JP2010229039A - Glyoxalase I inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a glyoxalase I-inhibiting agent that has a new skeleton different from that of a glutathione derivative and is highly specific to human glyoxalase I. <P>SOLUTION: A glyoxalase I-inhibiting agent and an anticancer agent, each represented by a predetermined formula, are provided. The glyoxalase I-inhibiting agent and the anticancer agent inhibit activity of human glyoxalase I with high specificity, and therefore are effectively utilized as a treating agent for various diseases such as an anticancer agent, a therapeutic agent for osteoporosis, or the like. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention preferably relates to a glyoxalase I inhibitor used as an anticancer agent and an osteoporosis therapeutic agent.

  In Japan, where the most common cause of death is cancer, the development of cancer drugs is an extremely important research issue. Currently, various anticancer drugs are used for treatment, but due to individual differences in drug sensitivity, administration of anticancer drugs by humans is not effective, and cancer cells are anticancer drugs. Acquiring resistance to is a big problem. For these reasons, there is a need for the development of cancer therapeutic agents that have high effects on anticancer drug-resistant cancer cell lines and that specifically act on cancer cells and have a new mechanism of action. .

  Glyoxalase I (GLO I) is an enzyme that catalyzes a reaction for producing SD-lactoyl glutathione from methylglyoxal (MG) and glutathione (GSH) (FIG. 1). Since methylglyoxal, a metabolic byproduct of glycolysis, is a highly reactive dicarbonyl compound, its excessive accumulation irreversibly modifies proteins, DNA, and RNA, and induces cell death (apoptosis) It is known. Here, glyoxalase I is uniformly expressed in all mammalian cells, but the expression level is extremely low. Interestingly, however, glyoxalase I is highly expressed in cancer cells such as colorectal cancer, liver cancer, and prostate cancer, and in cultured cancer cell lines that are resistant to anticancer agents. (Non-Patent Documents 1 to 3). Furthermore, there is a report that high expression of glyoxalase I is involved in acquiring resistance to anticancer agents such as etoposide and adriamycin.

  From these reports, it is speculated that cancer, which is an apoptosis-inhibiting disease, supplements the energy necessary for avoiding apoptosis and ensuring high disordered cell growth ability by an active anaerobic glycolysis system. Glyoxalase I is considered to act on the degradation of harmful methylglyoxal produced with active anaerobic glycolysis. Therefore, it is considered that by searching for a glyoxalase I inhibitor, an anticancer agent having a new mechanism of action that is effective for existing anticancer drug-resistant cancer cells can be developed.

  Representative examples of other diseases involving glyoxalase I include osteoporosis.

  For example, osteoporosis is a disease in which the bone resorption rate exceeds the bone formation rate and the bone density decreases due to the increased bone resorption function of osteoclasts. Here, osteoclasts are known to differentiate from monocyte / macrophage progenitor cells, but some glyoxalase I inhibitors have been reported to inhibit osteoclast differentiation. (Non-Patent Document 4). For this reason, the possibility that the glyoxal I inhibitor which can inhibit glyoxal I efficiently can be utilized as an effective therapeutic agent for the treatment of osteoporosis is suggested.

  Thus, it has been pointed out that glyoxalase I is associated with osteoporosis in addition to cancer, and affects the activity of glyoxalase I in the development of therapeutic agents for these diseases. It is expected to use compounds.

  So far, a glyoxalase I inhibitor having a glutathione skeleton as a substrate has been developed (Non-patent Document 5).

Davidson SD, Cherry JP, Choudhuri MS, Tasaki H, Mallouh C, Konno S. Glyoxalase I activity in human prosthesis cancer: a potential marker and importance in chemotherapy. J Urol. (1999) 161, 690-691 Ranganathan S, Walsh ES, Tew KD. Glyoxalase I in detoxification: studies using a glycoxalase I transfectant cell line. Biochem J. et al. (1995) 309, 127-131. Sakamoto H, Masima T, Kizaki A, Dan S, Hashimoto Y, Naito M, Tsuruo T. Glyoxalase I is involved in resistance of human leukemia cells to anti-agent agent-induced apoptosis. Blood. (2000) 9, 3214-3218 Kawawatani M, Okumura H, Honda K, Kanoh N, Muroi M, Dohmae N, Takami M, Kitagawa M, Futamura Y, Imoto M, Osada H. The identification of an osteoclastogenesis inhibitor through the inhibition of glyoxalase. Proc Natl Acad Sci USA. (2008), 105 (33), 11691-11696. Creighton DJ, Zheng ZB, Holewinski R, Hamilton DS, Eiseman JL. Glyoxalase I inhibitors in cancer chemotherapy. Biochem Soc Trans. (2003) 31, 1378-1382.

