JP2013159600A - Glyoxalase i inhibitor and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a more practical glyoxalase I inhibitor.SOLUTION: A glyoxalase I inhibitor includes a compound represented by formula (1). In the formula: R, R, R, R, R, R, R, R, Rand Rare each a hydrogen atom, a hydroxyl group, or a 1-4C alkyl group; and R, Rand Rare each independently a hydrogen atom or a 1-4C alkyl group which may have a substituent.

Description

  The disclosure herein relates to glyoxalase I inhibitors and uses thereof.

  As a compound exhibiting antitumor activity, for example, a flavone compound having glyoxalase I inhibitory activity is known (Patent Document 1). Glyoxalase I (GLO I: Glyoxalase I) produces SD-lactoylglutathione (SD) from methylglyoxal (MG) and glutathione (GSH) produced during intracellular anaerobic sugar metabolism It is an enzyme. MG is a glycolytic metabolic byproduct, but it is known that MG irreversibly modifies proteins, DNA, RNA, etc. to induce cell death (apoptosis). That is, MG is considered to have cell death inducing activity.

  Since cancer cells are generally in an anaerobic environment, energy production depends on an anaerobic glycolysis system, and MG is thus generated. In cancer cells, the expression of GLOI for detoxifying the MG thus generated is increased. Since GLOI activity is low in normal cells, GLOI inhibitors are expected to act specifically on cancer cells, promote MG accumulation in cancer cells, and induce cancer cell apoptosis.

JP 2011-219384 A

  The flavone compound disclosed in Patent Document 1 is a kind of flavonoid. Flavonoids are compounds composed of a C6-C3-C6 carbon skeleton having a structure close to the phenylchroman ring widely distributed in the plant kingdom. Yes. For this reason, the correlation between the structure of the flavonoid and the GLOI activity is not known at all, and it has been difficult to predict. Therefore, new GLOI inhibitors belonging to flavonoids have not yet been provided.

  Accordingly, an object of the present disclosure is to provide a more practical GLOI inhibitor and use thereof.

  The inventors of the present invention have conducted various studies including isoflavone compounds derived from plants, particularly leguminous plants, including screening using GLOI inhibitory activity as an index. As a result, isoflavones having GLOI inhibitory activity in cells. The compound was found. The disclosure herein is provided based on these findings.

According to the indication of this specification, the GLOI inhibitor containing 1 or more types selected from the compound represented by the following formula | equation (1) and these pharmaceutically acceptable salts is provided.
(In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ may each independently be a hydrogen atom, hydroxyl group or substituted. Represents a good alkyl group having 1 to 4 carbon atoms, and R ₃ , R ₆ and R ₁₀ each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 4 carbon atoms.)

Moreover, according to the indication of this specification, the antitumor agent containing 1 or more types selected from the compound represented by the following formula | equation (1) and its pharmaceutically acceptable salt is provided.
(In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ may each independently be a hydrogen atom, hydroxyl group or substituted. Represents a good alkyl group having 1 to 4 carbon atoms, and R ₃ , R ₆ and R ₁₀ each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 4 carbon atoms.)

  According to the indication of this specification, the pharmaceutical composition which contains the said glyoxalase I inhibitor as an active ingredient is provided.

  According to the disclosure of the present specification, there is provided a method for screening a glyoxalase inhibitor, comprising a step of evaluating glyoxalase I inhibitory activity of a test compound which is a compound having an isoflavonoid skeleton.

According to the disclosure of the present specification, the step of evaluating the glyoxalase I inhibitory activity of a test compound having an isoflavonoid skeleton,
An antitumor agent screening method is provided.

According to the disclosure of the present specification, an antitumor agent comprising a step of evaluating one or more glyoxalase I inhibitory activities selected from a test compound represented by the following formula (1) and derivatives thereof: Screening method.
(In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ may each independently be a hydrogen atom, hydroxyl group or substituted. Represents a good alkyl group having 1 to 4 carbon atoms, and R ₃ , R ₆ and R ₁₀ each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 4 carbon atoms.)

It is a figure which shows the result of the inhibitory activity with respect to hGLOI in 100 micromol of 14 types of isoflavone compounds and BBG. It is a figure which shows the evaluation result of the antiproliferative activity with respect to human leukemia cell line HL-60 of an inhibitor candidate compound. It is a figure which shows the result of having measured the amount of MG in the culture supernatant of the HL-60 cell processed with the inhibitor candidate compound. It is a figure which shows the observation result of the apoptosis in HL-60 cell processed with the inhibitor candidate compound 11.

