Macrophage polarizer containing an organic germanium compound and its use

Abstract

To provide novel means for inducing the polarization of macrophages toward the M1 type.SOLUTION: The present invention provides an M1 polarization agent and a SIRPα expression inhibitor for macrophages, which contain at least one selected from among compounds of the general formula (I) in the figure, pharmaceutically acceptable salts and esters thereof, and polymers thereof. The present invention also provides the use of the M1 polarization agent and M1-polarized macrophages in the treatment of diseases or conditions, particularly in the treatment of cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to macrophage M1 polarization agents and SIRPα expression inhibitors containing organic germanium compounds, as well as the use of M1 polarization agents and M1-polarized macrophages in the treatment of diseases or conditions, particularly cancer. [Background technology]

[0002] Macrophages are an important component of the immune system and possess plasticity, allowing them to significantly change their morphology and function in response to local environmental signals. Macrophages are broadly classified into pro-inflammatory M1 types and anti-inflammatory M2 types. M1 macrophages have high antigen-presenting and phagocytic abilities, secrete inflammatory cytokines, and contribute to the removal of pathogens such as bacteria and viruses, as well as damaged tissue. M2 macrophages play a role in wound healing and immune tolerance by secreting anti-inflammatory cytokines and growth factors. M1 and M2 macrophages maintain a constant balance with each other, and when this balance is disrupted, various inflammatory diseases occur.

[0003] Macrophages play a crucial role in cancer treatment. M1 macrophages exhibit enhanced immune responses and phagocytic activity against tumors, and suppress cancer progression and metastasis, leading to various advancements in cancer therapy utilizing M1 macrophages. However, cancer therapy using M1 macrophages faces the challenge that SIRPα (signal regulator protein α) on macrophages binds to CD47 on target cells, suppressing macrophage phagocytosis. Various types of cancer cells express high levels of CD47 compared to normal cells, and it is thought that they avoid phagocytosis by macrophages by emitting phagocytosis avoidance signals via the CD47-SIRPα immune checkpoint pathway. Inhibition of the CD47-SIRPα immune checkpoint using neutralizing antibodies is known to enhance macrophage phagocytosis of cancer cells and suppress cancer progression.

[0004] Furthermore, macrophages called TAMs (Tumor-Associated Macrophages) present within or near tumors possess M2-like properties. They suppress anti-tumor immunity through the secretion of immunosuppressive factors such as anti-inflammatory cytokines, promote cancer metastasis through the secretion of TGF-β, and enhance angiogenesis through the secretion of VEGF. Thus, TAMs provide a favorable microenvironment for cancer cell proliferation, making TAM suppression an important issue in cancer treatment.

[0005] On the other hand, Ge-132 (poly-trans-[(2-carboxyethyl)germasesquioxane], also known as repagermanium or asai germanium) is an organic germanium compound with various physiological effects, including immunostimulatory, antitumor, anti-inflammatory, analgesic, and synergistic effects with morphine. The antitumor effect of Ge-132 is exerted via its hydrolysate, 3-(trihydroxygermyl)propanoic acid (THGP), and in vivo studies using mice and rats have revealed that this is due to increased secretion of IFN-γ induced by NK cell activation and the subsequent activation of macrophages (Non-patent documents 1-3). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] H Aso et al., Microbiol Immunol. 1985;29:65-74. [Non-Patent Document 2] Suzuki, Fujio et al., Cancer and Chemotherapy, January 1987, 14(1):127-34. [Non-Patent Document 3] F Suzuki et al., J Interferon Res. Spring 1984;4(2):223-33. [Disclosure of the Invention] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a novel means for inducing the polarization of macrophages to the M1 type (M1 polarization). [Means for solving the problem]

[0008] The inventors have discovered that THGP induces M1 polarization of macrophages under specific conditions, suppresses the expression of SIRPα on macrophages, and further, through M1 polarization of macrophages, leads to the inhibition of epithelial-mesenchymal transition in cancer cells and suppression of CD47 expression, thereby completing the following invention.

[0009] (1) General formula (I) [ka] A compound of, In the formula, R 1 and R 2 These are hydrogen, halogen, nitro, hydroxy, cyano, and C, independently of each other. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4Haloalkenyl, C 3-4 Alkynyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 [[ID=1​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ (2) In general formula (I), R 1 , R 2 and R 3 However, they are independent of each other: hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinil, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 The polarizing agent according to (1), comprising an alkylsulfonyl compound, a pharmaceutically acceptable salt or ester thereof, or a polymer thereof. (3) In general formula (I), R 1 , R 2 and R 3 A polarizing agent according to (1) or (2), comprising a compound in which all are hydrogen, or a pharmaceutically acceptable salt or ester thereof, or a polymer thereof. (4) General formula (I) [ka] A compound of, In the formula, R 1 and R 2 These are hydrogen, halogen, nitro, hydroxy, cyano, and C, independently of each other. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4is an alkylsulfonyl or, together with two carbon atoms to which they are attached, forms a 4- to 10-membered monocyclic or polycyclic saturated ring, a 4- to 10-membered monocyclic or polycyclic partially saturated ring, a 6- to 10-membered monocyclic or polycyclic aromatic ring, or a 5- to 10-membered monocyclic or polycyclic heterocyclic ring containing 1 to 4 atoms selected from nitrogen, oxygen, and sulfur, these rings are unsubstituted or substituted by one or more substituents from the group consisting of halogen, nitro, hydroxy, cyano, oxo, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C 3-4 haloalkynyl, C 1-4 alkoxy, C 1-4 haloalkoxy, C 1-4 alkylthio, C 1-4 alkylsulfinyl and C 1-4 alkylsulfonyl, R 3 is hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C 3-4 haloalkynyl, C 1-4 alkoxy, C 1-4 haloalkoxy, C 1-4 alkylthio, C 1-4 alkylsulfinyl or C 1-4 alkylsulfonyl, a macrophage SIRPα expression inhibitor containing the compound or a pharmaceutically acceptable salt or ester thereof or a polymer thereof. (5) In general formula (I), R 1 , R 2 and R 3 are, independently of one another, hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 alkyl, C 1-4 haloalkyl, C2-4 Alkenyl, C 2-4 Haloalkenyl, C 3-4 Alkynyl, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkylsulfinyl or C 1-4 An expression inhibitor according to (4), which contains a compound that is alkylsulfonyl, or a pharmaceutically acceptable salt or ester thereof, or a polymer thereof. (6) In the general formula (I), R 1 , R 2 and R 3 are all hydrogen, or a pharmaceutically acceptable salt or ester thereof, or a polymer thereof, and the expression inhibitor according to (4) or (5). (7) An expression inhibitor according to any one of (4) to (6), which is used to inhibit the CD47-SIRPα immune checkpoint. (8) The compound of the general formula (I)