  However, since glutathione derivatives have low specificity for glyoxalase I, they are unsuitable as lead compounds that serve as the parent nucleus of the inhibitor structure, and clinical application is considered difficult.

  Therefore, an object of the present invention is to provide a glyoxalase I inhibitor having a novel skeleton different from a glutathione derivative and having high specificity for human glyoxalase I.

  The present inventors have found that a compound represented by a predetermined general formula exhibits specific inhibitory activity for glyoxalase I, and have completed the present invention.

  Specifically, the present invention provides the following.

［上記一般式（１）及び（１’）において、Ｘ及びＹは、それぞれ独立に、窒素原子、酸素原子、又は硫黄原子であり；Ｒ^１は炭素数１又は２の炭化水素鎖であり；Ｒ^２は、水酸基、炭素数１以上４以下のアルコキシ基、ヒドロキシアミノ基、又はアルコキシアミノ基であり；Ｒ^３、Ｒ^４、及びＲ^５は、それぞれ独立に水素原子、水酸基、ハロゲン原子、若しくは炭素数１以上５以下のアルコキシ基、又は、Ｒ^４とＲ^３又はＲ^５とが結合して形成される、ヘテロ原子を有していてもよい環状炭化水素基であり；Ｒ^６は、水素原子、炭素数１以上７以下のアルキル基、炭素数１以上７以下のアルコキシ基、アミノ基、又はハロゲン原子であり；点線と実線の二重線は単結合又は二重結合を示す。］ [1] A glyoxalase I inhibitor represented by the following general formula (1) or (1 ′).

[In the above general formulas (1) and (1 ′), X and Y are each independently a nitrogen atom, an oxygen atom, or a sulfur atom; R ¹ is a hydrocarbon chain having 1 or 2 carbon atoms; R ² is a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms, a hydroxyamino group, or an alkoxyamino group; R ³ , R ⁴ , and R ⁵ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, or An alkoxy group having 1 to 5 carbon atoms, or a cyclic hydrocarbon group which is formed by combining R ⁴ and R ³ or R ⁵ and may have a hetero atom; R ⁶ is hydrogen An atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 7 carbon atoms, an amino group, or a halogen atom; a double line with a dotted line and a solid line represents a single bond or a double bond. ]

  [2] X is a nitrogen atom, Y is an oxygen atom or a sulfur atom, and is bonded to X in the general formula (1), and a bond indicated by a dotted line and a solid line is a double bond. The glyoxalase I inhibitor according to [1].

  [3] A glyoxalase I inhibitor represented by the above general formula (1), which binds only to a carbon atom in the general formula (1) and has a bond represented by a dotted line and a solid double line. The glyoxalase I inhibitor according to [1] or [2], which is a double bond.

[4] The glyoxalase I inhibitor according to any one of [1] to [3], wherein R ⁴ is bonded to R ³ or R ⁵ to form a ring structure that may have a hetero atom. .

[5] A glyoxalase I inhibitor represented by the above general formula (1), wherein R ¹ is a methylene chain, [1] to [4] .

[6] The glyoxalase I inhibitor according to any one of [1] to [5], wherein R ² is a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms.

  [7] The glyoxalase I inhibitor according to any one of [1] to [6], which is used as an anticancer agent and a therapeutic agent for osteoporosis.

［上記一般式（１）及び（１’）において、Ｘ及びＹは、それぞれ独立に、窒素原子、酸素原子、又は硫黄原子であり；Ｒ^１は炭素数１又は２の炭化水素鎖であり；Ｒ^２は、水酸基、炭素数１以上４以下のアルコキシ基、ヒドロキシアミノ基、又はアルコキシアミノ基であり；Ｒ^３、Ｒ^４、及びＲ^５は、それぞれ独立に水素原子、水酸基、ハロゲン原子、若しくは炭素数１以上５以下のアルコキシ基、又は、Ｒ^４とＲ^３又はＲ^５とが結合して形成される、ヘテロ原子を有していてもよい環状炭化水素基であり；Ｒ^６は、水素原子、炭素数１以上７以下のアルキル基、炭素数１以上７以下のアルコキシ基、アミノ基、又はハロゲン原子であり；点線と実線の二重線は単結合又は二重結合を示す。］ [8] An anticancer agent represented by the following general formula (1) or (1 ′) and used for a cancer tissue that highly expresses glyoxalase I.

