Protein disulfide isomerase fluorescence inhibitor and use thereof

Abstract

To provide a novel compound that is suitable for a variety of applications including inhibition of PDI activity, and to provide, using the novel compound, a novel PDI activity inhibitor, a novel apoptosis inducer, a novel anticancer agent, a novel agent for treating infectious diseases, or a novel blood coagulation inhibitor.SOLUTION: A compound represented by General Formula (1) below, wherein X1 is a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group; Y1 to Y4 are independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom; Z1 to Z3 are independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group, or a nitro group, with at least one of Z1 to Z3 being a nitro group; and Z4 to Z7 are independently a hydrogen atom or an alkyl group.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to fluorescent inhibitors of protein disulfide isomerase and uses thereof. [Background technology]

[0002] Protein disulfide isomerase (PDI) is an enzyme present in the endoplasmic reticulum of eukaryotes and the periplasm of bacteria, which catalyzes the formation and cleavage of disulfide bonds between cysteine ​​residues in proteins during protein folding. In recent years, PDI inhibitors have been attracting increasing attention as they have been shown to have potential uses as anti-cancer drugs, infection prevention agents, and blood coagulation inhibitors.

[0003] Non-Patent Document 1 describes that PDI inhibitors are promising cancer therapeutic agents, and Non-Patent Document 2 describes that the PDI inhibitor LOC14 reduces influenza-induced inflammatory responses in mice. Furthermore, Non-Patent Document 3 describes that there is increasing evidence that PDIs play an important role in the onset and progression of cardiovascular diseases such as thrombosis and vascular inflammation, and that the effectiveness of PDI inhibitors needs to be further investigated, and that it would be interesting to investigate whether they have antithrombotic effects. Therefore, there is a strong demand for providing novel compounds suitable for various applications including inhibition of PDI activity, novel PDI activity inhibitors using the novel compounds, novel apoptosis inducers, novel anticancer agents, novel infectious disease treatment agents, or novel blood coagulation inhibitors. [Prior art documents] [Patent documents]

[0004] [Non-Patent Document 1] Cancer Med.2021 10:2812-2825 [Non-patent document 2] Int J Mol Sci. 2022 23:1078 [Non-patent document 3] Exp Mol Med. 2020 52(3):390-399 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to solve the above-mentioned problems of the prior art and to achieve the following objectives: to provide novel compounds suitable for various applications including inhibition of PDI activity, and to provide novel PDI activity inhibitors using the novel compounds, as well as novel apoptosis inducers, novel anticancer agents, novel infectious disease treatment agents, or novel blood coagulation inhibitors. [Means for solving the problem]

