JPWO2010128663A1 - Interleukin-2 production inhibitor - Google Patents

Abstract

A compound represented by any one of formulas (I) to (V) or a pharmaceutically acceptable salt thereof is used as an active ingredient of an interleukin-2 production inhibitor. In the formula, R1, R2, and R3 represent the same group selected from hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents a halogen. In the formula, R4 represents hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

Description

  The present invention relates to an interleukin-2 (IL-2) production inhibitor and an immunosuppressant comprising the same.

During the past several decades, many immunosuppressive agents have been developed and used for the purpose of reducing anti-inflammatory agents and rejection during organ transplantation (Non-patent Document 1). Many immunosuppressive drugs target lymphocytes, but are classified into several types depending on the mechanism of action. For example, glucocorticoids exert an immunosuppressive effect mainly by suppressing gene expression of IL-2 and other mediators. Cyclophosphamide metabolites alkylate DNA bases and preferentially suppress immune responses from B lymphocytes. Cyclosporin A (CsA) and tacrolimus (FK506) suppress the production of IL-2 and other cytokines by suppressing the calcineurin activity of T cells.
However, when these immunosuppressants are administered to patients, it is always necessary to be wary of side effects, albeit to varying degrees. The side effects vary depending on the type of immunosuppressant, but glucocorticoids have side effects such as diabetes, osteoporosis, glaucoma and increased infection rates when administered at high concentrations. In addition, CsA and FK506 have clinical effects by suppressing rejection at the time of organ transplantation and delaying the progression of disease states in patients with rheumatoid arthritis. The toxicity of the system has been observed clinically, clearly reducing their usefulness.
In the future, the development of drugs with different mechanisms of action and drugs with fewer side effects is awaited.

The cellular slime mold Dictyostelium discoideum (hereinafter referred to as slime mold) is a lower eukaryote that inhabits the fallen leaves of the forest and forms a fruiting body similar to mold. However, slime molds and molds (fungi) are a group of organisms that have evolved apart from each other, and the present inventors have thought that "slime molds = drug resources (a treasure house of antibiotics)" as well as fungi. In fact, several drug candidate substances have been reported.
DIF-1 (differentiation-inducing factor-1) is a low molecular weight compound containing chlorine that has been isolated and identified as a slime mold differentiation factor. DIF-3 isolated at the same time has low differentiation-inducing activity and is known to be a degradation product of DIF-1.
In recent years, the present inventors have investigated the pharmacological action of various DIF derivatives on mammalian cells and have reported that DIF-related compounds (DIFs) have an antitumor activity and an activity to promote cell glucose metabolism. In addition, analysis of “chemical structure-activity relationship” has shown that “antitumor activity” and “sugar metabolism promoting activity” possessed by DIFs can be separated by modification of DIF side chains (Non-patent Document 2). ~ 5).

Allison, A.C.Immunosuppressive drugs: the first 50 years and a glance forward.Immunopharmacol. 47, 63-83 (2000) Biochem Biophys Res Commun. 1997 Jul 18; 236 (2): 418-22 Cancer Res. 2004, 64, 2568-2571 Biochem. Pharmacol. 2005, 70, 676-685 FEBS Journal 2007, 274, 3392-3404

  An object of the present invention is to provide an IL-2 production inhibitor useful as an immunosuppressant or the like.

  The present inventor has intensively studied to solve the above problems. As a result, it was found that the compound represented by any one of formulas (I) to (V) has an IL-2 production inhibitory action. From these facts, it was found that these compounds are useful as immunosuppressants and the like, and the present invention has been completed.

That is, the present invention relates to an IL-2 production inhibitor comprising as an active ingredient a compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof.
The present invention also relates to an immunosuppressive agent comprising the IL-2 production inhibitor.
The present invention also relates to a compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof for suppressing IL-2 production or immunosuppression.
The present invention also relates to the use of a compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof in the production of IL-2 production suppression or immunosuppression.
The present invention also relates to an IL-2 production suppression method or immunosuppression method including a step of administering a compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof.

  Compounds represented by formulas (I) to (V) are drugs that modulate the activity of mammalian immune system cells, anti-inflammatory agents, therapeutic agents for autoimmune diseases and allergies, inhibitors for rejection during organ transplantation, etc. Can be suitably used. It can also be suitably used as a reagent for basic research such as IL-2 expression and cytokine research.