  The disclosure of the present specification relates to a GLOI inhibitor containing a compound having an isoflavone skeleton (hereinafter referred to as an isoflavone compound) included in a group of compounds collectively called flavonoids, or a pharmaceutically acceptable salt thereof, and use thereof. .

  The isoflavone compound disclosed herein (hereinafter referred to as the present isoflavone compound) can exhibit GLOI inhibitory activity. The isoflavone compound can also preferably exhibit antiproliferative activity based on GLOI inhibitory activity against cancer cells. Since GLOI is hardly expressed in normal cells, but overexpressed in cancer cells, GLOI inhibitors can act with high selectivity on cancer cells and induce cell death. .

  Hereinafter, the GLOI inhibitor containing this isoflavone compound, an antitumor agent, a pharmaceutical composition, the screening method of a GLOI inhibitor, etc. are demonstrated.

(GLOI inhibitor)
This isoflavone compound is represented by the following formula (1).

In the formula (1), R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ each independently have a hydrogen atom, a hydroxyl group or a substituent. It may represent an alkyl group having 1 to 4 carbon atoms which may be used.

  The alkyl group having 1 to 4 carbon atoms which may have a substituent may be linear or branched, but is preferably linear. Examples of such an alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, and a 2,2-dimethoxyethyl group. Preferred are an ethyl group and a propyl group, more preferred are a methyl group and an ethyl group, and even more preferred is a methyl group. Examples of the substituent include a hydroxyl group, a hydroxyalkyl group, an alkyl ether group, an alkylcarbonyl group, an amino group, an alkylamino group, a nitro group, a nitrile group, and a halogen atom. The alkyl group in these substituents preferably has 1 to 4 carbon atoms, more preferably a methyl group.

In the formula (1), R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R _12, and R ₁₃ are 5 of these 10 substituents. The number may be 6 or more, 6 or more, 7 or more, 8 or more, 9 or more, and all may be hydrogen atoms. May be. R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₁₂ and R ₁₃ may be 5 or more hydrogen atoms out of these 8 substituents, and 6 or more There may be seven or more, and all may be hydrogen atoms.

In the formula (1), R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ are methyl groups among these 10 substituents. May be 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more. R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R _12, and R ₁₃ may be 2 or more of these 8 substituents, and 3 or more It may be 4 or more, 5 or more, or 6 or more.

In Formula (1), R < ₃ >, R < ₆ > and R < ₁₀ > can represent the C1-C4 alkyl group which may have a hydrogen atom or a substituent each independently. The alkyl group having 1 to 4 carbon atoms which may have a substituent is as described above. Of these three substituents, R ₃ , R ₆ and R ₁₀ may be one or more of the above alkyl groups, may be two or more, and all may be the above alkyl groups. . R ₃ , R _6, and R ₁₀ may include 1 to 3 hydrogen atoms among these three substituents.

In the formula (1), R _{1 to} R ₈ , R ₁₀ , R ₁₂ and R ₁₃ represent a hydrogen atom, and a compound in which R ₉ and R ₁₁ are methyl groups is represented by the following formula (2). This compound is a natural isoflavone compound present in the bark of a plant belonging to the leguminous plant Erythrina variegata, and is called Erythrinin C. This isoflavone compound includes this natural isoflavone compound Erythrinin C, and includes a compound that is regulated within a certain range by substitution with a hydroxyl group of the hydrogen atom site or an alkyl group having 1 to 4 carbon atoms in the structure of Erythrinin C. .

  This isoflavone compound is obtained as a compound represented by the formula (2) obtained from the bark of a plant belonging to Erythrina variegata. In addition to this compound, the compound is appropriately modified organically or enzymatically. Can be obtained. This isoflavone compound can also be obtained by total synthesis or by semi-synthesis using other naturally derived compounds as starting materials.

  For example, the following method can be employed to obtain the compound represented by the formula (2) from the bark of a plant belonging to Erythrina variegata. The bark is cut into small pieces and cooled in acetone, and then the solvent is distilled off under reduced pressure to obtain an oily substance. This oily substance is washed with, for example, a hydrophobic solvent of n-hexane, and then partitioned between a hydrophobic solvent such as dichloromethane and water, and the hydrophobic solvent phase is distilled off under reduced pressure to obtain a residue. The residue is subjected to silica gel column chromatography (for example, silica gel 500 g, 10 × 13 cm) using chloroform-acetone (1 → 10: 1) or the like to obtain each fraction. Fraction presumably containing a compound represented by formula (2) is repeatedly subjected to silica gel column chromatography with benzene-ethyl acetate (5: 1) or the like (for example, 150 g of silica gel, 4.5 The compound represented by the formula (2) can be obtained by purifying with 10 g of silica gel and 1.0 x 27 cm after use.

