) and dimethylformamide (DMF; 1.8 mL, 23.7 mmol) were dissolved in chloroform (10 mL) and POCl ₃ (1.7 mL, 18.2) was dissolved at 0 ° C. mmol) was added and stirred for 5 minutes. Thereafter, the reaction mixture was heated to reflux overnight and cooled to room temperature. 2N Aqueous sodium hydroxide solution was added to adjust pH = 7, and the mixture was extracted with methylene chloride. The obtained organic layer was washed with water, then with saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and then the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 80: 1) to obtain Compound 2 (0.675 g, 3.74 mmol) (yield 47%). ¹ H NMR (400 MHz; CDCl ₃ ) δ10.2 (s, 1H), 8.18-8.15 (m, 1H), 7.52-7.51 (m, 1H), 7.50-7.42 (m, 2H).

(2) Synthesis scheme of (3,4-dihydro-2H-benzo [b] [1,4] dioxepin-7-yl) hydrazine hydrochloride (compound 5)
<Synthesis Method of Compound 3>
4-Nitrobenzene-1,2-diol (0.296 g, 1.9 mmol) was dissolved in DMF (10 mL), and potassium carbonate (1.31 g, 9.5 mmol) and 1,3-dibromopropane (0.3 mL, 2.85 mmol) were added. In order, the reaction mixture was stirred at 110 ° C. overnight. The reaction mixture was cooled to room temperature, ethyl acetate (20 mL) was added, washed with water, washed with saturated brine, and dried over anhydrous sodium sulfate. After distilling off the solvent under reduced pressure, Compound 3 was obtained in a crude yield of 0.29 g (1.49 mmol, 78% yield). ¹ H NMR (400MHz; CDCl3): δ 7.87-7.82 (m, 2H), 7.03 (d, J = 8.8 Hz, 1H), 4.40 (t, J = 6.0 Hz, 2H), 4.34 (t, J = 6.0 Hz, 2H), 2.30 (quint, J = 6.0 Hz, 2H).

<Synthesis Method of Compound 4>
The obtained compound 3 (0.29 g, 1.49 mmol) was dissolved in a mixed solvent of methanol (10 mL) and THF (5 mL), Pd / C (0.106 g) was added, and the mixture was overnight at room temperature under a hydrogen atmosphere. Stir. The palladium catalyst was filtered through Celite, and the solvent was distilled off under reduced pressure to obtain Compound 4 in a crude yield of 0.225 g (1.36 mmol, 91% yield). ¹ H NMR (400 MHz; CDCl ₃ ) δ 6.82 (d, J = 8.4 Hz, 1H), 6.37 (d, J = 2.7 Hz, 1H), 6.29 (dd, J = 8.4, 2.7 Hz, 1H), 4.17 (t, J = 5.4 Hz, 2H), 4.11 (t, J = 5.4 Hz, 2H), 3.49 (bs, 2H), 2.16 (quint, J = 5.4 Hz, 2H).

<Synthesis Method of Compound 5>
6N hydrochloric acid was added to compound 4 (0.225 g, 1.36 mmol), and NaNO ₂ (0.112 g, 1.63 mmol) dissolved in 0.5 mL of water was added with stirring at 0 ° C. SnCl ₂ · 2H ₂ O (0.614 g, 2.72 mmol) dissolved in 1 mL of concentrated hydrochloric acid was added and stirred for 30 minutes, and then the reaction was stopped with an aqueous sodium hydroxide solution. The reaction mixture was extracted with ethyl acetate, and the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, then dissolved in diethyl ether, and 1N hydrochloric acid ether solution was added to obtain Compound 5 as a solid. ¹ H NMR (400 MHz; CDCl ₃ ) δ 7.02 (d, J = 8.4 Hz, 1H), 6.71 (d, J = 2.7 Hz, 1H), 6.66 (dd, J = 8.4, 2.7 Hz, 1H), 4.14 (t, J = 5.4 Hz, 2H), 4.10 (t, J = 5.4 Hz, 2H), 2.11 (quint, J = 5.4 Hz, 2H).

(3) Synthesis scheme of 1-phenyl-1H-benzofuro [2,3-c] pyrazole derivative (compounds 13 to 16)
<Method for Synthesizing Compound 9>
Compound 2 (0.081 g, 0.45 mmol) and compound 5 (0.115 g, 0.53 mmol) are dissolved in ethanol (1.5 mL), sodium acetate (0.043 g, 0.53 mmol) is added, and microwave irradiation is performed at 80 ° C. for 10 minutes. It was. After filtering the solid, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane: ethyl acetate = 15: 1) to obtain Compound 9 in 0.099 g (0.29 mmol) ( Yield 64%). ¹ H NMR (400 MHz; CDCl ₃ ) δ 8.27 (d, J = 8.5 Hz, 1H), 7.82 (s, 1H), 7.63 (s, 1H), 7.47-7.37 (m, 3H), 6.99 (d, J = 8.6 Hz, 1H), 6.85 (d, J = 2.9 Hz, 1H), 6.70 (dd, J = 8.6, 2.9 Hz, 1H), 4.26 (t, J = 5.4 Hz, 2H), 4.19 (t, J = 5.4 Hz, 2H), 2.22 (quint, J = 5.4 Hz, 2H).