The figure which shows the structure of various DIF related compounds. The figure which shows the effect with respect to IL-2 mRNA amount (A), IL-2 protein amount (B), and cell proliferation (C) of various DIF related compounds (each 5 micromol) in Jurkat cell. 0.1% EtOH (vehicle) was used as a control, and 1 μM CsA and FK506 were used as positive controls. * Indicates that there is a significant difference in p <0.05 versus 0.1% EtOH (by t-test). Shows concentration-dependent effects of TH-DIF-1, TM-DIF-1, Bu-DIF-3 and CP-DIF-3 on IL-2 mRNA levels, IL-2 protein levels, and cell proliferation in Jurkat cells Figure. The figure which shows the concentration dependence of the effect of TH-DIF-1, TM-DIF-1, Bu-DIF-3 and CP-DIF-3 with respect to the activity of AP-1, NF-AT and NF-κB in Jurkat cells. The figure which shows the effect of TH-DIF-1, TM-DIF-1, Bu-DIF-3, and CP-DIF-3 (each 5 micromol) with respect to the amount of INF-γ mRNA and INF-γ protein in Jurkat cells. 0.1% EtOH (vehicle) was used as a control, and 1 μM CsA and FK506 were used as positive controls. * Indicates that there is a significant difference in p <0.05 versus 0.1% EtOH (by t-test). (A) The figure which shows the effect with respect to the proliferation of K562 human leukemia cell and mouse | mouth 3T3-L1 cell of various DIF related compounds (each 5 micromol). (B) The figure which shows the effect with respect to sugar uptake | capture of various DIF related compounds (each 5 micromol) of Confluent mouse | mouth 3T3-L1 cell. CsA and FK506 were also evaluated. * Indicates that there is a significant difference in p <0.05 versus 0.1% EtOH (by t-test). The figure which shows the effect of TM-DIF-1 with respect to the raise of IL-2 in a mouse | mouth serum by concanavalin A (ConA) stimulation. Graph values are average (n = 4) and standard deviation (Bars). * P <0.03 versus Control. ** P <0.0001 versus Control (by ANOVA, post hoc Fisher's protected least significant difference).

The present invention is described in detail below.
The IL-2 production inhibitor of the present invention comprises a compound represented by any one of formulas (I) to (V) or a pharmaceutically acceptable salt thereof as an active ingredient.

式中、Ｒ¹、Ｒ²、Ｒ³は水素または炭素数１〜５のアルキル基から選ばれる同一の基を示す。ＸはＣｌ、Ｂｒ、Ｉなどのハロゲンを表し、Ｃｌがより好ましい。尚、本明細書において、アルキル基はシクロアルキル基を含む。

^{Wherein, R 1, R 2, R} 3 represent the same group selected from alkyl groups of 1 to 5 hydrogen or carbon. X represents a halogen such as Cl, Br, or I, and Cl is more preferable. In the present specification, the alkyl group includes a cycloalkyl group.

As the compound of formula (I), the following compounds are preferred.

式中、Ｒ⁴は水素または炭素数１〜５のアルキル基を示す。ＸはＣｌ、Ｂｒ、Ｉなどのハロゲンを表し、Ｃｌがより好ましい。

In the formula, R ⁴ represents hydrogen or an alkyl group having 1 to 5 carbon atoms. X represents a halogen such as Cl, Br, or I, and Cl is more preferable.

As the compound of formula (II), the following compounds are preferred.

  The compounds of the above formulas (I) to (V) can be synthesized by the method described in Biochem. Pharmacol. 2005, 70, 676-685.

  The compounds of formulas (I) to (V) or pharmaceutically acceptable salts thereof have an IL-2 production inhibitory effect. Therefore, it can be used as an active ingredient of an immunosuppressive agent. The IL-2 production inhibitory effect means an effect of inhibiting IL-2 production at at least one of mRNA level and protein level, and preferably suppresses production at least at the protein level.

  Examples of the pharmaceutically acceptable salt of the compounds of formulas (I) to (V) include metal salts such as sodium, potassium, magnesium and calcium, ammonium salts and the like. The compounds of formulas (I) to (V) may be hydrates.

A medicament comprising a compound of formula (I) to (V) or a pharmaceutically acceptable salt thereof is prepared according to a known means generally used in a method for producing a pharmaceutical preparation. Pharmaceutically acceptable salt as it is or mixed with a pharmacologically acceptable carrier, for example, tablets (including sugar-coated tablets and film-coated tablets), powders, granules, capsules (including soft capsules) It can be safely administered orally or parenterally (eg, topical, rectal, intravenous administration, etc.) as a pharmaceutical preparation such as a liquid, injection, suppository, sustained-release agent and the like.
The content of the compound of formulas (I) to (V) or a salt thereof in the IL-2 production inhibitor or immunosuppressant is about 0.01 to about 100% by weight of the whole preparation.
The dose of the compound of formulas (I) to (V) or a salt thereof may be an amount effective for suppressing IL-2 production or immunosuppression, and varies depending on the administration subject, target organ, symptom, administration method, etc., and is particularly limited. Generally, however, it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg per day for a patient (assuming a body weight of 60 kg).

