LIN28B inhibitors

Abstract

To provide a composition applicable for prevention, treatment, or treatment of poor prognosis of various cancers involving LIN28B protein activity (e.g. hepatocellular carcinoma, pancreatic cancer, lung cancer, ovarian cancer, colon adenocarcinoma, chronic myelogenous leukemia, pancreatic ductal adenocarcinoma, malignant kidney tumor, breast cancer, and melanoma).SOLUTION: A benzaldehyde compound that inhibits expression of LIN28B protein is provided. The LIN28B inhibitor of the present invention is also useful for diabetes, pneumonia, hepatic cirrhosis, renal disorders, nephrosclerosis, and cardiotoxicity, and effectively serves as a component in nutritional compositions, supplements, foods, and beverages.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a LIN28B inhibitor containing a benzaldehyde compound, and a pharmaceutical composition, supplement composition, or anticancer composition containing the inhibitor. [Background technology]

[0002] Benzaldehyde and its derivatives were discovered to have anti-cancer activity by Dr. Mutsuyuki Toho (see, for example, Patent Document 1). Subsequent research by Toho and his colleagues has revealed that various benzaldehyde compounds (e.g., benzaldehyde, 5,6-O-benzylidene-L-ascorbic acid sodium salt, 4,6-O-benzylidene-D-glucopyranose, etc.) have anti-cancer activity, anti-HIV activity, reactive oxygen species scavenging activity (antioxidant activity), influenza treatment activity, and preventive and therapeutic effects against neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, etc.), epilepsy, heart disease, diabetic diseases, etc. (see, for example, Patent Documents 2 to 7 and Non-Patent Documents 1 to 3). Benzaldehyde is the simplest aromatic aldehyde and is inexpensive, and has little cytotoxicity to normal cells, making it a substance with great potential for reducing the pain caused by chemotherapy.

[0003] The LIN28B protein is known to function as one of the reprogramming factors from somatic cells to pluripotent stem cells (Non-Patent Document 4). It is also known that LIN28B can be used as a tumor marker (Patent Document 8). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 54-962 [Patent Document 2] Specification of Japanese Patent Publication No. 63-10685 [Patent Document 3] Patent Publication No. 7-242632 [Patent Document 4] Specification of Special Publication No. 3-33127 [Patent Document 5] JP 8-217675 A [Patent Document 6] Specification of Japanese Patent Publication No. 12747-1969 (Patent No. 560349) [Patent Document 7] Patent Publication No. 2017-43616 [Patent Document 8] Special Table 2014-525584 [Non-patent literature]

[0005] [Non-Patent Document 1] Takeuchi, S. et al., Agric Biol Chem 42: 1449-1451, 1978 [Non-patent document 2] Kochi, M. et al., The 13th International Cancer Congres, Seattle, 1982 [Non-patent document 3] Kochi, M. et al., The 14th International Cancer Congress, Budapest, 1986 [Non-patent document 4] Zhang, J. et al., Cell Stem Cell 19, 66-80, 2016 Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, benzaldehyde or its derivatives (hereinafter also referred to as "benzaldehyde compounds") have various useful activities and are considered to be useful substances. In this situation, it is desirable to develop new uses for benzaldehyde compounds based on their novel mechanism of action. [Means for solving the problem]

[0007] The present invention has discovered that benzaldehyde compounds inhibit the expression of LIN28B protein, and provides the following inventions.

[0008] That is, the present invention provides a LIN28B inhibitor, a pharmaceutical composition containing the same, a supplement composition or anticancer composition, a combination agent containing the LIN28B inhibitor, and the like according to the following aspects.

[0009] (1) A LIN28B inhibitor comprising a benzaldehyde compound or a pharmaceutically acceptable salt thereof. (2) The benzaldehyde compound is represented by the following general formula (I): [ka] (In the formula, R 1 -CHO, -CXO (where X is a halogen group), dioxolanyl group, dioxanyl group, [ka] and;R 2 are each independently a hydrogen atom, a halogen group, a lower alkyl group, a lower alkenyl group, a lower alkynyl group, or a lower alkoxy group; and n represents an integer of 1 to 5. (3) The benzaldehyde compound is a compound of general formula (I), wherein R 1 is -CHO, a 1,3-dioxolanyl group, a 1,3-dioxanyl group, or [ka] and;R 2 and each independently represent a hydrogen atom or a halogen group. (4) The inhibitor according to any one of (1) to (3) above, wherein the benzaldehyde compound is benzaldehyde, 5,6-O-benzylidene-L-ascorbic acid, 5,6-O-benzylidene-L-ascorbic acid monosodium salt (hereinafter sometimes abbreviated as SBA), 4,6-O-benzylidene-D-glucopyranose, or N-benzylideneethylamine. (5) A composition containing the inhibitor described in (1) to (4) above. (6) The composition described in (5) above, which is a pharmaceutical composition or a supplement composition. (7) The pharmaceutical composition according to (6) above, which is used for the prevention or treatment of a disease associated with LIN28B protein activity. (8) The pharmaceutical composition according to (7) above, wherein the disease associated with LIN28B protein activity is cancer, diabetes, nephropathy, renal fibrosis, nephrosclerosis, diabetic nephropathy, cirrhosis, nonalcoholic fatty liver disease, idiopathic interstitial pneumonia, or anthracycline anticancer drug-induced myocardial damage. (9) The pharmaceutical composition according to (8) above, wherein the cancer is liver cancer, pancreatic cancer, lung cancer, ovarian cancer, colon adenocarcinoma, chronic myeloid leukemia, pancreatic ductal adenocarcinoma, malignant renal tumor, Wilms' tumor, breast cancer, melanoma, gastric cancer, prostate cancer, cholangiocarcinoma, gallbladder cancer, head and neck cancer, osteosarcoma, diffuse midline glioma, glioblastoma, medulloblastoma, glioma, diffuse large B-cell lymphoma, follicular lymphoma, multiple myeloma, thyroid cancer, uterine cancer, EGFR-TKI therapy-resistant cancer, or radiotherapy-resistant cancer. (10) The composition described in (6) above, which is used to suppress epithelial-mesenchymal transition (EMT) of cancer cells or self-renewal or differentiation of cancer stem cells in the cancer. (10a) The composition according to (6) above, which is used for treating poor prognosis or inhibiting metastasis of the cancer. (11) A cancer metastasis inhibitor comprising the inhibitor according to any one of (1) to (4) above. (12) A combination drug comprising a LIN28B inhibitor according to any one of (1) to (4) above and an anticancer agent having an action mechanism unrelated to LIN28B protein activity. (13) The LIN28B inhibitor according to (1) to (4), which is used in combination with one or more EGFR inhibitors selected from osimertinib, gefitinib, erlotinib, aquatinib, dacomitinib, and pharmaceutically acceptable salts thereof, and / or one or more immune checkpoint inhibitors selected from nivolumab, pemprolizumab, and pharmaceutically acceptable salts thereof. (14) A combination drug comprising a LIN28B inhibitor according to any one of (1) to (4) above and an EGFR inhibitor. [Effects of the Invention]

[0010] The LIN28B inhibitor of the present invention can be used to prevent or treat various diseases in which LIN28B protein activity is involved, by inhibiting the expression of LIN28B protein. Diseases in which LIN28B protein activity is involved include, for example, various cancers (e.g., liver cancer, pancreatic cancer, lung cancer, ovarian cancer, colon adenocarcinoma, chronic myeloid leukemia, pancreatic ductal adenocarcinoma, malignant renal tumor, Wilms' tumor, breast cancer, melanoma, gastric cancer, prostate cancer, cholangiocarcinoma, gallbladder cancer, head and neck cancer, osteosarcoma, diffuse midline glioma, glioblastoma, medulloblastoma, glioma, diffuse large B-cell lymphoma, follicular lymphoma, multiple myeloma, thyroid cancer, uterine cancer, EGFR-TKI therapy-resistant cancer, and radiotherapy-resistant cancer), diabetes, nephropathy, renal fibrosis, nephrosclerosis, diabetic nephropathy, cirrhosis, non-alcoholic fatty liver disease, idiopathic interstitial pneumonia, and anthracycline anticancer drug-induced myocardial injury.