  Pharmaceutically acceptable salts of this compound include, for example, pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like.

  Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate and phosphate, and organic acid salts such as acetate, maleate, fumarate and citrate. Examples of the pharmaceutically acceptable metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of the pharmaceutically acceptable ammonium salt include salts such as ammonium and tetramethylammonium. Examples of the pharmaceutically acceptable organic amine addition salt include addition salts such as morpholine and piperidine. Examples of acceptable amino acid addition salts include addition salts of glycine, phenylalanine, lysine, aspartic acid, glutamic acid and the like.

The GLOI inhibitory activity of the present compound can be measured by a known method or can be measured by a method disclosed in Examples described later. The GLOI inhibitory activity of the present compound means an inhibitory activity against GLOI purified to a normal degree at least in vitro. The degree of activity is not particularly limited, but the IC ₅₀ obtained according to the examples described below is a known GLOI inhibitor Sp-bromo-benzylglutathione (BBG) (in the formula (3), R = H. It is preferably 50% or more, more preferably 70% or more, and still more preferably 80% or more.

Further, the GLOI inhibitory activity of the present isoflavone compound may further mean an antiproliferative activity of cancer cells. The antiproliferative activity of cancer cells can be evaluated using a known method, and can also be evaluated as antiproliferative activity against human leukemia cell line HL-60 according to the examples described later. The antiproliferative activity in this method is such that the IC ₅₀ is preferably 20 μM or less, more preferably 15 μM or less, even more preferably 10 μM or less, even more preferably 5 μM or less, and even more preferably 3 μM or less.

  In addition to being used as a GLOI inhibitor, this compound can also be used as an apoptosis inducer (particularly for cancer cells) or an antitumor agent. In particular, the present compound is useful as an antitumor agent when it induces apoptosis of cancer cells as one embodiment of GLOI inhibitory activity and has antiproliferative activity.

  The antitumor agent and pharmaceutical composition disclosed in the present specification include the present isoflavone compound as an active ingredient, an excipient, a stabilizer, a wetting agent, an emulsifier, an absorption accelerator, a pH adjuster, a surfactant, It may contain additives such as diluents, carriers, solubilizers, flavoring agents, preservatives, fragrances, colorants, coating agents, and water.

  The antitumor agent and the pharmaceutical composition disclosed in the present specification can employ any method of oral administration or parenteral administration, and can be in the form of a pharmaceutical preparation suitable for each. Examples of pharmaceutical preparations include liquid agents such as liquids, syrups, injections, liquid inhalants, emulsions, and solid agents such as tablets, powders, granules, capsules, ointments, solid inhalants, suppositories, etc. Can be mentioned.

  The content of the present isoflavone compound in the antitumor agent and pharmaceutical composition disclosed in the present specification varies depending on the use, dosage form, formulation purpose, etc., but generally 1 to 100% by mass in the total amount of the composition is Preferably, it is 10-50 mass%.

  Antitumor agent and pharmaceutical composition of the present invention The dosage of the pharmaceutical composition of the present invention and the like may be appropriately determined according to the age, weight, administration method and the like of the administration subject. For example, usually 0.001 mg to 100 mg, preferably 0.01 mg to 10 mg of the present isoflavone compound can be administered once a day or in several divided doses per kg of body weight per day for an adult.

(Screening method)
The screening method for a GLOI inhibitor disclosed in the present specification can comprise a step of evaluating the GLOI inhibitory activity of a test compound that is an isoflavone compound. According to this screening method, a useful GLOI activity inhibitor can be screened from an isoflavone compound having an isoflavonoid skeleton. In addition, seed compounds for antitumor agents can be screened. In addition, the isoflavone compound may be obtained by synthesis in addition to a naturally derived compound. Naturally derived compounds can be obtained from legumes. The source plant is preferably a plant belonging to the genus Erythrina such as Erythrina variegata, Erythrina suberosa, Erythrina poeppigiana, Erythrina x bidwillii, and more preferably Erythrina variegata.

  The GLOI inhibitory activity can be evaluated by employing a method disclosed in Examples described later in addition to a known method. In this evaluation step, the GLOI inhibitory activity can be evaluated, for example, by comparing with the GLOI inhibitory activity of BBG as a control. Typically, if it has a GLOI inhibitory activity of 50% or more, more preferably 70% or more, and more preferably 80% or more of BBG, it can be positively evaluated as having a GLOI inhibitory activity.