Chemical formula

[0010] According to the present invention, M1 polarization of macrophages can be induced, and M1-polarized macrophages can be produced. These macrophages have high phagocytic activity, suppressed SIRPα expression, and can inhibit epithelial-mesenchymal transition and suppress CD47 expression in cancer cells, making them useful in cancer treatment. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1a shows bright-field microscopy images of RAW 264.7 cells cultured for 0, 1, 7, 10, 45, and 90 days in a medium containing 500 μM THGP. Figure 1b shows bright-field microscopy images of RAW 264.7 cells cultured for 10 days in control medium (Ctrl) or a medium containing 500 μM THGP (arrows indicate spindle-shaped cells), and Figure 1c is a graph showing the percentage of spindle-shaped cells among all cells. Figure 1d is a graph showing the cell proliferation rate of RAW 264.7 cells cultured for 0, 1, 10, 30, and 40 days in THGP. Figure 1e shows fluorescence microscopy images of RAW 264.7 cells cultured for 20 to 120 days in control medium (Ctrl) or medium containing THGP 500 μM, after immunostaining for CD86 or CD206, or nuclear staining with DAPI. Figure 1f is a graph showing the M1 / M2 ratio (percentage of CD86-positive cells / percentage of CD206-positive cells). [Figure 2]Figure 2a is a graph showing the relative gene expression levels of M1 macrophage markers in RAW 264.7 cells cultured for 20 days in control medium (Ctrl, white bars) or medium containing 500 μM THGP (black bars), and Figure 2b is a graph showing the relative gene expression levels of M2 macrophage markers in these RAW 264.7 cells. Figure 2c is an image showing the results of Western blotting analysis of CD86 and CD206 protein expression in RAW 264.7 cells cultured for 20 to 120 days in control medium (Ctrl) or medium containing 500 μM THGP, and Figures 2d and 2e are graphs showing the relative protein expression levels of CD86 and CD206 quantified from the Western blotting results. [Figure 3] This graph shows the relative gene expression levels of the M1 macrophage marker iNOS in RAW 264.7 cells cultured for 4, 7, or 10 days in control medium (Ctrl) or medium containing THGP 50, 500, or 5000 μM. [Figure 4] Figure 4a shows fluorescence microscopy images of RAW 264.7 cells cultured for 10 days in control medium (Ctrl) or medium containing 50, 500, or 5000 μM THGP, after incorporating FITC-labeled beads. Figure 4b is a graph of relative fluorescence intensity with the fluorescence intensity of Ctrl set to 1. Figure 4c shows fluorescence microscopy images of RAW 264.7 cells cultured for 20 to 120 days in control medium (RAW C) or RAW 264.7 cells cultured for 20 to 120 days in medium containing 500 μM THGP (RAW T), which were fluorescently labeled and co-cultured with B16 4A5 cells labeled with another fluorescence in the presence or absence of 500 μM THGP. Figure 4d is a graph showing the percentage of B16 4A5 cells (double-stained cells) phagocytosed by the co-culture. [Figure 5]Figure 5a is a graph showing the percentage of B16 4A5 cells damaged when co-cultured with RAW C or RAW T in the presence or absence of 500 μM THGP. Figure 5b is a graph showing the cell proliferation of B16 4A5 cells cultured in control medium, medium containing 500 μM THGP, and conditional medium prepared from the culture supernatants of RAW C and RAW T, respectively. Figure 5c is a graph showing the viability of B16 4A5 cells cultured in control medium (Ctrl) or medium containing 500 μM THGP. Figure 5d is a graph showing the viability of B16 4A5 cells co-cultured with RAW C or RAW T in the presence of 1.25 μM 5-FU. Figure 5e is a graph showing the viability of B16 4A5 cells cultured in control medium (NC) or conditional medium prepared from the culture supernatants of RAW C and RAW T respectively (RAW C CM and RAW T CM) in the presence or absence of 1.25 μM 5-FU (5-FU). [Figure 6] Figure 6a shows bright-field microscopy images of B16 4A5 cells co-cultured with RAW C or RAW T. Figure 6b is a graph showing the relative gene expression levels of SIRP-α in RAW C (Ctrl) and RAW T (THGP). Figure 6c is a graph showing the relative gene expression levels of CD47 in B16 4A5 cells cultured in control medium (Ctrl), RAW C CM, or RAW T CM. [Figure 7] Figure 7a is a graph showing the TGF-β concentration in the culture supernatants of RAW C (Ctrl) and RAW T (THGP). Figures 7b and 7c are graphs showing the relative gene expression levels of N-cadherin and Vimentin in B16 4A5 cells cultured in control medium (Ctrl), medium containing 500 μM THGP, and RAW C CM or RAW T CM. Figure 7d is an image showing the results of Western blotting analysis of N-cadherin protein expression in these B16 4A5 cells, and Figure 7e is a graph showing the relative protein expression level of N-cadherin quantified from the Western blotting results. [Figure 8] Figure 8a shows the results of a migration assay of B16 4A5 cells cultured in control medium (Ctrl), medium containing 500 μM THGP, and RAW C CM or RAW T CM. Figure 8b is a graph showing the migratory ability of each B16 4A5 cell calculated from the results in Figure 8a, and Figure 8c is a graph showing the MMP-2 and MMP-9 activity of each B16 4A5 cell. Figure 8d is a graph showing the adhesion of B16 4A5 cells cultured in RAW C CM or RAW T CM. Figure 8e is a graph showing the invasiveness of B16 4A5 cells co-cultured with RAW C or RAW T. [Figure 9] Figure 9a shows bright-field microscopy images of B16 4A5 cells cultured in control medium (Ctrl), TGF-β-containing medium, or TGF-β·THGP-containing medium. Figure 9b shows fluorescence microscopy images of these B16 4A5 cells after immunostaining for N-cadherin or nuclear staining with DAPI. Figure 9c is a graph showing the relative protein expression levels of N-cadherin quantified from Western blotting results of these B16 4A5 cells. Figure 9d is a graph showing the invasiveness of these B16 4A5 cells. [Modes for carrying out the invention]

[0012] A first aspect of the present invention is general formula (I) [ka] This invention relates to a macrophage M1 polarization agent containing at least one of the following compounds, pharmaceutically acceptable salts or esters thereof, or polymers thereof.

[0013] compound The compounds used in the present invention are compounds of general formula (I), pharmaceutically acceptable salts or esters thereof, or polymers thereof. Here, the polymer is a polymer that yields a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof by hydrolysis.

[0014] In compounds of general formula (I), R 1 and R 2 These are, independently of each other: hydrogen; halogens such as fluorine, chlorine, or bromine; nitro; hydroxy; cyano; and linear, branched, or cyclic alkyl groups having 1 to 4 carbon atoms. 1-4 Alkyl; a linear, branched, or cyclic alkyl group having 1 to 4 carbon atoms, substituted with one or more halogens. 1-4 Haloalkyl; a linear, branched, or cyclic alkenyl with 2 to 4 carbon atoms. 2-4 Alkenyl; a linear, branched, or cyclic alkenyl with 2 to 4 carbon atoms, substituted with one or more halogens. 2-4 Haloalkenyl; a straight-chain or branched alkynyl with 3-4 carbon atoms. 3-4 Alkynyl; a linear or branched alkynyl with 3-4 carbon atoms substituted with one or more halogens. 3-4 Haloalkynyl;-OC 1-4 C represented by alkyl 1-4 Alkoxy;-OC 1-4 C represented by haloalkyl 1-4 Haloalkoxy;-SC 1-4 C represented by alkyl 1-4 Alkylthio;-SO-C 1-4 C represented by alkyl 1-4 Alkyl sulfinyl; or -SO2-C 1-4 C represented by alkyl 1-4 It can be an alkylsulfonyl.

[0015] Also, R 1 and R 2 Together with the two carbon atoms to which they are bonded, they may form a 4-10 membered monocyclic or polycyclic saturated ring, a 4-10 membered monocyclic or polycyclic partially saturated ring, a 6-10 membered monocyclic or polycyclic aromatic ring, or a 5-10 membered monocyclic or polycyclic heterocycle containing 1-4 atoms selected from nitrogen, oxygen, and sulfur.

[0016] A 4-10 membered monocyclic or polycyclic saturated ring is a saturated carbocyclic ring having one or more ring structures with 4-10 carbon atoms as ring constituent atoms. Examples include cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclooctyl, spirooctyl, and the like.

[0017] A 4-10 membered monocyclic or polycyclic partially saturated ring is a partially saturated carbon ring having one or more ring structures with 4-10 carbon atoms as ring constituent atoms. Examples include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, bicyclooctenyl, and the like.

[0018] A monocyclic or polycyclic aromatic ring with 6 to 10 members is an aromatic ring having one or more ring structures with 6 to 10 carbon atoms as ring constituent atoms, and examples include phenyl, naphthyl, and indyl.

[0019] A heterocycle containing 1 to 4 atoms selected from the group consisting of nitrogen, oxygen, and sulfur in a monocyclic or polycyclic structure with 5 to 10 members is a saturated ring, partially saturated ring, or aromatic ring having one or more ring structures with 6 to 10 atoms as ring constituent atoms, wherein 1 to 4 of the ring constituent atoms are heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur, and the other ring constituent atoms are carbon atoms. Examples include furanyl, thiophenyl, pyrrolyl, imidazolyl, pyranyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, indolyl, quinolyl, isoquinolyl, etc.

[0020] R 1 and R 2 The rings formed by this are halogen, nitro, hydroxy, cyano, oxo, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl and C 1-4 It may be substituted with one or more substituents from the group consisting of alkylsulfonyl groups. Details of each group are shown in R 1 and R 2 As stated in the explanation.

[0021] In compounds of general formula (I), R 3 These are hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 It is an alkylsulfonyl group. For details of each group, see R 1 and R 2 As stated in the explanation.

[0022] The compounds preferably used in the present invention are those in general formula (I) where R 1 , R 2 and R 3 However, they are independent of each other: hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinil, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4The compound is an alkylsulfonyl compound, or a pharmaceutically acceptable salt or ester thereof, or a polymer thereof.

[0023] Compounds of general formula (I) that are more preferably used in the present invention include R 1 ~R 3 Compounds in which all are hydrogen (THGP);R 1 and R 2 However, together with the two carbon atoms to which they are bonded, they form a 5-membered monocyclic saturated ring, R 3 A compound in which hydrogen is present (2-(trihydroxygelmyl)cyclopentanecarboxylic acid); R 1 and R 2 However, together with the two carbon atoms to which they are bonded, they form a six-membered monocyclic saturated ring, R 3 A compound in which hydrogen is present (2-(trihydroxygelmyl)cyclohexanecarboxylic acid); R 1 and R 2 However, together with the two carbon atoms to which they are bonded, they form an 8-membered polycyclic saturated ring, particularly bicyclo[2.2.2]octane, R 3 Compounds in which hydrogen is present (for example, 3-(trihydroxygelmyl)bicyclo[2.2.2]octane-2-carboxylic acid) can be cited.

[0024] In the present invention, particularly preferred compounds are THGP or pharmaceutically acceptable salts or esters thereof, or polymers thereof.

[0025] The present invention encompasses the use of pharmaceutically acceptable salts or esters of compounds of general formula (I). Such salts include salts with conventional bases, such as alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium and magnesium salts), ammonium salts, or organic amines (e.g., ethylamine, diethylamine, triethylamine, DIPEA, monoethanolamine, diethanolamine, triethanolamine, dimethylaminoethanol, diethylaminoethanol, tris(hydroxymethyl)aminomethane, choline (2-hydroxy-N,N,N-trimethylethaneaminium), procaine, dicyclohexylamine, dibenzylamine, N-methylmorpholine, N-methylpiperidine, arginine, lysine, and 1,2-ethylenediamine).

[0026] pharmaceutically acceptable esters of compounds of general formula (I) are esters of carboxylic acids of general formula (I) that are hydrolyzable in vivo. Examples of such esters include methyl, ethyl, tert-butyl esters, etc. 1-4 Alkyl esters are preferred.