[In the above general formulas (1) and (1 ′), X and Y are each independently a nitrogen atom, an oxygen atom, or a sulfur atom; R ¹ is a hydrocarbon chain having 1 or 2 carbon atoms; R ² is a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms, a hydroxyamino group, or an alkoxyamino group; R ³ , R ⁴ , and R ⁵ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, or An alkoxy group having 1 to 5 carbon atoms, or a cyclic hydrocarbon group which is formed by combining R ⁴ and R ³ or R ⁵ and may have a hetero atom; R ⁶ is hydrogen An atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 7 carbon atoms, an amino group, or a halogen atom; a double line with a dotted line and a solid line represents a single bond or a double bond. ]

  [9] X is a nitrogen atom, Y is an oxygen atom or a sulfur atom, and is bonded to X in the general formula (1), and the bond indicated by the dotted and solid double lines is a double bond. [8] The anticancer agent according to [8].

  [10] An anticancer agent represented by the above general formula (1), wherein in the above general formula (1), it binds only to a carbon atom, and a bond represented by a dotted line and a solid double line is double. The anticancer agent according to [8] or [9], which is a bond.

[11] The anticancer agent according to any one of [8] to [10], wherein R ⁴ is bonded to R ³ or R ⁵ to form a ring structure that may have a hetero atom.

[12] The anticancer agent according to any one of [8] to [11], which is the anticancer agent represented by the general formula (1), wherein R ¹ is a methylene chain.

[13] The anticancer agent according to any one of [8] to [12], wherein R ² is a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms.

  The glyoxalase I inhibitor and anticancer agent of the present invention can inhibit the activity of human glyoxalase I with high specificity. For this reason, it can utilize effectively as a therapeutic agent of various diseases, such as an anticancer agent and an osteoporosis therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS It is drawing which shows the outline | summary of the catalytic reaction of glyoxalase I. BRIEF DESCRIPTION OF THE DRAWINGS It is drawing which shows the result of evaluation of the inhibitory activity with respect to the glyoxalase I of the glyoxalase I inhibitor in Example 1. FIG. It is drawing which shows the result of evaluation of the inhibitory activity with respect to the glyoxalase I of the glyoxalase I inhibitor in Example 2 and 3. FIG. It is drawing which shows the result of evaluation of the inhibitory activity with respect to the glyoxalase I of the glyoxalase I inhibitor in Example 2 and 3. FIG. It is drawing which shows the result of evaluation of the growth inhibition with respect to the cancer cell of the glyoxalase I inhibitor in Example 4. FIG. It is drawing which shows the result of the evaluation of the apoptosis induction activity with respect to the cancer cell of the glyoxalase I inhibitor in Example 4. FIG. 6 is a drawing showing the results of evaluating the inhibitory activity of glyoxalase I inhibitor on glyoxalase I in Example 5. It is drawing which shows the result of evaluation of the inhibitory activity with respect to the glyoxalase I of the glyoxalase I inhibitor in Example 6. FIG. 6 is a drawing showing the results of evaluation of the inhibitory activity of glyoxalase I inhibitor on glyoxalase I in Example 7. It is drawing which shows the inhibitory activity of the glyoxalase I inhibitor of this invention.

  Hereinafter, embodiments of the present invention will be described in detail.

［上記一般式（１）及び（１’）において、Ｘ及びＹは、それぞれ独立に、窒素原子、酸素原子、又は硫黄原子であり；Ｒ^１は炭素数１又は２の炭化水素鎖であり；Ｒ^２は、水酸基、炭素数１以上４以下のアルコキシ基、ヒドロキシアミノ基、又はアルコキシアミノ基であり；Ｒ^３、Ｒ^４、及びＲ^５は、それぞれ独立に水素原子、水酸基、ハロゲン原子、若しくは炭素数１以上５以下のアルコキシ基、又は、Ｒ^４とＲ^３又はＲ^５とが結合して形成される、ヘテロ原子を有していてもよい環状炭化水素基であり；Ｒ^６は、水素原子、炭素数１以上７以下のアルキル基、炭素数１以上７以下のアルコキシ基、アミノ基、又はハロゲン原子であり；点線と実線の二重線は単結合又は二重結合を示す。］ <Glyoxalase I inhibitor>
[Compound represented by general formula (1)]
The glyoxalase I inhibitor of the present invention is a compound represented by the following general formula (1) or (1 ′).