  The screening method for an antitumor agent disclosed in the present specification can comprise a step of evaluating glyoxalase I inhibitory activity of a test compound that is an isoflavone compound. GLOI inhibitory activity is the basic activity of antitumor agents based on GLOI inhibitory activity. For this reason, according to the said evaluation process, the isoflavone compound (seed compound) which has a possibility as an antitumor agent can be screened widely and easily. In addition, the GLOI inhibitory activity in this screening method can be implemented by the same aspect as the evaluation process of GLOI inhibitory activity.

The screening method for an antitumor agent disclosed in the present specification can further comprise a step of evaluating the antiproliferative activity against cancer cells for a test compound affirmed to have GLOI inhibitory activity. By providing such an evaluation step, a more practical antitumor agent or its seed compound can be screened. The antiproliferative activity of a cancer cell is performed, for example, by supplying a test compound to the cancer cell and measuring an index relating to live cells or dead cells of the cultured mother particles for a certain period of time. More specifically, IC ₅₀ may be measured by varying the addition concentration of the test compound. In this evaluation step, if the antiproliferative activity of the test compound on cancer cells can be confirmed, it can be positively evaluated as having antiproliferative activity.

  It should be noted that the screening method for an antitumor agent disclosed in the present specification is based on the MG secreted by the cancer cells in the presence of the test compound for a test compound affirmed to have GLOI inhibitory activity and cancer cell antiproliferative activity And a step of evaluating the amount. By providing the MG amount evaluation step, it can be confirmed that growth of cancer cells is inhibited by GLOI inhibition in cancer cells. For the evaluation of the amount of MG, a known method such as HPLC for this type of compound can be adopted. For example, MG can be converted to 2-methylquinoxaline by adding o-phenylenediamine to a culture solution of cancer cells, and then loaded on HPLC for detection at a detection wavelength of 315 nm.

  The screening method for an antitumor agent disclosed in the present specification comprises one or more glyoxalase I inhibitory activities selected from a compound represented by formula (1) (present isoflavone compound) and a test compound that is a derivative thereof. The step of evaluating can be provided. According to this screening method, an antitumor agent (lead compound) more useful as a seed compound can be screened from the test compound represented by formula (1) and its derivatives. Various aspects of the screening method for an antitumor agent already described can be applied to this screening method. Although the range of the derivative of this isoflavone compound is not specifically limited, For example, about various R in Formula (1), the modification to substituents other than prescribed | regulated by Formula (1) can be included.

(Method for producing GLOI activity inhibitor and antitumor agent)
The method for producing a GLOI activity inhibitor or antitumor agent disclosed in the present specification comprises a step of separating a compound represented by formula (1) from a plant belonging to Erythrina variegata or a part thereof as a raw material. be able to. According to this production method, a compound represented by the formula (1) or a raw material thereof can be obtained efficiently using a plant body as a raw material.

  The plant body is preferably used as a raw material as a strip of about several millimeters. Moreover, it is preferable that the site | part is a bark. Extraction and separation of the compound represented by the formula (1) can be appropriately carried out by those skilled in the art. For example, as described above, an oily substance obtained by distilling off the solvent under reduced pressure after immersion in acetone. After removing the lipid from the mixture and obtaining a non-polar compound fraction using a general solvent partitioning technique, the fraction is further purified using liquid chromatography using a hydrophobic mobile phase with a polar filler such as silica. Separate and fractionate. Furthermore, a compound represented by the formula (2) is obtained by repeatedly purifying a fraction presumed to contain a compound represented by the formula (2) by silica gel column chromatography repeatedly. Can do.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.

(Acquire inhibitor candidate compounds)
In this example, 14 types of flavonoid compounds were obtained from leguminous Erythrina genus plants. The derived plant name and compound number are shown below, and the structure corresponding to the compound number is shown below.

(Acquisition of inhibitor candidate compound Erythrinin C (compound 11))
The bark (2.55 Kg) of Erythrina variegata was cut into small pieces, soaked in acetone, and the acetone was distilled off under reduced pressure to obtain an oil (133.7 g). The oil was washed with n-hexane and then partitioned between dichloromethane and water. The dichloromethane soluble part was distilled off under reduced pressure to obtain a reddish brown residue (50 g). This residue was subjected to silica gel column chromatography (silica gel 500 g, 10 × 13 cm) using chloroform-acetone (1 → 10: 1) to obtain 81 fractions (A1-A81, 200 mL each). Fraction A69-A70 (brown oil: 4.82 g) was repeatedly subjected to silica gel column chromatography using benzene-ethyl acetate (5: 1) (first time silica gel 150 g, 4.5 x 24 cm, second time silica gel 10 g, 1.0 x To give Erythrinin C (167 mg).

(Acquisition of other inhibitor candidate compounds)
In addition, other compounds were also obtained in accordance with Erythrinin C by appropriately changing the chromatography conditions.

