JP2019182845A - Kynurenine aminotransferase 2 (kat2) inhibitor - Google Patents

Abstract

Disclosed is a pharmaceutical composition useful for the treatment of diseases caused by increased production of KYNA, such as schizophrenia.
[Solution]

^[R 1 is, -OH, -OR ', - OCOR ', - OCO (CH 2) n COOH, -OCO (CH 2) n COOR ', glucuronic acid, Jigurukuron group, R' is halogen, amino in an optionally substituted _{C 1-6} alkyl, n represents 1 to 3; ^{R 2} is hydrogen, halogen, or a salt thereof represented by is _{C 1-6} alkyl optionally] substituted with amino.
[Selection figure] None

Description

  The present invention relates to kynurenine aminotransferase 2 (KAT2) inhibitors. More specifically, the present invention relates to a pharmaceutical composition for treating a disease caused by increased production of kynurenic acid (KYNA), comprising glycyrrhizic acid or a derivative thereof.

  There are four types of isozymes (KAT1 to KAT4) in kynurenine aminotransferase (KAT). One of the isozymes, KAT2, is an enzyme that converts L-kynurenine (L-KYN) to kynurenic acid (KYNA) in the tryptophan-kynurenine metabolic pathway.

  Kynurenic acid (KYNA) has antagonism against NMDA-type receptors and neuronal nicotinic acetylcholine, and when its concentration is increased, memory, learning, cognitive function is reduced, and in schizophrenic patients, the brain It has been reported that the concentration of KYNA increases (Non-Patent Documents 1 to 3). Furthermore, patients with epilepsy, amyotrophic lateral sclerosis, cerebral malaria, HIV encephalopathy down syndrome, bipolar disorder and Alzheimer's disease have also been reported to have increased KYNA levels (Non-Patent Documents 4 to 10). .

Glycyrrhizic acid and its derivatives are widely used for the treatment of peptic ulcers such as gastric ulcers, but there are no reports of KAT2 inhibitory activity, and schizophrenia, epilepsy, amyotrophic lateral sclerosis, brain There are no reports of being effective in treating diseases caused by increased KYNA production such as malaria, HIV encephalopathy, Down's syndrome, bipolar disorder and Alzheimer's disease.
Furthermore, although drugs such as PF-04859989, BFF-122, and S-ESBA ((S)-(4-ethylsulfonyl) benzoylalanine) are commercially available as KAT2 inhibitors, these drugs are glycyrrhizic acid and its derivatives. And the structure is very different.

Kynurenic acid levels are elevated in the cerebrospinal fluid of patients with schizophrenia. Neurosci Lett 313: 96-98 The kynurenic acid hypothesis of schizophrenia. Physiol Behav 92: 203-209 Elevated levels of kynurenic acid in the cerebrospinal fluid of male patients with schizophrenia. Schizophr Res 80: 315-322 Kynurenate and 7-chlorokynurenate formation in chronically epileptic rats.Epilepsia 46: 1010-1016 Endogenous protectant kynurenic acid in amyotrophic lateral sclerosis. Acta Neurol Scand 107: 412-418 Metabolites of the kynurenine pathway of tryptophan metabolism in the cerebrospinal fluid of Malawian children with malaria.J Infect Dis 188: 844-849 Quinolinic acid and kynurenine pathway metabolism in inflammatory and non-inflammatory neurological disease.Brain 115 (Pt 5): 1249-1273 Increased kynurenic acid levels and decreased brain kynurenine aminotransferase I in patients with Down syndrome.Life Sci 58: 1891-1899 Elevated levels of kynurenic acid in the cerebrospinal fluid of patients with bipolar disorder.J Psychiatry Neurosci. 2010, 35, 195-199. Kynurenine metabolism in Alzheimer ’s disease. J Neural Transm. 1999, 106, 165-181.

  An object of the present invention is to provide a pharmaceutical composition useful for the treatment of diseases caused by increased production of KYNA, such as schizophrenia.