[0027] Polymers of compounds of general formula (I) are of general formula (II) [ka] It can be expressed as follows: R in general formula (II) 1 ~R 3 As explained in general formula (I), n is an integer greater than or equal to 2.

[0028] Polymers can be obtained by a polymerization reaction that causes intermolecular dehydration condensation by drying a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in an aqueous solution, similar to the production of Ge-132 from THGP. The polymer may consist of all identical or different constituent units. The former polymer can be obtained by drying an aqueous solution containing one type of compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof, while the latter polymer can be obtained by drying an aqueous solution containing multiple types of compounds of general formula (I) or a pharmaceutically acceptable salt or ester thereof. Since the polymerization reaction is reversible, the polymer is hydrolyzed by dissolving it in an aqueous medium such as water, yielding the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof.

[0029] Examples of polymers of compounds of general formula (I) include Ge-132, the water-soluble organic germanium compound Poly-[(2-carboxyethyl-hydroxygermanium)oxide] which is a linear polymer described in Japanese Patent Publication No. 57-102895, and the water-soluble organic germanium compound Propagermanium (3-oxygermylpropionic acid polymer) which has a ladder-like structure (a cyclic structure composed of eight atoms of germanium and oxygen) described by Mizuno et al. (J. Pharm. Sci., 2015, 104 (8), 2482-2488).

[0030] Further examples of polymers of compounds of general formula (I) are shown in general formula (III). [ka] The compound is such that R4, R5, and R6 are independently hydrogen or a lower alkyl group, and X is an alkali metal cation, an ammonium cation, or a quaternary ammonium cation.

[0031] In general formula (III), R4, R5, and R6 are independently hydrogen or a lower alkyl group. A lower alkyl group is a linear or branched alkyl group having 1 to 6 carbon atoms, preferably 1 to 4, and more preferably 1 to 3 carbon atoms. In a preferred embodiment, R4, R5, and R6 are all hydrogen.

[0032] In general formula (III), X is Na + , K + These are alkali metal cations, ammonium cations, or quaternary ammonium cations. A quaternary ammonium cation is a cation in which four alkyl and / or aryl groups are bonded to nitrogen. In the present invention, preferred quaternary ammonium cations include cations in which four linear or branched alkyl groups, having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 3 carbon atoms, are bonded to nitrogen. Examples include tetramethylammonium cation, tetraethylammonium cation, tetrapropylammonium cation, ethyltrimethylammonium, triethylmethylammonium, and the like. In preferred embodiments, X is Na + That is the case.

[0033] A suitable example of a compound of general formula (III) is one in which R4 to R6 are all hydrogen and X is Na. + Therefore, 1,7,9,15-tetra(2'-sodium carboxyethylgermanium)-3,5,11,13-tetra-[sodium propanato(2-)-C 3’ , O']-germanium-2, 4, 6, 8, 10, 12, 14, 16, 17, 18, 19, 20-dodecaoxa-pentacyclo [8.1 1, 5 . 1 7, 11 . 1 9, 13 . 1 3, 15 One example is icosane (hereinafter referred to as the THGP octamer).

[0034] The compound of general formula (III) can be produced by crystallizing the compound of general formula (IV) (wherein R4 to R6 are as described in the explanation of general formula (III)) in a mixed solvent of water and a water-miscible organic solvent. [ka]

[0035] Compounds of general formula (IV) can be synthesized using an acrylic acid derivative of general formula (V) and trichlorogermane, as shown in the scheme below. [ka]

[0036] Hydrogelmation of an acrylic acid derivative of general formula (V) with trichlorogerman can be carried out at a temperature of approximately 25-40°C using, for example, concentrated hydrochloric acid, diethyl ether, or chloroform as a solvent. Then, the compound obtained by hydrogelmation can be reacted with an alkali metal hydroxide or amine in the presence of water, typically in an aqueous solution, to hydrolyze and neutralize it, thereby preparing a compound of general formula (IV).

[0037] Alternatively, compounds of general formula (IV), in which R4 to R6 are hydrogen atoms, can be prepared by reacting a known THGP polymer such as Ge-132 with an alkali metal hydroxide or amine in the presence of water.

[0038] The resulting compound of general formula (IV) is then crystallized in a mixed solvent of water and a water-miscible organic solvent. The mixing ratio of water to organic solvent in the mixed solvent can be 1:3 to 1:20 (v / v), preferably 1:3 to 1:5 (v / v). Examples of organic solvents that can be used include acetone, methanol, ethanol, 1-propanol, or 2-propanol, with ethanol being particularly preferred.

[0039] A crystallization mixture is prepared by adding the above organic solvent in an amount such that the mixing ratio falls within the above range to an aqueous solution of the compound of general formula (IV). The concentration of the compound of general formula (IV) in this mixture should be 2% to 12% (w / v), preferably 8% to 12% (w / v).

[0040] After thoroughly stirring the above mixture, crystals of the compound of general formula (III) can be precipitated by allowing it to stand at room temperature for 12 hours or more, preferably 24 hours or more. To promote crystallization, crystals of the compound of general formula (III) may also be added as seed crystals. The obtained crystals can be isolated and purified from the mixture by known means, such as filtration, washing with an organic solvent such as a lower alcohol, and vacuum drying.

[0041] Macrophage polarization Macrophage polarization refers to the significant changes in the morphology and function of macrophages in response to local environmental factors. Unstimulated macrophages are normally in a resting state (M0 macrophages), but upon external stimulation, they polarize into two distinct subsets: M1 type or M2 type (M2a, M2b, M2c, and M2d exist).

[0042] Macrophage M1 polarization is induced by IFN-γ alone, or by the synergistic action of IFN-γ with other cytokines (TNF-α, GM-CSF, etc.) or bacterial-derived components such as LPS. M1 macrophages possess high antigen-presenting and phagocytic abilities, and in addition, they produce inflammatory cytokines such as IL-12, IL-1, IL-6, and TNF-α, as well as effector molecules such as nitric oxide and reactive oxygen species. As a result, M1 macrophages promote the elimination of pathogens and cancer cells, and also promote the removal of damaged tissue.

[0043] In contrast, M2 polarization of macrophages is induced by various stimuli, including IL-4, IL-13, TLR agonists, IL-1R, IL-10, TGF-β, and glucocorticoids. M2 macrophages promote tissue repair and suppress immune responses through the production of anti-inflammatory cytokines such as IL-10 and TGF-β. TAMs have an M2-like phenotype and are known to promote cancer cell proliferation and metastasis, enhance angiogenesis, and reduce the drug sensitivity of cancer cells.

[0044] M1 macrophages and M2 macrophages can be defined using known molecular markers that characterize each. Examples of molecular markers for M1 macrophages include cell surface markers such as CD80 and CD86, cytokines such as TNF-α, IL-1β, and IL-6, and nitric oxide synthase (iNOS). Examples of molecular markers for M2 macrophages include cell surface markers such as CD206 and CD163, cytokines such as TGF-β and IL-10, chemokines such as CXCL2, and arginase, an arginine-degrading enzyme involved in ornithine production.

[0045] Macrophage polarization refers to a state in which a particular subset of macrophages becomes dominant within a population of macrophages. Polarization only requires that a specific subset be dominant over other subsets; it does not necessarily involve a decrease in the number of macrophages of other subsets or a decrease in the expression of molecular markers.

[0046] M1 Polarizing Agent The M1 polarization agent of the present invention (also simply called a polarization agent) can polarize macrophages to the M1 type. As described above, it was conventionally thought that IFN-γ was necessary for M1 polarization, but the M1 polarization agent of the present invention can induce M1 polarization of macrophages even under conditions in which IFN-γ is absent.

[0047] Macrophage M1 polarization is performed by treating macrophage source cells with an M1 polarization agent (polarization treatment). Macrophage source cells may be macrophage progenitor cells such as monocytes, or they may be unpolarized macrophages (M0 macrophages). Furthermore, since polarized macrophages can be repolarized into different subtypes, macrophage source cells may be macrophages polarized to a subset other than M1, typically M2 macrophages.

[0048] When macrophages that have undergone M1 polarization are intended for administration to a mammalian organism, the macrophage source cells may be from the same organism as the organism to which the administration is intended, or from a different organism; in other words, the macrophage source cells may be autologous or allogeneic. Preferably, the macrophage source cells are from the same organism, i.e., autologous. Macrophage source cells can be prepared in advance by collecting them from the blood, bone marrow, spleen, etc., of a mammalian organism.

[0049] In vitro polarization can be performed by culturing macrophage source cells for 7 days or more in a medium containing a compound of general formula (I) in a concentration of 50 μM to 10 mM or a pharmaceutically acceptable salt or ester thereof. The medium used in polarization is not limited as long as it can culture macrophage source cells, and examples include DMEM, MEM, α-MEM, and RPMI1640. The medium may contain additives such as fetal bovine serum (FBS), but it is not necessary to contain M1 polarization inducers such as IFN-γ.

[0050] The concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the culture medium may be 50 μM to 10 mM, for example, 50 μM to 5 mM, preferably 500 μM to 5 mM. When using a polymer of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof as a polarization agent, an amount should be added to the culture medium such that the concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the culture medium becomes 50 μM to 10 mM when added to the culture medium.

