WO2023090366A1 - Anticancer agent and cardiac-function-improving agent - Google Patents

Abstract

The present invention addresses the problem of providing an anticancer agent and/or a cardiac-function-improving agent having a novel action mechanism. A pharmaceutical composition containing, as an active ingredient, a substance that inhibits binding between a Kruppel-like factor 5 (KLF5) and a ubiquitin-specific peptidase 3 (USP3), in particular, any one of the compounds represented by formulas (I)-(III) or a pharmaceutically acceptable salt thereof, is useful as an anticancer agent and/or a cardiac-function-improving agent.

Description

Anticancer agents and cardiac function improving agents

The present invention relates to a novel cardiac function-improving agent, particularly to a cardiac function-improving agent having anticancer activity. The present invention also relates to a novel anticancer agent, and particularly to an anticancer agent having an effect of improving cardiac function.

Many cancers are caused by genetic and epigenetic instability of key proteins, including transcription factors. Transcription factors are attractive cancer therapeutic targets because of their frequent overactivation and dysfunction. Therapeutic regulation of transcription factors has been successful with nuclear receptors. However, most transcription factors are intrinsically disordered proteins (IDPs) and lack rigid small-molecule binding pockets, making them more difficult to target.
Against this background, target searches for oncogenic transcription factors using low-molecular-weight compounds have been conducted in order to disrupt the underlying oncogenic pathway.

Kruppel-like factor 5 (KLF5) belongs to the zinc finger-containing transcription factor family responsible for various cellular functions and mediates various cellular functions (Non-Patent Document 1). The present inventors have previously used mice in which Wnt-induced tumorigenesis was blocked by intestinal stem cell-specific ablation of the KLF5 gene, and found that KLF5 is an essential regulator of intestinal carcinogenesis at the stem cell level. have reported that (Non-Patent Document 2). Furthermore, the present inventors have elucidated that KLF5 plays an important role in inflammatory diseases such as heart failure, arteriosclerosis, obesity and cancer (Patent Document 1). From this, it was expected that KLF5 inhibitors would be effective in treating cardiovascular diseases and the like. A key hurdle in the development of KLF5 inhibitors is the lack of information about the three-dimensional (3D) structure of this class of proteins due to their inherent disordered nature (native denaturation). is. However, many naturally occurring proteins (IDPs) are thought to undergo disorder-to-order transitions upon binding protein partners called molecular recognition functions (MoRFs). It further suggests that the α-helices in the IDP are involved in protein-protein interactions (PPI).

In recent years, PPI inhibitors have attracted attention in drug discovery. One of the targets of PPI inhibitors is α-helices, which constitute a large class of protein secondary structure, since surface-exposed α-helices of proteins frequently interact with other proteins. Typical low molecular weight-α-helix mimetic compounds mimic amino acid residues i, i+4, i+7 located on one side of the α-helix. Based on this, several low-molecular-weight compounds with α-helix-like structures have been developed to inhibit disease-related molecules (Non-Patent Documents 3-5).

The present inventors hypothesized that the pharmacological interference of PPIs occurring in KLF5 might be a drug discovery target, especially under conditions in which KLF5 is overactivated. I had a big problem. In the absence of the 3D structure of KLF5, direct structure-based design of KLF5 is not possible. Also, the development of effective inhibitors of nuclear factors is more difficult than inhibitors of cytoplasmic molecules, since they must pass through both the cytoplasm and the nuclear membrane. Therefore, there is a need for a new approach to identify compounds that target transcription factors having an IDP structure such as KLF5.

The present inventors previously demonstrated that KLF5 is an essential regulatory factor for intestinal carcinogenesis at the stem cell level. It has been shown that genetic ablation of the KLF5 gene completely inhibits Wnt signal-driven tumorigenesis in mouse intestinal stem cells (Non-Patent Document 2). In addition, it has been clarified that KLF5 expression is increased in human colorectal cancer compared to normal colorectal epithelium, and that genomic amplification of the KLF5 gene is also accumulated in human colorectal cancer. (Non-Patent Document 2, Non-Patent Document 6). These results suggested that the development of a KLF5 inhibitor could serve as a therapeutic target for colorectal cancer.
The present inventors hypothesized that a low-molecular-weight KLF5 mimic inhibits protein-protein interaction and may serve as a new anticancer agent (Non-Patent Document 5, Non-Patent Document 7 ), and attempted to search for compounds that effectively suppress the proliferation of cancer cells.

WO2005/010185

The purpose of the present invention is to provide a compound that effectively suppresses the proliferation of cancer cells, and to provide a compound that improves cardiac function.

In view of the above problems, the present inventors have conducted intensive research and found that a bicyclic structure mimicking the main chain structure and four side chain structures of the four amino acid sequence (VAIF) of KLF5 A pyrazinooxadiazine-4,7-dione derivative is
(i) inhibit the proliferation and survival of human colon cancer cells and other cancer cells, but not the proliferation and survival of non-cancer cells;
(ii) decrease protein levels of the Wnt signaling pathway as well as KLF5 protein; and (iii) inhibiting the interaction of KLF5 with Ubiquitin-specific peptidase 3 (USP3), which is known to stabilize KLF5; and (iii) in vivo, without apparent side effects. effectively suppressing colon cancer cells transplanted in;
I found
Furthermore, the present inventors have completed the present invention by finding that these compounds have an effect of improving cardiac function.
That is, the present invention provides the following.
[1] An anticancer agent and/or an agent for improving cardiac function, comprising a substance that inhibits the binding of Kruppel-like factor 5 (KLF5) and Ubiquitin-specific peptidase 3 (USP3).
[2] The substance that inhibits the binding of KLF5 and USP3 has the following formulas (I) to (III):

The agent according to [1] above, which is a compound represented by any of or a pharmaceutically acceptable salt thereof.
[3] A method for screening an anticancer agent and/or a cardiac function improving agent, comprising the steps of measuring the KLF5-USP3 binding inhibitory activity of a test substance, and selecting compounds having the binding inhibitory activity.
[4] Formulas (I) to (III) below:

A compound represented by any of or a pharmaceutically acceptable salt thereof.
[5] Formulas (I) to (III) below:

A pharmaceutical composition comprising a compound represented by any of or a pharmaceutically acceptable salt thereof.
[6] The pharmaceutical composition according to [5] above, which is a KLF5 inhibitor.
[7] The pharmaceutical composition according to [5] or [6] above, which is an anticancer agent and/or an agent for improving cardiac function.
[8] The pharmaceutical composition of [7] above, wherein the anticancer agent is for colorectal cancer.
[9] The pharmaceutical composition of [7] above, wherein the agent for improving cardiac function is a therapeutic agent for heart failure.
[10] A method for preventing and/or treating cancer (eg, colon cancer), which comprises administering an effective amount of a substance that inhibits the binding of KLF5 and USP3 to a subject in need thereof.
[11] A method for improving cardiac function (eg, treating heart failure), which comprises administering an effective amount of a substance that inhibits the binding of KLF5 and USP3 to a subject in need thereof.
[12] The substance that inhibits the binding of KLF5 and USP3 has the following formulas (I) to (III):

The method according to [10] or [11] above, which is a compound represented by any of or a pharmaceutically acceptable salt thereof.

The compound of the present invention suppresses the proliferation and survival of cancer cells in vivo and in vitro, but does not suppress the proliferation and survival of non-cancer cells; it has cardiac function improving action.
Therefore, the compounds of the present invention or pharmaceutically acceptable salts thereof are useful as anticancer agents or cardiac function improving agents.