  In order to solve the above problems, the present inventors screened about 13,000 kinds of compounds, and as a result of earnestly examining the KAT2 inhibitory action of existing compounds, surprisingly among the test compounds having any structure, glycyrrhizin Finding that common acids and derivatives thereof can have an excellent CAT2 inhibitory action, and also having excellent therapeutic effects of glycyrrhizic acid and its derivatives against diseases caused by increased KYNA production, such as schizophrenia The present invention has been completed.

That is, the present invention provides the following aspects of the invention.
[1] Formula (I):
[In the ^{formula, R} 1, -OH, -OR ', - OCOR ', - OCO (CH 2) n COOH, -OCO (CH 2) n COOR ', a glucuronic acid or Jigurukuron acid groups, wherein , R ′ is C _1-6 alkyl optionally substituted with halogen or amino; n is 1-3; and R ² is C ₁ optionally substituted with hydrogen or halogen or amino. _-6 alkyl]
A pharmaceutical composition for treating a disease caused by increased production of kynurenic acid (KYNA), comprising a compound represented by the formula:
[2] R ¹ is —OH, —OCH ₃ , —OCOCH ₃ , —OCOCH ₂ COOH, —OCOCH ₂ CH ₂ COOH, —OCOCH ₂ COOCH ₃ , —OCOCH ₂ CH ₂ COOCH ₃ or a diglucuronic acid group; The pharmaceutical composition according to [1], wherein R ² is hydrogen or CH ₃ .
[3] The compound is:
[1] or [2] pharmaceutical composition selected from.
[4] The disease caused by the increased production of KYNA is schizophrenia, epilepsy, amyotrophic lateral sclerosis, cerebral malaria, HIV encephalopathy, Down's syndrome, bipolar disorder or Alzheimer's disease. [3] The pharmaceutical composition according to any one of [3].
[5] The pharmaceutical composition according to [4], wherein the disease caused by increased production of KYNA is schizophrenia.
[6] Formula (I):
[In the ^{formula, R} 1, -OH, -OR ', - OCOR ', - OCO (CH 2) n COOH, -OCO (CH 2) n COOR ', a glucuronic acid or Jigurukuron acid groups, wherein , R ′ is C _1-6 alkyl optionally substituted with halogen or amino; n is 1-3; and R ² is C ₁ optionally substituted with hydrogen or halogen or amino. _-6 alkyl]
Or a salt thereof, or a kynurenine aminotransferase 2 (KAT2) inhibitor.
[7] KYNA production comprising administering to a patient in need of treatment a therapeutically effective amount of a compound represented by the formula (I) of any one of [1] to [3] or a salt thereof Treatment method for diseases caused by the increase.
[8] The disease caused by the increased production of KYNA is schizophrenia, epilepsy, amyotrophic lateral sclerosis, cerebral malaria, HIV encephalopathy, Down's syndrome, bipolar disorder or Alzheimer's disease Method of treatment.
[9] The treatment method according to [8], wherein the disease caused by increased production of KYNA is schizophrenia.
[10] Use of the compound represented by the formula (I) of any one of [1] to [3] or a salt thereof in the manufacture of a medicament for treating a disease caused by increased production of KYNA.
[11] The compound represented by the formula (I) of any one of [1] to [3] or a salt thereof for use in the treatment of a disease caused by increased production of KYNA.
[12] A kynurenine amino acid, characterized by administering to a patient in need of treatment a therapeutically effective amount of a compound represented by the formula (I) of any one of [1] to [3] or a salt thereof Method for inhibiting transferase 2 (KAT2).

  According to the present invention, glycyrrhizic acid or a derivative thereof effectively inhibits kynurenine aminotransferase 2 (KAT2), thereby causing diseases caused by increased KYNA production such as schizophrenia, epilepsy, amyotrophic lateral sclerosis. It can be expected to exert excellent therapeutic effects on cerebral malaria, HIV encephalopathy, Down syndrome, bipolar disorder, Alzheimer's disease and the like.