[0051] The incubation period for the polarization treatment may be 7 days or longer, preferably 10 days or longer. The incubation period may be long, and as shown in the examples described later, the inventors have confirmed that M1 polarization continues even when incubated for 120 days. From an economic standpoint, incubation may be terminated when M1 polarization is confirmed. Therefore, the incubation period may be, for example, 7 to 120 days. The incubation period may also be 7 to 90 days, 7 to 45 days, 7 to 40 days, 7 to 30 days, or 7 to 20 days. Preferably, the incubation period is 10 to 120 days, 10 to 90 days, 10 to 45 days, 10 to 40 days, 10 to 30 days, or 10 to 20 days.

[0052] If the incubation period is less than 10 days, the concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the culture medium is preferably 500 μM to 10 mM, because low concentrations may result in variability in the degree of M1 polarization.

[0053] As long as the culture medium contains the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof at the above concentration, the culture medium may be changed or subcultured during the polarization treatment.

[0054] The culture temperature and gas concentration should be suitable for the individual macrophage raw material cells; for example, culture can be carried out at 37°C under 5% CO2.

[0055] In vivo polarization treatment can be performed, for example, by administering to the subject an amount of the compound of general formula (I), or a pharmaceutically acceptable salt or ester thereof, or a polymer thereof, at a rate of approximately 5 to 500 mg / kg body weight per day for 2 to 30 days in the case of oral administration.

[0056] M1 polarization of macrophages after polarization treatment can be confirmed by the fact that the number of M1 macrophages in the macrophage cell population is greater than the number of M2 macrophages. Alternatively, M1 polarization can be confirmed by the degree of increase in the expression of M1 macrophage molecular markers in the entire macrophage cell population being greater than the degree of increase in the expression of M2 macrophage molecular markers, for example, by the expression of M1 macrophage molecular markers being elevated and the expression of M2 macrophage molecular markers remaining unchanged or decreasing.

[0057] As shown in the examples described below, the polarization agent of the present invention can increase M1 macrophages and decrease M2 macrophages. Accordingly, the present invention also provides, in other embodiments, an M1 macrophage induction promoter comprising a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof, and an M2 macrophage induction inhibitor comprising a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof.

[0058] Macrophage SIRPα expression inhibitors and CD47-SIRPα immune checkpoint inhibitors In another embodiment, the present invention provides a macrophage SIRPα expression inhibitor containing a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof.

[0059] SIRPα is a receptor-type membrane protein highly expressed on the cell membranes of myeloid cells such as macrophages and nerve cells, and its physiological ligand is CD47. CD47 is highly expressed on the cell membranes of many cancer cells and is known to suppress macrophage phagocytosis by binding to SIRPα on macrophages. This intercellular signaling system, the CD47-SIRPα system, is attracting attention as an immune checkpoint in the innate immune system, and inhibitors of the CD47-SIRPα immune checkpoint are expected to be novel molecular targeted drugs for cancer.

[0060] Human SIRPα has two isoforms, while mouse SIRPα has six isoforms. Similarly, human CD47 has three isoforms, and mouse CD47 has five isoforms. Table 1 shows the amino acid sequences of each isoform and the nucleotide sequences of the cDNAs encoding them, as registered in the National Center for Biotechnology Information (NCBI) Reference Sequence Database. [Table 1]

[0061] The SIRPα expression inhibitor of the present invention can suppress SIRPα expression in macrophages, and macrophages with reduced SIRPα expression can be produced. Suppression of SIRPα expression can be achieved by culturing macrophage source cells for 7 days or more in a culture medium containing 50 μM to 10 mM of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof. The macrophage source cells, the concentration of the compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof in the culture medium, the culture time, and other culture conditions are as described in the section on polarization treatment.

[0062] Suppression of SIRPα expression can be confirmed at either the gene level or the protein level. Suppression at the gene level can be confirmed by hybridization using the nucleotide sequence information of the gene encoding SIRPα, quantitative PCR, RNA sequencing, or other common methods capable of detecting or quantifying specific gene expression. Suppression at the protein level can be confirmed by ELISA, RIA, insight hybridization, Western blot analysis, or other common methods capable of detecting or quantifying specific protein expression using specific antibodies.

[0063] Macrophages with reduced SIRPα expression are expected to evade the CD47-SIRPα immune checkpoint because their binding to CD47 on cancer cells is suppressed. Therefore, the present invention provides, in another embodiment, a CD47-SIRPα immune checkpoint inhibitor containing a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof.

[0064] Inhibition of the CD47-SIRPα immune checkpoint can be confirmed by ELISA, which can detect binding between CD47 and SIRPα, or by cell-based reporter assays, which can detect signal transduction from CD47-bound SIRPα, or by other common methods that can detect or quantify binding or signal transduction between specific proteins.

[0065] composition As described above, M1 polarized macrophages derived from compounds of general formula (I), pharmaceutically acceptable salts or esters thereof, or polymers thereof, possess high phagocytic activity and can inhibit the CD47-SIRPα immune checkpoint by suppressing SIRPα expression. The inventors have further discovered that these M1 polarized macrophages can inhibit epithelial-mesenchymal transition in cancer cells via their secretions and can suppress CD47 expression on cancer cells.

[0066] Epithelial-mesenchymal transition (EMT) is a phenomenon in which epithelial cells lose their epithelial characteristics and acquire mesenchymal characteristics. In addition to wound healing and the associated tissue fibrosis, it is also involved in cancer invasion and metastasis. Cancer cells acquire high metastatic, invasive, and tumorigenetic abilities, as well as stress resistance, through EMPLT. Therefore, inhibiting EMPLT in cancer cells can suppress cancer metastasis and improve drug resistance.

[0067] Furthermore, it is thought that the suppression of CD47 expression on cancer cells, combined with the suppression of SIRPα expression on macrophages, strongly inhibits the CD47-SIRPα immune checkpoint.

[0068] Therefore, it is thought that by allowing a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof to coexist with macrophage raw material cells in the body for an extended period, M1 polarization of macrophages and suppression of SIRPα expression are induced in the body. Furthermore, the induced M1 macrophages inhibit epithelial-mesenchymal transition in cancer cells and suppress CD47 expression, thus enabling treatment of cancer.

[0069] While the antitumor effect of Ge-132, a polymer of THGP, is well known, its mechanism is believed to be the increased secretion of IFN-γ induced by NK cell activation, and the subsequent induction of M1 macrophages. It was thought that Ge-132 could not fully exert its antitumor effect in patients who did not produce enough IFN-γ or in patients with low responsiveness to IFN-γ stimulation, where M1 polarization of macrophages by IFN-γ stimulation was unlikely to occur.

[0070] However, according to the present invention, by exposing THGP to macrophage source cells for an extended period, M1 polarization of macrophages can be induced even in the absence of IFN-γ. This means that THGP may be effective even in cancer patients who were previously thought not to exhibit the antitumor effects of THGP.

[0071] Thus, the present invention provides a composition containing a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof, for use in treating a disease or condition in subjects where an amount of IFN-γ sufficient to induce M1 polarization of macrophages is absent or in subjects in which M1 polarization of macrophages is not induced by IFN-γ.

[0072] Furthermore, M1 polarized macrophages prepared by co-occurring a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof with macrophage source cells in vitro for an extended period are also expected to exert similar effects upon administration to a target. Accordingly, the present invention also provides a composition for use in treating a disease or condition, which contains M1 polarized macrophages prepared by a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof, or polymers thereof.

[0073] In addition, the culture supernatant of M1 polarized macrophages contains various humoral factors produced by macrophages, which are also considered effective in treating diseases or conditions. Therefore, in the present invention, a composition containing the culture supernatant of M1 polarized macrophages, prepared by co-occurring a compound of general formula (I) or a pharmaceutically acceptable salt or ester thereof with macrophage raw material cells in vitro for an extended period of time, can also be used as a composition for treating diseases or symptoms. The culture supernatant may be the culture supernatant from the polarization treatment culture, or the culture supernatant obtained by newly culturing M1 polarized macrophages.

[0074] Compositions containing the compound of general formula (I) above, or pharmaceutically acceptable salts or esters thereof, or polymers thereof, compositions containing M1 polarized macrophages, and compositions containing the culture supernatant of M1 polarized macrophages are preferably cancer treatment compositions used for cancer patients, and in particular, pharmaceutical compositions for cancer treatment. Cancer treatment compositions can be used to enhance phagocytosis of cancer cells by macrophages, to suppress epithelial-mesenchymal transition of cancer cells, and to inhibit the CD47-SIRPα immune checkpoint.

[0075] Furthermore, the above composition can also be used in the treatment of various diseases or conditions other than cancer that are expected to be improved or prevented by M1 macrophages, such as in the treatment of infectious diseases and injuries, and in the treatment of diseases or conditions involving chronic inflammation, such as chronic inflammatory diseases, allergic diseases, autoimmune diseases, arteriosclerotic diseases (ischemic heart disease, stroke, etc.), neurodegenerative diseases (Alzheimer's disease), metabolic syndrome / lifestyle-related diseases (obesity, diabetes, dyslipidemia, chronic kidney disease, non-alcoholic steatohepatitis, etc.), and urinary disorders.

[0076] The above compositions are applicable to mammals that require treatment for a disease or condition, such as rodents including mice, rats, hamsters, and guinea pigs; primates including humans, chimpanzees, and rhesus monkeys; livestock including pigs, cattle, goats, horses, and sheep; and companion animals including dogs and cats. Humans are a preferred subject.