(Evaluation of inhibitory activity against purified hGLOI enzyme activity)
In this example, GLOI inhibitory activity was measured for the various flavonoid compounds obtained in Example 1. That is, an inhibitor candidate compound dissolved in dimethyl sulfoxide (DMSO) is added to a reaction solution containing a GLOI enzyme substrate or the like (reaction solution after equilibration described later) to a final concentration of 100 μM, and then purified hGLOI enzyme solution 5 μL (1 μg protein amount) was added, and each activity was measured to determine specific activity. The specific activity was compared with BBG (control) as an inhibitor to evaluate the inhibitory effect (% inhibition).

GLOI activity was performed by partially modifying the method of Oray et al. (Bedii Oray and Scott, J. Norton, Method in Enzymology, 90, 542-546 (1982)). That is, since the substrate MG passes through the intermediate hemimercapatal in the presence of the coenzyme GSH and is converted into SD-lactoylglutathione by hGLOI, SD-lactoylglutathione (ε _{240 nm} = 3370 M ⁻¹ cm ⁻¹⁾ generated by the enzyme reaction. ) Was measured and the enzyme activity was calculated.
<Reaction solution>
200 mM imidazole ・ HCl, 16 mM MgSO ₄ (pH 7.0) 2.73 mL
100 mM methylglyoxal (MG) 237 μL
100 mM reduced glutathione (GSH) 30 μL
Total 3.0 mL

  Each reaction was prepared and mixed and then incubated at 25 ° C. for 90 minutes to ensure equilibration. After equilibration, the reaction was started by adding 5 μL of hGLO I purified enzyme solution (1 μg protein amount) to the 3.0 mL reaction solution, and the increase in absorbance at 240 nm was measured at 25 ° C. for 2 minutes. The initial velocity was determined by the slope of the straight line in the absorbance versus time plot. The blank used was 5 μL of storage buffer instead of the enzyme solution.

The resulting enzyme activity and specific activity than the initial speed .DELTA.A ₂₄₀ was calculated from the following equation. Furthermore, the inhibition rate% relative to the blank was determined from the specific activities of BBG and inhibitor candidate. In addition, 1 unit of the enzyme activity GLO I activity of BBG was defined as the amount of enzyme that catalyzes the production of 1 μmol of S-D-lactoylglutathion per minute under the above-mentioned enzyme activity measurement conditions. The specific activity was defined as the enzyme activity per 1 mg of protein. The results are shown in FIG.
Enzyme activity = ΔA ₂₄₀ × 3 × 10 ³ / ε
Specific activity = enzyme activity / protein amount (mg)

(Calculation of IC ₅₀ value)
The inhibitor candidate compound dissolved in DMSO was added to the reaction solution after equilibration so as to have final concentrations of 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, and 100 μM. Subsequently, 5 μL of hGLOI purified enzyme solution (1 μg in protein amount) was added, and the activity was measured in the same manner as above to determine the specific activity. The specific activity was compared with BBG which is the control of the inhibitor and graphed (concentration vs.% inhibition) to obtain an IC ₅₀ value, and the concentration dependency of the inhibitory activity was evaluated. The results are shown in Table 3.

As shown in FIG. 1 and Table 2, it was found that all of the 14 types of isoflavone compounds, except for Compound 11, showed almost the same or higher inhibitory activity as BBG and had strong GLOI inhibitory activity. . In addition, as shown in Table 3, all of the compounds showed lower IC ₅₀ values than BBG, indicating that they have potent GLOI inhibitory activity.

In addition, hGLOI was prepared as follows.
(1) In order to amplify the amino acid coding region (SEQ ID NO: 1) of hGLOI using a human lung cDNA library as a template, a sense primer containing an initiation codon and an antisense primer containing a termination codon were designed.

sense primer: 5'-TAG CATATG ATG GCAGAACCGCAGCCCCCGTC-3 '(SEQ ID NO: 3)
Nde I start codon
antisense primer: 5'-CAC GTCGAC CTCACAGCA CTA CATTAAGGTTGCC-3 '(SEQ ID NO: 4) Sal I stop codon

(2) Using human lung cDNA as a template, PCR was carried out under the following conditions to amplify the amino acid coding region using a sense primer containing an initiation codon and an antisense primer containing a termination codon.
<Reaction mixture>
cDNA (human lung cDNA) 1 μL
10 × LA buffer (Mg ²⁺ free) 2.5 μL
2.5 mM dNTP 4 μL
25 mM MgCl ₂ 2.5 μL
LA Taq DNA polymerase (5units / μL) 0.25 μL
Sense primer (10μM) 2.5 μL
Antisense primer (10μM) 2.5 μL
QW 9.75 μL
Total volume 25μL
<Reaction conditions>
Equipment: TaKaRa PCR Thermal Cycler SP
After 3 min at 94 ° C
After 35 cycles of 94 seconds at 94 ° C., 1 minute at 58 ° C. and 1 minute at 72 ° C., the temperature was set to 3 minutes at 72 ° C.