FIG. 1 is a graph showing the results of selective suppression of proliferation of human colon cancer cells by the compound of the present invention. The compounds of the present invention inhibited the growth of human colon cancer cells (SW480 and HCT116) without affecting the growth of non-cancer cells (CCD841). The score indicated in each graph indicates the _IC50 concentration (μM) in SW480 cells examined 72 hours after treatment. Among the compounds, the compound of formula (I) inhibited the proliferation of SW480 cells at the lowest concentration. ICG-001 showed growth inhibitory effects on both cancer cells and non-cancer cells. FIG. 2 is a graph showing the result that the compound of the present invention inhibited cell proliferation of 3D spheroids of HCT116 cells, which are human colon cancer cells. Formula (II) compounds and formula (III) compounds were used as the compounds of the present invention. A Cell3iMager Duos system (SCREEN) was used for the measurement. FIG. 3 shows the results of examining the effects of the compounds of the present invention on KLF5 and Wnt proteins. Top (A): The compounds of the present invention decreased the protein levels of KLF5 and Survivin in SW480 cells after 24 hours treatment at 10 μM, but not in human normal cell line CCD841. Bottom (B): The compound of the present invention decreased the protein level of TCF4 in SW480 cells after 24 hours treatment at 10 μM, but did not change in normal human cell line CCD841 after the same treatment. FIG. 4 shows the results of examining the effects of the compounds of the present invention on KLF5 and Wnt proteins. Cyclin D1 and KLF5 protein levels and β-catenin phosphorylation at Ser675 were examined in SW480 cells treated with the compound of the present invention (compound of formula (I)) for 0, 6, 12, 18 and 24 hours. Treatment with the compound of the present invention decreased the phosphorylation of Cyclin D1 and KLF5, and Ser 675 of β-catenin. In contrast, in CCD841 cells, treatment with the compounds of the present invention for 24 hours did not significantly reduce protein levels and phosphorylation. FIG. 5 shows the results of examining the effects of the compounds of the present invention on KLF5 and Wnt proteins. Upper row (A): In SW480 cells treated with the compound of the present invention (10 μM) for 24 hours, phosphorylation of Ser 552 of β-catenin was suppressed. However, these compounds did not decrease the levels of β-catenin in SW480 cells. Bottom (B): Treatment with the compound of the present invention (10 μM) for 24 hours decreased the protein levels of KLF5, Survivin, and TCF4, and inhibited the phosphorylation of β-catenin at Ser552. In particular, the compound of formula (I) showed the most effective inhibitory effect. Figure 6 shows that compounds of formula (I) inhibit the formation of FLAG/HA-KLF5 protein complexes. (A) Using SW480 cells expressing FLAG/HA-KLF5 protein, the FLAG/HA-tagged KLF5 protein complex was purified by immunoprecipitation using sequential anti-FLAG tag antibody and anti-HA tag antibody. Immunoprecipitation-Western blotting confirmed that the FLAG/HA tag-KLF5 protein complex contained co-precipitated β-catenin protein. 10 µg of protein lysate was applied to each lane of the original lysate (left). For FLAG-tagged Ab immunoprecipitation, precipitated eluate from 4200 μg of original lysate was applied to each lane (middle). For tandem affinity purification by FLAG followed by HA immunoprecipitation, precipitated eluate from 16800 μg of original lysate was applied to each lane (right). (B) Graph showing the results of examining the relative amount of β-catenin protein per FLAG/HA-tagged KLF5 protein in the FLAG/HA-tagged KLF5 protein complex. Treatment with 10 μM compound of formula (I) for 24 hours decreased the protein level of β-catenin in the FLAG/HA-KLF5 protein complex. Quantification was performed under conditions where co-immunoprecipitated β-catenin protein was confirmed by blotting. FIG. 7 is a graph showing the results of examining the effects of the compounds of the present invention on KLF5 mRNA. Compounds of the present invention did not decrease KLF5 mRNA levels when SW480 cells were treated at 10 μM for 24 hours. Figure 8 shows that compounds of formula (I) inhibit USP3-KLF5 protein complex formation. (A) Using HEK293T cells overexpressing Myc-tag-USP3 protein and FLAG/HA-KLF5 protein, the Myc-tag-USP3 protein complex was purified by immunoprecipitation using an anti-Myc-tag antibody. Immunoprecipitation-Western blotting confirmed that the Myc-tag-USP3 protein complex contained co-precipitated KLF5 protein. (B) Graph showing the relative amount of KLF5 protein per Myc tag-USP3 protein in the Myc tag-USP3 protein complex. Treatment with compound of formula (I) at 20 μM for 4 hours decreased the protein level of KLF5 in the USP3 protein complex. Quantification was performed under conditions where KLF5 protein was confirmed by blotting. Formula (I) compound treatment under the same conditions (20 μM, 4 hours) did not suppress KLF5 protein levels. (A) 13 nude mice were subcutaneously implanted with HT29 human colon cancer cells (5×10 ⁶ ) and treated with compound of formula (I) (ip, 2 mg×2/day). The effect of the compound of formula (I) on implanted cancer cells was evaluated by measuring tumor volume. Tumor volume was calculated as ² ×major axis/2. Tumor size was significantly smaller than in untreated mice (n=13) and no side effects were observed. (B) shows the results of hematoxylin-eosin staining and immunohistological staining of KLF5 and Ki67 in the large intestine. It was shown that the intestinal tissue of mice treated with compounds of formula (I) did not show significant cellular damage. The upper graph is a graph showing the results of examining changes in left ventricular ejection fraction values by administering the compound of formula (I) at varying doses. The compound of formula (I) was varied at three concentrations including 25, 50 and 100 mg/kg (***: p<0.0001). The lower panel is a graph showing the results of varying doses confirming the effect of the compound of formula (I) on hypertrophic changes in the heart in mice receiving TAC. Compounds of formula (I) were varied at four concentrations including 12.5, 25, 50, 100 mg/kg. Cardiac hypertrophy is indicated by an increase in heart weight/body weight ratio (HW/BW) (*: p<0.05). 1 is a graph showing the cardiac function-improving action of the compound of formula (I). Treatment with compounds of formula (I) improved left ventricular ejection fraction values and significantly improved left ventricular ejection fraction in mice with established left ventricular pressure overload-induced heart failure (*** : p<0.0001). 1 is a graph showing the cardiac function-improving action of the compound of formula (I). Brain natriuretic peptide (BNP) levels, a marker of heart failure, were significantly decreased in TAC mice treated with compound of formula (I) (***: p<0.0001). Figure 10 is a graph showing that compounds of formula (I) inhibit hypertrophic changes in the heart in mice subjected to TAC. Cardiac hypertrophy is indicated by an increase in heart weight/body weight ratio (HW/BW) 4 weeks after TAC (**: p<0.01). Figure 2 is a graph showing that compounds of formula (I) ameliorate cardiac fibrosis induced by pressure overload (***: p<0.0001). FIG. 2 shows the protocol of the mouse survival study. Fig. 3 is a graph showing survival curves using pressure-overloaded heart failure model mice. Administration of the compound of formula (I) was confirmed to prolong survival.

The present invention provides an anticancer agent and/or cardiac function improving agent containing a substance that inhibits the binding of Kruppel-like factor 5 (KLF5) and interacting protein (especially USP3). A detailed description will be given below.

(Substance that inhibits binding between KLF5 and USP3)
KLF5 is a transcription factor and is involved in cell proliferation and cell cycle control. In pathological conditions, it is known to be expressed in activated smooth muscle cells and fibroblasts in damaged defects and atherosclerotic lesions, suggesting involvement in cardiovascular remodeling. It is KLF5 is involved in tissue fibrosis through proliferation and activation of fibroblasts.
USP3 is known to stabilize KLF5 by binding to and deubiquitinating KLF5 (Wu Y, et al. (2019) USP3 promotes breast cancer cell proliferation by deubiquitinating KLF5. J Biol Chem 294(47 ): 17837-17847).
In the present invention, the "substance that inhibits the binding of KLF5 and USP3" (hereinafter also simply referred to as a KLF5 inhibitor) is a substance that can inhibit (control) the interaction between KLF5 and USP3, and its origin is particularly limited. It may be a low-molecular-weight compound or a high-molecular-weight compound. Here, the low-molecular-weight compound is a compound having a molecular weight of less than about 1000, and includes, for example, organic compounds and derivatives thereof that can be commonly used as pharmaceuticals. It refers to organic compounds and derivatives thereof that can be preferably used as pharmaceuticals. Moreover, the polymer compound is a compound having a molecular weight of about 1000 or more, and includes proteins, polynucleic acids, polysaccharides, and combinations thereof.