[0077] As used herein, treatment of a disease or condition encompasses all types of interventions permitted when applying pharmaceuticals, quasi-drugs, foods or cosmetics for the purpose of curing, temporarily relieving, improving, or preventing a disease or condition, including, for example, delaying or halting the progression of the disease or condition, regression or disappearance of lesions, prevention of onset, or prevention of recurrence.

[0078] The above composition contains an effective amount of a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof, or an effective amount of M1 polarized macrophages, or an effective amount of M1 polarized macrophage culture supernatant. Here, "effective amount" means an amount effective for treating a disease or condition, and is determined appropriately by those skilled in the art depending on the method of use, the age of the subject, the nature of the disease or condition, and other conditions.

[0079] In a preferred embodiment, the effective amount of the compound of general formula (I), or a pharmaceutically acceptable salt or ester thereof, or a polymer thereof, is, for example, 10 μg to 300 mg, preferably 500 μg to 200 mg, and more preferably 1 mg to 100 mg per kg of body weight of the subject administered orally, and the effective amount of the culture supernatant is, for example, 0.001 mg to 100 mg, preferably 0.002 mg to 50 mg, and more preferably 0.005 mg to 20 mg per kg of body weight of the subject administered intravenously. In another preferred embodiment, the effective amount of M1 polarized macrophages is, in terms of M1 macrophages, 1 × 10⁶ per kg of body weight of the subject administered intravenously. 4 cells ~1×10 9 Cells, preferably 1 × 10 5 cells ~1×10 8 These are cells. These effective doses can be administered in one or multiple divided doses.

[0080] The above compositions may contain pharmaceutically acceptable carriers, buffers, stabilizers, preservatives, excipients, and other components, and can be used as pharmaceutical compositions, quasi-drug compositions, food and beverage compositions, or cosmetic compositions. The acceptable components in these compositions are well known to those skilled in the art for each purpose of the composition, and those skilled in the art can appropriately select and use components from those listed in the 17th edition of the Japanese Pharmacopoeia and other standards, for example, in the case of pharmaceutical compositions, within the scope of their ordinary capacity to implement.

[0081] The above compositions can be used in combination with additional means depending on their purpose. For example, if a composition is used as a pharmaceutical for the treatment of a disease or condition, it can be used in combination with other means effective for the treatment of said disease or condition. If a composition is used as a pharmaceutical for the treatment of cancer, it can be used in combination with chemotherapy, radiotherapy, surgery, or immunotherapy.

[0082] The dosage form of a composition containing a compound of general formula (I), a pharmaceutically acceptable salt or ester thereof, or a polymer thereof is arbitrary, but examples include oral preparations (tablets, capsules, powders, granules, fine granules, pills, suspensions, emulsions, liquids, syrups, etc.), injections, and topical preparations (sprays, topical solutions, inhalants, ointments, patches, etc.). The route of administration of the composition is not particularly limited and is determined appropriately depending on the dosage form. In one preferred embodiment, the composition can be administered orally, intravenously, intraperitoneally, or percutaneously.

[0083] Furthermore, while the dosage form of the composition containing M1 polarized macrophages and the composition containing the culture supernatant of M1 polarized macrophages is arbitrary, parenteral formulations such as injections are preferred. These compositions can be administered, for example, intravenously, intraperitoneally, or locally to the affected area.

[0084] The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. [Examples]

[0085] material and method ·Cell culture The mouse macrophage-derived cell line RAW 264.7 and the mouse melanoma-derived cell line B16 4A5 were provided by Riken Cell Bank. The cells were cultured at 37°C under 5% CO2 in Dulbecco's modified Eagle medium (DMEM) (Nissui Pharmaceutical Co., Ltd.) supplemented with 10% fetal bovine serum (FBS). Once the cells reached subconfluence, they were detached with a scraper or 0.25% trypsin / 1mM EDTA and seeded every 3-4 days at a ratio of 1:4 to 1:8. THGP processing RAW 264.7 cells were treated with THGP by subculturing them every 3 days in 10% FBS DMEM containing 50, 500, or 5000 μM THGP. The seeding rate of Raw 264.7 cells during subculturing was 1 × 10⁶ for a 10 cm dish. 6 For cells, use 5.0 × 10 for 6-well plates. 5 It was cells / wells.

[0086] Preparation of Conditional Medium RAW 264.7 cells cultured in 10% FBS DMEM containing 500 μM THGP for 10 to 120 days, and RAW 264.7 cells cultured in 10% FBS DMEM without THGP for 10 to 120 days, were each placed in a 10 cm dish in a 1 × 10⁶ dish. 6 Cells were seeded and cultured in 10% FBS DMEM for 3 days. The culture supernatant was collected and stored at -30°C until use. At the time of use, the culture supernatant was mixed with 10% FBS DMEM in a 1:1 ratio to prepare the conditional medium.

[0087] MTS assay The MTS assay was performed by measuring the absorbance at 490 nm using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega).

[0088] • Immunofluorescence staining Cells were fixed with 4% paraformaldehyde in PBS (Wako Pure Chemical Industries), permeabilized with 0.2% Triton X-100 at room temperature for 10 minutes, and then blocked with 1.5% BSA at room temperature for 30 minutes. The primary antibody reaction was carried out overnight at 4°C, and the secondary antibody reaction was carried out at room temperature for 1 hour. As primary antibodies, anti-B7-2 (CD86) antibody (sc-28347) (Santa Cruz), anti-Mannose Receptor (CD206) antibody (ab64693) (Abcam), and anti-N-Cadherin antibody (Cell signaling) were used. As secondary antibodies, Goat Anti-Mouse IgG H&L (TexasRED) or Goat Anti-Rabbit IgG H&L (FITC) (Abcam) were used. Antibodies were diluted with blocking buffer according to the manufacturer's recommended concentration. Nuclei were stained with DAPI (Dojin Chemical). Fluorescence observations were performed using a Nikon Eclipse TS100 fluorescence microscope and a Nikon DS-Fi3 (Nikon), and the images were analyzed using the NIS-element (Nikon) image analysis software.

[0089] • Real-time RT-PCR RNA was extracted from cells using Isogen (Nippon Gene). Using 1 μg of the extracted RNA as a template, reverse transcription was performed using Super Script III (Invitrogen) at 50°C for 1 hour and 95°C for 5 minutes. PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio) at 95°C for 5 seconds and 60°C for 30 seconds for 40 cycles. The primers used in the PCR reaction are shown in Table 2. RPS18 was used as an internal control for correction. The primer sets for CD47 amplification and SIRPα amplification were designed to amplify sequences common to all isoforms of CD47 and SIRPα, respectively. [Table 2]

[0090] Western blot Proteins were extracted from cells using RIPA buffer. Protein quantification was performed by the Bradford method (Bio-Rad). SDS-PAGE was performed using 7 μg (for RAW 264.7 cells) or 10 μg (for B16 4A5 cells) of protein. After transfer to a PVDF membrane, blocking was performed with 5% skim milk in TBS-T (Morinaga Milk Industry). The primary antibody reaction was performed overnight at 4°C, and the secondary antibody reaction was performed at room temperature for 1 hour. The primary antibodies used were anti-B7-2 (CD86) antibody (sc-28347) (Santa Cruz), anti-Mannose Receptor (CD206) antibody (ab64693) (Abcam), anti-N-Cadherin antibody (Cell signaling), and anti-β-actin antibody (Abcam). Furthermore, Goat Anti-Mouse IgG H&L (HRP) (ab6789), Goat Anti-Rabbit IgG H&L (HRP) (ab205718), and Donkey Anti-Goat IgG H&L (HRP) (ab205723) (Abcam) were used as secondary antibodies. The antibodies were diluted in blocking buffer according to the manufacturer's recommended concentration. Each band was quantified using the image analysis software Image-Lab (BioRad) and corrected with β-actin.

[0091] • Phagocytosis of latex beads Phagocytosis activity was evaluated using a phagocytosis assay kit (Cayman Chemical Company). Rabbit IgG FITC-latex beads were diluted 1:200 and phagocytosed in RAW 264.7 cells for 1 hour. The nuclei were stained with Hoechst 33452 (Dojin Chemical) and observed under a fluorescence microscope. The captured images were analyzed using NIS-element (Nikon), and the intensity of fluorescence emitted by FITC was used as an indicator of phagocytic activity.

[0092] • Phagocytosis of cancer cells (fluorescence staining) Raw 264.7 cells stained with Cell Tracker Green CMFDA Dye (Thermo Fisher) were placed in a 6-well plate with coverslips in a 2x10⁶ arrangement. 6 Cells were seeded per well and cultured in 10% FBS DMEM for 24 hours. Then, B16 4A5 cells stained with Cell Tracker Blue CMAC Dye (Thermo Fisher) were placed on RAW 264.7 culture cells in 2 × 10⁶ layers. 6 Cells were seeded per well. After incubation at 37°C for 2 hours, the cells were fixed with 4% paraformaldehyde in PBS (Wako Pure Chemical Industries), observed with a fluorescence microscope, and the captured images were analyzed using NIS-element (Nikon). Cells co-stained with blue and green were designated as RAW 264.7 cells that had phagocytosed B16 4A5 cells.