[In the above general formulas (1) and (1 ′), X and Y are each independently a nitrogen atom, an oxygen atom, or a sulfur atom; R ¹ is a hydrocarbon chain having 1 or 2 carbon atoms; R ² is a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms, a hydroxyamino group, or an alkoxyamino group; R ³ , R ⁴ , and R ⁵ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, or An alkoxy group having 1 to 5 carbon atoms, or a cyclic hydrocarbon group which is formed by combining R ⁴ and R ³ or R ⁵ and may have a hetero atom; R ⁶ is hydrogen An atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 7 carbon atoms, an amino group, or a halogen atom; a double line with a dotted line and a solid line represents a single bond or a double bond. ]

  Here, it is preferable that X is a nitrogen atom and Y is an oxygen atom or a sulfur atom. In addition, when X is a nitrogen atom, it connects with X and the bond represented by the double line of a dotted line and a solid line turns into a double bond.

R ¹ preferably has 1 carbon atom. In particular, when the glyoxalase I inhibitor of the present invention is represented by the general formula (1), R ¹ is preferably a methylene chain.

Examples of the alkoxy group having 1 to 4 carbon atoms represented by R ² include a methoxy group, an ethoxy group, a propoxy group, an iso-propoxy group, and a butoxy group, and the alkoxyamino group includes N-methoxy. A methaneamino group etc. can be mentioned. R ² is preferably a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms, and more preferably a hydrosyl group. In the case of administering the compound represented by the general formula (1) in vivo, from the viewpoint of permeability of the biological membrane, it is preferred that R ² is an alkoxy group having 1 to 4 carbon atoms.

Examples of the alkoxy group having 1 to 5 carbon atoms represented by R ³ , R ⁴ , and R ⁵ include a methoxy group, an ethoxy group, a propoxy group, an iso-propoxy group, a butoxy group, and a pentoxy group. . R ^{3, R 4,} and either ^{R 5} is a hydroxyl group, a halogen atom, or a number of 1 to 5 of the alkoxy group having a ^carbon, R 3, ^{R 4,} and any one is a hydroxyl group of ^{R 5,} halogen It is preferably an atom or an alkoxy group having 1 to 5 carbon atoms, and the remaining substituent is preferably a hydrogen atom.

Although it does not specifically limit as a cyclic hydrocarbon group which may have a hetero atom formed by combining R < ⁴ > and R < ³ > or R < ⁵ >, For example, R < ³ >, R < ⁴ >, and R < ⁵ > A naphthyl group, an anthracenyl group, a phenanthrenyl group, etc. formed with the benzene ring which has couple | bonded can be mentioned.

R ³ , R ⁴ , and R ⁵ are preferably formed by combining R ⁴ and R ³ or R ⁵ to form a cyclic hydrocarbon group that may have a hetero atom, and R ⁴ and R 5 More preferably, ³ or R ⁵ is bonded to form a naphthyl group together with the benzene ring to which these are bonded.

Examples of the alkyl group having 1 to 7 carbon atoms represented by R ⁶ include methyl group, ethyl group, propyl group, iso-propyl group, butyl group, t-butyl group, pentyl group, hexyl group, and heptyl group. Can be mentioned. Examples of the alkoxy group represented by R ⁶ having 1 to 7 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, an iso-propoxy group, a butoxy group, a pentoxy group, a hexoxy group, and a heptoxy group. Can do. R ⁶ is preferably a hydrogen atom.

  Moreover, in the said General formula (1), it couple | bonds only with a carbon atom and it is preferable that the coupling | bonding shown by the double line of a dotted line and a continuous line is a double bond.

Specific examples of the compound represented by the general formula (1) that satisfy the above conditions are shown in the following chemical formulas (1-1) to (1-12) and (1′-1).

  Among these compounds, compounds represented by chemical formulas (1-1) to (1-4), (1-8), (1-11), (1-12) and (1′-1) are preferable. And compounds represented by chemical formulas (1-2), (1-11), and (1-12) are more preferable.