[0006] The means for solving the above problems are as follows: <1> The compound is represented by the following general formula (1) or (2). [ka] [ka] However, in the general formula (1) or the general formula (2), X1 and X2 each independently represent a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring is a double bond); Y1, Y2, Y3, and Y4 each independently represent a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom; Z1, Z2, and Z3 each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and at least one of Z1, Z2, and Z3 is a nitro group; Z4, Z5, Z6 and Z7 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. <2> wherein X1 and X2 are hydroxy groups; <1> It is a compound described in <3> wherein Y1, Y2, Y3 and Y4 are hydrogen atoms; <1> It is a compound described in <4> wherein Z1 is a nitro group, and Z2 and Z3 are hydrogen atoms; <1> It is a compound described in <5> wherein Z4, Z5, Z6 and Z7 are hydrogen atoms; <1> It is a compound described in <6> The apoptosis inducer comprises a compound represented by the following general formula (1) or (2): [ka] [ka] However, in the general formula (1) or the general formula (2), X1 and X2 each independently represent a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring is a double bond); Y1, Y2, Y3, and Y4 each independently represent a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom; Z1, Z2, and Z3 each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and at least one of Z1, Z2, and Z3 is a nitro group; Z4, Z5, Z6 and Z7 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. <7> The anticancer agent is characterized by containing a compound represented by the following general formula (1) or the following general formula (2). [ka] [ka] However, in the general formula (1) or the general formula (2), X1 and X2 each independently represent a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring is a double bond); Y1, Y2, Y3, and Y4 each independently represent a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom; Z1, Z2, and Z3 each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and at least one of Z1, Z2, and Z3 is a nitro group; Z4, Z5, Z6 and Z7 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. <8> The PDI activity inhibitor is characterized by containing a compound represented by the following general formula (1) or (2). [ka] [ka] However, in the general formula (1) or the general formula (2), X1 and X2 each independently represent a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring is a double bond); Y1, Y2, Y3, and Y4 each independently represent a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom; Z1, Z2, and Z3 each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and at least one of Z1, Z2, and Z3 is a nitro group; Z4, Z5, Z6 and Z7 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. <9> The infectious disease treatment drug comprises a compound represented by the following general formula (1) or (2): [ka] [ka] However, in the general formula (1) or the general formula (2), X1 and X2 each independently represent a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring is a double bond); Y1, Y2, Y3, and Y4 each independently represent a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom; Z1, Z2, and Z3 each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and at least one of Z1, Z2, and Z3 is a nitro group; Z4, Z5, Z6 and Z7 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. <10> The blood coagulation inhibitor comprises a compound represented by the following general formula (1) or (2): [ka] [ka] However, in the general formula (1) or the general formula (2), X1 and X2 each independently represent a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring is a double bond); Y1, Y2, Y3, and Y4 each independently represent a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom; Z1, Z2, and Z3 each independently represent a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and at least one of Z1, Z2, and Z3 is a nitro group; Z4, Z5, Z6 and Z7 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a novel compound suitable for various applications including inhibition of PDI activity, and a novel PDI activity inhibitor, a novel apoptosis inducer, a novel anticancer agent, a novel infectious disease treatment agent, or a novel blood coagulation inhibitor using the novel compound. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of infrared spectroscopic analysis of FTNA. [Figure 2] FIG. 2 shows the results of infrared spectroscopy by FTMA. [Figure 3] FIG. 3 shows the results of 1H NMR of FTNA. [Figure 4] FIG. 4 shows the results of 1H NMR of FTMA. [Figure 5] FIG. 5 shows the results of 13C NMR of FTNA. [Figure 6] FIG. 6 shows the results of 13C NMR of FTMA. [Figure 7] FIG. 7 shows the results of electrospray ionization mass spectrometry analysis of FTNA. [Figure 8] FIG. 8 shows the results of electrospray ionization mass spectrometry analysis of FTMA. [Figure 9] FIG. 9 shows apoptosis induction by FTNA. [Figure 10] FIG. 10 is a diagram (part 1) showing the effect of FTNA on the cell cycle. [Figure 11] FIG. 11 is a diagram (part 2) showing the effect of FTNA on the cell cycle. [Figure 12] FIG. 12 shows proteins that exhibit fluorescence upon FTNA treatment. [Figure 13] FIG. 13 shows the results of CBB staining of the gel used for detecting proteins that exhibit fluorescence due to FTNA treatment. [Figure 14] FIG. 14 shows the identified proteins that exhibit fluorescence upon FTNA treatment. [Figure 15] FIG. 15 shows the results of SDS-PAGE of the PDI recombinant protein reacted with FTNA. [Figure 16] FIG. 16 shows the activity inhibition of fluorescently labeled PDI with FTNA. [Figure 17] FIG. 17 shows the inhibition of blood coagulation by FTNA. DETAILED DESCRIPTION OF THE INVENTION

[0009] (compound) The compound is represented by the following general formula (1) or (2). [ka] [ka]

[0010] In the general formula (1) or (2), X1 and X2 each independently represent a hydroxy group (-OH), an oxygen atom (=O), an ethylamino group (-NH(CH2CH3)), or a diethylamino group (-N(CH2CH3)2) (provided that when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring is a double bond); Y1, Y2, Y3, and Y4 each independently represent a hydrogen atom (-H), a methyl group (-CH3), a fluorine atom (-F), a chlorine atom (-Cl), or a bromine atom (-Br); Z1, Z2, and Z3 each independently represent a hydrogen atom (-H), a fluorine atom (-F), a chlorine atom (-Cl), a bromine atom (-Br), an alkyl group having 1 to 4 carbon atoms, or a nitro group (-NO2), and at least one of Z1, Z2, and Z3 is a nitro group; Z4, Z5, Z6 and Z7 each independently represent a hydrogen atom (-H) or an alkyl group having 1 to 4 carbon atoms.

[0011] The compound is not particularly limited and can be appropriately selected depending on the purpose, but is preferably one represented by the general formula (1). The compound is preferably a fluorescent compound.

[0012] In the general formula (1) or (2), X1 and X2 are not particularly limited as long as they are each independently a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (however, when X1 or X2 is an oxygen atom, the bond between the oxygen atom X1 or X2 and the benzene ring becomes a double bond, and X1 or X2 becomes an oxygen atom constituting a carbonyl group), and can be appropriately selected depending on the purpose. However, it is preferable that X1 or X2 is a hydroxy group, and it is more preferable that X1 and X2 are hydroxy groups.

[0013] In the general formula (1) or (2), Y1, Y2, Y3, and Y4 are not particularly limited as long as they are each independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom, and can be appropriately selected depending on the purpose. However, it is preferable that at least one of Y1, Y2, Y3, and Y4 is a hydrogen atom, it is more preferable that at least two of Y1, Y2, Y3, and Y4 are hydrogen atoms, and it is even more preferable that Y1, Y2, Y3, and Y4 are hydrogen atoms.

[0014] In the general formula (1) or (2), Z1, Z2, and Z3 are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and are not particularly limited and can be appropriately selected depending on the purpose, as long as at least one of Z1, Z2, and Z3 is a nitro group. However, it is preferable that Z1 is a nitro group, and it is more preferable that Z1 is a nitro group and Z2 and Z3 are hydrogen atoms. The alkyl group having 1 to 4 carbon atoms is not particularly limited and can be appropriately selected depending on the purpose, but is preferably an alkyl group having 1 to 3 carbon atoms, more preferably an alkyl group having 1 or 2 carbon atoms, and even more preferably a methyl group.