Examples of pharmacologically acceptable carriers include excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers in liquid formulations. And soothing agents. If necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts. Examples of the excipient include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid and the like. Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica and the like. Examples of the binder include crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, sodium carboxymethylcellulose and the like. Examples of the disintegrant include starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl starch sodium, L-hydroxypropyl cellulose, and the like. Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil and the like. Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like. Examples of the suspending agent include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; for example, polyvinyl alcohol, polyvinylpyrrolidone, Examples thereof include hydrophilic polymers such as sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose. Examples of isotonic agents include glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like. Examples of soothing agents include benzyl alcohol. Examples of the preservative include parahydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfite, ascorbic acid, α-tocopherol and the like.
The immunosuppressive agent of the present invention may be used in combination with other drugs.

  The following examples illustrate the present invention more specifically. However, the present invention is not limited to the following examples.

1) Effect of various DIF derivatives (DIFs) on IL-2 production in Jurkat cells In a series of experiments, an in vitro culture system of Jurkat cells (T cell model cells) was used. Dispense 1 mL (10 ⁶ cells / mL) of Jurkat cells suspended in RPMI1640 medium containing 10% fetal bovine serum into each well of a 12-well plastic plate. Vehicle (0.1% EtOH: Control) or DIFs (each 5 μM) was added thereto, 30 minutes later, ConA (Concanavalin A: 25 μg / mL) was added, and the expression level of IL-2 mRNA after 3 hours and 12 hours The amount of IL-2 protein produced later (the amount of IL-2 secreted into the medium) was examined (FIG. 2). As positive controls, 1 μM CsA and FK506 were used. IL-2 protein production was measured by ELISA using an anti-IL-2 antibody, and IL-2 mRNA expression was measured by Real Time RT-PCR.

  As a result, it was revealed that several derivatives inhibit IL-2 production (FIGS. 2A and 2B).

  Furthermore, a detailed study was performed using DIF derivatives TH-DIF-1, TM-DIF-1, Bu-DIF-3 and CP-DIF-3, which well inhibit IL-2 production (FIG. 3). As a result, these derivatives were found to inhibit IL-2 production in a concentration-dependent manner.

  At the same time, the effect of each compound on cell viability 12 hours after the addition of ConA was examined by the MTT method, but almost no cytotoxicity of 5 μM DIFs was observed (FIG. 2C).

  These results suggest that DIF derivatives may be used for basic research as IL-2 production regulators, and may be clinically applicable as immunosuppressants.

2) Effect of DIFs on AP-1, NFκB, NFAT activity
ConA binds to the T-cell receptor on the surface of Jurkat cells, regulates various enzyme activities in the cell, and finally activates transcription factors AP-1, NFκB, and NFAT to express IL-2 Is considered to be controlling. Therefore, in order to clarify the mechanism of action of DIFs, the effect of DIFs on AP-1, NFκB, and NFAT activities was examined. A vector in which a reporter (luciferase) gene is linked to the promoter region to which each factor binds is introduced into Jurkat cells. After stimulation with ConA in the presence or absence of DIFs (5 μM each), the lucifer of each cell Race activity was measured. Furthermore, detailed examination was performed using TH-DIF-1, TMDIF-1, Bu-DIF-3 and CP-DIF-3 (FIG. 4).

  As a result, TH-DIF-1 and TM-DIF-1 inhibited AP-1 and NFAT, and Bu-DIF-3 and CP-DIF-3 inhibited NFAT and NFkB activity. . These results suggest that there are multiple points of action in the mechanism of IL-2 expression regulation by DIFs.

3) Effect of DIFs on IFN-γ expression (Figure 5)
Next, we examined the effect of DIFs on Interferon-γ (IFN-γ) mRNA and protein expression (by ConA stimulation) in Jurkat cells. The cells are cultured for 30 minutes in the presence of 5 μM DIFs, and further, ConA is added, and the expression level of IFN-γ mRNA after 3 hours and the amount of IFN-γ protein after 12 hours (IFN-γ protein secreted into the medium) Amount). 0.1% EtOH (vehicle) was used as a control, and 1 μM CsA and FK506 were used as positive controls. IFN-γ protein production was measured by ELISA using an anti-IFN-γ antibody, and IFN-γ mRNA expression was measured by Real Time RT-PCR.