[0011] Furthermore, since the inhibitor of the present invention exerts anticancer activity by regulating LIN28B protein activity, it can be used as an effective anticancer agent for patients who have been treated with anticancer agents having a mechanism of action unrelated to LIN28B protein activity but have not achieved sufficient therapeutic effect, as a drug for suppressing cancer metastasis, and as a drug for treating poor prognosis after cancer treatment.

[0012] In addition to the above-mentioned anti-cancer activity, the LIN28B inhibitor of the present invention (or the above-mentioned benzaldehyde compound) also has antioxidant activity (see JP 8-99880 A); anti-HIV activity (see JP 8-3038 A); influenza prophylactic and therapeutic activity (see JP 8-217675 A); prophylactic and therapeutic activity against neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, multiple sclerosis, etc.), epilepsy, heart disease, or diabetic disease (see JP 2017-43616 A), and AXL inhibitory activity (see JP 2022-171109 A), and can therefore be effectively used as an ingredient in nutritional compositions, supplements, foods, beverages, etc. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of RT-PCR analysis of LIN28B expression in lung cancer cell line A549 cells, comparing the results when cells were treated with and without benzaldehyde (500 μM). [Figure 2] FIG. 2 shows the results of Western blot analysis of LIN28B protein expression in lung cancer cell line A549 cells, comparing the results when cells were treated with and without benzaldehyde (500 μM). [Figure 3] FIG. 3 shows the results of Western blot analysis of LIN28B protein expression in pancreatic cancer cell line BxPC-3 cells, comparing the results when cells were treated with and without benzaldehyde (500 μM). [Figure 4] FIG. 4 shows photographs of tumor tissues formed by orthotopically transplanting the KPC mouse cell line into the pancreas of a C57BL / 6 mouse. [Figure 5] FIG. 5 shows the results of measuring the volume of tumor tissue obtained in FIG. 4 above. [Figure 6A] FIG. 6A shows the results of analyzing LIN28B protein expression in the tumor tissues obtained in FIG. 4 above. [Figure 6B] FIG. 6B shows the results of analyzing ID1 protein expression in the tumor tissues obtained in FIG. 4 above. [Figure 6C] FIG. 6C shows the results of analyzing EpCAM protein expression in the tumor tissues obtained in FIG. 4 above. [Figure 6D] FIG. 6D shows the results of analyzing E-cadherin protein expression in the tumor tissues obtained in FIG. 4 above. [Figure 7A] FIG. 7A shows the results of tissue staining with anti-EpCAM antibody in lung tissues from the control group of KPC mice obtained in FIG. 4 above. [Figure 7B] FIG. 7B shows the results of tissue staining with anti-EpCAM antibody in lung tissues from the CDBA-administered group of KPC mice obtained in FIG. 4 above. [Figure 7C] FIG. 7C shows an enlarged view of the area marked with a star in FIG. 7A. [Figure 7D] FIG. 7D shows an enlarged view of the area marked with a star in FIG. 7A. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. LIN28B inhibitors of the present invention The present invention provides a LIN28B inhibitor comprising a benzaldehyde compound (benzaldehyde or a derivative thereof) or a pharmaceutically acceptable salt thereof. The LIN28B inhibitor is an agent that suppresses the intracellular expression of the LIN28B protein.

[0015] (1) LIN28B The human LIN28 family includes two paralogs, LIN28A (also referred to simply as "LIN28") and LIN28B. LIN28A and LIN28B are specifically expressed in undifferentiated embryonic stem cells (ESCs), while being dramatically down-regulated in most normal adult tissues (Gewalt, T. et al., Oncology Research 2023 31(2): 101-115).

[0016] LIN28B is an RNA-binding protein that promotes malignant tumorigenesis by inhibiting the maturation of the let-7 miRNA family, a family of tumor suppressors (Gewalt, T. et al., supra). The let-7 miRNA family consists of 12 members: let-7a-1, let-7a-2, let-7a-3; let-7b; let-7c; let-7d; let-7e; let-7f-1, let-7f-2; let-7g; let-7i; and miR-98 (Piskounova, E. et al., Cell 147, 1066-1079, 2011).

[0017] In humans, LIN28A consists of 209 amino acid residues (GenBank accession number: AIC52303.1), the LIN28B long isoform (hereafter simply referred to as "LIN28B") consists of 250 amino acid residues (GenBank accession number: AAZ38897.1), and the LIN28B short isoform consists of 180 amino acid residues. LIN28A and the LIN28B long isoform share approximately 76% amino acid identity and a cold shock domain (CSD) and a CCHC Zn finger (or zinc finger knuckle (ZFK)) domain. Within cells, Lin28A is primarily located in the cytoplasm, while Lin28B is primarily located in the nucleus (Piskounova, E. et al., supra). Furthermore, the short isoform of Lin28B lacks the cold shock domain and has been found to support let-7 miRNA maturation by competitively inhibiting the long isoform of LIN28B (Misuno, R. et al., Mol Cancer Res; 16(3) 2018, 403-415).

[0018] Ectopic expression of LIN28 has been observed in a wide range of advanced tumors, including liver cancer, lung cancer, ovarian cancer, colon adenocarcinoma, and chronic myeloid leukemia. In addition, LIN28A and LIN28B have been reported to be involved in interstitial pneumonia and liver fibrosis, which are related to epithelial-mesenchymal transition (EMT), as well as in the maturation of pancreatic beta cells and diabetes, as well as in diabetic nephropathy, nephrosclerosis, and cardiotoxicity (Zhu, H. et al., Cell. 2011 Sep 30; 147(1): 81-94; Zhou, X. et al., Stem Cell Reports. 2020 Jan 14; 14(1): 9-20; Liang, H. et al., J. Molecular Medicine (2016), vol 94, 655-665; Lekka, E. et al., Nature Communications (2022) vol 13, Article number: 7940; Dadras, F. et al., J. Renal Injury Prevention (2018) 7(1), 1-6; Grande, M. T. et al., (2015) Nature Medicine, 21(9), 989-997; Kardooni, A. et al., Diagnostic & Prognostic Approach. Molecular Biotechnology (2023) vol 65, 1403-1413).

[0019] Recently, new roles for LIN28B have been discovered. For example, it has been shown to promote tumor progression and metastasis in solid tumors. Specifically, the correlation between LIN28B expression and its inhibitor, let-7, suggests that high levels of LIN28B promote cell proliferation, epithelial-mesenchymal transition (EMT), oncogenesis, and angiogenesis in cancer cells, whereas high levels of let-7 promote cell differentiation. Furthermore, it has been shown that undifferentiated cells with high levels of LIN28B and low levels of let-7 can escape immune responses by expressing the immune checkpoint molecule PD-L1 (Gewalt, T. et al., supra). In addition, the proportion of diseases caused by overexpression of LIN28B has been reported to be 8-12% in liver cancer, pancreatic ductal adenocarcinoma, Wilms' tumor, colorectal adenocarcinoma, bladder cancer, and urothelial carcinoma, and approximately 15% in breast cancer (Gewalt, T. et al., supra).