[Method for Obtaining Compound Represented by General Formula (1)]
The compound represented by the general formula (1) can be obtained as a commercial product. Specific examples include NS-01538790 (manufactured by Namiki Shoji), NS-01538793 (manufactured by Namiki Shoji), NS-00197058 (manufactured by Namiki Shoji), NS-01576505 (manufactured by Namiki Shoji). it can.

[Use of glyoxalase I inhibitor]
Examples of the use of the glyoxalase I inhibitor include use as a therapeutic agent for diseases associated with enhancement of glyoxalase I, such as an anticancer agent and a therapeutic agent for osteoporosis. In particular, when used as an anticancer agent, the expression level of glyoxalase I is confirmed in the cancer tissue to which the glyoxalase I inhibitor is administered, and the expression of glyoxalase I is enhanced. It is preferred to use a glyoxalase I inhibitor only for patients with cancerous tissue.

  Since the expression level of glyoxalase I is very small in normal cells, it can exhibit apoptosis-inducing activity specifically in cancer cells that express glyoxalase I. For this reason, when a glyoxalase I inhibitor is used as an anticancer agent for cancer tissues that show signs of increased expression of glyoxalase I, an anticancer agent with few side effects and a strong action is used. Can be provided.

  In the present specification, when simply referring to glyoxalase I, it refers to human glyoxalase I, but the glyoxalase I inhibitor of the present invention is merely an enhancement of glyoxalase I in humans. It can be used not only as a therapeutic agent for diseases associated with, but also as a therapeutic agent for diseases associated with increased glyoxalase I in non-human animals. Specific examples of such non-human animals include mice, rats, cats, dogs, pigs, cows, monkeys and the like.

<Anticancer agent>
When the glyoxalase I inhibitor of the present invention is used as an anticancer agent, the compound represented by the general formula (1) (hereinafter also referred to as “the compound of the present invention”) is used as a pharmaceutically acceptable carrier. (For example, excipients and diluents) and the like, and appropriately formulated into any conventional dosage forms such as liquid preparations, powders, granules, tablets, capsules, syrups, injections, and aerosols And can be administered orally or parenterally.

  Examples of the pharmaceutically acceptable carrier include excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; cellulose, methylcellulose, hydroxypropylcellulose, polypropyl Binders such as pyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol-starch, sodium bicarbonate, calcium phosphate, and calcium citrate; Lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; citric acid, menthol, glycyrrhizin ammonium salt, glycine, orange powder, etc. Preservatives such as sodium benzoate, sodium bisulfite, methylparaban, and propylparaban; Stabilizers such as citric acid, sodium citrate, and acetic acid; Suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate A dispersant such as a surfactant; a diluent such as water, physiological saline, and orange juice; and a base wax such as cacao butter, polyethylene glycol, and white kerosene, but is not limited thereto. It is not a thing.

  The drug of the present invention is preferably an oral preparation or an injection preparation. A suitable oral preparation is a solution in which an effective amount of the compound of the present invention is dissolved in a diluent such as water, physiological saline, or orange juice; a capsule or sachet containing the effective amount of the compound of the present invention as a solid or granule. Or suspensions in which the compound of the present invention is dispersed in a suitable dispersion medium; and emulsions in which an effective amount of the compound of the present invention is dissolved and dispersed in a suitable dispersion medium is there.

  As injection preparations, aqueous and non-aqueous isotonic sterile injection solutions are suitable, and such injections contain antioxidants, buffers, antibacterial agents, isotonic agents and the like. May be. It is also preferred to use aqueous and non-aqueous sterile suspensions, which include suspensions, solubilizers, thickeners, stabilizers, preservatives, and the like. Also good. The preparation of the present invention can be enclosed in a container such as an ampoule or a vial in a unit dose or multiple doses. In addition, the compound of the present invention and a pharmaceutically acceptable carrier can be lyophilized and stored, and dissolved or suspended in a suitable sterile vehicle just before use.

  The dose of the anticancer agent of the present invention depends on the effective amount of the compound of the present invention, which is an active ingredient, the cytotoxicity of the compound of the present invention, the degree of disease progression, the drug acceptability of the administration subject, body weight, age, etc. Although it is different, it is usually 30 μg / kg body weight or more and 30 mg / kg body weight or less per day for an adult, preferably 300 μg / kg body weight or more and 3 mg / kg body weight or less. This amount can be administered once or divided into several times it can.

  Hereinafter, the present invention will be described in detail with reference to examples. In addition, this invention is not limited to the Example shown below at all.