A KLF5 inhibitor may be a pharmaceutically acceptable salt thereof.
"Pharmaceutically acceptable" means generally safe, non-toxic, and useful for the preparation of pharmaceutical compositions that are not biologically or otherwise undesirable, and This includes being acceptable for veterinary use as well as for pharmaceutical use.
"Pharmaceutically acceptable salt" means a salt of a compound of the invention, as defined above, that is pharmaceutically acceptable and that possesses the desired pharmacological activity. Such salts include inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acids; Acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfone acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2. 2.2] Oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert- Acid addition salts formed with organic acids such as butylacetate, laurylsulphate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid and muconic acid are included.

Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

When a KLF5 inhibitor has isomers such as optical isomers, stereoisomers, positional isomers and rotational isomers, any one isomer or a mixture of isomers can be used as a KLF5 inhibitor in the present invention. Available.

"Isomers" means any compounds that have identical molecular formulas but differ in the nature or order of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers". Stereoisomers that are not mirror images of one another are termed "diastereomers", and stereoisomers that are non-superimposable mirror images are termed "enantiomers", or sometimes termed "optical isomers". A carbon atom bonded to four different substituents is termed a "chiral center". A compound with one chiral center has two enantiomeric forms of opposite chirality. A mixture of two enantiomeric forms is called a "racemic mixture". A compound with more than one chiral center has 2 ⁿ⁻¹ enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as individual diastereomers, ethers, or as mixtures of diastereomers (termed "diastereomeric mixtures"). When one chiral center is present, stereoisomers can be characterized by the absolute configuration of that chiral center. Absolute configuration refers to the spatial arrangement of the substituents attached to the chiral center. Enantiomers are characterized by the absolute configuration of their chiral centers and are dictated by the R- and S-ranking rules of Cahn, Ingold and Prelog. Rules for stereochemical nomenclature, methods for determination of stereochemistry and methods for separating stereoisomers are well known in the art (see, for example, "Advanced Organic Chemistry", 4th edition, March, Jerry, John Wiley & Sons, New York, 1992).

The KLF5 inhibitor may be crystalline or amorphous. When the KLF5 inhibitor is a crystal, it can be used as the KLF5 inhibitor in the present invention whether it is a single crystal form or a mixture of crystal forms. Crystals can be produced by applying a crystallization method known per se to crystallize.

The KLF5 inhibitor may be a solvate (eg, hydrate, etc.) or a non-solvate, both of which can be used as the KLF5 inhibitor of the present invention.

The KLF5 inhibitor may be labeled with an isotope (eg, ³ H, ¹⁴ C, ³⁵ S, ¹²⁵ I, etc.).

As the KLF5 inhibitor used in the present invention, preferably the following formulas (I) to (III)

or a pharmaceutically acceptable salt thereof (hereinafter collectively referred to as "the compounds of the present invention").
The compounds of the present invention are novel compounds and can be prepared, for example, by the methods described in Examples 1-3.
A derivative of the compound of the present invention can be used as the KLF5 inhibitor of the present invention. Here, a derivative is an organic compound that has been modified by introduction of functional groups, oxidation, reduction, substitution or addition of atoms, etc., to the extent that the structure or properties of the base are not significantly changed. It means a compound that

A KLF5 inhibitor can inhibit the KLF5-USP3 interaction by inhibiting the binding of KLF5 to USP3. Therefore, KLF5 inhibitors show various pharmacological effects on mammals (e.g., humans, monkeys, cats, pigs, horses, cows, mice, rats, guinea pigs, dogs, rabbits, etc.), and KLF5-USP3 interaction is associated with diseases such as cancer and cardiovascular diseases, and is useful as a preventive and/or therapeutic drug and a cardiac function improving agent (hereinafter collectively referred to as "the drug of the present invention").
Treatment of cancer is understood to include reducing or eliminating cancer progression (eg, cancer growth and metastasis). Prevention of cancer is understood to include preventing or delaying the onset of cancer. Various types of cancer can be treated or prevented by the present invention. They include lung cancer, breast cancer, colon cancer (colorectal cancer), stomach cancer, pancreatic cancer, liver cancer, uterine cancer, ovarian cancer, glioma, melanoma, lymphoma, and leukemia. but not limited to these. The target cancer is preferably colon cancer. A subject in need of treatment may be a mammal, particularly a non-human primate or other animal, with various types of cancer. A preventive and/or therapeutic agent for cancer is synonymous with an anticancer agent.
As used herein, the term "cardiovascular disease" refers to diseases that affect the heart or blood vessels, or both. In particular, cardiovascular diseases include arrhythmia (atrial or ventricular, or both); atherosclerosis and its sequelae; angina pectoris; cardiac rhythm disturbances; myocardial ischemia; vasculitis, stroke; peripheral occlusive arteriopathy of limbs, organs, or tissues; post-ischemic reperfusion injury of the brain, heart, kidneys, or other organs or tissues; endotoxin, surgical, or trauma hypertension, valvular heart disease, heart failure, abnormal blood pressure; shock; vascular stenosis (including those associated with migraine); vascular abnormalities, inflammation, failure confined to a single organ or tissue. . Treatment of cardiovascular disease is understood to include reducing or eliminating progression of the disease. Prevention of cardiovascular disease is understood to include preventing or delaying the onset of said disease. Cardiovascular disease to be targeted is preferably heart failure.

For the treatment of cancer and/or cardiovascular disease, a therapeutically effective amount or a prophylactically effective amount of the compound of the present invention can be administered to a subject.

"An effective amount for treatment" means an amount sufficient to achieve such treatment for a disease when administered to an animal to treat the disease.

"Effective amount for prevention" means an amount sufficient to achieve such prevention for the disease when administered to an animal to prevent the disease.

"Effective amount" is the same as "effective amount for treatment" and "effective amount for prevention".

"Treatment" or "treating" means any administration of a compound of the invention and includes:
(1) prevention of disease development in animals that may be susceptible to the disease but have not yet experienced or exhibited the pathology or symptomatology of the disease;
(2) inhibition of the disease (i.e., halting further progression of the pathology and/or symptomatology) in animals experiencing or exhibiting disease pathology or symptomatology; or (3) disease pathology or symptomatology. Amelioration of the disease (ie, reversal of pathology and/or symptomatology) in animals experiencing or exhibiting symptomatology.

In a further aspect of the invention, the present invention provides a method of screening a library for biological activity (in the present invention, activity that inhibits the KLF5-USP3 interaction) and isolating biologically active library members. provide a way to Therefore, the present invention provides screening for anticancer agents and/or cardiac function improving agents, which comprises the step of measuring the activity of a test substance to inhibit the KLF5-USP3 interaction, that is, the activity of inhibiting the binding of KLF5 and USP3. provide a way.

In one embodiment, bioactivity data can be determined by binding assays. The method can comprise, for example, providing a composition comprising KLF5, the KLF5 interacting protein USP3, and a test substance. The method further comprises detecting changes in binding between KLF5 and USP3 due to the presence of the test agent, and then characterizing the effect of the test agent on binding. Assays may be performed by any method that can measure the effect of a test substance on the binding between two proteins. Many such assays are known in the art.

Test substances to be screened are not particularly limited, and examples thereof include proteins, nucleic acids, peptides, polypeptides, synthetic compounds, or libraries thereof. Libraries include synthetic compound libraries, peptide libraries, cDNA libraries, and the like.
The test substance can be tested at a number of different concentrations, with the concentrations chosen depending in part on the assay conditions. Examples of assay conditions are the concentrations of KLF5 and USP3. Concentrations in the range of about 0.1 to 100 μM may be used. In one embodiment, the assay evaluates the relative utility of two compounds to affect binding interactions between two proteins, wherein at least one of the two compounds is a compound of the present invention. is a compound of A more effective compound can serve as a reference compound to study the relationship between compound structure and compound activity.

The present invention also relates to prodrugs using the compounds of the present invention. Prodrugs are typically designed to release the active drug in the body during or after absorption by enzymatic and/or chemical hydrolysis. The prodrug approach is an effective way to enhance oral bioavailability or intravenous (i.v.) administration of poorly water-soluble drugs by chemical derivatization to more water-soluble compounds. It is a means. The most commonly used prodrug approach to increase the water solubility of drugs with hydroxyl groups is to prepare esters containing ionizable groups, e.g., phosphate, carboxylate, alkylamino groups ( Fleisher et al., Advanced Drug Delivery Reviews, 115-130, 1996; Davis et al., Cancer Res., 7247-7253).