[0020] Furthermore, overexpression of LIN28B has been reported to be a strong predictor of poor prognosis, negatively correlated with clinical outcome and patient survival from primary breast tumors (Kugel, S. et al., Cell 165, 1401-1415, June 2, 2016; Frances, JW et al., Nat Commun. 2020 Jul 3;11(1):3303), and to promote not only the progression but also metastasis of pancreatic ductal adenocarcinoma (Frances, JW et al., Nature Communications 11, Article number: 3303 (2020); Wang, Y. et al., Oncotarget, 2017, Vol.8, No.36, 60414-60428).

[0021] LIN28A, like LIN28B, induces epithelial-mesenchymal transition (EMT). Specifically, LIN28A induces EMT in breast cancer cells through downregulation of let-7a, resulting in higher mammosphere formation rates and increased ALDH activity. It has also been reported that overexpression of LIN28A has been found in metastatic breast cancer and strongly predicts poor prognosis in breast cancer (Liu, Y. et al., PLoS One. 2013 Dec 11;8(12):e83083). The LIN28B inhibitors of the present invention can inhibit LIN28B expression and thus may alleviate or treat poor prognosis in breast cancer.

[0022] As described above, LIN28B and LIN28A have attracted much attention as targets for antitumor agents.

[0023] (2) Epithelial-mesenchymal transition (EMT) Epithelial-to-mesenchymal transition (EMT or E / MT) is a differentiation program commonly observed in higher species, including vertebrates, during the process of epithelial cell differentiation (dedifferentiation). In normal tissues, EMT is observed during development, during gastrulation, and during wound fibrosis. In abnormal tissues, such as cancer, EMT is observed during increased cancer cell motility, migration, invasion, and metastasis. A reverse form of EMT, mesenchymal-epithelial transition (MET), is also known. For example, EMT is thought to be involved in the early stages of cancer cell metastasis, while MET is thought to be involved in the invasion of metastatic sites (Gewalt, T. et al., supra).

[0024] Typical morphological changes that occur during EMT include the following: First, epithelial cells disassemble intercellular junctions, such as tight junctions, adherens junctions, desmosomes, and gap junctions. They lose their apical-basal polarity and acquire an anterior-posterior organization. Next, the actin cytoskeleton is reorganized to form actin stress fibers and cell extensions, such as lamellipodia, filopodia, and invadopodia, enabling directional migration. Finally, the resulting cells acquire the ability to degrade extracellular matrix (ECM) proteins, which promotes invasive behavior. These changes are mediated by complex signaling cascades and transcription factor activity (e.g., Jang, J. et al., Nature Reviews (2020) 21 341-352).

[0025] LIN28B has attracted attention as an intracellular factor involved in the EMT (Gewalt, T. et al., supra). Furthermore, studies using LIN28B knockout mice have shown that LIN28B plays an important role in the metastasis of pancreatic ductal adenocarcinoma (PDAC) (Frances, JW et al., supra). Furthermore, it is known that in squamous cell carcinoma, E-cadherin, EpCAM, claudins, and occludin are elevated in epithelial cell states, while N-cadherin, fibronectin, and vimentin are elevated in mesenchymal cell states (Scanlon, CS et al., J Dent Res 92(2): 114-121, 2013). Therefore, increases or decreases in these cell adhesion molecules can be used as indicators of the EMT or MET state. Furthermore, it is known that important transcription factors involved in EMT include Snai1 (Snail), Snai2 (Slug), Zeb1, Zeb2 (SIP1), and Twist (Jang, J. et al., Nature Reviews (2020) 21 341-352).

[0026] (3) Functions and characteristics of cancer stem cells Tumor tissues are thought to contain cancer stem cells with self-renewal and pluripotency, as well as various types of cancer cells formed by the differentiation of these cancer stem cells. More specifically, cancer stem cells, like normal stem cells, self-renew while also differentiating into various cancer-constituting cells through pluripotency, serving as a "source" of cancer stem cells and cancer cells (Bonnet, D. et al., Nat. Med. 3, 730-737, (1997)). Furthermore, once differentiated, cancer cells can acquire mesenchymal properties through the epithelial-mesenchymal transition (EMT) described above, transforming into cancer stem cells (Clarke, M.F. et al., Cancer Res. 66, 9339-9344, (2006)). Furthermore, cancer stem cells have been reported to be associated with cancer treatment resistance due to their high drug efflux capacity and DNA damage repair capacity (Hanahan, D. et al., Cell 144, 646-674, (2011)). Therefore, inhibiting the functions of cancer stem cells, such as self-renewal, pluripotency, and resistance to therapy, can suppress the source of cancer, be effective in treating cancer, and contribute to the suppression of cancer metastasis and recurrence.

[0027] Regarding cell surface markers associated with cancer stem cells in various cancers, for example, CD24 in human breast cancer - , CD44 + , EpCAM + In human pancreatic cancer, CD24 + , CD44 + , EpCAM + EpCAM in human hepatocellular carcinoma + , CD44 + , CD133 + , CD90 + It is known that: (Clevers, H., Nature medicine, 2011, vol.17, p.316). Among the above markers, for example, EpCAM (epithelial cell adhesion molecule, also known as CD326) is a type I transmembrane glycoprotein that functions as an intercellular adhesion molecule specific to epithelial cells. Because it is highly expressed in various carcinomas, it is also used as a diagnostic marker and prognostic marker for carcinomas (Cancer Treat Rev. 2012 Feb;38(1):68-75, etc.).

[0028] In addition, ID1 (Inhibitor of DNA binding / differentiation) is known to be one of the intracellular factors whose expression levels are elevated in cancer stem cells. In normal cells, ID1 expression is elevated in embryonic stem cells (ESCs) and neural stem cells, where it controls the self-renewal of stem cells. On the other hand, in several types of cancer cells, ID1 is overexpressed, inducing cancer-induced angiogenesis and EMT, as well as conferring resistance to chemotherapy and radiation therapy (Zhao, Z. et al., Int. J. Med. Sci. 2020; 17(8): 995-1005). Human ID1 is 155 amino acids long (GenBank accession number: P41134.3) and contains a helic-loop-helic (HLH) structure within the molecule. It is known to inhibit the function of various transcription factors with basic helic-loop-helic (bHLH) motifs (e.g., MyoD, myogenin, Myf5, MRF4) by acting in a dominant-negative manner (e.g., Benezra, R. et al., Cell, 61(1), 49-59 (1990)). Regarding the mechanism and action of ID1 in various cancer types, for example, it is known to promote epithelial-mesenchymal transition (EMT) and metastasis in breast cancer by inhibiting cyclin D1 and S100A9 (Tobin, NP et al., BMC Cancer 2011, 11:417; Gumireddy, K. et al., Mol Cancer Res; 12(9), 1334-43 (2014)). Therefore, ID1 has been shown to be a useful target for antitumor agents (Zhao, Z. et al., Int. J. Med. Sci. 2020; 17(8): 995-1005, etc.).

[0029] (4) LIN28B protein inhibitor As used herein, "LIN28B protein activity" refers to an action involving LIN28B protein, such as the action of binding to Pri-let-7 miRNA and inhibiting its conversion to Pre-let-7 miRNA, or the action of binding to Pre-let-7 miRNA and inhibiting its conversion to mature let-7 miRNA, the action of inhibiting the formation of mature let-7 miRNA so as not to inhibit the translation of target mRNAs of let-7 miRNA (mRNAs such as SOX2, HMGA2, OCT4, and IGFBP1), and the regulation of intracellular signal transduction.