[0093] • Cell damage to cancer cells (MTS assay) RAW 264.7 cells and B16 4A5 cells were placed in 5 × 10⁶ well plates. 3 cells / well, 2.5 × 10 3 Cells were seeded per well and co-cultured in 10% FBS DMEM for 48 hours. Raw 264.7 cells and B16 4A5 cells were also cultured individually in the same manner. Cell counts were evaluated by the MTS assay. The percentage of B16 4A5 cells damaged by RAW 264.7 cells was calculated by subtracting the absorbance of the co-culture from the sum of the absorbances of RAW 264.7 and B16 4A5 cells cultured individually, and then dividing this result by the absorbance of B16 4A5 cells cultured individually.

[0094] · ELISA The amount of TGF-β in the culture supernatant was measured using the LEGEND MAX Mouse Latent TGF-β ELISA Kit (BioLegend).

[0095] • Migration assay B16 4A5 cells were placed in a 24-well plate in a 3.0 × 10⁶ arrangement. 5 Cells were seeded per well and cultured in 10% FBS DMEM for 24 hours. After scraping the cells using a 1000 μl blue tip, they were washed twice with PBS (-) and test medium was added. Cell images were taken at 0 hours and 48 hours after addition and analyzed using the image analysis software Image J / Fiji (NIH). The area of ​​cells present in the scraped area after 48 hours was calculated relative to the area of ​​cells scraped off and used as an index for evaluating migratory ability.

[0096] • MMP-2 and MMP-9 activity 30 μl of culture supernatant was applied to a 10% acrylamide gel containing gelatin, and SDS-PAGE was performed. The gel after electrophoresis was used to evaluate the activity of MMP-2 and MMP-9 by gelatin zymography. The degree of gelatin degradation by MMP-2 and MMP-9 on the gel was measured by image analysis using Image J / Fiji, and this was used as the activity of MMP-2 and MMP-9.

[0097] ·Cell adhesion B16 4A5 cells were cultured in test medium for 72 hours, and then harvested. The harvested cells were placed in a 96-well plate coated with type I Collagen (Wako Pure Chemical Industries) in a 5.0 × 10⁶ layer. 4 Cells were seeded in wells. After one hour, the wells were washed twice with PBS (-) to remove non-adherent cells, and the number of adhered cells was evaluated by the MTS assay.

[0098] ·Invasiveness The invasiveness of B16 4A5 cells was evaluated using the Boyden chamber method with 8 μm pore-size PET inserts (BD falcon) coated with type I collagen. RAW 264.7 cells were stained with Cell Tracker Green CMFDA Dye (Thermo Fisher), and B16 4A5 cells were stained with Cell Tracker Blue CMAC Dye (Thermo Fisher). 1 × 10⁶ RAW 264.7 cells were then sampled. 5 cells, B16 4A5 cells 5 x 10 4 Cells were seeded in the upper layer of a Boyden chamber. The upper layer was cultured in serum-free medium, and the lower layer in 10% FBS DMEM for 24 hours. After removing any remaining cells without invasiveness using a cotton swab, the cells were fixed in 4% paraformaldehyde in PBS (Wako Pure Chemical Industries) and observed under a fluorescence microscope. Cells stained blue were designated as B16 4A5 cells, and the number of cells visible in one field of view was counted. ·Statistical analysis Unless otherwise specified, data was obtained with n=6 and is shown as mean ± standard deviation. For statistical significance testing between means, the statistical software Excel Statistics was used. Student's t-test was used for two-group comparisons, and Dunnett's test was used for multiple-group comparisons. A p-value less than 0.05 was considered statistically significant between groups (*P < 0.05, **P < 0.01).

[0099] Example 1: M1 polarization of macrophages by THGP RAW 264.7 cells were cultured in a medium containing 500 μM THGP for up to 120 days and then treated with THGP. As a control, cells were cultured in a medium without THGP in parallel. The morphology of RAW264.7 cells changed over time to a spindle shape upon THGP treatment (Figure 1a; cells on Day 120 are not shown in the figure, but showed a similar morphological change to those on Day 90). After 10 days of THGP treatment, the proportion of spindle-shaped cells among all cells increased approximately twice compared to the proportion in the control medium (Figures 1b and 1c). Since the spindle shape is the cell morphology of M1 macrophages, this suggests that RAW 264.7 cells differentiated into M1 macrophages upon THGP treatment.

[0100] Cells treated with THGP on days 1, 10, 30, and 40 were passaged, and cells on the day of passage, 1 day later, and 2 days later were subjected to an MTS assay to measure cell proliferation rate. A decrease in cell proliferation rate was observed with THGP treatment for 10 days or more, and the longer the duration of THGP treatment, the greater the decrease in proliferation rate (Figure 1d). M1 macrophages have been reported to proliferate slowly, and this decrease in proliferation rate suggests that RAW 264.7 cells differentiated into M1 macrophages upon THGP treatment.

[0101] Immunofluorescence staining was used to detect the expression of CD86, a marker for M1 macrophages, and CD206, a marker for M2 macrophages, in cells treated with THGP for 20 to 120 days. THGP treatment reduced the proportion of CD206-positive cells in the total cell population and increased the M1 / M2 ratio (Figure 1e, Figure 1f). This indicates that THGP treatment polarized macrophages to M1.

[0102] Furthermore, gene expression of M1 macrophage markers (iNOS, CD80, CD86, TNF-α, IL-1β) and M2 macrophage markers (arginase, CD206, CD163, TGF-β, CXCL2) was measured by real-time RT-PCR in cells treated with THGP for 20 days. THGP treatment resulted in an increase in the gene expression levels of M1 macrophage markers and a decrease in the gene expression levels of M2 macrophage markers (Figures 2a and 2b), confirming M1 polarization of macrophages by THGP treatment. Protein expression of CD86 and CD206 in cells treated with THGP for 20 to 120 days also showed a similar trend to gene expression (Figures 2c to 2e).

[0103] Example 2: Effect of THGP concentration on macrophage M1 polarization RAW 264.7 cells were cultured in medium containing 50, 500, or 5000 μM THGP for 4, 7, or 10 days and then treated with THGP. As a control, cells were cultured in medium without THGP in parallel. The gene expression level of the M1 macrophage marker iNOS in the cultured cells was measured by real-time RT-PCR. The results are shown in Figure 3. At a THGP concentration of 50 μM, iNOS expression in THGP-treated cells was higher than that of control cells at all time points. Expression levels in THGP-treated cells varied considerably on days 4 and 7, but this variation decreased by day 10. At THGP concentrations of 500 or 5000 μM, iNOS expression in THGP-treated cells on day 4 was approximately the same as that of the control, but expression levels on days 7 and 10 were higher than the control, showing a dose-dependent increase. This indicates that M1 macrophages can be induced at THGP concentrations of 50 μM or higher, but it is preferable to treat for 10 days or more to minimize variability.

[0104] Example 3: Injury to cancer cells by M1 polarized macrophages prepared by THGP treatment. RAW 264.7 cells were cultured for 10 days in medium containing 50, 500, or 5000 μM THGP and then treated with THGP. As a control, cells were cultured concurrently in medium without THGP. Rabbit IgG FITC-latex beads were added to the cultured cells, and their phagocytic activity towards foreign substances was evaluated. THGP-treated cells showed enhanced phagocytic activity at all THGP concentrations, and the degree of enhancement was dose-dependent (Figures 4a and 4b).

[0105] Furthermore, RAW 264.7 cells were cultured in a medium containing 500 μM THGP for 20 to 120 days and designated as RAW T. As a control, RAW 264.7 cells were cultured in a medium without THGP for 20 to 120 days and designated as RAW C. These cells were stained with CMFDA, and their phagocytic activity against cancer cells was evaluated by co-culturing them with B16 4A5 cells stained with CMAC. For comparison, a group of RAW C and B16 4A5 cells co-cultured in the presence of 500 μM THGP (RAW C + THGP) was established.

[0106] The results are shown in Figures 4c and 4d. In co-culture with RAW C, B16 4A5 cells were hardly phagocytosed. In co-culture with RAW C in the presence of THGP, approximately 10% of B16 4A5 cells were phagocytosed. In contrast, in co-culture with RAW T, more than 60% of B16 4A5 cells were phagocytosed, confirming that M1 polarized macrophages prepared by THGP treatment have high phagocytic activity.

[0107] The cytotoxic activity of M1 polarized macrophages prepared by THGP treatment against B16 4A5 cells was evaluated by MTS assay under co-culture conditions, and the results showed a similar trend to the fluorescence staining results shown in Figure 4d (Figure 5a). To elucidate the mechanism in more detail, 5 × 10⁶ cells were used in a 6-well plate. 5B16 4A5 cells were seeded in cells / well and cultured for 24 hours. These cells were then cultured for a further 48 hours in conditional medium prepared from the culture supernatants of RAW C and RAW T, a medium containing 500 μM THGP, or a medium without THGP. Cell proliferation was evaluated by the MTS assay. Both RAW C and RAW T culture supernatants suppressed B16 4A5 cell proliferation, but no significant difference was observed between the two (Figure 5b). This indicates that the high cytotoxicity of M1 polarized macrophages prepared by THGP treatment against B16 4A5 cells is due to direct phagocytosis, not through the cytotoxic cytokines they secrete. Furthermore, B16 4A5 cells cultured in THGP-containing medium showed similar cell proliferation to those cultured in THGP-free medium (Figures 5b and 5c), indicating that THGP itself does not affect B16 4A5 cell proliferation.