Relationship between log equivalent concentration (nM) of commercially available KAT2 inhibitor (PF-04859989, BFF-122 and (S) -ESBA) and the compound of the present invention (glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone) and the inhibition rate and IC ₅₀ Values (μM) are shown. The relationship between the substrate concentration (mM) of PF-04859989 and control and the reaction rate (μM / min) (left figure) and KAT2 inhibition (right figure) of PF-04859989 by Lineweaver-Burk plot are shown. The relationship figure (left figure) of substrate concentration (mM) and reaction rate (micromol / min) of glycyrrhizic acid and control, and KAT2 inhibition (right figure) of glycyrrhizic acid by Lineweaver-Burk plot are shown. The relationship figure (left figure) of substrate concentration (mM) and reaction rate (micromol / min) of glycyrrhetinic acid and control, and KAT2 inhibition (right figure) of glycyrrhetic acid by Lineweaver-Burk plot are shown. The relationship between the substrate concentration (mM) of carbenoxolone and control and the reaction rate (μM / min) (left diagram) and KAT2 inhibition of carbenoxolone by the Lineweaver-Burk plot (right diagram) are shown. The mean value (± standard deviation) of serum kynurene concentration (nM) in mice in the control (0 mg / kg) group, the glycyrrhizic acid (25 mg / kg) administration group, and the glycyrrhizic acid (50 mg / kg) administration group is shown.

  The present invention is described in detail below.

  In the present invention, “halogen” means fluorine, chlorine, bromine and iodine.

In the present invention, “C _1-6 alkyl” means a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms. Specific examples of “C _1-6 alkyl” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1, Examples thereof include 1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like. Preferably, it is methyl or ethyl.

In the present invention, “glycyrrhizic acid or a derivative thereof” refers to the formula (I):
[In the ^{formula, R} 1, -OH, -OR ', - OCOR ', - OCO (CH 2) n COOH, -OCO (CH 2) n COOR ', a glucuronic acid or Jigurukuron acid groups, wherein , R ′ is C _1-6 alkyl optionally substituted with halogen or amino; n is 1-3; and R ² is C ₁ optionally substituted with hydrogen or halogen or amino. _-6 alkyl]
Or a salt thereof. Glycyrrhizic acid is an active ingredient contained in licorice root and has the following structure having effects as peptic ulcer and expectorant:
It is a compound which has this.
Glycyrrhetinic acid is one of β-amylin (oleanan) -based pentacyclic terpenoid derivatives obtained by hydrolysis of glycyrrhizic acid, and has the following structure:
It is a compound which has this. It is used as a seasoning to relieve the bitterness of drugs such as aloe and quinine, is effective in treating gastric ulcers, and also has properties as an expectorant. Inhibits enzymes that metabolize PGE-2 and PGF-2α.
Carbenoxolone has the following structure:
It is a compound which has this.

  In the present invention, glycyrrhizic acid and derivatives thereof may be in a salt form, and are not particularly limited as long as they are pharmaceutically acceptable salts. Examples of the salt include salts of alkali metals (eg, potassium, sodium, lithium, etc.), salts of alkaline earth metals (eg, calcium, magnesium, etc.), ammonium salts (eg, tetramethylammonium salt, tetrabutylammonium salt) Etc.), organic amines (for example, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) methylamine, lysine, arginine, N-methyl-D- Salt of glucamine and the like).

  Glycyrrhizic acid and its derivatives can be produced by known methods. Moreover, you may use a commercial item.

  In the present invention, “disease caused by increased production of kynurenic acid (KYNA)” refers to a disease or disorder caused by an increase in kynurenic acid concentration and its symptoms in the tryptophan-kynurenine metabolic pathway. Specific examples include, but are not limited to, schizophrenia, epilepsy, amyotrophic lateral sclerosis, cerebral malaria, HIV encephalopathy, Down's syndrome, bipolar disorder, Alzheimer's disease, and symptoms thereof. is not.

  In the present invention, “treatment” means cure and / or amelioration of a disease or disorder and its symptoms in mammals, particularly humans. It also includes preventing, alleviating and / or alleviating diseases resulting from increased production of NYNA.

