Cancer metastasis inhibitors containing pyrrolimidazole polyamide compounds as active ingredients

Abstract

The present invention pertains to: a pyrrole-imidazole polyamide compound that has a specific structure, or a salt thereof; a membrane protein gene expression inhibitor that includes the compound or a salt thereof; and a cancer metastasis inhibitor that includes the compound or a salt thereof as an effective component thereof.

Description

[Technical Field]

[0001] The present invention relates to a pyrrolimidazole polyamide compound having a specific structure or a salt thereof, a membrane protein gene expression inhibitor containing said compound or a salt thereof, and a cancer metastasis inhibitor containing said compound or a salt thereof as an active ingredient. [Background technology]

[0002] In cancer treatment, there is a need for technological development to prevent cancer metastasis. Cancer metastasis is a serious factor that threatens the lives of cancer patients, and inhibiting / suppressing it is important. Examples of cancer metastasis inhibitors include a pancreatic cancer cell invasion and metastasis inhibitor that can effectively suppress the invasion and metastasis of pancreatic cancer cells by utilizing RNA interference (Patent Document 1), a cancer cell metastasis inhibitor that contains an antibody that recognizes the extracellular domain of the human CLCP1 antigen and has metastasis inhibitory activity of cancer cells that express CLCP1 (Patent Document 2), and a cancer metastasis inhibitor that contains an antibody that specifically binds to human ADAM28 and has activity to inhibit the enzymatic activity of human ADAM28 (Patent Document 3).

[0003] Cancer-associated fibroblasts (CAFs) are cells present in the tumor microenvironment that play a crucial role in tumor growth and development. They are involved in the modification and degradation of the extracellular matrix (ECM) as well as the secretion of numerous chemokines, cytokines, and growth factors. CAFs are recognized to play a major role in the proliferation and metastasis of cancer cells.

[0004] Pyrrolimidazole polyamide compounds are known to be usable as RUNX inhibitors used as anticancer drugs (Patent Document 4, Non-Patent Document 1). Pyrrolimidazole polyamides are synthetic oligomers and are known to bind to the minor groove of DNA and recognize specific sequences through opposing pyrrole (P) and imidazole (I) pairs linked by hairpin ligation (Patent Document 4).

[0005] However, to date, there are no reports of cancer metastasis inhibitors utilizing pyrrolimidazole polyamide compounds. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-079280 [Patent Document 2] Japanese Patent Publication No. 2019-068805 [Patent Document 3] Japanese Patent Publication No. 2018-148910 [Patent Document 4] International Publication No. 2018 / 021200 brochure [Non-patent literature]

[0007] [Non-Patent Document 1] Rina Maeda et al., J Am ChemSoc . 2019 Mar13;141(10):4257-4263 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention aims to provide pyrrolimidazole polyamide compounds and their uses that are useful for the treatment or prevention of cancer metastasis. [Means for solving the problem]

[0009] As a result of diligent research to solve the above problems, the inventors of the present invention have discovered that the compound represented by formula (I) (hereinafter also referred to as compound (I)) has the effect of binding to membrane protein genes and suppressing the expression of membrane proteins, as well as inhibiting cancer metastasis, and have completed the present invention. [ka] In other words, the present invention consists of the following: 1. A compound represented by formula (I) or a salt thereof. 2. A pharmaceutical composition comprising the compound or a salt thereof according to item 1 above. 3. An inhibitor of membrane protein gene expression comprising the compound or a salt thereof according to item 1 above. 4. A cancer metastasis inhibitor comprising the compound or a salt thereof according to item 1 above as an active ingredient.

Advantages of the Invention

[0010] According to the present invention, it is possible to provide a pyrrole imidazole polyamide compound that suppresses the expression of membrane protein genes related to cancer metastasis and is useful for the treatment of cancer metastasis, and uses thereof.