[0030] As used herein, the terms "LIN28B protein inhibitor," "LIN28B inhibitor," or "inhibitor" relating to LIN28B mean suppressing expression such as transcription or translation of LIN28B protein, suppressing the mature let-7 miRNA formation function of LIN28B protein, etc. As used herein, "suppressing" the above-mentioned expression, activity, etc. relating to an "inhibitor" means that, for example, the LIN28B expression level, such as the amount of LIN28B mRNA transcription or the amount of LIN28B protein translation, or the protein activity level, is reduced by 1% or more, preferably 5% or more, more preferably 10% or more, even more preferably 20% or more, and even more preferably 30% or more, compared to when the inhibitor is not used. LIN28B protein and LIN28B mRNA can be detected by procedures known to those skilled in the art, such as Western blot and RT-qPCR.

[0031] As used herein, the term "anticancer agent" refers to an agent used for the purposes of tumor reduction, suppression of tumor cell proliferation, prevention of cancer metastasis, prevention of cancer recurrence, prevention of carcinogenesis, etc. Preferably, the anticancer agent of the present invention is a drug that exerts anticancer activity by inhibiting LIN28B protein activity.

[0032] The structure of the benzaldehyde (BA) compound as a LIN28B protein inhibitor preferably used in the present invention is not particularly limited as long as it has the inhibitory function. For example, the benzaldehyde (BA) compound may be represented by the following general formula (I):

[0033] [ka] (In the formula, R 1 -CHO, -CXO (where X is a halogen atom), dioxolanyl group, dioxanyl group, -CH=NR 3 (R 3 is a lower alkyl group), [ka] R 2are each independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkenyl group, a lower alkynyl group, or a lower alkoxy group; and n is an integer of 1 to 5. Here, the "dioxolanyl group" refers to a 5-membered heterocyclic group having two oxygen atoms as heteroatoms, preferably a 1,3-dioxolanyl group in which the carbon atom at position 2 is bonded to the phenyl group of formula (I). The remaining atoms at positions 4 and 5 may be unsubstituted or substituted with one or more halogen atoms, alkyl groups, alkenyl groups, alkoxy groups, hydroxyl groups, etc. Here, the "dioxanyl group" refers to a 6-membered heterocyclic group having two oxygen atoms as heteroatoms, and is preferably a 1,3-dioxanyl group in which the carbon atom at position 2 is bonded to the phenyl group of formula (I). The remaining atoms at positions 4 to 6 may be unsubstituted or substituted with one or more halogen atoms, alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, hydroxyl groups, etc. Here, the term "halogen atom" refers to a group derived from a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. Moreover, "lower" means having 1 to 6 carbon atoms.

[0034] As used herein, the term "alkyl group" means a monovalent straight-chain or branched-chain hydrocarbon group, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, and the like.

[0035] As used herein, the term "alkenyl group" refers to monovalent straight-chain and branched-chain hydrocarbon groups containing a double bond, including, but not limited to, ethenyl, 1- and 2-propenyl, 2-methyl-1-propenyl, 1- and 2-butenyl, and the like. As used herein, the term "alkynyl group" refers to monovalent straight-chain and branched-chain hydrocarbon groups containing a triple bond, including, but not limited to, ethynyl, propynyl, butynyl, and the like.

[0036] As used herein, the term "alkoxy" refers to -O-alkyl, including, but not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, and the like.

[0037] The compounds of general formula (I) are known substances or can be synthesized from known starting materials using various known reactions. For example, in the compound of general formula (I), R 1 is -CHO and R 2 The compound in which R are all hydrogen atoms is benzaldehyde, which is a known compound. The anticancer activity of benzaldehyde is described, for example, in Japanese Patent Publication No. 54-962. In the compound of general formula (I), R 1 but [ka] and;R 2 The compound in which all of are hydrogen atoms is 5,6-O-benzylidene-L-ascorbic acid, which is also known. A method for producing 5,6-O-benzylidene-L-ascorbic acid is described in Steroids, Vol. 12, p. 309 (1968), and other methods are disclosed in Japanese Patent Publication No. 3-33127 and U.S. Patent No. 5,036,103. Furthermore, the anticancer activity of 5,6-O-benzylidene-L-ascorbic acid or its sodium, potassium, and calcium salts, and methods for producing these salts are described in Japanese Patent Publication No. 3-33127 and U.S. Patent No. 5,036,103.

[0038] In the compounds of general formula (I), R 1 but [ka] and R 2The compound in which all of are hydrogen atoms is 4,6-benzylidene-D-glucopyranose, which is also a known compound. The structure, anticancer activity, etc. of this compound are described, for example, in Japanese Patent Publication No. 10685 / 1988. In the compounds of general formula (I), R 1 Ga-CN-R 3 (R 3 is an ethyl group), and R 2 A compound in which all of are hydrogen atoms is N-benzylideneethylamine. The anticancer activity of this compound is described, for example, in Japanese Patent Publication No. 54-70428. Compounds of general formula (I) other than the above compounds can be synthesized by taking into consideration the common knowledge of synthetic chemists and the like, in addition to the synthesis method for benzaldehyde compounds described in the above document.

[0039] Preferred benzaldehyde compounds for use in the present invention include benzaldehyde, 5,6-O-benzylidene-L-ascorbic acid, 4,6-O-benzylidene-D-glucopyranose, N-benzylideneethylamine, 2-phenyl-1,3-dioxolane, 2-phenyl-4-methyl-1,3-dioxolane, and 2-phenyl-1,3-dioxane. More preferred benzaldehyde compounds for use in the present invention are benzaldehyde and 5,6-O-benzylidene-L-ascorbic acid. The structural formulas of 2-phenyl-1,3-dioxolane, 2-phenyl-4-methyl-1,3-dioxolane, and 2-phenyl-1,3-dioxane are as follows: [ka]

[0040] When the active ingredient of the pharmaceutical composition of the present invention has at least one asymmetric center, it may exist in various optical isomers or configurations. Thus, the compounds of the present invention may exist as separate optically active (+) and (-) isomers, as well as racemates or (±) mixtures. Furthermore, in the case of compounds with two or more asymmetric centers, diastereomers of each optical isomer may also exist. The present invention encompasses all of these forms. For example, diastereomers can be separated by methods well known to those skilled in the art, such as fractional crystallization, and optically active isomers can be obtained by well-known organic chemistry techniques for this purpose.

[0041] Furthermore, the benzaldehyde compound, which is an active ingredient of the present invention, can be converted into a "pharmaceutically acceptable salt" if necessary. Examples of "pharmaceutically acceptable salts" include mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, sulfate, and nitrate; sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate; and acid addition salts such as oxalate, tartrate, citrate, maleate, succinate, acetate, benzoate, mandelate, ascorbate (e.g., sodium ascorbate, potassium ascorbate, etc.), lactate, gluconate, and malate; and organic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, methanesulfonate, p-toluenesulfonate, oxalate, tartrate, citrate, acetate, and lactate. In the present invention, sodium or potassium 5,6-O-benzylidene-L-ascorbate is a preferred salt form.

[0042] Furthermore, the benzaldehyde compound, which is the active ingredient of the present invention, may absorb water or become a hydrate by adsorbing water when freeze-dried from an aqueous solution or by recrystallization, and such salts are also included in the active ingredient of the present invention. Furthermore, the benzaldehyde compound can be used in the form of an inclusion compound containing the benzaldehyde compound using choleric acid, cyclodextrin, or the like. Preferably, the inclusion compound of the benzaldehyde compound is CDBA (β-cyclodextrin compound benzaldehyde: a β-cyclodextrin inclusion compound of benzaldehyde). Therefore, a LIN28B inhibitor containing an inclusion compound containing a benzaldehyde compound is also one embodiment of the LIN28B inhibitor containing the benzaldehyde compound according to the present invention. The LIN28B inhibitor according to the present invention (or the benzaldehyde compound or its inclusion compound) can be used as an active ingredient in pharmaceutical compositions, nutritional compositions, foods (e.g., nutritional supplements), supplements, etc., depending on the application.

