JP5424960B2 - Inhibitor of interaction between CCR2B or CCR5 and front protein - Google Patents

Description

  The present invention relates to an inhibitor of interaction between CCR2B or CCR5 and a front protein.

  The chemokine receptors CCR2 and CCR5 are trimeric G protein-coupled seven-transmembrane receptors (G-PCRs), and when chemokines are bound, the trimeric G protein is coupled to the receptor. Is activated, and the released G subunit activates phosphatidylinositol-3-kinase (PI3K) and induces localization to the tip. Activated PI3K induces the localization of the low molecular weight G protein Rac / Cdc42 to the tip of the active form, forming foliate and filopodia in the direction of cell progression, and contracting in the cell tail The polarity of the cell occurs and the cytoskeleton is reconstructed to allow cell migration.

  The front protein is a cytoplasmic protein that associates directly and specifically with the Pro-12-C terminal region (intracellular C-terminal region) of CCR2, and forms a cluster with CCR2 after CCL2 stimulation.

  When phylogenetic analysis based on homology of amino acid sequences of 20 types of chemokine receptors is performed, CCR2B and CCR5 have very high homology, and even when compared with the amino acid sequence of the intracellular C-terminal region, CCR5 is the closest located receptor. The front protein binds to the intracellular C-terminal regions of both CCR2B and CCR5 having high homology (Patent Document 1). This has been clarified by the so-called yeast two-hybrid method (hereinafter referred to as “Y2H”). CCR2 has two isoforms, CCR2A and CCR2B, having different C-terminal regions, and CCR2B is a major CCR2 isoform widely expressed in cells.

  As a result of exhaustively preparing more than 20 kinds of various CCR2 partial deletion mutant expression vectors and analyzing the binding activity to the front protein using Y2H, 16 amino acids in the intracellular C-terminal region of the chemokine receptor CCR2B It was found to be a protein binding region (= CCR2 Pro-C). It was possible to reconstruct the front protein-CCR2 interaction in vitro in a cell-free system using peptides in this region. Furthermore, the Pro-C region of CCR5 also showed binding activity to the front protein, suggesting that both receptors bind to the front protein via the Pro-C region. The amino acid sequence of the front protein is described in SEQ ID NOS: 1 and 2 in the sequence listing described below.

  CCR2B and CCR5 have been shown to be involved in inflammatory diseases such as rheumatoid arthritis and multiple sclerosis, association with neuropathic pain, and pathogenesis such as cancer. CCR5 not only plays a role in cell migration, but also functions as a co-receptor (co-receptor) when HIV-1 virus infects host cells. R5 virus is a virus that dominates in the early stages of infection, and X4 virus appears as it progresses. Therefore, blocking R5 virus infection is important to suppress the spread of HIV infection at an early stage of infection. The possibility that the front protein is involved in these known CCR2B and CCR5-related diseases is also fully considered. In recent years, it has also been reported that front proteins are involved in various diseases and biological phenomena such as congestive heart failure (Non-patent document 1), prostate metastatic cancer (Non-patent document 2), and mesenchymal stem cell homing control (Non-patent document 3). Has been.

  There are currently no anti-inflammatory drugs that target chemokine receptors, not limited to CCR2B and CCR5. An approach has been taken in which the function and interaction of chemokines and chemokine receptors are inhibited extracellularly by antibodies and small molecule antagonists. CCR2B and CCR5 have been shown to be involved in arteriosclerosis and multiple sclerosis, and clinical trials for small molecule antagonists and neutralizing antibodies are underway. As for CCR5, almost all T cells express CCR5 in the rheumatoid arthritis synovial fluid, and clinical trials for rheumatoid arthritis have been conducted for maraviroc, which is marketed as an anti-HIV drug. Inhibiting the intracellular signaling mechanism of CCR2B or CCR5 has not been performed because no molecule that specifically controls signals in the cell has been found so far, but the front protein is not CCR2B or CCR5. It is believed that such an approach was made possible for the first time by the inventors' knowledge that they interact and promote cell migration signals.

  Regarding inhibition experiments with siRNA, it has been reported in Non-Patent Document 4 that siRNA against the front protein is introduced into a mouse pre-B cell line and cell migration toward CCR5 is reduced.