  In the present invention, “patient” means humans and animals such as dogs, cats, horses and the like. Among these, human is preferable.

As used herein, “therapeutically effective amount” means the amount of an active ingredient that elicits a biological or pharmaceutical response as defined by a researcher or physician in a tissue, system, animal or human. "Therapeutically effective amount" also includes the meaning of the amount of an active ingredient that provides a therapeutic effect against a disease when administered to a patient to treat the disease. The “therapeutically effective amount” will vary depending on the active ingredient, the type of disease and its severity, and the age, weight, etc., of the mammal to be treated.
Such effective amounts include the amount of glycyrrhizic acid or its derivatives alone and / or the amount of glycyrrhizic acid or its derivatives in combination with other active ingredients having a KAT2 inhibitory action, such as PF-04859989.

  The kynurenine aminotransferase 2 (KAT2) inhibitor of the present invention inhibits the function of KAT2 involved in the conversion of L-KYN to KYNA in the tryptophan-kynurenine metabolic pathway, and inhibits the production of KYNA. It means the drug that exerts.

The pharmaceutical composition and KAT2 inhibitor of the present invention only need to contain glycyrrhizic acid or a derivative thereof as an active ingredient, and may further contain other pharmaceutically active ingredients as long as the effects of the active ingredient are not hindered. . For example, existing KAT2 inhibitors such as PF-04859989, BFF-122, and S-ESBA may be used in combination. The ratio of glycyrrhizic acid or a derivative thereof in the pharmaceutical composition of the present invention is not particularly limited.
In addition, the pharmaceutical composition of the present invention may be composed only of glycyrrhizic acid or a derivative thereof.

  The pharmaceutical composition and KAT2 inhibitor of the present invention can be administered orally or parenterally (for example, subcutaneous, intravenous, topical, nasal, pulmonary, rectal, etc.).

  The dosage form of the present invention can be appropriately selected and prepared according to the physical condition, health condition, etc. of the subject. For example, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, dispersible tablets, dissolving tablets), capsules, granules (effervescent granules), powders, oral solutions (elixirs, suspensions, emulsions, limonades) , Syrup (syrup preparation), oral jelly, oral tablet (troche, sublingual tablet, buccal tablet, adhesive tablet, gum), oral spray, oral semisolid preparation, mouthwash, injection (Infusion, implantable injection, continuous injection), dialysis agent (peritoneal dialysis agent, hemodialysis agent), inhalant (inhalation powder, inhalation solution, inhalation aerosol), suppository, rectal half Solid preparations, enema preparations, eye drops, eye ointments, ear drops, nasal drops (nasal powders, nasal drops), vaginal tablets, vaginal suppositories, external solid preparations (external powders), external liquids (Liniment, lotion), spray (external aerosol, pump spray), soft , Creams, gels, patches (tape, cataplasms) in dosage forms, such as can be prepared by known methods.

  The pharmaceutical composition of the present invention comprises, as necessary, excipients (for example, lactose, sucrose, D-mannitol and microcrystalline cellulose), disintegrants (for example, carmellose, carmellose sodium and low-substituted hydroxypropylcellulose), Binders (eg, hydroxypropylcellulose, hydroxypropylmethylcellulose, povidone and crystalline cellulose), lubricants (eg, magnesium stearate, calcium stearate and talc), solvents (eg, water, ethanol and propylene glycol), buffers ( For example, trisodium phosphate, sodium hydrogen phosphate and sodium dihydrogen phosphate), suspending agents (eg, gum arabic, tragacanth and sodium carboxymethyl cellulose), emulsifiers (eg, glycerin fatty acid ester) It may include a Le and sorbitan fatty acid esters) such as one or more pharmaceutically acceptable carriers.

  The administration period of glycyrrhizic acid or a derivative thereof and a concomitant drug is not particularly limited, and these may be administered simultaneously to the administration subject, or may be administered with a time difference. Glycyrrhizic acid or a derivative thereof may be administered to the concomitant drug first, simultaneously or later. Alternatively, glycyrrhizic acid or a derivative thereof and a combination drug may be used.