[0015] In the general formula (1) or (2), Z4, Z5, Z6, and Z7 are not particularly limited as long as they are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and can be appropriately selected depending on the purpose. However, it is preferable that Z4, Z5, Z6, and Z7 are a hydrogen atom.

[0016] Specific examples of the compound are not particularly limited and can be appropriately selected depending on the purpose. However, those represented by any of the following structural formulae (1) to (24) are preferred, those represented by any of the following structural formulae (1) to (12) are more preferred, those represented by any of the following structural formulae (1) to (10) are even more preferred, those represented by any of the following structural formulae (1) to (8) are particularly preferred, and those represented by the following structural formula (1) (3',6'-dihydroxy-2-{[(5-nitrothiophen-2-yl)methylidene]amino}spiro[isoindol-1,9'-xanthene]-3(2H)-one: FTNA) are most preferred.

[0017] [ka] [ka]

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[0018] As shown in the examples, the compound is extremely stable. As shown in the examples, the compounds are capable of inducing apoptosis. As shown in the examples, the compound can inhibit the proliferation of cancer cells. As shown in the Examples, the compound can bind to PDI (protein disulfide isomerase) and inhibit the activity of PDI. As shown in the examples, the compounds are capable of inhibiting blood coagulation.

[0019] The PDIs (protein disulfide isomerases) PDI1, PDIA3, and PDIA4 are thought to be therapeutic targets for influenza viruses (Chamberlain N, et al., Redox Biol. 22, 101129, 2019; Non-Patent Document 2; Kim Y, Chang KO, Virus Res. 247, 26-33, 2018). Furthermore, because inhibiting PDIs on the surface of HIV target cells prevents HIV from entering cells, PDI inhibitors are expected to be used as prophylactic and therapeutic drugs for infectious diseases (Markovic I. et al., Blood. 103, 1586-1594, 2004). Furthermore, PDIA4 is also involved in the uncoating of the human astrovirus genome during cell entry (Aguilar-Hernandez N, Viruses. 13, 53, 2020). Thus, the compounds can prevent or treat infectious diseases such as viruses.

[0020] -Method for synthesizing compounds- The compound can be synthesized by adding dropwise an ethanol solution of 5-nitrothiophene-2-carboxaldehyde or an analogue of 5-nitrothiophene-2-carboxaldehyde to a stirred ethanol solution of fluorescein hydrazide or an analogue of fluorescein hydrazide, refluxing for 24 hours, returning the reaction mixture to room temperature, filtering the resulting precipitate, washing with diethyl ether, and air-drying it. The resulting product can be recrystallized from ethanol.

[0021] The fluorescein hydrazide or fluorescein hydrazide analogue may be a commercially available product or may be synthesized by a known method. Examples of the fluorescein hydrazide include commercially available products from MedChemExpress, BOC Sciences, ChemSheen LLC, and the like. Commercially available analogues of fluorescein hydrazide include rhodamine G6 hydrazide (MedChemExpress, BOC Sciences, Chemsheen LLC, etc.) and rhodamine B hydrazide (BOC Sciences, Cayman Chemical Company, Tokyo Chemical Industry Co., Ltd., etc.).

[0022] The 5-nitrothiophene-2-carboxaldehyde or analogue of 5-nitrothiophene-2-carboxaldehyde may be a commercially available product or may be synthesized by a known method. Examples of the 5-nitrothiophene-2-carboxaldehyde include commercially available products from Merck (Sigma-Aldrich), Thermo Fisher Scientific, and Fujifilm Wako Pure Chemical Industries, Ltd. Commercially available analogues of the 5-nitrothiophene-2-carboxaldehyde include 4-bromo-5-nitrothiophene-2-carboxaldehyde (Merck) and 5-nitrothiophene-3-carboxaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0023] The fluorescein hydrazide is represented by the following structural formula (25). [ka]

[0024] The fluorescein hydrazide analogue is not particularly limited and can be appropriately selected depending on the purpose, but is preferably one represented by any one of the following structural formulae (26) to (36). [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0025] The 5-nitrothiophene-2-carboxaldehyde is represented by the following structural formula (37). [ka]

[0026] The analog of 5-nitrothiophene-2-carboxaldehyde is not particularly limited and can be appropriately selected depending on the purpose, but is preferably represented by the following structural formula (38) or (39). [ka] [ka]

[0027] (Apoptosis inducer) The apoptosis inducer contains a compound (a compound represented by general formula (1) or the following general formula (2)), and may further contain other components. The compounds are as described above in the "(Compound)" section.

[0028] (Anticancer drug) The anticancer agent contains a compound (a compound represented by general formula (1) or the following general formula (2)), and may further contain other ingredients. The compounds are as described above in the "(Compound)" section.

[0029] (PDI activity inhibitor) The PDI activity inhibitor contains a compound (a compound represented by general formula (1) or the following general formula (2)), and may further contain other components. The compounds are as described above in the "(Compound)" section.