International Publication No. WO2003 / 070946

J Card Fail. 13 (2): 114-119, 2007 J Cell Biochem. 104: 1587-1597, 2008 Cell Stem Cell. 5: 2 (6): 566-575, 2008 Journal of Immunology 183: 6387-6394, 2009

  A substance that inhibits the interaction between the front protein and CCR2B or CCR5 has not been known so far. Accordingly, an object of the present invention is to obtain a new concept anti-inflammatory agent having such an inhibitory action.

  In order to find a substance that inhibits the interaction between the front protein and CCR2B or CCR5, a large number of known compound groups are tested, and from these compound groups, a compound having an effective inhibitory action is found. Completed.

  That is, in the first embodiment, the present invention provides a compound represented by the following formula I (wherein x1 and x2 are the same or different from each other and R is lower alkyl) or a salt thereof: Provided is an inhibitor of the interaction between CCR2B or CCR5 and a front protein, which is contained as an active ingredient.

In that embodiment, R in formula I above is methyl or ethyl.
In another embodiment, x1 and x2 in Formula I above are both chlorine or bromine.

In yet another embodiment, the compound is a compound of formula II:

In the second aspect, the present invention also provides an anti-inflammatory agent comprising the compound represented by the formula I as an active ingredient.
In that embodiment, the compound is a compound of formula II as defined above.

  In another embodiment, the anti-inflammatory agent of the invention is for the treatment of an inflammatory disease associated with the front protein, CCR2B or CCR5.

  By inhibiting the interaction between a specific front protein in a cell and a specific chemokine receptor CCR2B or CCR5, it is possible to inhibit a limited one of the various signal pathways elicited by the chemokine receptor. become. This is expected to reduce side effects more than blocking receptor activation by chemokine binding itself.

This figure is a figure which shows the interaction inhibitory activity between front protein and CCR2 by the compound represented by Formula II. This figure is a conceptual diagram of the TAXISscan method. This figure is a figure which shows the result of a migration activity inhibition test. This figure is a figure which shows the thickening degree of the sole in a delayed hypersensitivity model mouse. The number in parentheses after Formula II indicates the concentration (mg / L) of the compound of Formula II administered. This figure is a figure which shows the result of a toxicity evaluation test.

<Active ingredient>
The active ingredient of the inhibitor and anti-inflammatory agent of the present invention is the compound represented by the above formula I or a salt thereof. This compound or a salt thereof has a property of strongly inhibiting the interaction between the front protein and CCR2B.

  In the compounds of formula I, x1 and x2 are the same or different halogens and R is lower alkyl.

  Halogen is any of fluorine, chlorine, bromine or iodine, and preferred halogen is chlorine or bromine.

  Further, the halogens x1 and x2 may be the same halogen or different from each other, but the same halogen is preferable.

  Lower alkyl is a saturated or unsaturated linear or branched alkyl having 1 to 6 carbon atoms, and includes, for example, methyl, ethyl, ethenyl, propyl, propenyl, butyl, butenyl, pentyl, hexyl and the like. Preferred lower alkyl is a saturated linear alkyl having 1 to 4 carbon atoms, and more preferred lower alkyl is methyl or ethyl.

  Specific examples of compounds of formula I are the compounds of formula II above. This compound is a compound in which, in formula I, x1 and x2 are chlorine and R is methyl.

  The compound of formula II is a substance that is commercially available from Enamin (Ukraine), product number (EnamineID) is T5223244.

  Furthermore, the salt of the compound of formula I and formula II is a salt of the compound of formula I or formula II and any acid or base, and the acid or base is any pharmaceutically acceptable. Good. The acid is an inorganic acid or an organic acid. Examples of the inorganic acid include, but are not limited to, acids such as hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids include, but are not limited to, for example, Acids such as acetic acid, propionic acid, citric acid, oxalic acid and succinic acid are included. On the other hand, the base includes, but is not limited to, a base containing an alkali metal such as sodium or potassium, such as sodium hydroxide or potassium hydroxide. The salt formation can be carried out by contacting the phenolic hydroxyl group or cyclic amino group of the compound of formula I or formula II with an equivalent amount of acid or base in a suitable solvent.

  For the preparation of a compound of formula I or formula II, for example, a ketone compound can be derived by a Friedel-Crafts reaction of one benzoyl chloride derivative and the other heterocyclic compound.