In another aspect, the invention provides pharmaceutical compositions containing the compounds of the invention or pharmaceutically acceptable salts thereof. These compositions can be used in various methods of the invention (eg, treatment of cancer, cardiovascular disease), which are described in detail below.

The pharmaceutical composition of the present invention can be formulated according to the intended administration route. Examples of routes of administration include parenteral (eg, intravenous), intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous administration may contain the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol. , or other synthetic solvents), antibacterial agents (e.g., benzyl alcohol or methylparaben), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffering agents (acetate, citric acid salts, or phosphates), and tonicity agents (eg, sodium chloride or dextrose). Additionally, pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenterals can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASE, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved free from contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, liquid-phase polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved with various antibacterial and antifungal agents (eg, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols (eg, mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of an injectable composition can be effected by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared, for example, by incorporating the compound of the present invention in the required amount in an appropriate solvent with the above-described ingredients, alone or in combination, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the required other ingredients from those enumerated above and the dispersion medium. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization to prepare a powder of the active ingredient plus any desired additional ingredients from a previously sterile-filtered solution. is. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.

Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is applied orally and swished or expectorated. or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients or compounds of similar nature: binders (such as microcrystalline cellulose, tragacanth, or gelatin), excipients. (e.g. starch, lactose), disintegrants (e.g. alginic acid, Primogel, or corn starch), lubricants (e.g. magnesium stearate or Sterotes), glidants (e.g. colloidal silicon dioxide), Sweetening agents such as sucrose, saccharin, or flavoring agents such as peppermint, methyl salicylate, or orange flavoring may be included.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant (eg, a gas such as carbon dioxide) or propellant.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the dosage form. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are generally formulated into ointments, salves, gels, or creams known in the art.

The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing the desired therapeutic effect to produce the desired therapeutic effect. It contains a predetermined amount of active compound calculated in association with the required pharmaceutical carrier. The specifics of the dosage unit forms of the present invention are determined by the unique characteristics of the active compound, the particular therapeutic effect sought to be achieved, and the limitations inherent in the art of combining such active compounds for individual therapy. and directly depend on them.

For example, in certain embodiments, preferred pharmaceutical compositions of the invention are those suitable for oral administration in unit doses, eg, tablets or capsules containing from about 1 mg to about 1 g of a compound of the invention. In some other embodiments, the pharmaceutical compositions of the invention are suitable for intravenous, subcutaneous or intramuscular injection. A patient may receive, for example, from about 1 μg/kg to about 1 g/kg of a compound of the invention intravenously, subcutaneously or intramuscularly. Intravenous, subcutaneous and intramuscular doses may be given by means of bolus injection. Alternatively, the intravenous dose may be given by continuous infusion over a period of time. Alternatively, the patient receives a daily oral dose approximately equivalent to the daily parenteral dose and the composition is administered 1 to 4 times per day.

Preferably, the compounds of the present invention can be administered to mammals, including humans, parenterally (particularly preferably by continuous infusion or rapid intravenous administration).

In that case, the dosage is appropriately selected depending on various factors such as the weight and/or age of the patient, and/or the degree of symptoms and route of administration. For example, in general, doses of the compounds of the present invention for parenteral administration range from 1 to 10,000 mg/day/m ² of human body surface area, preferably from 1 to 5,000 mg/day/m 2 of human body surface area, by continuous infusion. /day/m ² and more preferably 10-5000 mg/day/m ² of human body surface area.

A pharmaceutical composition containing the compound of the present invention or a pharmaceutically acceptable salt thereof can be used to treat diseases regulated by the KLF5-USP3 interaction, specifically cancer and cardiovascular diseases.

The present invention will be described in more detail below with examples and test examples, but the present invention is not limited to these.

Example 1
Synthesis of compounds of formula (I)

Reagents and reaction conditions used in each step.
a) NaBH(OAc) ₃ , dichloroethane;
b) HATU, _DIPEA , _CH2Cl2 (DCM);
c) DEA, DCM;
d) BnBr, _Cs2CO3 , _{CH3CN ;}
e) _Ph3P , DIAD, DCM;
f) hydrazine hydrate, DCM;
g) DMAP, DCM;
h) _H2 , Pd/C, MeOH;
i) HATU, DIEA, DCM;
j) _HCO2H

The synthesis of the compound of formula (I) is as follows. Amine moiety (6) was prepared by condensing Fmoc-Ile (4) with amine (3) derived from benzaldehyde (1) and amine (2) and deprotecting the Fmoc group. Hydroxylcarboxylic acid (7) was esterified, Mitsunobu reaction and deprotected to obtain hydroxylamine (11). The hydroxylamine (11) was converted to the acid moiety (14) by reaction with isobutyl chloroformate and hydrogenolysis. Amine moiety (6) was coupled with acid moiety (14) using a condensing reagent and treated with formic acid to give the desired compound of formula (I).

Details of the synthesis of compounds of formula (I) are as follows.
All starting materials and reagents were purchased from commercial suppliers. Thin layer chromatography (TLC) was performed on 0.2 mm silica gel 60 F254 plates (Merck). Analytical HPLC/MS data were collected on a Waters 2795 instrument; LC/MS conditions were a gradient of _CH3CN / _H2O from 5% _CH3CN to 0.1% formic acid, then 100% _CH3CN . The gradient was 5 minutes at a flow rate of 1.0 mL/min on a Develosil C30-UG-5 column (50×4.6 mm, Nomura Chemical Co., Ltd.). ¹ H-NMR spectra were recorded on a Bruker Biospin AVANCE III HD 500 spectrometer (500 MHz). Chemical shifts are reported in ppm using TMS as internal standard.

Preparation of N-benzyl-2,2-diethoxyethan-1-amine (3) Benzaldehyde in 2,2-diethoxyethan-1-amine (2) (10.3 mL, 70.5 mmol, 1.5 eq) A solution of (1) (5.0 g, 47.0 mmol) was stirred at room temperature for 30 minutes. The reaction mixture was diluted with dichloroethane (110 mL), triacetoxyborohydride (NaBH(OAc) ₃ , 30.0 g, 141.0 mmol, 3.0 eq) was added portionwise and stirred at room temperature for 3 hours. . The reaction mixture was then washed with saturated aqueous _NaHCO3 , water and brine. _The collected organic phases were dried over _Na2SO4 and filtered. The filtrate was concentrated in vacuo and the residue was washed with petroleum ether to give N-benzyl-2,2-diethoxyethan-1-amine (3) (7.9 g, 75%) as a white solid.

(9H-fluoren-9-yl)methyl ((2S,3S)-1-(benzyl(2,2-diethoxyethyl)amino)-3-methyl-1-oxopentan-2-yl)carbamate (5) Preparation of compound (3) (2.5 g, 11.0 mmol, 1.1 eq) was treated with (((9H-fluoren-9-yl)methoxy)carbonyl)-L-isoleucine (4 ) (3.5 g, 10.0 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 4. 6 g, 12.0 mmol, 1.2 eq) and diisopropylethylamine (DIPEA, 3.5 mL, 20.0 mmol, 2.0 eq). The mixture was stirred at room temperature for 3 hours. The solution _was then washed with water and the organic phase was dried over _Na2SO4 and filtered. The filtrate is concentrated in vacuo and the residue is purified by column chromatography (petroleum ether:AcOEt=10:1) to give (9H-fluoren-9-yl)methyl ((2S,3S)-1-(benzyl ( 2,2-diethoxyethyl)amino)-3-methyl-1-oxopentan-2-yl)carbamate (5) (4.5 g, 81%) was obtained as a pale yellow oil.

Preparation of (2S,3S)-2-amino-N-benzyl-N-(2,2-diethoxyethyl)-3-methylpentanamide (6) Compound ( 5) (280.0 mg) in dichloromethane (5 mL) , 0.5 mmol) was added with diethylamine (DEA, 2 mL) and stirred at room temperature for 2 hours. After confirming the completion of the reaction by TLC, DCM was distilled off under reduced pressure. The resulting product was purified by column chromatography (DCM:MeOH=30:1) to give (2S,3S)-2-amino-N-benzyl-N-(2,2-diethoxyethyl)-3- Methylpentanamide (6) (150 mg, 89%) was obtained as a pale yellow oil.