(3) The PCR product was subjected to 1% agarose gel electrophoresis in the presence of ethidium bromide (0.5 μg / mL), and the gel was cut while confirming the target band (amino acid coding region; about 580 bp) by UV irradiation. Thereafter, DNA was purified by a conventional method. After treating pGEM-TEasy Vector incorporating this DNA fragment with NdeI and SalI, an expression vector was prepared using pET-Vector (Novagen) so as to express hGLOI as a fusion protein with His-tag.

(4) His-tag fused hGLOI protein expression vector was introduced into E. coli BL21 (DE3) strain by electroporation, applied to LA agar plate medium and cultured overnight at 37 ° C, and His-tag fused hGLO. Colonies incorporating the I protein expression plasmid were obtained. This colony was inoculated into 2.5 mL of LA medium and cultured overnight at 37 ° C. with shaking. Next, inoculate 250 ml of this culture medium into LA culture medium and incubate with shaking at 37 ° C for 4 hours. Add IPTG (final concentration 1 mM), and further shake culture for 4 hours to express protein. Guidance was performed. The culture after induction of protein expression is collected by centrifugation at 7,000 rpm for 5 minutes at 4 ° C, then added with Homogenize buffer (30 mM Tris-HCl (pH 7.5), 1 mM DTT), and sonication (30 sec x 6 times). ) Thereafter, the supernatant obtained by centrifugation at 100,000 rpm and 4 ° C. for 15 minutes was used as the hGLO I crude enzyme solution. Dehydrate the hGLO I crude enzyme solution using a PD-10 gel filtration column (GE Healthcare), and replace the solvent with a Binding buffer (20 mM Na-PB, 500 mM NaCl, 20 mM Imidazol (pH 7.4)). After that, affinity chromatography was performed under the following conditions to purify hGLO I. The recovered hGLO I is desalted with a PD-10 gel filtration column, and the solvent is replaced with a storage buffer (50 mM Imidazol, 20% glycerol, 20 mM GSH, 5 mM MgSO ₄ (pH 7.2)), and purified enzyme solution It was.

<Measurement condition>
Column: HisTrap ^TM HP 5 mL (GE Healthcare)
Controller: Gradi Frac (GE Healthcare)
Detector: Optical Unit UV-1 (280 nm)
Mobile phase:
・ Binding buffer (20 mM Na-PB, 500 mM NaCl, 20 mM Imidazol (pH7.4))
・ Elution buffer (20 mM Na-PB, 500 mM NaCl, 500 mM Imidazol (pH7.4))

(Evaluation of antiproliferative activity against human leukemia cell line HL-60)
In this example, among isoflavone compounds that were shown to have a potent GLOI inhibitory action in vitro, compounds 8, 9 and 14, and also compound 11 that showed a weaker GLOI inhibitory activity than BBG were also observed in cultured cells. Antiproliferative activity was measured.

  In this example, BBG, which is a known inhibitor of GLOI, is poor in cell membrane permeability and is considered to have no effect when added to a culture solution. Therefore, in order to increase cell membrane permeability, BBG cyclopentyl is used. The effect on cultured cells was evaluated using BBGC which is an ester form. It is considered that BBGC is converted into BBG after permeation of the cell membrane, where the ester group is hydrolyzed in the cell.

Human leukemia cell line HL-60 cells were seeded on a 96-well plate (8.25 × 10 ³ cells, 100 μL / well) and cultured at 37 ° C. for 24 hours. Thereafter, the inhibitor candidate compound and BBGC (control) prepared at each concentration were dissolved in a medium, added at 80 μL / well, and cultured for 48 hours. DMSO was used for dissolution and dilution of the compound, and a chemical solution was prepared so that the final DMSO concentration was 0.2 to 0.5%.

  Next, 30 μL of MTS reagent (CellTiter 96 Aqueous One Solution reagent; Promega) and 10 μL of medium (10% FBS / RPMI) were prepared per well of the cell culture solution in the 96-well plate after chemical treatment. Was added and incubated for 1.5 hours under subculture conditions. Thereafter, the absorbance of the produced formazan dye at 490 nm was measured with a multi-label counter (Wallac 1420 ARVOsx, PerkinElmer). Since MTS is reduced by dehydrogenase contained in mitochondria in living cells to produce brown formazan, the absorbance at 490 nm is proportional to the number of living cells. The results are shown in FIG.