<Method for Synthesizing Compound 10>
By the method similar to the synthesis of Compound 9, Compound 10 was obtained in 55% yield (0.07 g, 0.21 mmol) from Compound 2 (0.068 g, 0.38 mmol) and Compound 6 (0.093 g, 0.46 mmol). ¹ H NMR (400MHz; CDCl ₃ ) δ 8.27 (d, J = 7.6 Hz, 1H), 7.81 (bs, 1H), 7.46-7.36 (3H, m), 6.85 (d, J = 8.4 Hz, 1H), 6.77 (bs, 1H), 6.60 (bs, 1H), 4.32-4.27 (m, 4H).

<Method for Synthesizing Compound 13>
Compound 9 (0.099 g, 0.29 mmol) was dissolved in N-methyl-2-pyrrolidone (NMP, 0.5 mL), and copper (I) iodide (6 mg, 0.03 mmol), (1R, 2R) -cyclohexane-1, 2-diamine (7 μL, 0.06 mmol) and potassium carbonate (0.08 g, 0.58 mmol) were added, and microwave irradiation was performed at 100 ° C. for 10 minutes. The reaction mixture was cooled to room temperature, water (10 mL) was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, and dried over anhydrous sodium sulfate. After distilling off the solvent under reduced pressure, the obtained residue was purified by silica gel column chromatography (hexane: ethyl acetate = 20: 1) to obtain Compound 13 in a yield of 19% (0.017 g, 0.054 mmol). ¹ H NMR (400 MHz; CDCl ₃ ) δ 8.02 (d, J = 8.4 Hz, 2H), 7.87 (s, 1H), 7.69 (d, J = 8.0 Hz 1H), 7.58-7.51 (m, 3H), 7.36-7.29 (m, 3H); ¹³ C NMR (100 MHz; CDCl ₃ ) δ160.0, 156.4, 138.0, 131.1, 129.5, 126.1, 124.1, 124.1, 121.7, 120.2, 118.0, 112.5, 109.5.

<Method for Synthesizing Compound 14>
By the method similar to the synthesis of Compound 13, Compound 14 was obtained in 13% yield (0.0083 g, 0.028 mmol) from Compound 10 (0.07 g, 0.21 mmol). ¹ H NMR (400 MHz; CDCl ₃ ) δ 7.84 (s, 1H), 7.69 (d, J = 8.7 Hz, 1H), 7.57-7.56 (m, 2H), 7.50 (dd, J = 2.5, 8.7 Hz, 1H), 7.35-7.32 (m, 2H), 7.03 (d, J = 8.7 Hz, 1H), 4.35 (s, 4H); ¹³ C NMR (100 MHz; CDCl ₃ ) δ 160.0, 144.0, 142.0, 132.0, 130.7, 124.0, 123.9, 121.8, 120.2, 117.8, 112.4, 111.5, 109.2, 107.8.

<Method for Synthesizing Compound 15>
After obtaining Compound 11 from Compound 2 (0.1 g, 0.55 mmol) and Compound 7 (0.124 g, 0.66 mmol) by the same method as the synthesis of Compound 9, Compound 15 was prepared in the same manner as the synthesis method of Compound 13. Two step yield 3% (0.0046 g, 0.016 mmol) was obtained. ¹ H NMR (400MHz; CDCl ₃ ): δ 7.84 (s, 1H), 7.71-7.69 (m, 1H), 7.58-7.54 (m, 2H), 7.54-7.48 (m, 1H), 7.47-7.31 (m , 2H), 6.96 (d, J = 8.4 Hz, 1H), 6.07 (s, 2H).

<Method for Synthesizing Compound 16>
Similar to the synthesis of Compound 15, Compound 16 was obtained from Compound 2 (0.1 g, 0.55 mmol) and Compound 8 (0.054 g, 0.55 mmol) in a two-step yield of 31% (0.04 g, 0.17 mmol). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.85 (s, 1H), 7.71-7.67 (m, 2H), 7.60-7.56 (m, 2H), 7.37-7.31 (m, 2H), 7.14 (d, J = 8.7 Hz, 1H), 4.33 (t, J = 5.6 Hz, 2H), 4.28 (t, J = 5.6 Hz, 2H), 2.27 (quint, J = 5.6 Hz, 2H).

[Example 2] Cell growth inhibitory activity evaluation and luciferase assay Cell growth inhibitory activity was evaluated as follows.
After culturing HeLa cells in a 96-well multiwell plate at a density of 5 × 10 ³ cells / 100 μl for 6 hours, the compound was diluted with RPMI-1640 medium to a concentration of 60-0.2 μM, and 100 μl was added to the cells for 72 hours. Cultured at 37 ° C. Thereafter, an MTT (sigma) reagent solution (5 mg / ml) dissolved in PBS was added and cultured for 2 hours. After 2 hours, the supernatant was removed, 100 μl of DMSO was added, and the absorbance at 595 nm was measured with a plate reader. The concentration at which cell proliferation was inhibited by 50% was calculated by a semi-logarithmic graph with the absorbance of cells not administered with the drug being 100%, and shown as IC ₅₀ values (Table 1).