Brief Description of the Drawings

[0011] [Figure 1] Figure 1 shows that cancer-associated fibroblasts (CAFs) are found in cervical cancer, pancreatic cancer, breast cancer, lung cancer, and colorectal cancer. [Figure 2] Figure 2 shows that in a nude mouse orthotopic transplantation system, CAFs cause the growth promotion and lymph node metastasis of cervical cancer cells. [Figure 3] Figure 3 shows the results of comprehensive gene expression analysis of CCF-1 cells and CCF-TT cells by DNA array. The hTERT gene and the SV40 large T antigen gene were introduced into CCF-1 cells having the ability to metastasize cancer cells, and CCF-TT cells immortalizing CCF-1 cells were prepared. CCF-TT cells lost the metastatic ability that CCF-1 cells had. Comprehensive gene expression analysis was performed on CCF-1 cells and CCF-TT cells using a DNA array, and membrane protein genes TM4SF1 and TMEM132b with a difference in expression level were identified. X and Y are TM4SF1 and TMEM132b. <了 [Figure 4] Figure 4 is a schematic diagram showing that a pyrrole imidazole polyamide compound binds to a gene and suppresses the expression of membrane proteins X and Y. X and Y are TM4SF1 and TMEM132b.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The present invention relates to a compound represented by formula (I) or a salt thereof. The present invention also relates to a pharmaceutical composition containing the compound of the present invention or a salt thereof, an inhibitor of membrane protein gene expression containing the compound of the present invention or a salt thereof, and an inhibitor of cancer metastasis containing the compound of the present invention or a salt thereof as an active ingredient.

[0013] The compound of the present invention is a pyrrole imidazole polyamide compound, preferably a compound (PIP-Chb conjugate) in which pyrrole imidazole polyamide (PIP) is linked to chlorambucil (Chb) which is an alkylating agent, and most preferably, it is compound (I). Examples of the salt of compound (I) include alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (magnesium salt, calcium salt, etc.), other metal salts (aluminum salt, etc.), inorganic salts (hydrochloride, ammonium salt, amines, etc.), organic salts (glucosamine salt, etc.) and the like. When using the salt of compound (I) in a drug, it is preferable to use a pharmacologically acceptable salt.

[0014] Pyrroliimidazole polyamide (PIP) is a polyamide containing N-methylpyrrole units (Py), N-methylimidazole units (Im), and a γ-aminobutyric acid moiety, where Py, Im, and the γ-aminobutyric acid moiety are linked to each other by amide bonds (-C(=O)-NH-) (Traugeret al, Nature, 382, ​​559-61 (1996); White et al, Chem. Biol., 4, 569-78 (1997); and Dervan, Bioorg. Med. Chem., 9, 2215-35 (2001)). In pyrroliimidazole polyamide, the pyrrole (Py) / imidazole (Im) pair recognizes CG base pairs, the Py / Py pair recognizes AT or TA base pairs, and the Im / Py pair recognizes GC base pairs. Furthermore, when 3-hydroxypyrrole (Hp) is used, the Hp / Py pair recognizes the TA base pair, and the Py / Hp pair recognizes the AT base pair. The γ-aminobutyric acid portion acts as a linker, causing the entire molecule to fold into a U-shaped conformation (hairpin shape). In this U-shaped conformation, two strands containing Py and Im are arranged in parallel, flanking the linker. As a result, the pyrrole-imidazole polyamide can enter the minor groove of the DNA double helix, bind to any DNA double helix in a sequence-specific manner, and thereby inhibit the binding of transcription factors to that DNA double helix.

[0015] Pyrrolimidazole polyamides can be synthesized, for example, using Fmoc peptide solid-phase synthesis techniques with Py and Im derivatives having Fmoc-protected amino groups, as described in Non-Patent Document 1, as synthesis starting materials, but are not limited to this method.

[0016] Derivatives of pyrrolimidazole polyamide (PIP) can also be created by introducing various functional groups to the molecular ends. For example, compounds that have alkylating ability to DNA (alkylating agents), such as seco-CBI, chlorambucil, bleomycin, nitrogen mustard, pyrrolobenzodiazepine, duocalmycin, enediyne compounds, and their derivatives, can be introduced as needed. Alkylating agents, including chlorambucil, introduce alkyl groups to the guanine N-7 position or adenine N-3 position of DNA, causing adduct formation and crosslinking reactions between guanines or adenines.

[0017] The compound or salt of the present invention binds to the genes of the transmembrane proteins TM4SF1 and TMEM132b, repressing their gene expression and thereby suppressing the expression of these membrane proteins. The transmembrane protein TM4SF1 (Transmembrane-4 L six familymember-1) was discovered in 1986 by Hellstrom et al. as the "L6 antigen" or "tumor cell antigen" because it was abundantly expressed on many cancer cells. The transmembrane protein TMEM132b (transmembrane protein 132b) is known to be mainly expressed in the brain, thyroid gland, and two other tissues.