<Preparation Example 1; Production of human glyoxalase I recombinant protein>
Expression in which a DNA sequence encoding a human glyoxalase I protein is inserted into an Escherichia coli expression vector pET28-A partially encoding a His × 6 tag in order to produce a human glyoxalase I recombinant protein (rhGLO I) Plasmid pET28-A / hGLO I was prepared. This pET28-A / hGLO I was introduced into an E. coli strain (BL21), seeded on a kanamycin plate, and cultured at 37 ° C. overnight. Colonies were picked up, inoculated into 1 ml of 2 × YT medium and cultured overnight at 37 ° C. with shaking. This culture solution was added to 100 ml of 2 × YT medium and cultured with shaking until the absorbance at 600 nm reached 0.6.

  To this was added isopropyl-β-thiogalactopyranoside to 1 mM to induce expression, and the mixture was cultured with shaking at 37 ° C. for 3 hours. This Escherichia coli culture solution was recovered by centrifugation at 4000 rpm for 5 minutes. In the collected E. coli, 5 ml of lysis buffer (50 mM potassium phosphate buffer (pH 7.0), 0.1% nonidet P-40 (registered trademark, polyoxyethylene (8) octylphenyl ether, Wako Pure Chemical Industries, Ltd.) 1 mM phenylmethylsulfonyl fluoride, 500 mM NaCl) was added, and E. coli was disrupted by sonication. Centrifugation was performed after disruption, and TALON resin (registered trademark) was added to the supernatant to bind the human glyoxalase I recombinant protein bound to the 6 × His tag. Elution buffer (50 mM potassium phosphate buffer (pH 7.0), 0.1% nonidet P-40 (registered trademark, polyoxyethylene (8) octylphenyl ether, manufactured by Wako Pure Chemical Industries, Ltd.)), Elution with 1 mM methylphenylsulfonyl fluoride, 500 mM NaCl, 300 mM imidazole). The eluted human glyoxalase I recombinant protein was subjected to SDS polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue to confirm the degree of purification.

<Example 1>
About the compound represented by the said Chemical formula (1-1), the inhibitory activity of human glyoxalase I recombinant protein was examined as follows.

[Evaluation of glyoxalase I inhibitory activity]
Methylglyoxal was added at 7.9 mM, glutathione at 1 mM, magnesium sulfate at 14.6 mM, and imidazole hydrochloric acid at 182 mM, and a substrate solution made up to 25 μl with a pH 7.0 reaction buffer was added at 25 ° C. for 15 minutes. Pre-incubated. On the other hand, 25 μl of a mixed solution of glyoxalase I 1 μg / ml and a compound represented by the chemical formula (1-1) (six lines of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM) Was dispensed into 384 well plates and preincubated for 10 minutes at room temperature. Thereafter, 25 μl of the substrate solution was added to the mixed solution of the enzyme and the compound represented by the chemical formula (1-1) to start the reaction, and the absorbance at 240 nm was measured every 10 seconds at 25 ° C. The production amount of D-lactylutathione was measured and reacted for a total of 5 minutes. For each concentration of the compound represented by chemical formula (1-1), the inhibitory activity (%) of glyoxalase I is calculated, the X axis is the logarithm of the concentration of chemical formula (1-1), and the Y axis is glyoxalase. As the inhibitory activity (%) of I, the concentration (IC ₅₀ ) of the compound represented by the chemical formula (1-1) when the inhibitory activity against glyoxalase I was 50% was calculated.

  In addition, the density | concentration of each component shall show the final density | concentration in a reaction solution. The results are shown in FIG.

<Example 2>
In the compound represented by the general formula (1), groups that can be taken as R ³ , R ⁴ , and R ⁵ were appropriately changed, and changes in the inhibitory activity against human glyoxalase I recombinant protein were examined according to Example 1.

Specifically, in addition to the compound represented by the chemical formula (1-1) in which only R ⁴ is a methoxy group, it is represented by the following chemical formula (2-1) in which the methoxy group of the methoxyphenyl group is located in the ortho position. The inhibitory activity against glyoxalase I was evaluated for the compound represented by the chemical formula (1-4) in which only R ⁵ is a methoxy group.

Further, the compound represented by the chemical formula (1-2) in which the methoxy group of R ^{4 in} the compound represented by the chemical formula (1-1) is changed to a bromine atom, and the chemical formula (1-3) changed to a hydroxyl group. The inhibitory activity against glyoxalase I was also evaluated. The results are shown in FIGS.