  The dose of glycyrrhizic acid or a derivative thereof of the present invention varies depending on the administration method, the age of the subject, the degree of disease, symptoms, dosage form, etc., for example, administered at a dose of 0.001 mg to 10 g per day. Can do. The amount of glycyrrhizic acid or a derivative thereof per day may be administered once to several times. When used in combination with a drug other than glycyrrhizic acid or a derivative thereof, the dose of glycyrrhizic acid or a derivative thereof can be reduced according to the dose of a drug other than glycyrrhizic acid or a derivative thereof.

  The present invention will be described more specifically with reference to the following examples, but these examples do not limit the present invention.

Example 1: Screening for KAT2 inhibitor candidate compounds According to the following method, about 13,000 kinds of compounds obtained from Validated Compound Library and Core Library provided by the University of Tokyo Drug Discovery Organization were preliminarily screened. 39 types of preliminary compounds were selected, and compounds that could become KAT2 inhibitors were selected from the selected compounds.

[reagent]
150 mM 2-amino-2-methyl-1-propanol (AMP) buffer (pH 9.5): AMP (13.371 g; SIGMA, No. 08578) is mixed with ultrapure water and adjusted to pH 9.5 with HCl. After adjustment, 150 mM AMP buffer (pH 9.5) was prepared.
200 ng / μL recombinant human KAT2 (rhKAT2): The concentration of rhKAT2 purified by the baculovirus / insect cell expression system was measured and diluted with AMP buffer to prepare 200 ng / μL rhKAT2.
20 mM L-kynurenine (KYN): KYN (4.164 g; SIGMA, No. K8625) was dissolved in an AMP buffer to prepare 20 mM KYN.
20 mM 2 (α) -ketoglutaric acid (α-KG): α-KG (292.2 mg; Nacalai Tesque, No. 19807-95) was dissolved in AMP buffer to prepare 20 mM α-KG.
-10 mM pyridoxal-5'-phosphate (PLP): PLP (265 mg; Nacalai Tesque, No. 29606-32) was dissolved in AMP buffer to prepare 10 mM PLP.
-0.1% Tween-20: Tween-20 (100 μL; Nacalai Tesque, No. 23926-35) was mixed with AMP buffer to prepare 0.1% Tween-20.
-300 mM zinc acetate dihydrate (pH 5.0) solution: Zinc acetate dihydrate (65.85 g; Nacalai Tesque, No. 36911-15) is dissolved in ultrapure water and adjusted to pH 5.0 with acetic acid. A 300 mM zinc acetate dihydrate (pH 5.0) solution was prepared.
[machine]
・ FLUORAC, 384 well, microplate (Greiner, No.781076)
・ Multi detection mode plate reader PHERAstar Plus (BMG Labtech, No.470-101)
・ Multi-label plate reader ARVO (Perkin Elmer, No.2030)
・ Multiple cooling centrifuge (TOMY)

[Method]
1. Preliminary compound selection Preliminary screening was performed according to the following method to select preliminary compounds.
(1) 100 nL / well of each compound (2 mM) was dispensed onto a plate for enzyme inhibition measurement by POD dispensing (n = 1, final concentration of compound 10 μM, final concentration of DMSO 0.5%). For control and background, 100 nL / well of an aqueous solution containing the same concentration of DMSO was dispensed (n = 16).
(2) The following enzyme solution was added to the test compound, control, and background wells. For background, 1 μL / well of AMP buffer was added instead of rhKAT2.

Enzyme solution (for test compound and control)
200 ng / μL rhKAT2 1 μL
0.1% Tween-20 0.5μL
AMP buffer 8.5 μL
10 μL

Enzyme solution (for background)
0.1% Tween-20 0.5μL
AMP buffer 9.5 μL
10 μL

(3) The following substrate solution was added to the test compound, control and background wells.