Preparation of benzyl (S)-2-hydroxy-3-methylbutanoate (8) Cesium carbonate (Cs ₂ CO ₃ , 6.89 g, 21.2 mmol, 0.5 eq) was dissolved in CH ₃ CN (30 mL). A solution of (S)-2-hydroxy-3-methylbutanoic acid (7) (5.0 g, 42.3 mmol) was added at 0° C. and the resulting mixture was stirred for 40 minutes. Benzyl bromide (BnBr, 7.97 g, 46.6 mmol, 1.1 eq) was added and the reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was filtered and the filtrate was diluted with AcOEt. After the organic layer was washed with saturated NH ₄ Cl solution, saturated NaHCO ₃ solution and brine, it was dried over Na ₂ SO ₄ and the solvent was removed in vacuo. The crude product was purified by flash column chromatography using petroleum ether:AcOEt (10:1) as eluent to give benzyl (S)-2-hydroxy-3-methylbutanoate (8) (7. 9 g, 90%).

Preparation of benzyl (R)-2-((1,3-dioxoindolin-2-yl)oxy)-3-methylbutanoate (10) Compound ( 8) (7.9 g, 38.2 mmol), triphenylphosphine (PPh ₃ , 23.0 g, 87.8 mmol, 2.3 eq) and N-hydroxyphthalimide (9) (13.1 g, 80.2 mmol, 2.1 eq). Diisopropyl azodicarboxylate (DIAD, 17.8 g, 87.8 mmol, 2.3 eq) was added at -20°C to -40°C. After 40 minutes at the same temperature, the reaction mixture was concentrated and directly purified by flash column chromatography using petroleum ether:AcOEt (10:1 to 6:1) as eluent to give benzyl (R)-2-(( 1,3-dioxoindolin-2-yl)oxy)-3-methylbutanoate (10) (11.9 g, 88%) was obtained as a colorless oil.

Preparation of benzyl (R)-2-(aminooxy)-3-methylbutanoate (11) Hydrazine monohydrate (2.08 mL, 42.9 mmol, 1.1 eq) was dissolved in DCM (150 mL) in an ice bath. ) and stirred at the same temperature for 2 hours until the reaction was completed. The reaction mixture was filtered and the filtrate diluted with DCM. After washing the organic layer with brine, it was dried over Na ₂ SO ₄ and the solvent was removed in vacuo. The crude product was purified by flash column chromatography using petroleum ether:AcOEt as eluent to give benzyl (R)-2-(aminooxy)-3-methylbutanoate (11) (8.03 g, 95% ) as a colorless oil.

Preparation of benzyl (R)-2-(((isobutoxycarbonyl)amino)oxy)-3-methylbutanoate (13) Compound (11) (20. 0 g, 89.7 mmol, 2.0 eq) and 4-dimethylaminopyridine (DMAP, 1.67 g, 13.5 mmol, 0.15 eq) to isobutyl chloroformate (12) (22.6 mL, 179.4 mmol, 2.0 eq). ) was added and stirred at the same temperature for 2 hours until the reaction was completed.
The reaction mixture was concentrated and purified directly by flash column chromatography using petroleum ether:AcOEt as eluent to afford benzyl (R)-2-(((isobutoxycarbonyl)amino)oxy)-3-methylbutanoate. (13) (27 g, 93%) was obtained as a colorless oil.

Preparation of (R)-2-(((isobutoxycarbonyl)amino)oxy)-3-methylbutanoic acid (14) To a stirred solution of compound (13) (22 g, 68.1 mmol) in MeOH (150 mL) 10%. A palladium-carbon catalyst (Pd/C, 2.2 g) was added and hydrogen was displaced three times. The mixture was stirred at room temperature until completion of the reaction. (R)-2-(((isobutoxycarbonyl)amino)oxy)-3-methylbutanoic acid ( 14) (14.5 g, 91%) was obtained as a white solid.

isobutyl (((R)-1-(((2S,3S)-1-(benzyl(2,2-diethoxyethyl)amino)-3-methyl-1-oxopentan-2-yl)amino)-3 -methyl-1-oxobutan-2-yl)oxy)carbamate (15) To a solution of compound (14) (1.886 g, 8 mmol) in dry DCM (40 mL) was added compound (6) (2.691 g, 8 mmol, 1.0 eq), DIPEA (3 mL, 2.2 eq) and HATU (3.34 g, 8.8 mmol, 1.1 eq) were added. The reaction mixture was stirred overnight at room temperature. After removing the solvent, the reaction mixture was dissolved in AcOEt (150 mL) and washed with brine. _The organic phase was dried over _Na2SO4 and filtered. The filtrate was concentrated in vacuo, the residue was purified by silica gel column chromatography (n-Hex:AcOEt=95:5-65:35), isobutyl (((R)-1-(((2S,3S)- 1-(benzyl(2,2-diethoxyethyl)amino)-3-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)oxy)carbamate (15) (3.82 g, 87%) was obtained as a colorless oil. Developing solvent, n-Hex:AcOEt=2:1; color former, phosphomolybdic acid; Rf=0.51

isobutyl (3R,6S,9aS)-8-benzyl-6-((S)-sec-butyl)-3-isopropyl-4,7-dioxohexahydropyrazino[2,1-c][1,2 ,4] Preparation of oxadiazine-1(6H)-carboxylate (compound of formula (I)) Compound (15) (3.82 g, 6.92 mmol) was dissolved in HCO ₂ H (50 mL). The reaction mixture was heated at 45° C. overnight. After removing HCO ₂ H, the residue was purified by silica gel column chromatography (n-Hex:AcOEt=75:25) and isobutyl (3R,6S,9aS)-8-benzyl-6-((S)-sec -butyl)-3-isopropyl-4,7-dioxohexahydropyrazino[2,1-c][1,2,4]oxadiazine-1(6H)-carboxylate, compound of formula (I) (3. 07 g, 97%) as a colorless viscous oil. Developing solvent, n-Hex:AcOEt=4:1; color former, phosphomolybdic acid; Rf=0.35
¹ H-NMR (CDCl ₃ ) δ: 0.84(d, 3H, J=5.6Hz), 0.95(t, 3H, J=5.9Hz), 0.96(d, 6H, J=5.4Hz), 1.08(d, 3H, J=5.7Hz), 1.12(d, 3H, J=5.4Hz), 1.26(m, 1H), 1.56(m, 1H), 1.98(m, 1H), 2.20(m, 1H), 2.53( m, 1H), 3.32(dd, 1H, J=9.2, 3.5Hz), 3.69(t, 1H, J=8.9Hz), 4.01(d, 2H, J=5.2Hz), 4.45(br s, 1H) , 4.59(d, 1H, J=11.7Hz), 4.68(d, 1H, J=11.7Hz), 5.07(d, 1H, J=5.1Hz), 5.86(br d, 1H, J=5.7Hz), 7.22(d, 2H, J=5.4Hz), 7.27-7.35(m, 3H). MS (ESI): [M+H] ⁺ m/z 460.3.

Example 2
Synthesis of Formula (II) Compound The formula (II) compound was synthesized in the same manner as in Example 1.

Example 3
Synthesis of Formula (III) Compound The formula (III) compound was synthesized in the same manner as in Example 1.

Test Example 1: Anticancer action (material and method)

1. Cell culture experiments included human colon cancer cell lines SW480 (ATCC, RIKEN), HT29 (JCRB) and HCT116 (RIKEN), and human non-cancer cell lines CCD841CoN (ATCC) and HEK293T. was used. CCD841CoN that had been passaged 10 times or more after purchase was not used.