[0030] (infectious disease treatment drug) The infectious disease treatment drug contains a compound (a compound represented by general formula (1) or the following general formula (2)), and may further contain other ingredients. The compounds are as described above in the "(Compound)" section.

[0031] (blood coagulation inhibitors) The blood coagulation inhibitor contains a compound (a compound represented by general formula (1) or the following general formula (2)), and may further contain other components. The compounds are as described above in the "(Compound)" section. [Example]

[0032] Examples of the present invention will be described below, but the present invention is not limited to these examples in any way.

[0033] (Synthesis Example 1: Synthesis of Compound of the Present Invention (3',6'-dihydroxy-2-{[(5-nitrothiophen-2-yl)methylidene]amino}spiro[isoindol-1,9'-xanthen]-3(2H)-one: FTNA)

[0034] While stirring 10 mL of an ethanol solution of fluorescein hydrazide (0.3460 g, 1.0 mmol, MedChemExpress), 10 mL of an ethanol solution of 5-nitrothiophene-2-carboxaldehyde (0.1580 g, 1.0 mmol, Merck (Sigma-Aldrich)) was added dropwise, followed by refluxing for 24 hours. The reaction mixture was returned to room temperature, and the resulting precipitate was filtered, washed with diethyl ether, and air-dried to obtain a yellow powder. The product was recrystallized from ethanol. [ka]

[0035] Comparative Synthesis Example 1: Synthesis of a control analog of FTNA (3',6'-dihydroxy-2-{[(3-methylthiophen-2-yl)methylidene]amino}spiro[isoindol-1,9'-xanthen]-3(2H)-one: FTMA)

[0036] While stirring 10 mL of an ethanol solution of fluorescein hydrazide (0.3460 g, 1.0 mmol, MedChemExpress), 10 mL of an ethanol solution of 3-methylthiophene-2-carboxaldehyde (0.1260 g, 1.0 mmol, Merck (Sigma-Aldrich)) was added dropwise, followed by refluxing for 24 hours. The reaction solution was returned to room temperature, and the resulting precipitate was filtered, washed with diethyl ether, and dried under reduced pressure to obtain a pale yellow powder. The product was recrystallized from ethanol. [ka]

[0037] -Basic characteristics of FTNA and FTMA- The chemical formula, molar mass, optical properties, and solubility of FTNA synthesized in Synthesis Example 1 and FTMA synthesized in Comparative Synthesis Example 1 are shown in Table 1. "MeOH" is methanol, "THF" is tetrahydrofuran, "DMSO" is dimethyl sulfoxide, and "DMF" is N,N-dimethylformamide. FTNA showed no decrease in reactivity in DMSO solution at 4°C for more than three years.

[0038] [Table 1]

[0039] -Physicochemical properties of FTNA and FTMA- The melting point of the FTNA synthesized in Synthesis Example 1 was 298°C. The melting point of FTMA synthesized in Comparative Synthesis Example 1 was 290°C.

[0040] FTNA synthesized in Synthesis Example 1 and FTMA synthesized in Comparative Synthesis Example 1 were analyzed by infrared spectroscopy. 1 H NMR, 13 C NMR, electrospray ionization mass spectrometry (ESI-MS), and X-ray crystal structure analysis were performed.

[0041] Infrared spectroscopic analysis (IR) (KBr; cm) of FTNA synthesized in Synthesis Example 1 -1 The results are shown in Figure 1. Infrared spectroscopic analysis (IR) (KBr; cm) of FTMA synthesized in Comparative Synthesis Example 1 -1 The results are shown in Figure 2.

[0042] FTNA synthesized in Synthesis Example 1, 1 The results of analysis by H NMR (500 MHz, DMSO-d, TMS) are shown in Figure 3. FTMA synthesized in Comparative Synthesis Example 1, 1 The analysis by H NMR (500 MHz, DMSO-d, TMS) is shown in Figure 4.

[0043] FTNA synthesized in Synthesis Example 1, 13 The analysis by C NMR (125 MHz, DMSO-d, TMS) is shown in Figure 5. FTMA synthesized in Comparative Synthesis Example 1, 13 The analysis by C NMR (125 MHz, DMSO-d, TMS) is shown in Figure 6.

[0044] The results of electrospray ionization mass spectrometry (ESI-MS) analysis of the FTNA synthesized in Synthesis Example 1 are shown in FIG. The results of electrospray ionization mass spectrometry (ESI-MS) analysis of FTMA synthesized in Comparative Synthesis Example 1 are shown in FIG.

[0045] The FTNA synthesized in Synthesis Example 1 was subjected to X-ray crystal structure analysis. FTNA crystals suitable for X-ray measurements were obtained by slowly evaporating a mixed solution of ethanol and DMSO at room temperature. The crystal data of FTNA was as follows: crystal system,monoclinic;space group,I2 / a;a=14.711(3)Å;b=14.5553(15)Å;c=25.957(4)Å;α(°)=90.000;β(°)=98.96(2);γ(°)=90.000;V=5490.3(16)Å 3 ;Z=8;GOF=0.960;CCDC:2038565.