[0043] 2. Compositions containing LIN28B inhibitors The LIN28B inhibitor of the present invention or a composition containing the same can be used to prevent or treat various diseases associated with LIN28B protein activity by inhibiting the expression, activity, etc. of LIN28B protein. Diseases associated with LIN28B protein activity include, for example, various cancers, diabetes, nephropathy, renal fibrosis, nephrosclerosis, diabetic nephropathy, cirrhosis, nonalcoholic fatty liver disease, idiopathic interstitial pneumonia, and anthracycline anticancer drug-induced myocardial injury. Specific cancers to be treated include, for example, liver cancer, pancreatic cancer, lung cancer, ovarian cancer, colon adenocarcinoma, chronic myeloid leukemia, pancreatic ductal adenocarcinoma, malignant renal tumor, Wilms' tumor, breast cancer, melanoma, gastric cancer, prostate cancer, cholangiocarcinoma, gallbladder cancer, head and neck cancer, osteosarcoma, diffuse midline glioma, glioblastoma, medulloblastoma, glioma, diffuse large B-cell lymphoma, follicular lymphoma, multiple myeloma, thyroid cancer, uterine cancer, EGFR-TKI therapy-resistant cancer, radiation therapy-resistant cancer, and metastatic cancers and prognosis states of these cancers. Therefore, the LIN28B inhibitor of the present invention or a composition containing the same suppresses epithelial-mesenchymal transition (EMT) of cancer cells in the above-mentioned cancers, and can therefore suppress metastasis of the above-mentioned cancers, and can also be used for the treatment or prevention of poor prognosis of the above-mentioned cancers. Furthermore, because LIN28B inhibits the formation of let-7 miRNA, the LIN28B inhibitors of the present invention are useful for treating diseases associated with abnormalities in let-7 miRNA, such as brain diseases, neurodegenerative diseases, cardiovascular diseases (such as stroke), arteriosclerosis, inflammation, diabetes, immune disorders, and Alzheimer's disease (Bernstein, DL et al., Biomedicines 2021).

[0044] In addition to the above-mentioned anticancer activity and antiviral activity, the LIN28B inhibitor of the present invention or a composition containing the same has other properties, such as antioxidant activity (see Japanese Patent Application Laid-Open No. 8-99880); anti-HIV activity (see Japanese Patent Application Laid-Open No. 8-3038); prophylactic and therapeutic activity against influenza (see Japanese Patent Application Laid-Open No. 8-217675); and therapeutic activity against neurodegenerative diseases (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar degeneration, multiple sclerosis, Creutzfeldt-Jakob disease, progressive multifocal leukoencephalopathy, dementia with Lewy bodies). , corticobasal degeneration, amyotrophic lateral sclerosis, Parkinson's disease, Parkinson's syndrome, spinocerebellar degeneration, spastic paraplegia, spinocerebellar degeneration, multiple sclerosis, progressive multifocal leukoencephalopathy, non-herpetic acute limbic encephalitis, etc.), epilepsy, heart disease, or diabetic disease (see JP 2017-43616 A), and has anti-cancer activity and antiviral activity due to AXL inhibition (see JP 2022-171109 A), and can therefore be effectively used as an ingredient in nutritional compositions, supplements, and foods and drinks (foods, beverages, etc.).

[0045] As used herein, "foods and beverages" includes health foods, functional foods, foods for specified health uses, and other foods intended to promote health, as well as all foods to which the LIN28B inhibitors of the present invention can be added or formulated. Examples of foods and beverages of the present invention include tablets, powders, granules, energy drinks, liquid diets, tea drinks, soft drinks, dairy drinks, butter, mayonnaise, shortening, margarine, various salad dressings, breads, noodles, pasta, confectioneries such as cookies, chocolate, candy, and chewing gum. The foods and beverages of the present invention can be prepared according to conventional methods by blending other food ingredients, various nutrients, various vitamins, minerals, dietary fiber, and various additives, such as taste-imparting components, sweeteners, acidulants such as organic acids, stabilizers, and flavors, which are used in the preparation of the foods and beverages.

[0046] When the LIN28B inhibitor of the present invention is used in a supplement (also referred to as a "supplement composition"), the supplement formulation can be designed and manufactured based on known techniques. The supplement can be formulated into any form together with auxiliary agents and excipients by known methods and then orally ingested (administered). Examples of the formulation include granules, tablets, and capsules. The supplement of the present invention may contain one or more ingredients selected from GABA, glucosamine, biotin, DHA, EPA, lutein, vitamins, coenzyme Q10, minerals, calcium, lactic acid bacteria, bifidobacteria, sesamin, soy isoflavones, collagen, and placenta.

[0047] Various ingredients can be added when formulating supplements. Examples of such additives include dietary fiber and thickeners derived from sources other than vegetables, such as hemicellulose, lignin, guar gum, konjac mannan, isagol, alginic acid, agar, carrageenan, chitin, carboxymethylcellulose, and polydextrose; edible oils; minerals, such as calcium, iron, sodium, zinc, copper, potassium, phosphorus, magnesium, iodine, manganese, and selenium; fat-soluble or water-soluble vitamins, such as vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, niacin, folic acid, and pantothenic acid; glycerin, fatty acid esters, and the like. These include emulsifiers and dispersants such as cellulose, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, phospholipids, gum arabic, xanthan gum, tragacanth gum, and locust bean gum, bulking agents, excipients, preservatives and antioxidants, flavor adjusters and fragrances, flavorings such as sodium chloride, monosodium glutamate, glycine, succinic acid, and sodium lactate, acidulants such as citric acid, sodium citrate, acetic acid, adipic acid, fumaric acid, and malic acid, low-calorie sweeteners such as maltitol and aspartame, coloring agents, and calcium.

[0048] 3. Medicines, pharmaceutical compositions, supplement compositions, or combinations containing LIN28B inhibitors Anticancer drugs have diverse mechanisms of action. For this reason, while an anticancer drug with a certain mechanism of action is effective for a certain patient, an anticancer drug with a different mechanism of action may not be effective. This is because the mechanism of action of the anticancer drug used may not act effectively on cancer cells, taking into account the patient's genetic background, etc. Therefore, another embodiment of the present invention also provides an anticancer drug composition containing the LIN28B inhibitor of the present invention, for administration to a patient who has been treated with an anticancer drug whose mechanism of action is not involved in the expression or activity of the LIN28B protein.

[0049] The LIN28B inhibitors of the present invention regulate intracellular functions by inhibiting the expression and / or activity of LIN28B protein, thereby forming mature let-7 miRNA. Therefore, they can be used in combination with anticancer drugs that are not involved in the activity of LIN28B protein or let-7 miRNA, as combined drugs or concomitant drugs. A combined drug is a pharmaceutical product administered to a patient by combining two or more components, and each component can be administered simultaneously or separately. For the dosage and administration of known anticancer drugs used in combination, please refer to the package insert of the anticancer drug.

[0050] According to another embodiment of the present invention, the anticancer composition of the present invention can be used in combination with (a) the LIN28B inhibitor of the present invention and (B) an anticancer agent having a mechanism of action that is not involved in the expression and / or activity of the LIN28B protein.