As shown in FIG. 4C, the IC ₅₀ for each compound is represented by the chemical formula (2-1), IC ₅₀ = 55.9 μM, and the chemical formula (1-4). IC ₅₀ = 4.68 μM, the compound represented by the chemical formula (1-2) was IC ₅₀ = 6.93 μM, and the compound represented by the chemical formula (1-3) was IC ₅₀ = 16.5 μM. .

<Example 3>
In order to investigate the influence on the inhibitory activity of the hetero atom on the benzothiazole ring in the compound represented by the general formula (1), the compound represented by the chemical formula (1-7) in which all the hetero atoms of the benzothiazole ring are nitrogen atoms. About the compound to be tested, together with the compound represented by the chemical formula (1-1), the inhibitory activity against human glyoxalase I recombinant protein was evaluated according to Example 1. The results are shown in FIG. 3 and FIG. 4 (B). Note that, as shown in FIG. 4C, the IC ₅₀ of the compound represented by the chemical formula (1-7) was IC ₅₀ > 100 μM.

<Example 4>
In Example 2, the inhibitory activity against human glyoxalase I recombinant protein was examined, the compound represented by the chemical formula (1-4), the compound represented by the chemical formula (1-2), the chemical formula (1-3) The apoptosis-inducing activity for cultured cells was evaluated together with the compound represented by the chemical formula (1-1).

[Evaluation of apoptosis-inducing activity]
Cultured cancer cells (HL-60) were treated with the glyoxalase I inhibitor of the present invention, and cell viability was measured by MTS assay to evaluate apoptosis-inducing activity.

In the evaluation, HL-60 was seeded on a 96-well culture plate at 5 × 10 ⁴ cells / 100 μl / well. Immediately after seeding the cells in a 96-well culture plate, a glyoxalase I inhibitor was added to a final concentration of 0.1 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM, 300 μM, and 1000 μM and cultured for 24 hours. In these cells, 20 μl of MTS solution (3- (4,5-dimethyldimethylazolyl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium salt, Promega The product was reacted for 4 hours in a CO ₂ incubator, and the absorbance at 490 nm was measured.

Calculate the cell viability (%) with the following formula (3), and create a graph with the logarithm of the concentration of the glyoxalase I inhibitor of the present invention on the X axis and the cell viability (%) on the Y axis. The concentration of glyoxalase I inhibitor (IC ₅₀ ) when the cell viability was 50% was calculated.

  The “background” appearing in the above formula (3) is the absorbance of 100 μl of culture medium plus 20 μl of MTS solution.

  The cell viability when glyoxalase I inhibitor was evaluated at a concentration of 500 μM using HL-60 is shown in FIG. 5, and the relationship between the concentration of glyoxalase I inhibitor and the viability of HL-60 is shown in FIG. It is shown in FIG.

<Example 5>
In the compounds represented by the above general formulas (1) and (1 ′), in order to investigate the influence on the inhibitory activity of the hetero atom on the benzothiazole ring due to the difference in the position of the double bond bonded only to the carbon atom, In the general formula (1), the compound represented by the chemical formula (1-1), which is bonded to only a carbon atom and a bond represented by a dotted line and a solid line is a double bond; and the above general formula (1 ′ ), A compound represented by the chemical formula (1′-1) which is bonded only to a carbon atom and the bond represented by a dotted line and a solid line is a double bond, according to Example 1. The inhibitory activity against the ase I recombinant protein was evaluated. The results are shown in FIG.

  In addition, the sample used in this Example as the compound represented by the chemical formula (1′-1) is the compound represented by the chemical formula (1′-1) and the compound represented by the chemical formula (1-1). And 3: 1.

<Example 6>
In the same manner as in Example 1, the above chemical formulas (1-5), (1-6), (1-8) to (1-12), and (1′-1) and the following chemical formula (2-2) and About a mixture of compounds represented by (2-3) ((2-2) :( 2-3) = 3: 1), together with the compound represented by chemical formula (1-1), human glyoxalase I recombinant The inhibitory activity against the protein was evaluated. The inhibitory activity of glyoxalase I in 100 μM of each compound is shown in FIG. 8 (A), and the compounds represented by chemical formulas (1-8), (1-11), (1-12), and (1′-1) FIG. 8B shows the relationship between the concentration of and the glyoxalase I inhibitory activity.