[0046] The FTMA synthesized in Comparative Synthesis Example 1 was subjected to X-ray crystal structure analysis. Crystals of FTMA suitable for X-ray measurement were obtained by slowly evaporating a mixed solution of ethanol and DMF at room temperature. The FTMA crystal data were as follows: Crystal system,triclinic;space group,P-1;a=9.632(9)Å;b=11.051(10)Å;c=13.533(12)Å;α(°)=71.84(5);β(°)=73.59(6);γ(°)=88.71(5);V=1310(2)Å 3;Z=2;GOF=0.939;CCDC:2038564.

[0047] (Test Example 1: Cell Damage and Apoptosis Induction) We confirmed the cell damage and apoptosis induction caused by FTNA. -MTT assay- The MTT assay is a method for evaluating cell proliferation, viability, and toxicity by utilizing the reduction of MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) taken up into cells by mitochondrial dehydrogenase.

[0048] Various cancer cell lines (RAW264.7 cells (mouse leukemia monocyte-macrophage cell line, DS Pharma Biomedical), HeLa cells (human cervical cancer cell line, RCB0007, RIKEN BioResource Center), HepG2 cells (human liver cancer cell line, RCB1648, RIKEN BioResource Center), MCF-7 cells (human breast cancer cell line, RCB1904, RIKEN BioResource Center), CACO-2 cells (human colon cancer epithelial cell line, RCB0988, RIKEN BioResource Center)) and normal cells (MDCK cells (normal canine renal tubular epithelial cells, RCB0995, RIKEN BioResource Center), lung fibroblasts, and hepatocytes) were cultured at 1 × 10 cells / well in a 96-well cell culture microplate. 4 The cells were seeded at 1 cell / well and cultured in 10% FBS-DMEM medium containing 0.1 to 100 μmol / L of FTNA synthesized in Synthesis Example 1. After 24, 48, or 72 hours of culture, 10 μL of 5 mg / mL MTT solution (PBS solution) was added to each 100 μL of medium in each well, and the wells were further cultured for 3 hours. The medium was then removed, and 100 μL of DMSO was added to dissolve the formazan. The amount of formazan produced was determined by measuring the absorbance at 570 nm using a microplate reader. The FTNA concentration required to reduce cell viability by 50% (IC50 value) was determined from a graph showing the relationship between FTNA concentration and relative viability. The results are shown in Table 2.

[0049] The lung fibroblasts were isolated from mouse lungs according to the method of Seluanov A et al. (J Vis Exp. 2010 Oct 5; (44), 2033). Cell culture was performed using collagen I-coated plates in DMEM medium containing 10% FBS at 37°C in a cell culture incubator with 5% CO2 / 95% air. The hepatocytes were isolated from mouse livers by the in situ collagenase perfusion method according to the method of Severgnini M et al. (Cytotechnology. 2012 Mar;64(2):187-95). Cell culture was performed using collagen I-coated plates in DMEM medium containing 10% FBS at 37°C in a cell culture incubator with 5% CO2 / 95% air.

[0050] [Table 2]

[0051] The results in Table 2 show that the IC50 value of FTNA in RAW264.7 cells and other cells decreased rapidly after 48 hours of culture. The IC50 value of FTMA did not decrease over the 24 to 72 hour incubation period.

[0052] -Annexin V staining, DAPI staining- Annexin V is a protein that binds phosphatidylserine (PS), which is normally present on the inner surface of the cell membrane, to the surface of the cell membrane in the early stages of apoptosis. 2+ This can be used to detect apoptotic cells. DAPI is a cell membrane-impermeable nuclear staining fluorescent dye that stains the nuclei of apoptotic necrotic cells and cells with damaged cell membranes.

[0053] Apoptotic cell staining was performed using Alexa Fluor® 568 (ex / em=578 / 603 nm)-labeled Annexin V (Invitrogen) according to the experimental protocol provided by Invitrogen. Furthermore, DAPI was added to the medium to a final concentration of 1 μg / mL, and simultaneous observation with Annexin V staining was performed.

[0054] RAW264.7 cells (mouse leukemia monocyte-macrophage cell line, DS Pharma Biomedical) were cultured at 1 × 10 in an EZVIEW Culture Plate LB (glass bottom plate, 96 wells). 3 The cells were seeded at 1 cell / well and cultured in 10% FBS-DMEM medium containing 0.1 to 100 μmol / L of FTNA synthesized in Synthesis Example 1 or FTMA synthesized in Comparative Synthesis Example 1. After 24 to 72 hours of culture, the medium was removed from each well, and a buffer solution containing annexin V (10 mmol / L HEPES, 140 mmol / L NaCl, 2.5 mmol / L CaCl2, pH 7.4) was added and allowed to react for 15 minutes, after which the cells were washed with the same buffer solution.