[0051] Drugs that can be used in combination with the LIN28B inhibitor of the present invention include, for example, EGFR inhibitors or EGFR signaling inhibitors (e.g., gefitinib, erlotinib, osimertinib; Cell Death and Disease (2019) 10:361), anti-PD-1 antibodies (Oncotarget, 2017, Vol. 8, (No. 52), pp: 89761-89774), temozolomide (CNS Neurosci Ther. 2020;26:777), paclitaxel (Mol Cancer Ther; 16(12), pp: 2881-2891, December 2017), kinase inhibitors (sorafenib (British Journal of Cancer 120, 512-521 (2019)) and sunitinib ( (Oncogene 35, 2687-2697 (2016)) etc., but are not limited to these.

[0052] According to a preferred embodiment of the present invention, (a) a preferred LIN28B inhibitor is benzaldehyde or 5,6-O-benzylidene-L-ascorbic acid; and (b) an anticancer drug component used in combination therewith, such as gefitinib, irinotecan hydrochloride, topotecan hydrochloride, docetaxel, paclitaxel, vinblastine sulfate, vincristine sulfate, vindesine sulfate, etoposide, teniposide, vinorelbine tartrate, busulfan, carboquone, thiotepa, or cinnamoyl benzoate. Clofosfamide, melphalan, estramustine sodium phosphate, mechlorethamine oxide hydrochloride, ifosfamide, ranimustine, nimustine hydrochloride, bleomycin hydrochloride, peplomycin sulfate, zinostatin stimarat, actinomycin D, aclarubicin hydrochloride, doxorubicin hydrochloride, idarubicin hydrochloride, amrubicin hydrochloride, daunorubicin hydrochloride, pirarubicin, epirubicin hydrochloride, mitomycin, valrubicin, methotrexate , mercaptopurine, fludarabine phosphate, cladribine, fluorouracil, tegafur, cytarabine, gemcitabine hydrochloride, cytarabine ocfosfate, capecitabine, doxifluridine, carmofur, enocitabine, nedaplatin, carboplatin, cisplatin, fadrozole hydrochloride, anastrozole, exemestane, bicalutamide, flutamide, tamoxifen citrate, toremifene citrate, tretinoin, pentostatin , L-asparaginase, dacarbazine, procarbazine hydrochloride, mitoxantrone hydrochloride, sobuzoxane, trastuzumab, rituximab, imatinib mesylate, 5-fluoro-2'-deoxyuridine, ascleoside, carbocririne, quinolespan, krestin, erlotinib, aquatinib, dacomitinib, osimertinib, sorafenib, sunitinib, bortezomib, oxaliplatin, and picibanil. According to an alternative preferred embodiment of the present invention, (a) the preferred LIN28B inhibitor is benzaldehyde or 5,6-O-benzylidene-L-ascorbic acid; and (b) the anticancer drug component used in combination therewith includes, but is not limited to, fulvestrant, letrozole, finasunate, platins, finasunate, rapamycin, leucovorin, lapatinib, lonafarnib, camptothecin, bryostatin, adezelesin, anthracycline, carzelesin, bizelesin, dolastatins, auristatins, duocarmycins, eleutherobin, taxol, vincristine, prednisone or prednisolone, other alkylating agents (e.g., mechlorethamine, chlorambucil), antimetabolites (e.g., azathioprine), other microtubule inhibitors (e.g., vinca alkaloids such as taxanes), podophyllotoxins (etoposide phosphate and epipodophyllotoxin), topoisomerase inhibitors, other cytotoxins (e.g., actinomycin, edrecolomab, plicamycin), and pharmaceutically acceptable salts of the above agents, and Anti-cancer antibodies (e.g., cetuximab, bevacizumab, ibritumomab, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, anatumomab, apolizumab, bavituximab, belimumab, bivatuzumab mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, catumaxomab, cetuximab, sitatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan tetraxetan), conatumumab, dacetuzumab, daclizumab, detumomab, ecromeximab, edrecolomab, elotuzumab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, fresolimumab, galiximab, glenbatumumab vedotin, gemtuzumab, ibri Tumomab tiuxetan, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab, lumilisimab, mapatumumab, matuzumab, milatuzumab, mitumomab, nacolomab butafenatox tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, ofatumumab, olaratumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, lobatumumab, sibrotuzumab, tacatuzumab tetraxetan, taplitumomab paptoxpaptox), tenatumomab, ticilimumab, tigatuzumab, tositumomab or modified products thereof, tremelimumab, tuocuzumab-celmoleukin, veltuzumab, visilizumab, volociximab, votumumab, zalutumumab, zanolimumab, IGN-101, MDX-010, ABX-EGR, EMD72000, ior-t1, MDX-220, MRA, H-11 scFv, huJ591, TriGem, TriAb, R3, MT-201, G-250, ACA-125, Onyvax-105, CD:-960, Cea-Vac, BrevaRex AR54, IMC-1C11, GlioMab-H, ING-1, anti-LCG MAb, MT-103, KSB-303, Therex, KW2871, anti-HMI.24, anti-PTHrP2C4 antibody, SGN-30, TRAIL-RI MAb, prostate cancer antibody, H22xKi-r, ABX-Mai, Imuteran, Monopharm-C), and conjugates of the above drugs with antibodies (e.g., auristatins MMAE and MMAF, maytansinoids such as DM-1, calicheamicin, or various cytotoxins). When the cancer metastasis inhibitor according to the present invention is used, it can be used in combination with known anticancer agents such as those mentioned above.

[0053] Preferably, the EGFR inhibitor that can be used in combination with a BA in the present invention is one or more inhibitors selected from, for example, osimertinib, gefitinib, erlotinib, aquatinib, dacomitinib, and pharmaceutically acceptable salts thereof. Preferably, the immune checkpoint inhibitor that can be used in combination with a BA in the present invention is one or more inhibitors selected from, for example, nivolumab (trade name: Opdivo), pemprolizumab (trade name: Keytruda), and pharmaceutically acceptable salts thereof.

[0054] Furthermore, since the inhibitor of the present invention exerts anticancer activity by regulating the expression and / or activity of LIN28B protein, it can be used as an effective anticancer agent for patients who have been treated with anticancer agents having a mechanism of action that is not involved in the expression and / or activity of LIN28B protein but have not achieved sufficient therapeutic effects. Furthermore, the inhibitor according to the present invention is effective in preventing cancer metastasis and recurrence by regulating the expression and / or activity of the LIN28B protein. The cancer metastasis inhibitor (or composition) according to the present invention may be administered to a patient, for example, in an amount sufficient to inhibit cancer metastasis, simultaneously with an anticancer drug containing the above-mentioned component, in a regimen subsequent to a treatment regimen using an anticancer drug containing the above-mentioned component, or alternately with an anticancer drug containing the above-mentioned component. Such an administration regimen may be appropriately determined by the attending physician, taking into consideration the condition of the patient to be treated.

[0055] 4. Formulation of the inhibitor, cancer metastasis suppressor, or composition containing the same of the present invention The administration or ingestion form of the LIN28B inhibitor, cancer metastasis inhibitor, composition, or anticancer composition according to the present invention is not particularly limited, and they can be administered or ingested orally or parenterally. The composition containing the LIN28B inhibitor according to the present invention can be used as a pharmaceutical composition, nutritional composition, food, supplement, etc. The benzaldehyde compound used in the present invention may be formulated alone, or may be provided in the form of a desired composition product obtained by blending it with a pharmaceutically acceptable carrier, a formulation additive, a food additive, a supplement carrier or additive, etc. In this case, the benzaldehyde compound used in the present invention may be contained in an amount of 0.1 to 99.9 wt % in products such as formulations, foods, and supplements.