[0108] Furthermore, the cytotoxic activity of M1 polarized macrophages prepared by THGP treatment against B16 4A5 cells was evaluated by MTS assay in the same manner as the experiment shown in Figure 5a, except that 5-FU was added to the culture medium to a concentration of 1.25 μM during co-culture. Co-culture with RAW C in the presence of 5-FU reduced the viability of B16 4A5 cells more than co-culture alone in the presence of 5-FU, and co-culture with RAW T in the presence of 5-FU further reduced the viability of B16 4A5 cells more than co-culture with RAW C in the presence of 5-FU (Figure 5d). To elucidate the mechanism in more detail, 5-FU was added to conditional medium prepared from the culture supernatants of RAW C and RAW T, or to a control THGP-free medium to a concentration of 1.25 μM. B16 4A5 cells were cultured in these media for 48 hours, and the cell count was evaluated by MTS assay. The culture supernatant of RAW C increased the viability of B16 4A5 cells in the presence of 5-FU, but the culture supernatant of RAW T did not show such an effect (Figure 5e). This suggests that M1 polarized macrophages prepared by THGP treatment can effectively damage cancer cells when used in combination with anticancer drugs without conferring humoral factor-mediated drug resistance to cancer cells, as was observed with untreated macrophages.

[0109] Example 4: Suppression of CD47-SIRPα immune checkpoint molecule expression by THGP and M1 polarized macrophages prepared by THGP treatment. In co-culture with B16 4A5 cells, RAW T cells were observed to accumulate more around B16 4A5 cells than RAW C cells (Figure 6a), suggesting that RAW T cells have a higher recognition ability for B16 4A5 cells than RAW C cells. Focusing on CD47-SIRPα, one of the mechanisms by which macrophages recognize cancer cells, we measured SIRP-α gene expression in RAW C and RAW T cells using real-time RT-PCR, and found that SIRP-α gene expression was suppressed in RAW T cells (Figure 6b).

[0110] Also, 5 × 10 in a 6-well plate 5B16 4A5 cells were seeded in cells / well and cultured for 24 hours. These cells were then cultured for a further 48 hours in conditional medium prepared from the culture supernatants of RAW C and RAW T, respectively, or in THGP-free medium. Real-time PCR was used to measure CD47 gene expression in the cultured B16 4A5 cells. The culture supernatant from RAW C increased CD47 gene expression in B16 4A5 cells, but the culture supernatant from RAW T did not show such an effect (Figure 6c). This indicates that THGP suppresses SIRPα gene expression in macrophages and inhibits CD47 gene expression in cancer cells via humoral factors secreted by macrophages.

[0111] Example 5: Inhibition of epithelial-mesenchymal transition in cancer cells by M1 polarized macrophages prepared by THGP treatment. As shown in Figure 2b, TGF-β gene expression was suppressed in M1 polarized macrophages prepared by THGP treatment. To investigate TGF-β expression at the protein level, RAW C and RAW T were each placed in 96-well plates in 5.0 × 10⁶ units. 4 Cells were seeded per well and cultured for 48 hours. The amount of TGF-β in the recovered culture supernatant was suppressed, similar to gene expression (Figure 7a).

[0112] Next, the effect of M1 polarized macrophages prepared by THGP treatment on epithelial-mesenchymal transition was evaluated. 5 × 10⁶ macrophages were placed in a 6-well plate. 5 B16 4A5 cells were seeded in cells / well and cultured for 24 hours. These cells were then cultured for a further 48 hours in conditional medium prepared from the culture supernatants of RAW C and RAW T, respectively, or in medium containing 500 μM THGP or THGP-free medium. The gene expression of N-cadherin and Vimentin, mesenchymal cell markers, in the cultured B16 4A5 cells was measured by real-time RT-PCR.

[0113] The culture supernatant of RAW C increased the expression levels of N-cadherin and Vimentin genes in B16 4A5 cells, but the culture supernatant of RAW T did not show such an effect (Figures 7b and 7c). N-cadherin showed a similar trend at the protein level (Figures 7d and 7e). This indicates that M1 polarized macrophages prepared by THGP treatment do not induce epithelial-mesenchymal transition in cancer cells via humoral factors, unlike macrophages not treated with THGP. It is presumed that THGP treatment polarizes macrophages to M1, thereby suppressing the production of TGF-β, a major factor in inducing epithelial-mesenchymal transition, and thus inhibiting the induction of epithelial-mesenchymal transition in cancer cells.

[0114] Example 6: Suppression of cancer cell metastasis by M1 polarized macrophages prepared by THGP treatment. A migration assay of B16 4A5 cells was performed using conditional medium prepared from the culture supernatants of RAW C and RAW T, as well as medium containing 500 μM THGP or medium without THGP, as test media. The culture supernatant of RAW C enhanced the migration ability of B16 4A5 cells, but no such effect was observed in the culture supernatant of RAW T (Figures 8a and 8b).

[0115] Furthermore, the MMP-2 and MMP-9 activity of B16 4A5 cells was evaluated using conditional medium prepared from the culture supernatants of RAW C and RAW T, THGP-containing medium (500 μM), or THGP-free medium, all in serum-free test media. The culture supernatant of RAW C enhanced the MMP-2 and MMP-9 activity of B16 4A5 cells, but no such effect was observed in the culture supernatant of RAW T (Figure 8c).

[0116] Next, the adhesion of B16 4A5 cells was evaluated using conditional media prepared from the culture supernatants of RAW C and RAW T, respectively, as test media. The culture supernatant of RAW T reduced the adhesion of B16 4A5 cells compared to RAW C (Figure 8d). Furthermore, the invasiveness of B16 4A5 cells was evaluated by co-culturing RAW C or RAW T with B16 4A5 cells using a boyden chamber. RAW T suppressed the invasiveness of B16 4A5 cells compared to RAW C (Figure 8e).

[0117] These results indicate that M1-polarized macrophages prepared by THGP treatment do not enhance the metastatic potential of cancer cells via humoral factors, unlike macrophages not treated with THGP. It is hypothesized that THGP treatment polarizes macrophages to M1, suppressing the induction of epithelial-mesenchymal transition in cancer cells, and consequently inhibiting the enhancement of cancer cell metastasis.

[0118] Comparative example: Confirmation of the direct effects of THGP on epithelial-mesenchymal transition and metastatic potential of cancer cells. 5 × 10⁶ B16 4A5 cells in a 6-well plate 5B16 4A5 cells were seeded in cells / wells and cultured for one week in either a THGP-free medium containing recombinant mouse TGF-β 10 ng / ml (Biolegend) or a medium containing THGP 500 μM with the same concentration of TGF-β, thereby treating B16 4A5 cells with either TGF-β alone or a combination of TGF-β and THGP. Treatment with TGF-β alone increased the number of mesenchymal morphology cells (Figure 9a) and enhanced the protein expression of the mesenchymal cell marker N-cadherin, as confirmed by immunostaining and Western blot analysis of B16 4A5 cells (Figures 9b, 9c). The combined treatment of TGF-β and THGP showed similar effects to that of TGF-β alone. Furthermore, no difference was observed between TGF-β alone and the combined treatment of B16 4A5 cells in the evaluation of invasiveness using a boyden chamber. From this, it was concluded that THGP acts on cancer cells not directly, but indirectly through M1-polarized macrophages prepared by THGP treatment, thereby suppressing epithelial-mesenchymal transition and metastatic potential of cancer cells. [Sequence Listing Free Text]

[0119] Sequence ID 1: Nucleotide sequence of iNOS amplification forward primer Sequence ID 2: Base sequence of the reverse primer for iNOS amplification Sequence ID 3: Base sequence of CD80 amplification forward primer Sequence ID 4: Base sequence of reverse primer for CD80 amplification Sequence ID 5: Base sequence of CD86 amplification forward primer Sequence ID 6: Base sequence of the reverse primer for CD86 amplification. Sequence ID 7: Nucleotide sequence of forward primer for TNF-α amplification Sequence ID 8: Base sequence of a reverse primer for TNF-α amplification Sequence ID 9: Nucleotide sequence of forward primer for arginase amplification Sequence ID No. 10: Base sequence of reverse primer for arginase amplification. Base sequence of forward primer for amplifying CD163 with SEQ ID NO: 11 Base sequence of reverse primer for amplifying CD163 with SEQ ID NO: 12 Base sequence of forward primer for amplifying CD206 with SEQ ID NO: 13 Base sequence of reverse primer for amplifying CD206 with SEQ ID NO: 14 Base sequence of forward primer for amplifying CD47 with SEQ ID NO: 15 Base sequence of reverse primer for amplifying CD47 with SEQ ID NO: 16 Base sequence of forward primer for amplifying SIRPA with SEQ ID NO: 17 Base sequence of reverse primer for amplifying SIRPA with SEQ ID NO: 18 Base sequence of forward primer for amplifying IL-1β with SEQ ID NO: 19 Base sequence of reverse primer for amplifying IL-1β with SEQ ID NO: 20 Base sequence of forward primer for amplifying TGF-β with SEQ ID NO: 21 Base sequence of reverse primer for amplifying TGF-β with SEQ ID NO: 22 Base sequence of forward primer for amplifying CXCL2 with SEQ ID NO: 23 Base sequence of reverse primer for amplifying CXCL2 with SEQ ID NO: 24 Base sequence of forward primer for amplifying N-cadherin with SEQ ID NO: 25 Base sequence of reverse primer for amplifying N-cadherin with SEQ ID NO: 26 Base sequence of forward primer for amplifying Vimentin with SEQ ID NO: 27 Base sequence of reverse primer for amplifying Vimentin with SEQ ID NO: 28 Base sequence of forward primer for amplifying RPS18 with SEQ ID NO: 29 Base sequence of reverse primer for amplifying RPS18 with SEQ ID NO: 30

Claims

1. General formula (I) 【Chemistry 1】 A compound of, wherein R 1 and R 2 are, independently of each other, hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C 3-4 haloalkynyl, C 1-4 alkoxy, C 1-4 haloalkoxy, C 1-4 alkylthio, C 1-4 alkylsulfinyl or C 1-4 alkylsulfonyl, or together with the two carbon atoms to which they are attached, form a 4- to 10-membered monocyclic or polycyclic saturated ring, a 4- to 10-membered monocyclic or polycyclic partially saturated ring, or a 5- to 10-membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1 to 4 atoms selected from nitrogen, oxygen and sulfur, These rings are either unsubstituted or have halogen, nitro, hydroxy, cyano, oxo, or C rings. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl and C 1-4 It is substituted with one or more substituents from the group consisting of alkylsulfonyls, R 3 These are hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 It is an alkylsulfonyl, The aforementioned compound or a pharmaceutically acceptable salt thereof or its C 1-4 An agent containing alkyl esters or polymers thereof for inducing macrophage M1 polarization in subjects that do not produce an amount of IFN-γ sufficient to induce macrophage M1 polarization, or in subjects in which macrophage M1 polarization is not induced by IFN-γ.