<Composition>
As described above, CCR2B and CCR5 have very high homology, and CCR2B and CCR5 are also highly homologous receptors when compared with the amino acid sequence of the intracellular C-terminal region. The front protein binds to the intracellular C-terminal regions of both of these highly homologous CCR2B and CCR5 (International Publication No. WO2003 / 070946). In addition, CCR2B and CCR5 have been shown to be involved in inflammatory diseases such as (chronic) rheumatoid arthritis and multiple sclerosis, association with neuropathic pain, and pathological formation such as cancer. On the other hand, front proteins are also known to be involved in various diseases such as congestive heart failure, prostate metastatic cancer, and mesenchymal stem cell homing control.

  In inflammatory diseases, it is considered that tissue destruction proceeds when effector cells such as macrophages and monocytes are excessively accumulated and activated in the inflamed area. The intracellular protein that specifically activates the chemotaxis of these effector cells is the front protein, and by inhibiting its function, the cell is suppressed from moving to the inflammatory site, so CCR2B and CCR5 are It becomes possible to suppress inflammation of related diseases.

  The compound of formula I or formula II of the present invention is an interaction inhibitor between CCR2B or CCR5-frontal protein obtained by the yeast two-hybrid (Y2H) screening method, and has the ability to inhibit cell migration. (Examples 1 and 2 described later). In cell-level experiments, functional decline due to cytotoxicity is also considered to be a cause of migration inhibition.To eliminate this possibility, cytotoxicity was also evaluated and cell migration inhibition ability was confirmed (see below). Example 3). That is, the compound represented by Formula I or Formula II strongly inhibits the interaction and has passed the cytotoxicity test, and the inhibition of migration by the compound represented by Formula I or Formula II inhibits the interaction. It has been proved to be due to.

  Accordingly, the present invention provides an inhibitor of the interaction between CCR2B or CCR5 and a front protein, comprising a compound of formula I or formula II or a salt thereof as an active ingredient.

  The present invention further provides an anti-inflammatory agent comprising a compound of formula I or formula II or a salt thereof as an active ingredient.

  Preferred compounds of formula I are those in which R is methyl or ethyl and x1 and x2 are both chlorine or bromine. More preferred compounds of formula I are compounds of formula II.

  The compounds of Formula I or Formula II of the present invention may be used to inhibit the interaction of CCR2B or CCR5 with the front protein or through such inhibition, inflammatory diseases associated with the front protein, CCR2B or CCR5, and other diseases Can be used to treat.

  Such inflammatory diseases and other diseases include, but are not limited to, refractory inflammatory diseases such as arteriosclerosis, rheumatoid arthritis, multiple sclerosis, congestive heart failure, metastatic cancer, neuropathic pain, etc. These include diseases associated with CCR2B or CCR5 and diseases associated with front protein.

  The inhibitors of the present invention may be used as pharmaceuticals or as research reagents to inhibit the interaction between CCR2B or CCR5 and the front protein.

  When the compounds of formula I or formula II of the present invention are used as the above-mentioned inhibitors or anti-inflammatory agents as pharmaceuticals, they may be administered by any route of oral or parenteral administration. Parenteral administration includes, for example, intravenous administration, transdermal administration, transmucosal administration, intraperitoneal administration, and rectal administration.

  The preparation for that purpose is not particularly limited, and for example, solid preparation, granule preparation, powder preparation, solution preparation, suspension preparation, emulsion preparation, injection preparation, delayed release preparation, sustained release preparation, capsule preparation, sachet Includes various dosage forms such as formulations.

  In addition to the above active ingredients, such preparations include various additives such as excipients, carriers, diluents, disintegrants, suspending agents, emulsifiers, delayed release polymers and coatings, colorants, A flavoring agent etc. can be included. As such an additive, any pharmaceutically known agent can be used.