[0056] In the present invention, when an anticancer composition is prepared, the above-mentioned other anticancer agents can be further blended. In this case, the blending ratio of the benzaldehyde compound to the other anticancer agents is appropriately selected depending on the type of cancer, the patient's age and symptoms, the administration route, the purpose of treatment, etc., and can be selected, for example, within the range of 1:99 to 99:1. The pharmaceutical composition of the present invention can also be blended with two or more other anticancer agents. In this case, the blending ratio is also appropriately selected depending on the type of cancer, the patient's age and sex, the severity of symptoms, the administration route, the purpose of treatment, etc.

[0057] Examples of pharmaceutically acceptable carriers or additives, food additives, and supplement carriers or additives that can be used include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, pigments, diluents, solubilizers, solubilizers, isotonicity agents, pH adjusters, and stabilizers.

[0058] Examples of products suitable for oral administration include powders, tablets, capsules, microcapsules, fine granules, granules, liquids, and syrups. For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, and glycine can be used, along with starch, preferably corn, potato, or tapioca starch, and various disintegrants such as alginic acid and certain double silicates, and granulation binders such as polyvinylpyrrolidone, sucrose, gelatin, and gum arabic. Lubricants such as magnesium stearate, sodium lauryl sulfate, and talc are often very effective for tableting. Similar solid compositions can also be used by filling gelatin capsules. Suitable materials for this purpose include lactose or milk sugar, as well as high-molecular-weight polyethylene glycols. When aqueous suspensions and / or elixirs for oral administration are desired, the active ingredient may be combined with various sweeteners or flavorings, coloring agents or dyes, and, if necessary, emulsifying agents and / or suspending agents, and diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof may be used.

[0059] Because benzaldehyde compounds can be irritating, they are preferably used in the form of an inclusion compound containing a benzaldehyde compound using known techniques that utilize the inclusion ability of choleric acid, cyclodextrin, or the like (see, for example, Japanese Patent Application Laid-Open No. 55-038338). To impart stability and acid resistance to the inclusion compound for long-term storage and fully maintain its efficacy, a pharmaceutically acceptable coating can be applied to the inclusion compound and formulated into an anticancer drug composition with excellent stability. A cyclodextrin inclusion compound containing a benzaldehyde compound (cyclodextrin-encapsulated benzaldehyde compound (hereinafter sometimes abbreviated as "CDBA") can be obtained by, for example, adding 2 to 7 g of the compound to 1 liter of saturated aqueous β-cyclodextrin solution, mixing, and stirring for 3 to 7 hours, resulting in the precipitation of an inclusion compound. The precipitate can be dried under reduced pressure to obtain an inclusion compound of a benzaldehyde compound.

[0060] Formulations suitable for parenteral administration include, for example, injections and suppositories. For parenteral administration, the active ingredient of the present invention can be dissolved in either sesame oil or peanut oil, or in aqueous propylene glycol. The aqueous solutions should be suitably buffered (preferably to a pH of 8 or higher), if necessary, and the liquid diluent should first be rendered isotonic. Such aqueous solutions are suitable for intravenous injection, while oily solutions are suitable for intraarticular, intramuscular, and subcutaneous injection. The preparation of all of these solutions under sterile conditions can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art. Additionally, the active ingredient of the present invention can be administered topically, such as to the skin. In this case, topical administration is preferably carried out in the form of a cream, jelly, paste, or ointment, according to standard pharmaceutical practice.

[0061] The dose of the inhibitor, cancer metastasis suppressor, or composition of the present invention is not particularly limited, and an appropriate amount can be selected depending on various conditions, such as the condition of the subject (e.g., patient), the subject's age and symptoms, the route of administration, and the purpose of treatment. For example, in the case of oral administration, a dose in the range of 10 mg to 20 g, 100 mg to 10 g, and preferably 150 mg to 5 g per day for an adult (e.g., weighing 60 kg) can be applied. These daily doses may be administered, for example, in two to four divided doses. When the inhibitor or composition of the present invention is used as a nutritional supplement or a supplement, the dosage can be appropriately determined with reference to the above dosage. [Example]

[0062] The present invention will be explained in more detail below based on examples and reference examples, but the present invention is not limited to these examples in any way.

[0063] 1. Gene expression analysis by RT-qPCR LIN28B gene expression analysis in lung cancer cell line A549 cells 2 × 10 human lung cancer cell line A549 cells were placed in each well of a 6-well plate. 5 After 20 hours, cells were compared between two groups: 1) a group treated with DMSO, sealed with a seal, and cultured in a 1% O2 hypoxic incubator for 20 hours; and 2) a group treated with benzaldehyde (Tokyo Chemical Industry) (500 μM), sealed with a seal, and cultured in a 1% O2 hypoxic incubator for 20 hours. Because benzaldehyde (BA) is volatile, each well was sealed to maintain airtightness. Total RNA was isolated from cells in each well using TRIzol Reagent (Invitrogen) and treated with DNase I. A portion of the resulting RNA (approximately 500 ng) was reverse transcribed (RT) using the Two-Step cDNA synthesis method. Quantification by real-time PCR analysis was performed in triplicate using SYBR Premix Ex Taq II (Takara) and a Thermal Cycle Dice TP800 (Takara). The primers used were Lin28b Fw 5'-CATCTCCATGATAAACCGAGAGG-3' (SEQ ID NO: 1) and Lin28b Rv 5'-GTTACCCGTATTGACTCAAGGC-3' (SEQ ID NO: 2). The results of LIN28B expression analysis by RT-qPCR are shown in Figure 1. It was shown that benzaldehyde administration significantly suppressed LIN28B transcription. **P<0.01 (Student's t-test).

[0064] 2. Protein expression analysis by Western blot (1) LIN28B protein expression analysis in lung cancer cell line A549 cells In each well of a 6-well plate, 2 × 10 lung cancer cell line A549 cells were added. 5 After 20 hours, the following groups were compared: 1) an untreated group; 2) a group administered DMSO, sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 2 hours; 3) a group administered benzaldehyde (500 μM), sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 2 hours; 4) a group administered DMSO, sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 20 hours; and 5) a group administered benzaldehyde (500 μM), sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 20 hours. Extracts from each cell were collected in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 10% SDS, 5% glycerol, 5% β-mercaptoethanol, 10% bromophenol blue), and SDS-PAGE and Western blotting were performed according to standard procedures. The primary antibody used was LIN28B Antibody (1:1000 dilution; catalog #4196; Cell Signaling). The results of LIN28B protein expression analysis by Western blot in each group are shown in Figure 2. It was confirmed that LIN28B protein expression was suppressed by benzaldehyde administration in the 2-hour and 20-hour groups.

[0065] (2) Analysis of LIN28B protein expression in pancreatic cancer cell line BxPC3 cells 2 × 10 human pancreatic cancer cell line BxPC3 cells were placed in each well of a 6-well plate. 5 After 20 hours, the following groups were compared: 1) an untreated group; 2) a group administered DMSO, sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 2 hours; 3) a group administered benzaldehyde (500 μM), sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 2 hours; 4) a group administered DMSO, sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 20 hours; and 5) a group administered benzaldehyde (500 μM), sealed with a tape, and cultured in a 1% O2 hypoxic incubator for 20 hours. Each cell extract was recovered in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 10% SDS, 5% glycerol, 5% β-mercaptoethanol, and 10% bromophenol blue), and SDS-PAGE and Western blotting were performed according to standard procedures. The primary antibody used was anti-LIN28B antibody (1:1000 dilution; catalog #4196; Cell Signaling). The results of LIN28B protein expression analysis by Western blot in each group are shown in Figure 3. It was confirmed that LIN28B expression was suppressed by benzaldehyde administration in the 2-hour and 20-hour groups.