Substrate solution
20 mM KYN 1μL
20 mM α-KG 1μL
10 mM PLP 1 μL
0.1% Tween-20 0.5μL
AMP buffer 6.5 μL
10 μL

(4) The plate was spun down with a centrifuge and incubated at room temperature for 2 hours.
(5) A detection solution (300 mM zinc acetate dihydrate (pH 5.0) solution) was added at 20 μL / well, and the plate was spun down with a centrifuge.
(6) The fluorescence intensity of each test compound, control and background was measured with a multi-detection mode plate reader PHERAstar Plus (Ex 340 nm, Em 460 nm).
(7) S / B, CV (background and control) and Z ′ of the plate were calculated from the measured values of background and control based on the following formulas (n ≧ 4).

・ S / B = Mean _C / Mean _B
-CV (%) = 100x (SD _B / Mean _B )
100x (SD _C / Mean _C )
Z ′ = 1− (3SD _B + 3SD _C ) / (Mean _C− Mean _B )

Mean _B: average value of background Mean _C: average value of Control SD _B: standard deviation of the background SD _C: control the standard deviation of

(8) Based on the following formula, the inhibition rate (InH (%)) was calculated with Control as 100% reaction and Background as 0% reaction. The top 39 compounds with high calculated inhibition rates were selected as preliminary compounds.

InH (%) = 100 × {1- (Sample-Background) / (Control-Background)}

Sample: fluorescence intensity of test compound Control: average value of fluorescence intensity of control Background: average value of fluorescence intensity of background

2. Selection of candidate compounds for KAT2 inhibitor According to the following method, 39 selected compounds were screened to select compounds useful as KAT2 inhibitors.
(1) Each compound (2 mM) was dispensed at 100 nL / well by POD dispensing to a plate for enzyme inhibition measurement (Plate A) and a control plate (Plate B) for subtracting the fluorescence value due to autofluorescence of the compound (n = 4, final concentration of compound 10 μM, final concentration of DMSO 0.5%). For control and background, 100 nL / well of an aqueous solution containing the same concentration of DMSO was dispensed (n = 16).
(2) For Plates A and B, the following steps were performed.
(Plate A)
(A) 10 μL each of enzyme solution and substrate solution having the same composition as in 1 above was added to each well for test compound, control and background.
(B) The plate was spun down in a centrifuge and incubated for 2 hours at room temperature.
(C) 20 μl / well of a detection solution (300 mM zinc acetate dihydrate (pH 5.0) solution) was added, and the plate was spun down with a centrifuge.

(Plate B)
After the steps (a) to (c) were carried out in a test tube to complete the reaction, the solution was dispensed onto a plate.
(3) The fluorescence intensity of each test compound, control and background was measured with a multi-label plate reader ARVO (Ex 340 nm, Em 460 nm).
(4) S / B, CV (background and control) and Z ′ of each plate were calculated from the measured values of background and control on the basis of the above-mentioned formulas (n ≧ 4).
(5) Using the value obtained by subtracting the fluorescence intensity of Plate A from the fluorescence intensity of Plate B, using control as 100% reaction and background as reaction 0%, the inhibition rate (InH (%)) was calculated based on the following equation. . A compound having an InH (%) of 20% or more was selected as a candidate compound, and a compound having an InH (%) of 70% to 100% was selected as a useful compound as a KAT2 inhibitor.

InH (%) = 100x {(S _RawB- S _RawA ) / (Control-Background)}

S _RawB: average value of background average value -Plate B of the fluorescence intensity of the test compound Plate B S _RawA: Plate A in average -Plate A fluorescence intensity of test compound background average value Control: the Plate A Control Average value of fluorescence intensity of background: Background of Plate A: Average value of fluorescence intensity of background of Plate A

[result]
Table 1 shows the S / B ratio, CV (%) (control and background) and Z ′ value of each plate.

Table 2 shows the average values and standard deviations of InH (%) in 39 compounds used for screening.

  From the above results, 36, 37, and 38 with InH (%) of 70% to 100% were identified as candidate compounds for KAT2 inhibitor. These compounds were carbenoxolone, glycyrrhizic acid and glycyrrhetinic acid and were found to be very similar in structure. Surprisingly, it has been shown that glycyrrhizic acid and its derivatives can have an excellent KAT2 inhibitory action in common among test compounds having all structures. That is, it is considered that compounds similar in structure to these compounds may also be useful as KAT2 inhibitors.