The luciferase assay was performed as follows.
HeLa cells stably expressing HRE-luciferase using 96-well multiwell plates at a density of 5x10 ³ cells / 100 μl under hypoxic conditions (1% O ₂ , 94% N ₂ , 5% CO ₂ ) After culturing for 16 hours in RPMI-1640 medium containing various compound concentrations (0.03 to 30 μM), the culture supernatant was removed. Thereafter, luciferase assay was performed using Luciferase Assay System ^™ (Promega) according to the attached protocol. The concentration at which 50% inhibition of luciferase activity was calculated using a semilogarithmic graph, with the luciferase activity of cells not administered with the drug as 100%, was shown as the IC ₅₀ value (Table 1).

  As shown in Table 1, the benzofuropiazole derivative showed an HIF inhibitory activity equivalent to that of YC-1, which is a known HIF inhibitor. In particular, compounds 13 and 14 in which an alkylenedioxy group was bonded to the benzene ring and cyclized showed high inhibitory activity.

[Example 3] Western blot
HeLa cells are seeded in a 12-well multiplate at a concentration of 2.5 x 10 ⁵ cells / 1 ml. After adding Compound 13 or LW6, the cells are cultured in an aerobic atmosphere (20% O ₂ ) for 1 hour to reduce hypoxia. The cells were cultured for 4 hours in an atmosphere (1% O ₂ ). Remove the medium, lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 5 mM sodium fluoride, 2.5 mM p-nitrophenylene phosphate, 10 μg / ml phenylmethylsulfonylfluoride, 1 mM sodium Add 80 μl of vanadate, and 10 μg / ml leupeptin) to lyse the cells, and sample buffer (50 mM Tris, pH 7.4, 20% SDS, 50% glycerol, 20% 2-thioethanol, 50 μg / ml bromophenol blue) 20 μl was added and subjected to ultrasonic treatment and heat denaturation treatment. Proteins were separated from the cell lysate by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was treated with an anti-HIF-1α antibody (BD Transduction Laboratories, Lexington, KY), an anti-tubulin antibody (Santa Cruz Biotechnology, Inc.), and further treated with an HRP-conjugated secondary antibody. The membrane was treated with ECL kit (GE healthcare Buckinghashire, UK), and the protein was visualized with Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA) (FIG. 1).
As shown in FIG. 1, LW6 inhibited HIF-1α protein accumulation under hypoxic conditions at a concentration of 30 μM, whereas compound 13 did not affect HIF-1α protein accumulation.

Example 4 Immunostaining HeLa cells were seeded on a cover glass at a concentration of 1 × 10 ⁴ cells / 100 μl, placed in a 35 mm dish and cultured for 2 hours, and then 1 ml of the medium was added and cultured for 12 hours. The compound was added and treated for 8 hours, the medium was removed, and the cells were fixed by treatment with 4% paraformaldehyde solution for 15 minutes. Thereafter, the cells were treated with 0.4% Triton X for 5 minutes and immunoblock (DS Pharma Biomedical Co., Ltd.) for 5 minutes. Anti-HIF-1α antibody was added and treated with FITC-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) for 4 hours and with DAPI (Wako Pure Chemical Industries) for 5 minutes. Encapsulated with ProLong (registered trademark) Gold antifade reagent (Invitrogen) and observed with a fluorescence microscope (FIG. 2).
As shown in FIG. 2, HIF-1α protein was degraded under aerobic conditions (20% O ₂ ), but stabilized and transferred into the nucleus under hypoxic conditions (1% O ₂ ). . This result indicates that Compound 13 does not inhibit nuclear translocation of HIF-1α protein under hypoxic conditions.

  Since the present invention relates to a substance that can be used as a pharmaceutical, it can be used in industrial fields such as the pharmaceutical industry.

Claims (6)

The following general formula (I)
(Wherein R ¹ , R ² , R ³ and R ⁴ each represent a hydrogen atom, a methoxy group, an ethoxy group, a hydroxy group, an N-morpholyl urea group, or an N- (4-aminophenyl) urea group, Or adjacent ones are bonded to form an alkylenedioxy group, and R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are each a hydrogen atom, a methoxy group, a hydroxy group, a methoxycarbonyl group, a carboxyl group, An isopropyl group, an isobutoxy group, or adjacent ones are bonded to form an alkylenedioxy group. ]
A compound represented by The compound according to claim 1, wherein R ⁶ and R ⁷ in the general formula (I) are bonded to form an alkylenedioxy group. The R ⁶ and R ⁷ in the general formula (I) are combined to form a methylenedioxy group, an ethylenedioxy group, or a 1,3-n-propylenedioxy group. Compound.   A HIF inhibitor comprising the compound according to any one of claims 1 to 3.   A pharmaceutical composition comprising the compound according to any one of claims 1 to 3.   An anticancer agent comprising the compound according to any one of claims 1 to 3.