[0055] Annexin V fluorescence was observed using an inverted fluorescence microscope (Zeiss Axio Vert. A1 FL-LED, Carl Zeiss Microscopy, LLC) with filter set 43 (excitation BP 545 / 25, beam splitter FT 570, emission BP 605 / 70). For DAPI fluorescence observation, an inverted fluorescence microscope (Zeiss Axio Vert. A1 FL-LED, Carl Zeiss Microscopy, LLC) with Filter Set 49 (EX G 365, BS FT 395, EM BP 445 / 50) was used.

[0056] The results are shown in Figure 9. The upper panel of FIG. 9 shows control cells, the middle panel of FIG. 9 shows cells to which FTMA was added, and the lower panel of FIG. 9 shows cells to which FTNA was added. In each row, a bright-field image is shown on the left, a fluorescent image of FTMA or FTNA is shown in the center, and a fluorescent image of Annexin V staining and DAPI is shown on the right.

[0057] From the results in FIG. 9, no annexin V-positive cells were observed in the control cells to which neither FTMA nor FTNA was added. In the presence of FTMA, only a few Annexin V-positive cells were detected, but in the presence of FTNA, approximately half of the cells were Annexin V-positive. Among these Annexin V-positive cells, some showed positive DAPI staining. Cells that are positive only for Annexin V are in the early stage of apoptosis, while cells that are positive for Annexin V and DAPI staining are in the late stage of apoptosis and are considered to be dead cells in which the selective permeability of the cell membrane has been disrupted. These results indicated that FTNA-induced cell death in RAW264.7 cells was a cell death process similar to apoptosis.

[0058] (Test Example 2: Effect on cell cycle) The effect of FTNA on the cell cycle was confirmed using HeLa cells. HeLa cells (RCB0007, RIKEN BioResource Center) were cultured at 2.0 × 10 -3 DMEM (1.0 g / L glucose with sodium pyruvate, without L-Gln and Phenol Red, liquid, Nacalai Tesque, Inc.) medium containing 10% FBS-DMEM medium (1.0 g / L glucose with sodium pyruvate, without L-Gln and Phenol Red, liquid, Nacalai Tesque, Inc.) containing 100 U / mL L-Ala-L-Gln, 100 U / mL penicillin, and 100 U / mL streptomycin was used to culture 1 × 10 cells in a glass-bottom dish (GBCD15, Violamo). 4 The cells were seeded at 35 mm per dish and cultured at 37°C under 5% CO2 / 95% air conditions.

[0059] HeLa cells were cultured for 24 or 48 hours in the presence of FTNA at concentrations of 0.3 μmol / L to 10 μmol / L, as synthesized in Synthesis Example 1, and the cell morphology was observed using a microscope culture system, Stage Top Incubator (Tokai Hit). The results are shown in Figure 10. Furthermore, the time course of morphological changes during cell division was observed in the presence of FTNA at a concentration of 5 μmol / L, and the results are shown in FIG.

[0060] The results in Figure 10 show that in HeLa cells cultured for 24 hours, in control cells (0 μmol / L) to which no FTNA was added, almost no cells in metaphase of cell division (spherical cells with an equator line) or shrunken cells (dead cells) were observed, but as the concentration of FTNA increased, a larger number of cells in metaphase of cell division were observed, and as the concentration increased further, the number of dead cells also increased. Furthermore, in HeLa cells cultured for 48 hours, even in cells treated with a low concentration of FTNA (0.3 μmol / L), dead cells as well as metaphase cells were observed. The results in Figure 11 show that in the time course of cell division, in control cells (0 μmol / L) to which no FTNA was added, it took approximately 1 hour for a cell in metaphase to divide into two cells (control, cell a, cell b in Figure 11), whereas in the presence of 5 μmol / L FTNA, cell division stopped in metaphase M for more than 3 hours, after which the cells shrank and died (5 μM FTNA, cell b, cell c in Figure 11). Similar results to those obtained with HeLa cells were also obtained with CACO-2 cells and MCF-7 cells (not shown).

[0061] (Test Example 3: Identification of proteins that exhibit fluorescence upon FTNA treatment) Using RAW264.7 cells, endogenous proteins that exhibit fluorescence upon FTNA treatment were identified. RAW264.7 cells (a murine leukemia monocyte-macrophage cell line, DS Pharma Biomedical) were cultured at 1 × 10 in a culture dish (tissue culture dish (for adherent cells), 35 mm, AGC Technoglass-IWAKI). 5Cells were seeded in a petri dish and cultured for 24 hours in a medium containing 3 μM FTNA. The cells were then recovered from the culture dish, washed three times with PBS, and disrupted by repeated freeze-thawing in 20 mmol / L HEPES / NaOH, pH 7.4, 150 mmol / L NaCl. The resulting cell disruption solution was centrifuged at 80×g for 10 minutes, and the resulting supernatant was used as a cell extract.