  As a result, CsA and FK506 (1 μM), which are known immunosuppressants, well suppressed the expression of IFN-γ mRNA and protein, whereas the effect of the DIF derivative was small. This result suggests that the DIF derivative may be used as a factor that can control IL-2 expression relatively specifically, and as an immune regulator with fewer side effects.

4) Effect of DIFs on K562 human leukemia cells and mouse 3T3-L1 cells (Fig. 6)
As described above, DIF derivatives have antitumor activity (Kubohara, 1999; Shimizu et al. 2004; Gokan et al. 2005; etc.) and glucose metabolism promoting activity (Omata et al. 2007; Kubohara et al. 2008). Some have Therefore, the correlation between the chemical structure of DIFs and their pharmacological activity and the IL-2 expression regulating activity of the present invention was examined.
First, the number of cells after culturing K562 cells or 3T3-L1 cells in the presence of 0.1% EtOH, 1 μM CsA, 1 μM FK506, or 5 μM DIFs for 3 days was measured and compared. As a result, TH-DIF-1, TM-DIF-1 has almost no effect on the proliferation of K562 cells and 3T3-L1 cells (a model of normal cells), and Bu-DIF-3, CP-DIF-3 It was revealed that the proliferation of K562 cells was inhibited to some extent (FIG. 6A).
In addition, 3T3-L1 cells in a confluent state were cultured for several hours in the presence of 0.1% EtOH, 1 μM CsA, 1 μM FK506, or 5 μM DIFs, and the sugar consumption of the cells was measured. As a result, although the effect of TM-DIF-1 on the cell glucose metabolism was somewhat high, the effects of other DIFs were generally small, although to a different extent (FIG. 6B). Furthermore, it was also clarified that the known immunosuppressive agent CsA exhibits some cell growth inhibitory action and glucose metabolism inhibitory action.
These results indicate that the two known mechanisms of action and the IL-2 expression control mechanism of the present invention may be at least partially different. In particular, TH-DIF-1 has only been confirmed to have a high IL-2 expression inhibitory activity, and it can be particularly expected as a specific IL-2 expression inhibitor, a candidate substance for an immunosuppressant with few side effects, and a lead compound.

5) Effect of TM-DIF-1 on IL-2 rise in mouse serum induced by concanavalin A (ConA) stimulation
Control group and TM-DIF-1 group mice (n = 4 each) were administered intravenously with 5 mL / kg of Vehicle and TM-DIF-1 (10 mg / kg), and 15 minutes later, ConA (200 μg / individual) was administered intravenously.
FK506 was used as a positive control for IL-2 suppression. FK506 group mice (n = 4) were orally administered FK506 (50 mg / kg) and 120 minutes later, ConA (200 μg / individual) was intravenously administered.
For the Control group, TM-DIF-1 group, and FK506 group, whole blood was collected 4 hours after ConA stimulation, and the serum IL-2 concentration was measured.
As a result, TM-DIF-1 significantly suppressed IL-2 elevation caused by ConA stimulation. In addition, no acute toxicity or abnormal behavior of mice associated with TM-DIF-1 administration was observed.

Claims (10)

An interleukin-2 production inhibitor comprising a compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof as an active ingredient.

In the formula, R ¹ , R ² and R ³ represent the same group selected from hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

In the formula, R ⁴ represents hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

The interleukin-2 production inhibitor according to claim 1, wherein the compound represented by any one of formulas (I) to (V) is any of the following compounds.

The immunosuppressive agent containing the interleukin-2 production inhibitor of Claim 1 or 2. A compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof for inhibiting interleukin-2 production.

In the formula, R ¹ , R ² and R ³ represent the same group selected from hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

In the formula, R ⁴ represents hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

A compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof for immunosuppression.

In the formula, R ¹ , R ² and R ³ represent the same group selected from hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

In the formula, R ⁴ represents hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

The compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof, which is any of the following compounds:

A method for inhibiting interleukin-2 production, comprising a step of administering a compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof.

In the formula, R ¹ , R ² and R ³ represent the same group selected from hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

In the formula, R ⁴ represents hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

The method according to claim 7, wherein the compound represented by any one of formulas (I) to (V) is any of the following compounds.

An immunosuppressive method comprising a step of administering a compound represented by any one of the following formulas (I) to (V) or a pharmaceutically acceptable salt thereof.

In the formula, R ¹ , R ² and R ³ represent the same group selected from hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

In the formula, R ⁴ represents hydrogen or an alkyl group having 1 to 5 carbon atoms, and X represents halogen.

The method according to claim 9, wherein the compound represented by any one of formulas (I) to (V) is any one of the following compounds.

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