2. Anti- rat anti-KLF5 antibody used was a monoclonal rat anti-KLF5 antibody produced jointly with Kyowa Kirin (Japan). The following primary antibodies were also used. Survivin (Novus), GAPDH (Millipore), TCF4 (Cell signaling), β-Catenin (Cell signaling), phospho-β-Catenin (Ser 552) (Cell signaling), Cyclin D1 (Cell signaling), phos pho-β-catenin (Ser 675) (Cell signaling), β-Actin (Cell signaling), Ki67 (DAKO), USP3 (Abcam). The following secondary antibodies were used. ECL anti-rabbit IgG HRP (GE Healthcare), anti-mouse IgG HRP (GE Healthcare), and anti-rat IgG HRP (Santa Cruz, Cell Signaling) were used.

3. Determination of Relative Cell Viability Cells were seeded at a density of 1×10 ⁴ cells/100 μL per well in 96-well tissue culture plates (Falcon/Corning). Approximately 1-2 days after seeding the cells, the medium was changed to medium containing each concentration of compound or control (DMSO). After 72 hours of compound treatment, 10 μL of Cell Counting Kit 8 or Cell Counting Kit (Dojindo, Japan) reagent was added per well, and the absorbance at 450 nm was measured with an absorbance meter. The relative viability of cells at each concentration was calculated relative to a 0 μM control with DMSO (Wako).

4. Western blotting protein lysates were added to cOmplete tablets in modified RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM EDTA, 0.1% NP-40, 0.1% Triton-X, 10% glycerol). (Roche), by ultrasonic washing with a Bioraptor (Sonic Bio) or by hand homogenization with a Tenbroeck homogenizer (Wheaton). The protein concentration was measured using Protein Assay Rapid Kit (Wako), and the protein amount was adjusted to the same concentration. The protein lysate was mixed with 2-ME (Wako) and Bolt LDS Sample Buffer (x4) (Novex, Life Technologies) and incubated at 95°C for 5 minutes.
Proteins were subjected to electrophoresis in Bolt 4-12% Bis-Tris Plus gels (Invitrogen). After electrophoresis, proteins in the gel were transferred to Immobilon membrane (Millipore). Membranes were incubated in TBS supplemented with 0.05% Tween 20 containing primary antibody and 4% skimmed milk (Snow Brand). Membranes were washed with TBS containing 0.05% Tween 20. Membranes were incubated in TBS with 0.05 % Tween 20 and 4% skimmed milk containing anti-rabbit/mouse/rat IgG HRP conjugated (GE healthcare) or anti-rat IgG HRP conjugated (Santa Cruz) secondary antibodies.
Membranes were washed with TBS containing 0.05% Tween 20 and soaked in Immobilon Forte Western HRP Substrate (Millipore). Signals were detected and evaluated with an ImageQuant LAS (GE Healthcare) or ChemiDoc (BioRad).

5. RT-PCR
Total RNA was extracted from the cells using Isogen (Nippon Gene), and cDNA was prepared from the total RNA using Prime Script (Takara Bio). cDNA was subjected to PCR using the LightCycler 96 system (Roche). The primer sequences for KLF5 are 5'-ctgcctccagaggacctg-3' (SEQ ID NO: 1) and 5'-tctgtctatactttatgctggaat-3' (SEQ ID NO: 2). The primer sequences for GAPDH are 5'-agccacatcgctcagac-3' (SEQ ID NO:3) and 5'-gcccaatacgacaatcc-3' (SEQ ID NO:4).

6. Human Colon Cancer Cell Spheroid Formation and Measurement HCT116 cells were plated on PrimeSurface low-adhesion tissue culture plates (Sumitomo Bakelite). The size of spheroids was measured with Cell3iMager duos (Screen).

7. Implantation of tumor under administration of compound of formula (I) and measurement of tumor 5×10 ⁶ HT29 cells were subcutaneously implanted in a nude mouse (purchased from SLC Japan). Twice daily intraperitoneal administration of the compound of formula (I) (2 mg) was initiated 2 days after transplantation. The minor axis and major axis of the tumor were measured using vernier calipers. Tumor volume was evaluated by 2×major axis/2.

8. Histopathological Analysis Tissues were fixed in neutral buffered formalin. Fixed tissues were embedded in paraffin. Paraffin-embedded tissues were sliced and subjected to hematoxylin and eosin staining or subsequent immunohistochemistry.

9. Immunohistochemistry Immunohistochemistry for KLF5 was performed using neutral buffered formalin-fixed mouse intestinal sections and human post-operative specimens.
The anti-KLF5 mouse or rat monoclonal antibody was cloned jointly by the present inventors and Kyowa Kirin Co., Ltd. and purified.
Immunohistochemistry for Ki67 was performed using an anti-Ki67 antibody (Gene Tex).
Antigen retrieval was performed by autoclaving the sections in 10 mM sodium citrate (pH 6.0) at 121° C. for 15 minutes. After washing with PBS, sections were incubated with primary antibody. Thereafter, immunohistochemical staining was performed using an LSAB kit (DAKO) using 3,3'-diaminobenzidine as a chromogenic substrate, followed by counter staining with hematoxylin.

10. Purification of USP protein complex by immunoprecipitation pPyCAG-IP vector expressing FLAG/HA-hKLF5 and pPyCAG-IP vector expressing Myc-tag-hUSP3 were generated (H. Niwa, et al., Mol Cell Biol 22 , 1526-1536 (2002)). The pPyCAG-IP vector expressing FLAG/HA-hKLF5 and the pPyCAG-IP vector expressing Myc-tag-hUSP3 were co-transfected into HEK293T cells using Hilymax reagent (Dojindo, Japan). HEK293T cells expressing FLAG/HA-hKLF5 and HEK293T cells expressing Myc-tag-hUSP3 were treated with the compound of formula (I) (20 μM) or DMSO (control) for 4 hours. Cell pellets were suspended in modified RIPA buffer using cOmplete tablets (Roche). The suspended cell pellet was homogenized using Tenbrroeck homogenizers (Wheaton). The homogenized mixture was centrifuged at 15000 rpm for 30 minutes at 4° C. and the supernatant was obtained as a cell lysate.
Cell lysates were mixed with anti-Myc-tagged mAb magnetic beads (MBL, Japan) at 4°C for 4 hours. The magnetic beads were washed with bead wash buffer (100 mM KCl, 50 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 0.1% NP-40, and 10% glycerol). The magnetic beads were then mixed and spun using a modified RIPA buffer containing Myc-tagged peptides (MBL, Japan) to obtain an eluate of protein complexes bound to the anti-Myc-tagged mAb-magnetic beads. This eluate contained immunoprecipitated protein complexes of Myc-tag-USP3. The immunoprecipitated UPS3 protein complex and co-immunoprecipitated KLF5 protein were estimated by Western blotting.

11. Purification of KLF5 Protein Complex by Immunoprecipitation A FLAG/HA-hKLF5 expressing pMxs-Puro vector was prepared by inserting the FLAG/HA-hKLF5 sequence into an empty pMxs-Puro vector. This vector was transfected into Plat-A cells using Fugene reagent (Roche). Supernatant containing retrovirus was harvested from Plat-A cells transfected with the FLAG/HA-hKLF5 expression vector. The supernatant was added to SW480 cells and SW480 cells stably expressing FLAG/HA-KLF5 were selected by puromycin resistance.
SW480 cells expressing FLAG/HA-hKLF5 were treated with compound of formula (I) (10 μM) or DMSO (control) for 24 hours. Cell pellets were suspended in modified RIPA buffer using cOmplete tablets (Roche). The suspended cell pellet was homogenized using Tenbrroeck homogenizers (Wheaton). The homogenized mixture was centrifuged at 15000 rpm for 30 minutes at 4° C. and the supernatant was obtained as a cell lysate.
Cell lysates were mixed with anti-FLAG-tagged mAb magnetic beads (MBL, Japan) at 4° C. for 4 hours. The magnetic beads were washed with bead wash buffer (100 mM KCl, 50 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 0.1% NP-40, and 10% glycerol). The magnetic beads were then mixed and spun using a modified RIPA buffer containing 3x FLAG-tagged peptide (Sigma) to obtain an eluate of protein complexes bound to the anti-FLAG-tagged mAb-magnetic beads. This eluate contained immunoprecipitated protein complexes of FLAG/HA-tag-KLF5.
FLAG peptide eluate was subjected to sequential immunoprecipitation with anti-HA tagged mAb magnetic beads (MBL, Japan) and HA-tag peptide elution (MBL). Immunoprecipitation using HA tag was performed in the same manner as immunoprecipitation using FLAG tag. The HA bead eluate eluted after tandem affinity purification contained higher purity FLAG/HA-KLF5 protein complexes. Western blotting suggested that the KLF5 protein complex contained the immunoprecipitated FLAG/HA-KLF5 protein complex and the co-immunoprecipitated β-catenin protein.