[0062] Next, the obtained cell extract (500 μg protein) was separated by two-dimensional electrophoresis (2D-PAGE). In the first dimension, proteins were separated by isoelectric focusing using Immobiline™ DryStrips (pH 3-10, 13 cm, GE Healthcare) with an IPGphor Isoelectric Focusing System (Amersham Pharmacia Biotech). The strips were then loaded onto a 13% polyacrylamide gel (size: 14 cm). <w>×13cm <l>×1.0mm <t>) and separated by SDS-PAGE to develop the proteins in two dimensions. After 2D-PAGE, the gel was scanned (600V PMT, Ex / Em=488nm / 520nm) using a Typhoon 9410 (Amersham). The results are shown in Figure 12. The gel was then stained with CBB (Bio-Safe™ Coomassie G-250 Stain, CBB, Bio-Rad). The results are shown in Figure 13.

[0063] Proteins fluorescently modified by FTNA treatment, spot No. 1 to spot No. 10 shown in Figures 12 and 13 (fluorescently modified proteins in 10 spots in the FTNA-treated RAW264.7 cell extract separated by 2D-PAGE), were excised from the gel, and the proteins were identified by Peptide Mass Fingerprint analysis using MALDI-TOF-MS at Cosmo Bio Co., Ltd. The results are shown in Figure 14.

[0064] As shown in Figure 14, spots 1 to 3 were triosephosphate isomerase (TPI), a glycolytic enzyme. Spot 9 was lymphocyte cytoplasmic protein 1 (LCP1, also known as L-plastin), an F-actin-binding protein. Spots 4 to 8 and 10 were all PDI family proteins (PDIA1, PDIA3, PDIA4, PDIA6, ERP46).

[0065] In addition to RAW264.7 cells (a murine leukemia-monocyte-macrophage cell line, DS Pharma Biomedical), 2D electrophoresis was performed on various cancer cell lines (HeLa cells (a human cervical cancer cell line, RCB0007, RIKEN BioResource Center), HepG2 cells (a human liver cancer cell line, RCB1648, RIKEN BioResource Center), MCF-7 cells (a human breast cancer cell line, RCB1904, RIKEN BioResource Center), and CACO-2 cells (a human colon cancer epithelial cell line, RCB0988, RIKEN BioResource Center)), and normal cells (MDCK cells (normal canine renal tubular epithelial cells, RCB0995, RIKEN BioResource Center)).

[0066] Based on the molecular weight and isoelectric point of 2D-PAGE of each cell line, the endogenous proteins that exhibit fluorescence upon FTNA treatment are thought to be five types of proteins in the PDI family (PDIA3, PDIA4, PDIA5, PDIA6, and ERP46). The fluorescently modified proteins TPI1 (spots 1 to 3) and LCP1 (spot 9) observed in mouse-derived RAW264.7 cells were not observed in these human- or dog-derived cell lines.

[0067] Test Example 4: Inhibition of PDI activity 0.1 μg / mL of human PDI recombinant (P4HB, Peptide Institute) was heated at 37°C for 2 hours in 10 mmol / L HEPES, pH 7.4-75 mmol / L NaCl containing 0.0025-500 μmol / L of FTNA (synthesized in Synthesis Example 1) or 0.0025-500 μmol / L of FTMA (synthesized in Comparative Synthesis Example 1). Bacitracin (Enzo Life Sciences), a known PDI inhibitor, was used as a control for comparison of the PDI activity inhibitory effect. The resulting reaction mixture was subjected to SDS-PAGE analysis and PDI activity measurement.

[0068] -SDS-PAGE analysis- After SDS-PAGE, the gel was scanned (Ex / Em=488 nm / 520 nm) using a Typhoon 9410 imaging system (Amersham) to confirm the fluorescent modification of PDI by FTNA. The results are shown in the upper panel of FIG. After fluorescent imaging of the gel, the gel was stained with CBB. The results are shown in the lower part of FIG.

[0069] From the results in FIG. 15, the fluorescent modification of PDI by FTNA began to be detected at around 1 μmol / L, and the fluorescent signal became stronger as the FTNA concentration increased, and then reached a nearly constant fluorescence intensity.

[0070] -PDI activity measurement- PDI activity was measured using a PROTESEOSTAT PDI assay kit (Ebzo Life Science). The results are shown in Figure 16.

[0071] The results in FIG. 16 demonstrate that PDI activity decreases with a concentration-dependent increase in PDI fluorescence signal. The IC50 value of FTNA for PDI activity was approximately 20 μmol / L, which was approximately 1 / 50 of the IC50 value of 1 mmol / L of bacitracin, a known PDI inhibitor. FTMA, a control analog of FTNA, had almost no effect on PDI activity in the same concentration range as FTNA.

[0072] (Test Example 5: Inhibition of blood coagulation) It is known that the risk of thrombosis increases with aging, smoking, excessive stress, alcohol consumption, lifestyle-related diseases (high blood pressure, dyslipidemia, diabetes, etc.), and cancer treatment. In recent years, it has been revealed that PDI inhibitors inhibit PDI-dependent platelet aggregation reactions in the blood coagulation process, thereby suppressing thrombus formation. Since FTNA is a specific inhibitor of PDI, it may have a suppressive effect on thrombus formation. Therefore, we investigated the effect of FTNA on thrombus formation using mouse whole blood.