[0018] In this specification, "gene expression repression" means suppressing gene expression in transcription and / or post-transcriptional stages through the action of DNA-binding factors containing the compound. "Genes expression repression" means not only when the expression of a target gene is 100% suppressed, but also when it is suppressed by 75% or more, 50% or more, or 20% or more, as indicated by the expression level of the target gene's mRNA or protein. The degree of gene expression repression can be determined, for example, by comparing the amount of mRNA or protein produced by the gene in cells or individuals into which the compound of the present invention has been introduced with cells or individuals into which the compound has not been introduced or is unrelated. mRNA production can be measured by Northern hybridization or RT-PCR, and protein production can be measured by Western blotting, ELISA, protein activity measurement, or fluorescence intensity from a fluorescent protein, etc.

[0019] Cancer-associated fibroblasts (CAFs) are cells present in the tumor microenvironment, playing a crucial role in tumor growth and development. CAFs are found in numerous cancers, specifically cervical cancer, pancreatic cancer, breast cancer, lung cancer, and colorectal cancer (Figure 1). Cervical cancer, in particular, is rich in CAFs and is associated with cancer progression and treatment resistance. CAFs promote cancer cell proliferation and induce metastasis, and anti-CAF drugs are expected to be effective in other CAF-rich cancers as well. CAFs include, but are not limited to, CCF-1 cells used in the present invention.

[0020] The compounds of the present invention can be used to inhibit cancer metastasis. Examples of cancers whose metastasis is suppressed by the compounds of the present invention include those described below. In this specification, "inhibition of cancer metastasis" refers to both inhibiting tumor metastasis when the primary tumor has not yet metastasized, and inhibiting further tumor metastasis and / or tumor growth at the metastatic site when the tumor has already metastasized.

[0021] The present invention relates to a method for inhibiting cancer metastasis. The compounds of the present invention have the effect of treating cancer by inhibiting cancer metastasis. The method for inhibiting cancer metastasis according to the present invention comprises administering compound (I) or a salt thereof to a subject diagnosed with cancer in an amount effective in inhibiting cancer metastasis.

[0022] The compounds of the present invention, or pharmaceutical compositions containing them, suppress the metastasis of tumor cells and can therefore be applied to a variety of cancers. The cancers are preferably solid tumors, meaning tumors other than hematopoietic tumors. More preferably, the cancers include cervical cancer, pancreatic cancer, breast cancer, lung cancer, colorectal cancer, adrenocortical cancer, Kaposi's sarcoma, anal cancer, appendiceal cancer, malformed / rhabdoid tumors, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, bronchial tumors, carcinoid tumors, cardiac tumors, chordomas, chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, bile duct cancer, endometrial cancer, ependymoma, esophageal cancer, sensory neuroblastoma, Ewing's sarcoma, eye cancer, fallopian tube cancer, gallbladder cancer, stomach cancer, and gastrointestinal cancer. This includes, but is not limited to, carcinoid tumors, gastrointestinal stromal tumors, germ cell tumors, head and neck cancers, heart cancers, liver cancers, hypopharyngeal cancers, kidney cancers, laryngeal cancers, lip and oral cancers, melanomas, Merkel cell carcinomas, mesotheliomas, oral neoplasms, oral cancers, osteosarcomas, ovarian cancers, penile cancers, pharyngeal cancers, prostate cancers, rectal cancers, salivary gland cancers, skin cancers, small intestine cancers, soft tissue sarcomas, stomach cancers, testicular cancers, throat cancers, thyroid cancers, urethral cancers, uterine cancers, vaginal cancers, and vulvar cancers.

[0023] "Treatment" means eliminating, reducing, or halting the progression of a disease or its symptoms through a particular procedure. "Treatment" can also include preventing the onset of a disease.

[0024] The compounds according to the present invention can be prepared as pharmaceutical compositions containing a pharmaceutically acceptable carrier (pharmaceutical carrier) as needed.

[0025] Examples of pharmaceutical carriers include fillers, bulking agents, binders, humectants, disintegrants, lubricants, diluents, and excipients, which are commonly used depending on the dosage form of the formulation. These are selected and used as appropriate depending on the dosage form of the resulting formulation. More specifically, examples include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, acacia gum, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, and lactose. These are used individually or in combination of two or more as appropriate depending on the dosage form of the target drug. In addition, stabilizers, bactericides, buffers, isotonic agents, chelating agents, surfactants, and pH adjusters may be used as appropriate. Examples of stabilizers include human serum albumin, common L-amino acids, sugars, and cellulose derivatives. L-amino acids are not particularly limited and may include glycine, cysteine, glutamic acid, etc. Sugars are also not particularly limited and may include monosaccharides such as glucose, mannose, galactose, and fructose; sugar alcohols such as mannitol, inositol, and xylitol; disaccharides such as sucrose, maltose, and lactose; polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, and hyaluronic acid, and their derivatives. Cellulose derivatives are also not particularly limited and may include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose. Surfactants are also not particularly limited and can be either ionic or nonionic surfactants. Examples of surfactants include polyoxyethylene glycol sorbitan alkyl esters, polyoxyethylene alkyl ethers, sorbitan monoacyl esters, and fatty acid glycerides.Examples of buffering agents include boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid, and / or their corresponding salts (e.g., alkali metal salts and alkaline earth metal salts such as sodium, potassium, calcium, and magnesium salts). Examples of isotonic agents include sodium chloride, potassium chloride, sugars, and glycerin. Examples of chelating agents include sodium edetate and citric acid.