Example 2: Examination of KAT2 inhibitory activity of existing drugs and candidate compounds According to the same measurement method as Example 1, obtained by screening with commercially available KAT2 inhibitors PF-04859989, BFF-122 and S-ESBA The fluorescence intensity at each measured concentration of the obtained compound, glycyrrhetinic acid, glycyrrhizic acid and carbenoxolone was measured, and the KAT2 inhibition rate was calculated. A calibration curve was created based on the calculated inhibition rate and measured concentration (converted into log concentration (nM)), and the IC ₅₀ value was calculated.

Table 3 shows the average value (n = 3) of the fluorescence intensity at each measured concentration of PF-04859989, BFF-122, S-ESBA, glycyrrhetinic acid, glycyrrhizic acid and carbenoxolone.

The log concentration (nM) of each measured concentration of PF-04859989, BFF-122, S-ESBA, glycyrrhetinic acid, glycyrrhizic acid and carbenoxolone and the average value (n = 3) of the inhibition rate are shown in Table 4, and each The IC ₅₀ values (μM) of the compounds are shown in Table 5. Further, FIG. 1 shows the relationship between the log concentration of each compound and its inhibition rate and the IC ₅₀ value.

  From the above results, it was shown that glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone have superior KAT2 inhibitory activity compared to existing BFF-122 and (S) -ESBA.

Example 3: Kinetic study of KAT2 inhibitory activity of existing drugs and candidate compounds Using PF-04859989 as an existing drug and glycyrrhizic acid, glycyrrhizic acid and carbenoxolone as compounds of the present application, determination of inhibition mode by Lineweaver-Burk plot went. Specifically, in a 1.5 mL tube, in a DMSO solution () as a compound solution or control, 30 μL enzyme solution (same composition as the enzyme solution of Example 1 (for test compound and control)), 70 μL substrate solution ( The same composition as the substrate solution of Example 1, but the concentration of the substrate (KYN) was 10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM or 0.3125 mM), and DMSO solution as a compound solution or control And incubated at 37 ° C. for 30 minutes. Further, the protein removal solution (3% perchloric acid; 100 μL) was added to the tube and stirred, and then centrifuged at 14,000 rpm for 10 minutes to precipitate the protein. The supernatant was transferred to an HPLC tube, the kynurenic acid concentration was measured by high performance liquid chromatography (HPLC), and the KAT2 inhibition of each compound was analyzed by Lineweaver-Burk plot.

2 to 5 show graphs of substrate concentrations and enzyme reaction rates and Lineweaver-Burk plots of PF-04859989, glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone. As is clear from the Lineweaver-Burk plot of each compound, PF-04859989 inhibits KAT2 activity as a non-competitive inhibitor, whereas glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone have KAT2 inhibitory action as competitive inhibitors. Indicated.
That is, glycyrrhizic acid or its derivative is recognized to function as a competitive inhibitor, unlike existing drugs.

Example 4: Examination of KAT2 Selectivity of Candidate Compounds For glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone, the inhibitory activity of KAT1, KAT2, KAT3 and KAT4 was measured, and the KAT2 selectivity of candidate compounds was examined.
Except that high performance liquid chromatography (HPLC) was used for quantification of kynurenic acid, KAT1 (human KAT1) and KAT2 (human KAT2, glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone were prepared in the same manner as in Example 1. Inhibitory activity (IC ₅₀ value (μM)) against mouse KAT2) was measured. For measurement of KAT1 enzyme inhibitory activity, kynurenine and sodium pyruvate were used as substrates, and for measurement of KAT2 enzyme inhibitory activity (human and mouse), kynurenine and α-KG were used as substrates.
In addition, according to the same method as the measurement method of Example 3, the concentration of kynurenic acid in the KAT3 substrate solution and KAT4 substrate solution at each measurement concentration of glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone was measured, and GraphPad Prism v6.07 was The inhibitory activity (IC ₅₀ value (μM)) of each compound against KAT3 (human KAT3) and KAT4 (human KAT4) was measured from nonlinear regression.