As shown in FIG. 2, it was shown that all of compounds 8, 11 and 14 except for compound 9 have a cell growth inhibitory action. The IC ₅₀ values of compounds 8, 11 and 14 for the growth of HL-60 cells were 33.5 μM, 2.2 μM and 35.3 μM, respectively. In vitro, Compound 11, which showed only weaker GLOI inhibitory activity than BBG, showed stronger cell growth inhibitory activity than BBGC. This is considered to reflect the property that the compound 11 is hardly soluble in water and easily permeates the cell membrane. On the other hand, the fact that Compound 9, which was shown to have a stronger GLOI inhibitory activity than BBG in vitro, did not show cytostatic activity, suggests that Compound 9 has low cell permeability.

(Evaluation of MG content in HL-60 cell culture supernatant)
In this example, the amount of MG in the culture supernatant was evaluated in order to confirm that the antiproliferative activity of the isoflavone compound on cells was based on the GLOI inhibitory action.

(Exposure of cells to candidate inhibitor compounds)
HL-60 cells are seeded in a 6 cm dish (2.8 × 10 ⁵ cells, 5 mL / dish), cultured at 37 ° C. for 24 hours, and then the inhibitor candidate compound and BBGC (control) prepared at each concentration are added to a final concentration of 10 It was added so that it might become μM, and further cultured for 24 hours. DMSO was used for dissolution and dilution of the inhibitor candidate compound, and a chemical solution was prepared so that the final DMSO concentration was 0.2 to 0.5%.

(Measurement of MG in culture supernatant)
The supernatant obtained by centrifugation after recovering the culture solution after the chemical treatment was used as a sample. To 4.5 mL of the collected supernatant, 450 μL of 60% perchloric acid (PCA) was added (final concentration 0.45 M) and incubated on ice for 10 minutes. Thereafter, the supernatant obtained by centrifugation (12000 rpm, 15 min) was applied to a Sep-Pak® Light tC18 cartridge (Waters). The tC18 cartridge was pretreated by washing with 6-8 mL of acetonitrile and then flowing 6-8 mL of 10 mM KH ₂ PO ₄ (pH 2.5). In this eluate, o-phenylenediamine (o-PD) dissolved in PBS and 5-methylquinoxaline (5-MQ) dissolved in QW as an internal standard were respectively added to a final concentration of 25 μM (125 nmol) and 0.5 μM (2.5 nmol) and allowed to react at room temperature for 3.5 hours. Since o-PD reacts with MG in the culture medium to produce 2-methylquinoxaline (2-MQ), 2-MQ and 5-MQ were quantified by HPLC / UV method.

The sample after the reaction was applied again at a speed of about 1 to 2 mL / min using a tC18 cartridge, and 2-MQ and 5-MQ were retained on the tC18 cartridge. Next, after washing the tC18 cartridge with 10 mM KH ₂ PO ₄ (pH 2.5), the target compound was eluted with 2 mL of acetonitrile. The eluate was concentrated to about 100 μL (SpeedVac SPD1010; ThermoFisher SCIENTIFIC), filtered through a PVDF membrane filter (0.45 μm; MILLIPORE), and then quantified by the HPLC / UV method under the following conditions. The results are shown in FIG.
<Measurement condition>
Column: Develosil C30-UG-5 4.6 mm id × 150 mm
(Nomura Chemical)
Controller: Jasco PU-980 (JASCO Corporation)
Detector: Jasco UV-1570 (315 nm; JASCO Corporation)
Mobile phase: 20% acetonitrile, 80% 10 mM KH ₂ PO ₄ (pH 2.5)
Flow rate: 1.5 mL / min
Sample volume: 50 μL
<Recorder conditions>
Recorder: Chromatopack C-R5A (Shimadzu)
Chart speed: 0.2 cm / min

  As shown in FIG. 3, the MG concentration in the culture supernatant of the control (cells treated with DMSO) was 0.024 μM, whereas BBGC used as a positive control was 0.076 ± 0.016 μM. Further, 0.01 ± 0.002 μM and 0.016 μM MG were detected in the culture supernatants treated with compounds 8 and 14, respectively, and only about 10-20% MG was detected as compared with BBGC. On the other hand, 0.352 ± 0.012 μM MG was detected in Compound 11, and more than 4 times as much MG was detected as compared with BBGC. From these results, it was considered that Compound 11 inhibits GLOI in HL-60 cells, thereby causing MG accumulation and, as a result, inducing cell growth suppression.