As shown in FIG. 8C, the IC ₅₀ of the compounds represented by the chemical formulas (1-8), (1-11), (1-12), and (1′-1) is 13. 1 μM, 3.71 μM, 1.72 μM, and 26.5 μM.

<Example 7>
In the same manner as in Example 4, for the compounds represented by chemical formulas (1-11) and (1-12), the apoptosis-inducing activity of HL-60 was evaluated together with the compound represented by chemical formula (1-1). . The results are shown in FIG.

  The glyoxalase I inhibitory activity of each compound used in Examples 1 to 7 is summarized in FIG. As can be seen from FIG. 10, the glyoxalase I inhibitory activity is not limited to the compounds represented by the chemical formula (1-1), particularly the chemical formulas (1-4), (1-7), (1-11), and ( It can be seen that the compound represented by 1-12) has a high value.

Claims (13)

A glyoxalase I inhibitor represented by the following general formula (1) or (1 ′).

[In the above general formulas (1) and (1 ′), X and Y are each independently a nitrogen atom, an oxygen atom, or a sulfur atom; R ¹ is a hydrocarbon chain having 1 or 2 carbon atoms; R ² is a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms, a hydroxyamino group, or an alkoxyamino group; R ³ , R ⁴ , and R ⁵ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, or An alkoxy group having 1 to 5 carbon atoms, or a cyclic hydrocarbon group which is formed by combining R ⁴ and R ³ or R ⁵ and may have a hetero atom; R ⁶ is hydrogen An atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 7 carbon atoms, an amino group, or a halogen atom; a double line with a dotted line and a solid line represents a single bond or a double bond. ]   X is a nitrogen atom, Y is an oxygen atom or a sulfur atom, connected to X in the general formula (1), and a bond represented by a double line of a dotted line and a solid line is a double bond. The glyoxalase I inhibitor described in 1.   A glyoxalase I inhibitor represented by the above general formula (1), which binds only to a carbon atom in the above general formula (1), and a bond represented by a dotted line and a solid double line is a double bond. The glyoxalase I inhibitor according to claim 1 or 2. The glyoxalase I inhibitor according to any one of claims 1 to 3, wherein R ⁴ is bonded to R ³ or R ⁵ to form a ring structure which may have a hetero atom. The glyoxalase I inhibitor represented by the general formula (1), wherein R ¹ is a methylene chain. The glyoxalase I inhibitor according to any one of claims 1 to 5, wherein R ² is a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms.   The glyoxalase I inhibitor according to any one of claims 1 to 6, which is used as an anticancer agent and a therapeutic agent for osteoporosis. An anticancer agent represented by the following general formula (1) or (1 ′) and used for a cancer tissue that highly expresses glyoxalase I.

[In the above general formulas (1) and (1 ′), X and Y are each independently a nitrogen atom, an oxygen atom, or a sulfur atom; R ¹ is a hydrocarbon chain having 1 or 2 carbon atoms; R ² is a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms, a hydroxyamino group, or an alkoxyamino group; R ³ , R ⁴ , and R ⁵ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, or An alkoxy group having 1 to 5 carbon atoms, or a cyclic hydrocarbon group which is formed by combining R ⁴ and R ³ or R ⁵ and may have a hetero atom; R ⁶ is hydrogen An atom, an alkyl group having 1 to 7 carbon atoms, an alkoxy group having 1 to 7 carbon atoms, an amino group, or a halogen atom; a double line with a dotted line and a solid line represents a single bond or a double bond. ]   X is a nitrogen atom, Y is an oxygen atom or a sulfur atom, and is linked to X in the general formula (1), and the bond indicated by a dotted line and a solid line is a double bond. The anticancer agent described in 1.   It is an anticancer agent represented by the general formula (1), and in the general formula (1), it is bonded to only a carbon atom, and a bond represented by a dotted line and a solid double line is a double bond. The anticancer agent according to claim 8 or 9. The anticancer agent according to any one of claims 8 to 10, wherein R ⁴ is bonded to R ³ or R ⁵ to form a ring structure which may have a hetero atom. An anti-cancer agent represented by the above general formula (1), R ¹ is an anti-cancer agent according to any one of claims 8 11 is a methylene chain. The anticancer agent according to any one of claims 8 to 12, wherein R ² is a hydroxyl group or an alkoxy group having 1 to 4 carbon atoms.

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