12. Synthesis of Beads with Covalently Attached Compounds of Formula (I ) All side chains of compounds of formula (I) are occupied by hydrophobic structures with low chemical reactivity. Furthermore, the backbone of formula (I) is chemically surrounded by side chains, making the chemical structure inaccessible to the covalent linker. Therefore, it was difficult to covalently bind the compound of formula (I) to Sepharose beads using a linker with a reactive group.
Therefore, a photoaffinity reaction was used. Photoaffinity reactions can be used to generate random covalent bonds between less reactive chemical structures and Sepharose beads. This method was used to prepare beads covalently attached to compounds of formula (I) in random orientations (Kanoh, N. et al., Angew Chem Int Edit 44, (23), pp 3559-3562; Kawatani, M. et al. P Natl Acad Sci USA 105, 11691-11696).

13. Mass Spectrometry of Formula (I) Compound Bound Proteins Protein lysates were prepared from SW480 human colon cancer cells in modified RIPA buffer using a Tenbroeck homogenizer (Wheaton). The protein lysate was mixed overnight at 4° C. with beads to which the compound of formula (I) was covalently attached or control beads to which no compound of formula (I) was attached. The beads were washed and proteins bound to the compound of formula (I) covalently bound beads or control beads were eluted with LDS Sample Buffer (x4) (Invitrogen) supplemented with 2-ME (Wako).
Eluted proteins were subjected to LC/MS/MS analysis using Orbitrap (ThermoFisher) to identify proteins significantly enriched with compound of formula (I) covalently attached beads compared to control beads. A Tandem Mass Tag ^™ stem (Thermo Fisher) was used for protein quantification.

14. JFCR39 cancer cell panel JFCR (Japanese Foundation for Cancer Research) 39 Analysis using cancer cell panel has been reported (e.g., Nagai R, Friedman SL, & Kasuga M (2009) The biology of Kruppel-like factors (Springer Verlag) ).

(result)

1. Effect of Compounds of the Present Invention on Growth of Human Colon Cancer Cells The growth inhibitory effects of the compounds of the present invention (compounds of formula (I), compounds of formula (II) and compounds of formula (III)) on human colon cancer cells were investigated.
The compounds of the present invention inhibited the proliferation of human colon cancer cells (SW480 and HCT116) (Fig. 1). This effect was more clearly demonstrated when the effect on the proliferation of 3D spheroids of human colon cancer cells was examined (Fig. 2). In particular, the compound of formula (I) exhibited the most potent antiproliferative activity with an _IC50 of 2.5 μM.
Of particular interest is that the compounds of the present invention did not affect viability of non-cancer cells (CCD841) (Fig. 1). On the other hand, such characteristics were not observed in a compound (ICG-001) known to have Wnt signaling inhibitory activity.

2. The compounds of the invention reduce KLF5 and Wnt signaling proteins in colon cancer cells.
When the compounds of the present invention (formula (II) compound, formula (III) compound) are administered to human colon cancer cells, KLF5, TCF4, Survivin, β-catenin phosphorylated at Ser552 was significantly reduced (Fig. 3, Fig. 5). Compounds of formula (I) also reduced protein levels of Cyclin D1, a key cell cycle promoter whose transcription is regulated by KLF5 (Fig. 4). Furthermore, the phosphorylation of β-catenin at positions 552 or 675, which is a hallmark of Wnt signaling activation, was decreased by the compounds of the present invention (FIGS. 4 and 5). Furthermore, in SW480 cells, the protein level of β-catenin contained in the purified FLAG/HA-KLF5 protein complex was reduced by treatment with the compounds of the present invention (Fig. 6). Interestingly, the compounds of the present invention did not decrease intracellular KLF5 mRNA levels (FIG. 7).
A decrease in Wnt signal-related proteins by administration of the compounds of the present invention was observed in cancer cells, but had almost no effect on CCD841, a human non-cancer cell line (FIGS. 3 and 4). Furthermore, the anti-tumor profile of the compounds of the present invention was examined using the JFCR39 human cancer cell line. COMPARE analysis (National Cancer Institute, USA) showed that the anti-tumor fingerprint of compound of formula (II) was similar to that of Wnt pathway inhibitor LGK974.
These results suggest that the compounds of the present invention inhibit cancer cell growth by inhibiting the Wnt signaling pathway, which is the most important molecular pathway in human colorectal cancer.

3. Complex formation of the KLF5-USP3 protein is impaired by treatment with the compounds of the invention.
Proteins that bind to Formula (I) compounds were identified using Formula (I) compound-bound beads. The method of Kanoh et al. (Kanoh N, et al., Angew Chem Int Edit 44(28):4282-4282.(2005)) can irreversibly photocrosslink small molecules to affinity beads in a functional group-independent manner. was used. Then, they discovered that ubiquitin-specific peptidase 3 (USP3) is included in the proteins that bind to the compound of formula (I). Since USP3 is known to stabilize KLF5 by deubiquitination (Saltz LB, et al. N Engl J Med 343(13):905-914.(2000)), the USP3-KLF5 protein complex is It was investigated whether it is inhibited by treatment with compounds of formula (I). To detect this protein complex, we co-expressed FLAG/HA-KLF5 and Myc-tag-USP3 in HEK293T cells and purified the Myc-tag-USP3 protein complex from cell lysates by immunoprecipitation (IP). . As shown in FIG. 8, IP-Western blot analysis revealed that treatment with the compound of formula (I) reduced the amount of KLF5 in the Myc-tag-USP3 protein complex.

4. Suppression of In Vivo Implanted Tumor Growth by Compounds of the Present Invention In order to investigate the in vivo antitumor effect of compound of formula (I), compound of formula (I) was administered to nude mice subcutaneously implanted with HT-29 human colon cancer cells. was administered intraperitoneally.
As shown in Figure 9A, administration of the compound of formula (I) resulted in a significant reduction in tumor volume. Remarkably, the compound of formula (I) did not cause overt side effects, including intestinal and colonic histological abnormalities (Fig. 9B).

Test Example 2: Cardiac Function Improving Action (Materials and Methods)

1. Animals and pressure-overload heart failure model Eight-week-old male C57BL/6 mice were bred under temperature- and humidity-controlled conditions and used for experiments. Mice had free access to food and water in a room under a 12 hour light/dark cycle. Transverse aortic constriction surgery (TAC) was used to induce pressure overload in mice. Mice were randomly assigned to either the TAC group or the sham-operated group.
Aortic diameter was narrowed when ligated with a 27-gauge needle on the aortic arch of TAC mice and the needle was removed with 6-0 silk after ligation. The needle was quickly removed, leaving a stenosis in the transverse aorta. The sham-treated group underwent a similar surgical procedure without ligation.

2. Echocardiography Echocardiography was performed before TAC and 4 weeks after TAC. Briefly, echocardiography was performed using a Vevo2100 equipped with a 30 MHz linear transducer. Left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic diameter (LVEDd), end-diastolic interventricular septal thickness (IVSd), end-systolic interventricular septal thickness (IVS), end-diastolic left Posterior ventricular wall thickness (LVPWd), end-systolic LV posterior wall thickness (LVPW), heart rate (HR), and left ventricular ejection fraction (EF) were measured.
Images were acquired to calculate left ventricular ejection fraction and measure wall thickness. Mice were then euthanized and blood and heart tissue were collected.

3. Treatment After TAC surgery, mice were also randomly assigned to three groups containing TAC treated with the vehicle group (negative control) and the compound of formula (I) administration group.
The compound of formula (I) (50 mg/kg/day) was administered intraperitoneally for 10 days (twice daily; 25 mg/kg/day). Vehicle (DMSO) was administered intraperitoneally to sham and wild type groups.
After 4 weeks, hearts and blood were collected and heart weights were checked to compare heart weight/body weight (HW/BW, mg/g). Left ventricular (LV) tissue was then collected for subsequent experiments.