Table 6 shows the KAT1, KAT2, KAT3 and KAT4 inhibitory activities (IC ₅₀ values (μM)) of glycyrrhizic acid, glycyrrhetinic acid and carbenoxolone.

  Glycyrrhizic acid, glycyrrhetinic acid, and carbenoxolone were shown to have high inhibitory activity against KAT2 among KAT isozymes. That is, glycyrrhizic acid and derivatives are recognized to exhibit high KAT2 selectivity.

Example 5: Examination of KAT2 inhibitory action of glycyrrhizic acid in vivo In 8-week-old C57BL / 6N male mice, glycyrrhizic acid (25 mg / kg (n = 7) or 50 mg / kg (n = 8)) was orally administered for 3 days. As a control, physiological saline was orally administered for 3 days (n = 7). On the day after the last administration (the fourth day from the start of administration), blood is collected from the abdominal vena cava of the mouse, and the concentration of kynurenic acid in the serum is measured from the collected blood according to the same method as the measurement method of Example 3. did.

Table 7 and FIG. 6 show the mean value (± standard deviation) of serum kynurene concentration (nM) in mice of the control group, the glycyrrhizic acid (25 mg / kg) administration group, and the glycyrrhizic acid (50 mg / kg) administration group. .

  From the above results, it was shown that glycyrrhizic acid decreases the concentration of serum kynurenic acid in a concentration-dependent manner. That is, glycyrrhizic acid and its derivatives are recognized to show KAT2 inhibitory activity even in vivo.

  The present invention inhibits the function of CAT2 involved in the conversion of L-KYN to KYNA in the tryptophan-kynurenine metabolic pathway and suppresses the increase in KYNA production, thereby causing diseases caused by increased KYNA production, such as schizophrenia, It is useful for the treatment of epilepsy, amyotrophic lateral sclerosis, cerebral malaria, HIV encephalopathy, Down's syndrome, bipolar disorder, Alzheimer's disease and the like.

Claims (6)

Formula (I):

[In the ^{formula, R} 1, -OH, -OR ', - OCOR ', - OCO (CH 2) n COOH, -OCO (CH 2) n COOR ', a glucuronic acid or Jigurukuron acid groups, wherein , R ′ is C _1-6 alkyl optionally substituted with halogen or amino; n is 1-3; and R ² is C ₁ optionally substituted with hydrogen or halogen or amino. _-6 alkyl]
A pharmaceutical composition for treating a disease caused by increased production of kynurenic acid (KYNA), comprising a compound represented by the formula: R ¹ is —OH, —OCH ₃ , —OCOCH ₃ , —OCOCH ₂ COOH, —OCOCH ₂ CH ₂ COOH, —OCOCH ₂ COOCH ₃ , —OCOCH ₂ CH ₂ COOCH ₃ or a diglucuronic acid group; and R ² The pharmaceutical composition according to claim 1, wherein is hydrogen or CH ₃ . The compound is:
The pharmaceutical composition according to claim 1 or 2, selected from:   The disease caused by the increased production of KYNA is schizophrenia, epilepsy, amyotrophic lateral sclerosis, cerebral malaria, HIV encephalopathy, Down syndrome, bipolar disorder or Alzheimer's disease. The pharmaceutical composition according to one item.   The pharmaceutical composition according to claim 4, wherein the disease caused by the increased production of KYNA is schizophrenia. Formula (I):

[In the ^{formula, R} 1, -OH, -OR ', - OCOR ', - OCO (CH 2) n COOH, -OCO (CH 2) n COOR ', a glucuronic acid or Jigurukuron acid groups, wherein , R ′ is C _1-6 alkyl optionally substituted with halogen or amino; n is 1-3; and R ² is C ₁ optionally substituted with hydrogen or halogen or amino. _-6 alkyl]
Or a salt thereof, or a kynurenine aminotransferase 2 (KAT2) inhibitor.