2. In general formula (I), R 1 , R 2 and R 3 However, they are independent of each other: hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinil, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 A compound that is alkylsulfonyl or a pharmaceutically acceptable salt thereof or its C 1-4 The agent according to claim 1, comprising an alkyl ester or a polymer thereof.

3. In general formula (I), R 1 , R 2 and R 3 Compounds in which all are hydrogen, or pharmaceutically acceptable salts thereof, or their C 1-4 The agent according to claim 1 or 2, comprising an alkyl ester or a polymer thereof.

4. General formula (I) 【Chemistry 2】 A compound of, In the formula, R 1 and R 2 These are hydrogen, halogen, nitro, hydroxy, cyano, and C, independently of each other. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 They are alkylsulfonyls, or together with the two carbon atoms to which they are bonded, they form a 4-10 membered monocyclic or polycyclic saturated ring, a 4-10 membered monocyclic or polycyclic partially saturated ring, or a 5-10 membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1-4 atoms selected from nitrogen, oxygen, and sulfur. These rings are either unsubstituted or have halogen, nitro, hydroxy, cyano, oxo, or C rings. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl and C 1-4 It is substituted with one or more substituents from the group consisting of alkylsulfonyls, R 3 is hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C 3-4 haloalkynyl, C 1-4 alkoxy, C 1-4 haloalkoxy, C 1-4 alkylthio, C 1-4 alkylsulfinyl or C 1-4 alkylsulfonyl, The aforementioned compound or a pharmaceutically acceptable salt thereof or its C 1-4 An agent containing alkyl esters or polymers thereof for suppressing SIRPα expression in macrophages in subjects that do not produce an amount of IFN-γ sufficient to induce M1 polarization of macrophages, or in subjects in which M1 polarization of macrophages is not induced by IFN-γ.

5. In general formula (I), R 1 , R 2 and R 3 are, independently of one another, hydrogen, halogen, nitro, hydroxy, cyano, C 1-4 alkyl, C 1-4 haloalkyl, C 2-4 alkenyl, C 2-4 haloalkenyl, C 3-4 alkynyl, C3-4 haloalkynyl, C 1-4 alkoxy, C 1-4 haloalkoxy, C 1-4 alkylthio, C 1-4 alkylsulfinyl or C 1-4 alkylsulfonyl, or a pharmaceutically acceptable salt thereof, or a C 1-4 alkyl ester thereof, or a polymer thereof, the agent according to claim 4.

6. In general formula (I), R 1 , R 2 and R 3 Compounds in which all are hydrogen, or pharmaceutically acceptable salts thereof, or their C 1-4 The agent according to claim 4 or 5, comprising an alkyl ester or a polymer thereof.

7. An agent according to any one of claims 4 to 6, used to inhibit the CD47-SIRPα immune checkpoint.

8. General formula (I) 【Transformation 3】 A compound of, In the formula, R 1 and R 2 These are hydrogen, halogen, nitro, hydroxy, cyano, and C, independently of each other. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 They are alkylsulfonyls, or together with the two carbon atoms to which they are bonded, they form a 4-10 membered monocyclic or polycyclic saturated ring, a 4-10 membered monocyclic or polycyclic partially saturated ring, or a 5-10 membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1-4 atoms selected from nitrogen, oxygen, and sulfur. These rings are either unsubstituted or have halogen, nitro, hydroxy, cyano, oxo, or C rings. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl and C 1-4 It is substituted with one or more substituents from the group consisting of alkylsulfonyls, R 3 These are hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 It is an alkylsulfonyl, The aforementioned compound or a pharmaceutically acceptable salt thereof or its C 1-4 A composition comprising alkyl esters or polymers thereof for treating a disease or condition in which M1 macrophages are expected to be improved or prevented in subjects who do not produce an amount of IFN-γ capable of inducing M1 polarization of macrophages, or in subjects in whom M1 polarization of macrophages is not induced by IFN-γ, wherein the disease or condition is selected from the group consisting of cancer, infection, and trauma.

9. In general formula (I), R 1 , R 2 and R 3 However, they are independent of each other: hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinil, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 A compound that is alkylsulfonyl or a pharmaceutically acceptable salt thereof or its C 1-4 The composition according to claim 8, comprising an alkyl ester or a polymer thereof.

10. In general formula (I), R 1 , R 2 and R 3 Compounds in which all are hydrogen, or pharmaceutically acceptable salts thereof, or their C 1-4 The composition according to claim 8 or 9, comprising an alkyl ester or a polymer thereof.

11. A composition according to any one of claims 8 to 10, which is a pharmaceutical composition for cancer treatment.

12. A composition according to any one of claims 8 to 11 for enhancing phagocytosis of cancer cells by macrophages.

13. A composition according to any one of claims 8 to 12 for suppressing epithelial-mesenchymal transition of cancer cells.

14. A composition according to any one of claims 8 to 13 for inhibiting the CD47-SIRPα immune checkpoint.

15. General formula (I) for concentrations from 50 μM to 10 mM 【Chemistry 4】 A compound of, In the formula, R 1 and R 2 These are hydrogen, halogen, nitro, hydroxy, cyano, and C, independently of each other. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 They are alkylsulfonyls, or together with the two carbon atoms to which they are bonded, they form a 4-10 membered monocyclic or polycyclic saturated ring, a 4-10 membered monocyclic or polycyclic partially saturated ring, or a 5-10 membered monocyclic or polycyclic saturated or partially unsaturated heterocycle containing 1-4 atoms selected from nitrogen, oxygen, and sulfur. These rings are either unsubstituted or have halogen, nitro, hydroxy, cyano, oxo, or C rings. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl and C 1-4 It is substituted with one or more substituents from the group consisting of alkylsulfonyls, R 3 These are hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinyl, C 3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 It is an alkylsulfonyl, The aforementioned compound or a pharmaceutically acceptable salt thereof or its C 1-4 A method for producing M1-polarized macrophages, comprising culturing macrophage raw material cells in a medium containing an alkyl ester for seven days or more, wherein the medium does not contain IFN-γ and the culture does not involve co-culture with T cells.

16. The culture medium is R in general formula (I). 1 , R 2 and R 3 These independently produce hydrogen, halogen, nitro, hydroxy, cyano, and C. 1-4 Alkyl, C 1-4 Haloalkyl, C 2-4 Alkenil, C 2-4 Haloalkenil, C 3-4 Alkinil, C3-4 Haloalkynyl, C 1-4 Alkoxy, C 1-4 Haloalkoxy, C 1-4 Alkylthio, C 1-4 Alkyl sulfinyl or C 1-4 A compound that is alkylsulfonyl or a pharmaceutically acceptable salt thereof or its C 1-4 The method according to claim 15, comprising an alkyl ester.

17. The culture medium is R in general formula (I). 1 , R 2 and R 3 Compounds in which all are hydrogen, or pharmaceutically acceptable salts thereof, or their C 1-4 The method according to claim 15 or 16, comprising an alkyl ester.

18. The method according to any one of claims 15 to 17, wherein the macrophage source cells are macrophage progenitor cells, unpolarized macrophages, or macrophages polarized to a subset other than type M1.

19. The culture medium contains a compound of general formula (I) in a concentration of 500 μM to 5 mM, or a pharmaceutically acceptable salt thereof, or its C. 1-4 The method according to any one of claims 15 to 18, comprising an alkyl ester.

20. The method according to any one of claims 15 to 19, wherein the culture is carried out for 10 days or more.

21. A composition for use in treating a disease or condition that is expected to be improved or prevented by M1 macrophages, comprising M1 polarized macrophages or their culture supernatant produced by any one of claims 15 to 20, wherein the disease or condition is selected from the group consisting of cancer, infectious diseases and trauma.

22. The composition according to claim 21, which is a pharmaceutical composition for cancer treatment.

23. The composition according to claim 21 or 22 for enhancing phagocytosis of cancer cells by macrophages.

24. A composition according to any one of claims 21 to 23 for suppressing epithelial-mesenchymal transition of cancer cells.

25. A composition according to any one of claims 21 to 24 for inhibiting the CD47-SIRPα immune checkpoint.

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