4. Histology Hearts were collected 4 weeks after TAC surgery. Heart tissue was embedded in paraffin and sectioned at a thickness of 5 μm. Sections were transferred to microscope slides and stained with hematoxylin and eosin (HE) to assess cardiomyocyte cross-sectional area and with Masson and trichrome (MT) to assess the degree of fibrosis. Histopathological features of each slice were examined under a microscope (Keyence BZ-9000) to observe global changes in heart size and fibrosis. Areas of fibrosis were analyzed by Image-J.

5. RNA extraction and quantitative real-time PCR
Cardiac tissue was collected after TAC.
RNA was extracted from mouse LV tissue using the RNeasy Mini Kit® (QIAGEN) and DNA contamination was removed by RNase-free DNase digestion (QIAGEN, cat#79254) according to the manufacturer's protocol. Next, cDNA was synthesized by ReverTraAce® Reverse Transcriptase (Toyobo) according to the manufacturer's protocol. Real-time PCR was performed using the SYBR Premix Ex Taq II kit (Takara Biotechnology) and performed using the qPCR system (Stratagene Mx3005P). Relative expression levels of target genes were determined after normalization to the GAPDH gene and quantified using the comparative threshold cycle method.

6. Proteins were extracted from heart tissue using Western Blotting T-PER® Tissue Protein Extraction Reagent (Thermo Scientific). Protein concentration was then quantified by the Pierce ^™ BCA Protein Assay Kit® (Thermo Scientific) according to the manufacturer's protocol. Equal amounts of protein from each sample were fractionated by SDS-PAGE. Proteins were transferred from gel SDS-PAGE to membrane by a gel transfer apparatus. Membranes were then incubated with various primary antibodies for 1 hour at room temperature and secondary antibodies for 1 hour at room temperature. Protein expression levels were normalized to corresponding GAPDH (Thermo Scientific) levels.

7. Survival study 10 each of 40 mice, untreated group (WT) not undergoing transverse aortic constriction surgery (TAC), sham-operated group (Sham), vehicle group (Vehicle) and formula (I) compound administration group were divided into four groups. The vehicle group and the formula (I) compound dose group are receiving TAC. Mice in the WT, Sham, and vehicle groups were intraperitoneally administered DMSO twice daily for 10 days after sham operation or TAC. Each mouse in the compound of formula (I) administration group was administered compound of formula (I) twice daily at a dose of 25 mg/kg for 10 days after TAC. Mouse survival was monitored daily for 14 weeks. Kaplan-Meier plots in GraphPad Prism 6 software were used to assess survival, followed by Tukey's multiple comparison test for comparison between groups. The protocol is shown in FIG.

(result)
1. Varying Dosages of Compounds of Formula (I) to Prevent Heart Failure Compounds of Formula (I) are administered at four concentrations including 12.5, 25, 50, 100 mg/kg, or three doses including 25, 50, 100 mg/kg. was varied at one concentration. Mice were injected twice daily. Efficacy at a dose of 25 mg/kg (50 mg/kg/day) of the compound of formula (I) was found to be sufficient to prevent heart failure due to TAC pressure overload.
The compound of formula (I) (50 mg/kg/day) suppressed the decrease in left ventricular ejection fraction and reduced the increase in HW/BW in TAC mice (Fig. 10).

2. The compound of formula (I) enhances the left ventricular ejection fraction of TAC mice and improves cardiac function From 10 days to 1 month after TAC, few TAC mice died. The compound of formula (I) (50 mg/kg/day) was administered intraperitoneally for 10 days (twice daily) in a group of TAC mice treated with compound of formula (I).
Treatment with compounds of formula (I) greatly improved survival in mice with established left ventricular pressure overload-induced heart failure.
Four weeks after TAC, left ventricular ejection fraction was significantly decreased in the vehicle group compared to the Formula (I) compound-treated TAC mouse group. This means that the compound of formula (I) improved left ventricular ejection fraction values (Figure 11). In addition, echocardiography showed that end-systolic left ventricular dimension (LVID), end-diastolic left ventricular dimension (LVIDd), and left ventricular mass were compared with the compound of formula (I) in the treated TAC mouse group. A significant increase in correction (LV mass correction) was shown. Quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) also revealed that brain natriuretic peptide (BNP) levels, a marker of heart failure, were significantly higher in TAC mice treated with compound of formula (I) compared to TAC. decreased (Fig. 12). These data demonstrate that cardiac function was improved after TAC and treatment with compounds of formula (I).

3. Compounds of formula (I) attenuate cardiac hypertrophy in vivo and ameliorate myocardial fibrosis induced by pressure overload (Fig. 13). Hematoxylin and eosin staining (HE) of heart sections showed hypertrophic changes in mice receiving TAC, which were reduced in Formula (I) compound-treated TAC mice. These data demonstrate that compounds of formula (I) reduce cardiac hypertrophy after TAC. Heart tissue sections were then evaluated for fibrosis by Masson's trichrome staining (MT). An increase in interstitial fibrosis in cardiac tissue was observed in mice undergoing TAC surgery, but this increase was attenuated in TAC mice treated with compound of formula (I) (Figure 14). These data demonstrate that compounds of formula (I) ameliorate cardiac fibrosis induced by pressure overload.

3. The compound of formula (I) prolongs the survival period in pressure overload heart failure model The effect of the compound of formula (I) in pressure overload heart failure model mice (TAC mice) was evaluated by a survival test. The results are shown in FIG.
From the long-term survival curves, the mortality rate of TAC mice treated with compound of formula (I) was significantly lower than that of vehicle-treated TAC mice (n=10 in each group). In the formula (I) compound-administered group, the median survival time was 2.2 times longer than in the vehicle-administered group (11 weeks for the formula (I) compound-administered group and 5 weeks for the vehicle-administered group).

The compound of the present invention has the following characteristics.
(i) inhibit the growth and survival of cancer cells, but not the growth and survival of non-cancer cells;
(ii) reducing not only KLF5 protein levels but also protein levels of the Wnt signaling pathway, and inhibiting the interaction of KLF5 with Ubiquitin-specific peptidase 3 (USP3), which is known to stabilize KLF5;
(iii) effectively inhibits transplanted colon cancer cells in vivo without apparent side effects;
(iv) have a cardiac function-improving effect;
Therefore, the compounds of the present invention or pharmaceutically acceptable salts thereof are useful as anticancer agents or cardiac function improving agents.
Although only some exemplary embodiments of the present invention have been set forth in detail above, those skilled in the art will appreciate such exemplary embodiments without departing substantially from the novel teachings and advantages of the present invention. It will be readily understood that many variations in the embodiments are possible. Accordingly, all such modifications are intended to be included within the scope of this invention.

This application is based on Japanese Patent Application No. 2021-187420 (filing date: November 17, 2021) filed in Japan, the contents of which are all incorporated herein.

Claims (9)

1. An anticancer agent and/or cardiac function improving agent containing a substance that inhibits the binding of Kruppel-like factor 5 (KLF5) and Ubiquitin-specific peptidase 3 (USP3).
2. Substances that inhibit the binding of KLF5 and USP3 have the following formulas (I) to (III):
   

   

   The agent according to claim 1, which is a compound represented by any of or a pharmaceutically acceptable salt thereof.
3. A screening method for anticancer agents and/or cardiac function improving agents, comprising a step of measuring the KLF5-USP3 binding inhibitory activity of a test substance, and a step of selecting a compound having binding inhibitory activity.
4. Formulas (I) to (III) below:
   

   

   A compound represented by any of or a pharmaceutically acceptable salt thereof.
5. Formulas (I) to (III) below:
   

   

   A pharmaceutical composition comprising a compound represented by any of or a pharmaceutically acceptable salt thereof.
6. The pharmaceutical composition according to claim 5, which is a KLF5 inhibitor.
7. The pharmaceutical composition according to claim 5 or 6, which is an anticancer agent and/or an agent for improving cardiac function.
8. The pharmaceutical composition according to claim 7, wherein the anticancer agent is for colorectal cancer.
9. The pharmaceutical composition according to claim 7, wherein the agent for improving cardiac function is a therapeutic agent for heart failure.

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