(Confirmation of apoptosis-inducing effect of compound 11)
In this example, the apoptosis-inducing effect of Compound 11 was confirmed.

(Exposure of cells to compound 11)
HL-60 cells were seeded on a 24-well plate (4.0 × 10 ⁴ cells, 500 μL / well), compound 11 was dissolved in medium to a final concentration of 10 μM, added at 100 μL / well, and added at 37 ° C. Incubate for hours. Note that DMSO was used for dissolution and dilution of the natural compound, and a chemical solution was prepared so that the final DMSO concentration was 0.2 to 0.5%.

(Nuclear staining)
The HL-60 cells after chemical solution treatment were collected in a 1.5 mL microtube, washed twice with 1 mL of phosphate buffered saline (PBS), added with 1 mL of 2% formaldehyde / PBS, and allowed to stand for 20 minutes for fixation. Next, 20 μL of 100 μg / mL Hoechst33342 (DOJINDO) was added, and the mixture was inverted and mixed, and then left to stand for 40 minutes while being shielded from the nucleus. Subsequently, after centrifuging at 900 rpm for 3 minutes, the supernatant was discarded, and the cells were washed again by adding 1 mL of PBS, and this operation was performed three times. Subsequently, the cell suspension was dropped onto a slide glass, covered with a cover glass, and the periphery of the cover glass was fixed with nail polish. This was observed with an HS all-in-one fluorescence microscope (BZ-9000; KEYENCE). The results are shown in FIG.

  In FIG. 4, the arrow has shown the nuclear fracture location. As shown in FIG. 4, in the cells treated with compound 11, nuclear rupture characteristic of apoptosis was observed. It was found that the antiproliferative activity of Compound 11 is based on the cell growth inhibitory action through induction of apoptosis.

  As mentioned above, although embodiment and the Example of this invention were described in detail, these are only illustrations and do not limit a claim. The technology described in the claims includes various modifications and changes of the specific examples illustrated above.

  The technical elements described in this specification or the drawings exhibit technical usefulness alone or in various combinations, and are not limited to the combinations described in the claims at the time of filing. In addition, the technology exemplified in this specification or the drawings can achieve a plurality of objects at the same time, and has technical usefulness by achieving one of the objects.

  SEQ ID NOs: 3, 4: Primers

Claims (10)

A glyoxalase I inhibitor comprising a compound represented by the following formula (1):
(In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ may each independently be a hydrogen atom, hydroxyl group or substituted. Represents a good alkyl group having 1 to 4 carbon atoms, and R ₃ , R ₆ and R ₁₀ each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 4 carbon atoms.) _{R 1 ~R 8, R 10,} R 12 and R ₁₃ represents a hydrogen atom, R ₉ and R ₁₁ represents a methyl group, glyoxalase I inhibitor according to claim 1. An antitumor agent comprising a compound represented by the following formula (1).
(In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ may each independently be a hydrogen atom, hydroxyl group or substituted. Represents a good alkyl group having 1 to 4 carbon atoms, and R ₃ , R ₆ and R ₁₀ each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 4 carbon atoms.) The antitumor agent according to claim 3, wherein R _{1 to} R _8, R _10, R ₁₂ and R ₁₃ represent a hydrogen atom, and R ₉ and R ₁₁ represent a methyl group.   A pharmaceutical composition comprising the glyoxalase I inhibitor according to claim 1 or 2 as an active ingredient. Evaluating glyoxalase I inhibitory activity of a test compound having an isoflavonoid skeleton,
A screening method for a glyoxalase I inhibitor. Evaluating glyoxalase I inhibitory activity of a test compound having an isoflavonoid skeleton,
A screening method for an antitumor agent.   Furthermore, the screening method of Claim 7 provided with the process of evaluating the antiproliferative activity with respect to a cancer cell about the said test compound affirmed having glyoxalase I inhibitory activity.   The method further comprises the step of evaluating the amount of methylglyoxal secreted by the cancer cells in the presence of the test compound for the test compound affirmed to have glyoxalase I inhibitory activity and antiproliferative activity. 9. The screening method according to 8. A screening method for an antitumor agent, comprising a step of evaluating one or more glyoxalase I inhibitory activities selected from test compounds represented by the following formula (1) and derivatives thereof.
(In the above formula, R ₁ , R ₂ , R ₄ , R ₅ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ and R ₁₃ may each independently be a hydrogen atom, hydroxyl group or substituted. Represents a good alkyl group having 1 to 4 carbon atoms, and R ₃ , R ₆ and R ₁₀ each independently represent a hydrogen atom or an optionally substituted alkyl group having 1 to 4 carbon atoms.)