[0026] A pharmaceutical composition containing the compound according to the present invention may also contain, in addition to the compound, an additional active ingredient effective in treating cancer.

[0027] The dosage range of the pharmaceutical composition is not particularly limited and is appropriately selected according to the efficacy of the contained components, the form of administration, the route of administration, the type of disease, the characteristics of the subject (body weight, age, condition, and whether or not other medicines are being used, etc.), and the judgment of the attending physician. Generally, an appropriate dosage is preferably in the range of about 0.01 μg to 100 mg per kg of body weight of the subject, preferably about 0.1 μg to 1 mg. However, these dosages can be changed using general, standard experiments for optimization that are well known in this field. The above dosage can be administered in one to several doses per day, or intermittently at a rate of once every few days or weeks.

[0028] When administering the pharmaceutical composition according to the present invention, it may be used alone or in combination with other compounds, pharmaceuticals, or anticancer agents necessary for treatment. Preferably, it may be administered in combination with an anticancer agent. Here, administration in combination does not necessarily mean simultaneous administration; the pharmaceutical composition according to the present invention may be administered first and then the anticancer agent, or the anticancer agent may be administered first and then the pharmaceutical composition according to the present invention.

[0029] The administration route can be either systemic or local. In this case, the appropriate route of administration should be selected according to the disease, symptoms, etc. The drug according to the present invention can be administered by either oral or parenteral routes, but oral administration is more preferred. Parenteral routes include not only the usual intravenous and intra-arterial administration, but also subcutaneous, intradermal, and intramuscular administration.

[0030] The dosage form is not particularly limited and can be in various forms. For example, in addition to being usable as a solution formulation, it can also be freeze-dried to a storable state, and then dissolved in a buffer solution containing water or physiological saline to an appropriate concentration before use. It may also be in a sustained-release or sustained-release form.

[0031] Specifically, for oral administration, the formulations may be tablets, capsules, powders, granules, pills, liquids, emulsions, suspensions, solutions, alcoholic preparations, syrups, extracts, and elixirs. Parenteral preparations may include, but are not limited to, injections such as subcutaneous, intravenous, intramuscular, and intraperitoneal injections, transdermal or transdermal patches, ointments or lotions, sublingual preparations and oral patches for oral administration, and aerosols and suppositories for nasal administration. These preparations can be manufactured by known methods commonly used in the pharmaceutical manufacturing process.

[0032] When preparing oral solid dosage forms, after adding excipients, and optionally binders, disintegrants, lubricants, colorants, flavoring agents, and odor-masking agents to the above-mentioned active ingredients, tablets, coated tablets, granules, powders, capsules, etc., can be manufactured by conventional methods. Such additives may be those commonly used in the field. For example, excipients may include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid; binders may include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, and polyvinylpyrrolidone; disintegrants may include dried starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceride stearate, and lactose; lubricants may include refined talc, stearate, borax, and polyethylene glycol; and flavoring agents may include sucrose, orange peel, citric acid, and tartaric acid.

[0033] When preparing oral liquid formulations, the above-mentioned compounds can be combined with flavoring agents, buffering agents, stabilizers, odor-masking agents, etc., to produce oral liquid formulations, syrups, elixirs, etc., by conventional methods. In this case, the flavoring agents may be those listed above, sodium citrate may be used as a buffering agent, and tragacanth, gum arabic, gelatin, etc., may be used as stabilizers.

[0034] When preparing injectable preparations, pH adjusters, buffers, stabilizers, isotonic agents, local anesthetics, etc., can be added to the above compounds, and subcutaneous, intramuscular, and intravenous injectable preparations can be manufactured by conventional methods. Examples of pH adjusters and buffers in this case include sodium citrate, sodium acetate, and sodium phosphate. Examples of stabilizers include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycolic acid, and thiolactic acid. Examples of local anesthetics include procaine hydrochloride and lidocaine hydrochloride. Examples of isotonic agents include sodium chloride and glucose.