[0073] FTNA (0, 5, 10, or 50 μmol / L) was added to a tube containing 100 μL of whole blood drawn from the inferior vena cava of a mouse (SAMR1 / Toho, derived from Kyoto University and bred in-house). The blood was then left at room temperature for 30 minutes, and the size of the thrombus formed was compared. The results are shown in Figure 17. Therefore, we investigated the effect of FTNA on thrombus formation using mouse whole blood.

[0074] The results in FIG. 17 show that blood coagulation (blood clot formation) is inhibited as the FTNA concentration increases.

[0075] From the above, it was found that the compound of the present invention can be used as a novel apoptosis inducer, a novel anticancer agent, a novel PDI activity inhibitor, a novel infectious disease treatment agent, or a novel blood coagulation inhibitor.< / t> < / l> < / w>

Claims

1. A compound represented by the following general formula (1) or the following general formula (2): 【Chemistry 1】 【Chemistry 2】 However, in the general formula (1) or the general formula (2), X １ and X ２ are each independently a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that X １ or X ２ is an oxygen atom, X is an oxygen atom １ or X ２ and the benzene ring form a double bond), Y １ , Y ２ , Y ３ and Y ４ are each independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom, Z １ , Z ２ and Z ３ are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and Z １ , Z ２ and Z ３ at least one of is a nitro group; Z ４ , Z ５ , Z ６ and Z ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

2. The X １ and X ２ The compound of claim 1 , wherein is a hydroxy group.

3. The Y １ , Y ２ , Y ３ and Y ４ The compound according to claim 1 , wherein is a hydrogen atom.

4. Said Z １ is a nitro group, and the Z ２ and Z ３ The compound according to claim 1 , wherein is a hydrogen atom.

5. Said Z ４ , Z ５ , Z ６ and Z ７ The compound according to claim 1 , wherein is a hydrogen atom.

6. An apoptosis inducer comprising a compound represented by the following general formula (1) or (2): 【Transformation 3】 【Chemistry 4】 However, in the general formula (1) or the general formula (2), X １ and X ２ are each independently a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that X １ or X ２ is an oxygen atom, X is an oxygen atom １ or X ２ and the benzene ring form a double bond), Y １ , Y ２ , Y ３ and Y ４ are each independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom, Z １ , Z ２ and Z ３ are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and Z １ , Z ２ and Z ３ at least one of is a nitro group; Z ４ , Z ５ , Z ６ and Z ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

7. An anticancer agent comprising a compound represented by the following general formula (1) or (2): 【Transformation 5】 【Transformation 6】 However, in the general formula (1) or the general formula (2), X １ and X ２ are each independently a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that X １ or X ２ is an oxygen atom, X is an oxygen atom １ or X ２ and the benzene ring form a double bond), Y １ , Y ２ , Y ３ and Y ４ are each independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom, Z １ , Z ２ and Z ３ are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and Z １ , Z ２ and Z ３ at least one of is a nitro group; Z ４ , Z ５ , Z ６ and Z ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

8. A PDI activity inhibitor comprising a compound represented by the following general formula (1) or (2): 【Transformation 7】 【Transformation 8】 However, in the general formula (1) or the general formula (2), X １ and X ２ are each independently a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that X １ or X ２ is an oxygen atom, X is an oxygen atom １ or X ２ and the benzene ring form a double bond), Y １ , Y ２ , Y ３ and Y ４ are each independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom, Z １ , Z ２ and Z ３ are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and Z １ , Z ２ and Z ３ at least one of is a nitro group; Z ４ , Z ５ , Z ６ and Z ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

9. A therapeutic agent for infectious diseases, comprising a compound represented by the following general formula (1) or the following general formula (2): 【Chemistry 9】 【Chemistry 10】 However, in the general formula (1) or the general formula (2), X １ and X ２ are each independently a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that X １ or X ２ is an oxygen atom, X is an oxygen atom １ or X ２ and the benzene ring form a double bond), Y １ , Y ２ , Y ３ and Y ４ are each independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom, Z １ , Z ２ and Z ３ are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and Z １ , Z ２ and Z ３ at least one of is a nitro group; Z ４ , Z ５ , Z ６ and Z ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

10. A blood coagulation inhibitor comprising a compound represented by the following general formula (1) or (2): 【Chemistry 11】 【Chemistry 12】 However, in the general formula (1) or the general formula (2), X １ and X ２ are each independently a hydroxy group, an oxygen atom, an ethylamino group, or a diethylamino group (provided that X １ or X ２ is an oxygen atom, X is an oxygen atom １ or X ２ and the benzene ring form a double bond), Y １ , Y ２ , Y ３ and Y ４ are each independently a hydrogen atom, a methyl group, a fluorine atom, a chlorine atom, or a bromine atom, Z １ , Z ２ and Z ３ are each independently a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, an alkyl group having 1 to 4 carbon atoms, or a nitro group, and Z １ , Z ２ and Z ３ at least one of is a nitro group; Z ４ , Z ５ , Z ６ and Z ７ are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

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