  The dose of the active ingredient can vary depending on the patient's age, sex, condition (severity), weight, etc., and such dose should be determined by the attending physician. However, any dosage may be used as long as it exhibits the above-mentioned effects and does not have toxicity. Such a dose varies depending on the type of formulation, but is, for example, a dose of about 0.01 mg to about 10 mg per dosage unit.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
[Example 1]
Inhibitory activity of the front protein-CCR2 interaction GAL4 BD which is a transcription domain binding domain fusion protein expression vector containing the C-terminal sequence of the front protein GAL4 which is an activation domain fusion protein expression vector containing the C-terminal sequence of CCR2 AD was transformed into yeast strain Y190 by the lithium acetate / polyethylene glycol transformation protocol (see Ito et al, J. Bacteriol. 153: 163-168 (1983)). After culturing this Y190 / FNT-CCR2 yeast strain to the logarithmic growth phase, 0.5% Westase treatment was performed for 10 minutes to transiently protoplast the yeast. Yeast was suspended in a histidine-deficient medium, 3-aminotriazole (Sigma Chemical Co.) was added to a final concentration of 5 mM, and this was applied to a 96-well plate. Thereafter, the compound represented by the formula II was added to 50 μM, cultured for 24 hours, the absorbance (800 nm) was measured, and the interaction inhibition activity was verified with Y2H. About 50% of interaction inhibition was recognized by addition of the compound represented by Formula II with respect to the control which added DMSO (FIG. 1).

[Example 2]
Inhibition of cell migration ability The inhibition activity of cell migration ability by the compound represented by formula II was evaluated using the TAXIScan method (Nitta, et al., Journal of Immunological method; 320, 155-163 (2007)).

Here, the TAXISscan method is a cell migration ability analysis (TAXIScan) system originally developed by ECI. The TAXIScan system uses a silicon wafer chip created using the latest microfabrication technology to form a concentration gradient of a specific chemotactic factor and measures the migration activity of cells in the horizontal direction depending on the gradient. (FIG. 2). As a result of analyzing the images obtained using this system and quantifying the directionality (how much the migratory cells have advanced with respect to the high concentration factor of migratory factor), treatment with the compound represented by Formula II Directionality was suppressed in a concentration-dependent manner (FIG. 3). Note that the relative values in FIG. 3 indicate the difficulty of progression when the linear cell progression is 100 when the concentration of the compound represented by Formula II is 0%. In the Boyden chamber method, the compound represented by Formula II had an IC ₅₀ of 15 μM.

[Example 3]
Effectiveness evaluation test in animals Immunization is performed by intraperitoneal administration of 1 × 10 ⁶ sheep red blood cells to mice, and 2 × 10 ⁸ sheep red blood cells are re-administered subcutaneously in the footpad (foot sole). The intensity of the induced cellular immune response was measured by measuring the thickening of the sputum. 500 μl of a compound of formula II dissolved in DMSO and diluted 10-fold with physiological saline was intraperitoneally administered (20, 10 or 5 mg / kg) 30 minutes before re-administration to the footpad. The thickness of the ridge was measured with calipers.

  When the degree of thickening relative to the thickness of the footpad on the side where sheep red blood cells were not re-administered was compared with the DMSO-administered group, the thickness decreased in a dose-dependent manner and an anti-inflammatory effect was observed (FIG. 4).

[Example 4]
Toxicity evaluation test A compound represented by the formula II (100, 10, 1 or 0.1 μM) was added to Jurkat / CCR2 cells obtained by introducing CCR2 into Jurkat cells using the vector pcMGS-NEO, and 48 After the time culture, a toxicity evaluation test was performed by measuring the cell viability by the WST-1 method.

  As a result, no cytotoxicity was observed even at 10 μM in which a complete decrease in directionality was observed (FIG. 5).

  The compound represented by the formula II in the present invention has few side effects, and the species difference between mice and humans is small. Therefore, the test results of mice that are experimental animals can be applied to humans as they are. For this reason, application and utilization as a new concept anti-inflammatory agent different from conventional anti-inflammatory agents can be expected.

Claims (7)

CCR2B or CCR5 containing a compound represented by the following formula I (wherein x1 and x2 are the same or different from each other and R is lower alkyl) or a salt thereof as an active ingredient; Inhibitors of the interaction.

  Inhibitor according to claim 1, wherein in formula I, R is methyl or ethyl.   The inhibitor according to claim 1 or 2, wherein in the formula I, x1 and x2 are both chlorine or bromine. 4. Inhibitor according to any one of claims 1 to 3, wherein the compound is a compound of formula II below.

  An anti-inflammatory agent comprising as an active ingredient a compound of the formula I as defined in any one of claims 1 to 3 or a salt thereof.   6. The anti-inflammatory agent according to claim 5, wherein the compound is a compound of formula II as defined in claim 4.   The anti-inflammatory agent according to claim 5 or 6, which is used for treatment of an inflammatory disease associated with a front protein, CCR2B or CCR5.

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