[0035] The present invention will be described in detail below with reference to examples, but the technical scope of the present invention is not limited by the description of the following examples. Those skilled in the art can carry out many modifications and alterations based on the description herein without departing from the technical scope of the present invention. Unless otherwise specified, the following examples can be carried out in accordance with standard genetic engineering and molecular biological techniques known to those skilled in the art, as described, for example, Sambrook and Maniatis, in Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. When using a kit, follow the instructions provided with the kit. The contents of the references cited herein constitute part of the disclosures and contents of this specification. [Examples]

[0036] A portion of cancerous tissue (approximately 1 cm square) from a uterine cancer patient who visited the Department of Gynecology at Kawasaki Medical School Hospital was collected, placed in a 10 cm petri dish, and separated in 10% FBS and DMEM medium. The cells were cultured for 4 weeks at 37°C and 5% CO2. From the proliferating cells, a population of fibroblasts was morphologically selected and collected using a cell separation cylinder. These cultured cells were then isolated and used as primary fibroblast culture cells in the following experiments (hereinafter referred to as CCF-1 cells). GFP-labeled cervical cancer cell line ME180 (provided by the Hikada Laboratory, Research Institute for Microbial Diseases, Osaka University) was used in 1.5 × 10⁶ cells. 6 1 piece and 6.0 × 10 5One CCF-1 cell was co-transplanted subcutaneously into nude mice. Ten mice were selected for co-transplantation and ten for single-transplantation, and each nude mouse was subcutaneously injected into one site on the left dorsal side. Tumor volume was measured in three directions. After 5 weeks, the tumor volume of nude mice that received co-transplantation was significantly larger than that of nude mice that received ME180 cell single-transplantation, indicating a tumor growth promoting effect by CCF-1 cells (Figure 2). Furthermore, metastasis to the inguinal and mediastinal lymph nodes was observed in 5 out of 11 mice 10 weeks after transplantation (Figure 2, Table 1). On the other hand, no metastasis was observed in the 10 mice that received ME180 cell single-transplantation. The presence or absence of metastatic lesions was identified using the green fluorescence emitted by GFP as an indicator, the relevant tissue was fixed in formalin, paraffin-embedded sections were prepared, and HE staining was performed to pathologically confirm that they were lymph node metastases of transplanted cancer cells.

[0037] CCF-TT cells were created by introducing the hTERT gene and the SV40largeT antigen gene into CCF-1 cells, which have the ability to metastasize cancer cells, thereby immortalizing the CCF-1 cells. 2.5x10 7 Individual CCF-TT cells and 5x10 6 Each GFP-labeled ME180 cell was co-transplanted subcutaneously into six nude mice, and they were observed for 10 weeks. As a result, no metastasis was observed in either group. In other words, CCF-TT cells had lost the metastasis-constituting ability that CCF-1 cells possessed. To elucidate the genes responsible for this difference, comprehensive gene expression analysis of CCF-1 and CCF-TT cells was performed using DNA arrays, and membrane protein genes X and Y (TM4SF1 and TMEM132b, respectively) with differing expression levels were identified (Figure 3). Both genes TM4SF1 and TMEM132b were upexpressed in CCF-1 cells.

[0038] Compound (I), a pyrrolimidazole polyamide compound that binds to genes expressing TM4SF1 and TMEM132b, was obtained.

[0039] Reagents and solvents used in the synthesis of pyrrolimidazole polyamide: Fmoc-Py-COOH Fujifilm & Wako Pure Chemical Corporation Fmoc-Im-COOH Fujifilm & Wako Pure Chemical Corporation Fmoc-PyIm-COOH Fujifilm Wako Pure Chemical Corporation Fmoc-β-Ala-COOH Watanabe Chemical Industry Co., Ltd. Fmoc-Abu(γ)-COOH Watanabe Chemical Industry Co., Ltd. N,N-Dimethyl-1,3-propanediamine HCTU Peptide Institute DIEA Tokyo Chemical Industry Co., Ltd. NMP (Super Dehydrated) Fujifilm & Wako Pure Chemical Corporation DMF (for Peptide Synth.) Fujifilm & Wako Pure Chemical Corporation Piperidine 98% Fujifilm Wako Pure Chemical Corporation Reagents and solvents used in resin preparation: Oxime resin LL (100-200 mesh) Watanabe Chemical Industry Co., Ltd. Fmoc-Py-COOH Fujifilm & Wako Pure Chemical Corporation DCC Tokyo Chemical Industry Co., Ltd. HoBt Peptide Institute NMP (Super Dehydrated) Fujifilm & Wako Pure Chemical Corporation DIEA Tokyo Chemical Industry Co., Ltd. DMF (for Peptide Synth.) Fujifilm & Wako Pure Chemical Corporation Acetic Anhydride (Guaranteed Reagent) MeOH Fujifilm Wako Pure Chemical Corporation DCM nacalai tesque Reagents and solvents used in CHb coupling: Chorambucil SIGMA PyBOP Watanabe Chemical Industry Co., Ltd. DIEA Tokyo Chemical Industry Co., Ltd. DMF (low moisture solvent) nacalai tesque Solvent used for separation: TFA nacalai tesque Acetonitrile nacalai tesque Furthermore, Diethl Ether (Fujifilm Wako Pure Chemical Corporation) was used when the purified compound (I) was powdered.

[0040] Methods for synthesizing and purifying pyrroleimidazole polyamide: Synthesis: Without further purification of the reagents and solvents mentioned above, automated pyrrole-imidazole polyamide synthesis was performed using the PSSM-8 system (Shimadzu) by the Fmoc solid-phase synthesis method described in Non-Patent Document 1, etc. Oxime resin was used in all syntheses.

[0041] Analysis and purification: analysis: High-performance liquid chromatography (HPLC) analysis was performed using a JASCO PU-2080 Plus pump, a JSCO UV-2075 Plus UV / vis detector, and a Chemcobond 5-ODS-H 4.6 mm × 150 mm column (Chemo Plus Scientific). The mobile phase was acetonitrile-TFA (0.1% in water, v / v) at a flow rate of 1.0 mL / min. Matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOFMS) was obtained on a Microflex-KSII (Bruker Daltonics). Saturated HCCA (α-cyano-4-hydroxycinnamic acid) in TA30 (0.1% TFA / MeCN=7 / 3) was used as the matrix along with a peptide calibration standard.

[0042] purification: The crude product of the synthesized pyrrole imidazole polyamide was eluted with a linear gradient for 15 minutes at a flow rate of 1.0 mL / min using 0.1% TFA containing 0 - 75% acetonitrile on the HPLC system used for analysis, and a peak was observed at a retention time of 12.1 minutes (detected at 254 nm).

[0043] Confirmation of compound (I): When the MALDI - TOFMS spectrum of the purified peak fraction was measured, the measured value was 1524.434. m / z of compound (I) C 70 H 87 Cl2N 25 O 11 + [M + H] + Since it matched the calculated value of 1524.647, it was identified as the target product.

[0044] 1.5×10 6 GFP - labeled cervical cancer cells ME180 strain and 6.0×10 5 cancer - associated fibroblast CCF - 1 cells were co - transplanted subcutaneously into 11 nude mice. After 10 weeks, the whole - body metastasis was analyzed using the fluorescence of GFP as an index, and lymph node metastasis in the supraclavicular fossa or axillary lymph node metastasis was observed in 5 mice. On the other hand, in the same way, two types of cells, cervical cancer cells ME180 strain and cancer - associated fibroblast CCF - 1 cells, were subcutaneously transplanted into 9 nude mice, and compound (I) at 1 mg / kg was intraperitoneally administered once a week. After 10 weeks, the same analysis was performed, and as a result, no metastasis to lymph nodes or other organs was observed in all 9 mice (Table 1).

Table 1

[0045] The pyrrole imidazole polyamide compound of the present invention binds to the genes expressing TM4SF1 and TMEM132b and suppresses the expression of membrane proteins TM4SF1 and TMEM132b (Figure 4). In Figure 4, X and Y represent TM4SF1 and TMEM132b respectively.

Industrial Applicability

[0046] The pyrrolimidazole polyamide compounds of the present invention suppress the expression of membrane protein genes associated with cancer metastasis and can be applied to the treatment of cancer metastasis.

Claims

1. A compound represented by formula (I) or a salt thereof. 【Chemistry 1】

2. A pharmaceutical composition comprising the compound or a salt thereof as described in claim 1.

3. A membrane protein gene expression inhibitor comprising the compound or a salt thereof as described in claim 1.

4. A cancer metastasis inhibitor comprising the compound or a salt thereof as described in claim 1 as an active ingredient.

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