[0066] 3. Animal experiments using the KPC mouse cell line (1) Measurement of pancreatic tumor volume and evaluation of treatment efficacy All experiments using mice were approved by the Keio University Animal Care and Use Committee (approval number 19036) and further approved by the Genetic Modification Experimental Protocol Review Board (protocol number D2019-013, accession number D000239), and were conducted in accordance with established guidelines. KPC cells (C57 / BL6 genetic background, Pdx1-Cre, lox-stop-lox-KrasG12D / +, lox-stop-lox-tp53R172H / +; catalog #153474; Cancer Research UK, Glasgow Beatson Institute), derived from a pancreatic cancer model mouse, were cultured at 5 × 10 4 Twelve 7-week-old female C57BL / 6 mice were orthotopically transplanted with 1000 cells each into the pancreatic tissue via laparotomy. These 12 mice were divided into four groups of three mice each. Three days after the transplant, two of the four groups were randomly selected as treatment groups by a third party and administered CDBA (β-cyclodextrin compound benzaldehyde) intraperitoneally at 40 mg / kg / day, 6 days per week. On day 25 after the transplant, five mice from each treatment group (one mouse in the treatment group died 4 days after the transplant) and six mice from the control group were dissected, and tumor tissue was excised (Figure 4), and the pancreatic tumor volume was measured (Figure 5). For comparisons between the treatment and control groups, Student's t-test was used to test for significance of the quantitative data. A P value of 0.0006 indicated a statistically significant difference. The results of this experiment showed a significant inhibitory effect on pancreatic tumor volume in the CDBA-treated group (Fig. 5). Therefore, it was found that BA compounds such as CDBA are effective in reducing pancreatic tumor volume.

[0067] (2) Evaluation of the inhibitory effect of LIN28B and other genes in pancreatic tumors by immunohistochemistry Tumor tissues from the treated and control mice were immunohistochemically stained using anti-LIN28B antibody (1:50 dilution; catalog #16178-1-AP; Proteintech), anti-EpCAM antibody (1:100 dilution; catalog #ab71916; Abcam), anti-ID1 antibody (1:200 dilution; catalog #ab203202; Abcam), and E-cadherin antibody (1:200 dilution; catalog #610181; BD Biosciences) according to the manufacturer's protocol. The results are shown in Figure 6A-D. In the control group, cells with high expression of LIN28B, ID1, EpCAM, and E-cadherin were distributed exclusively around tumor blood vessels. Therefore, it is speculated that the cells around tumor blood vessels in the control group exhibit a hybrid E / M phenotype. On the other hand, in the CDBA-treated group, the expression of LIN28B, ID1, EpCAM, and E-cadherin was all suppressed in the cells around tumor blood vessels. When highly expressed in cancer cells, LIN28B is thought to promote cell proliferation, epithelial-mesenchymal transition (EMT), tumor formation, and angiogenesis (see above). Therefore, reducing the expression of LIN28B in cancer cells may be effective in suppressing these events. When ID1 is overexpressed in cancer cells, it is thought to induce angiogenesis and EMT, and also cause resistance to chemotherapy and radiation therapy (see above). Therefore, reducing ID1 expression in cancer cells may contribute to suppressing metastasis and treatment resistance based on these events. If EpCAM is a marker for cancer cells or cancer stem cells, as mentioned above, a decrease in its expression level may indicate a decrease in cancer stem cells. Furthermore, a decrease in cell adhesion molecules such as EpCAM is thought to indicate a decrease in cells in the epithelial mesenchymal plasticity (EMP) state (a partial EMT state in which epithelial cells (E) and mesenchymal cells (M) are mixed), which is thought to be particularly involved in the E / MT process, particularly in metastatic potential (Jang, J. et al., Nature Reviews (2020) 21 341-352). Furthermore, E-cadherin is normally expressed on the cell membrane surface of epithelial cells, but in the EMP state, it loses its original polarity and is expressed in areas other than the membrane surface. In the control group, high expression of E-cadherin that had lost polarity was confirmed in round cells with reduced cell adhesion, which are thought to be cells in the EMP state. In the BA-treated group, the reduction in E-cadherin is thought to indicate a decrease in EMP state cells.

[0068] (3) Evaluation of metastatic lesions using EpCAM in lung tissue by immunohistochemistry Lung tissues from the treated and control mice were immunohistochemically stained using an anti-EpCAM antibody (1:100 dilution; catalog #ab71916; Abcam), which is highly expressed in lung metastases, according to the manufacturer's protocol. The results are shown in Figures 7A-7D and Table 1 below. Numerous spots (lesions) stained with anti-EpCAM antibodies were observed in lung tissue from the control group. In particular, numerous hematogenous metastases (circled by solid circles) and pleural disseminations (circled by dotted circles) were observed (Figure 7A, Table 1 below). Figure 7C shows a magnified view of hematogenous metastases (★), and Figure 7D shows a magnified view of pleural disseminations (☆). Hematogenous metastases exhibited round, oval, or lobular EpCAM-positive cells with smoothly demarcated borders. Pleural disseminations exhibited EpCAM-positive cells that spread to the pleural surface from floating cells present in the exudate, implanted on the pleural surface, or spread as a thick crust through continuous or skip lesions. (Reference: Locatelli et al., "Mechanisms of Tumor Dissemination in Thoracic Neoplasms," "The Thorax, Cancer Dissemination Pathways," pages 1-33, Springer Nature Switzerland AG 2020) On the other hand, no hematogenous metastatic lesions were observed in the lung tissue of the CDBA-treated group, and only three pleural dissemination lesions were observed (Figure 7B, Table 1 below). As shown in the foci counts in Table 1 below, CDBA treatment clearly reduced hematogenous pulmonary metastatic lesions and pleural dissemination lesions, demonstrating the inhibitory effect of CDBA on cancer metastasis. [Table 1] These results from animal experiments using the KPC mouse cell line showed that CDBA suppressed the expression of LIN28B, ID1, and EpCAM, and inhibited lung metastasis from cancer tissue transplanted into the pancreas.

[0069] Therefore, it was found that BA compounds such as CDBA have the effect of suppressing the hybrid E / M phenotype of cancer cells in tumor tissues, particularly cancer cells around tumor blood vessels, i.e., they have the effect of suppressing EMT, inhibiting cancer metastasis, and even suppressing the function of cancer stem cells. These are thought to provide effects such as suppressing metastasis, treatment resistance, and recurrence of cancers such as pancreatic tumors. [Industrial Applicability]

[0070] The LIN28B inhibitor of the present invention or a composition containing the same can be used to prevent or treat various diseases associated with LIN28B protein activity (e.g., various cancers) by inhibiting the expression and / or activity of LIN28B protein. Furthermore, the benzaldehyde compound as the LIN28B inhibitor of the present invention has antiviral, antioxidant, anti-HIV, influenza prophylactic and therapeutic effects, and AXL inhibitory effects in addition to anticancer activity, and can therefore be effectively used as an ingredient in nutritional compositions, supplements, foods, beverages, and the like. [Sequence List Free Text]

[0071] SEQ ID NO: 1: Lin28B forward primer SEQ ID NO: 2: Lin28B reverse primer

Claims

1. A LIN28B inhibitor comprising a benzaldehyde compound or a pharmaceutically acceptable salt thereof, wherein the benzaldehyde compound is represented by the following general formula (I): 【Chemistry 1】 (In the formula, R 1 is —CHO, a 1,3-dioxolanyl group, a 1,3-dioxanyl group, or 【Chemistry 2】 and R 2 each independently represents a hydrogen atom or a halogen group; and n represents an integer of 1 to 5.

2. A LIN28B inhibitor comprising a benzaldehyde compound selected from benzaldehyde, 5,6-O-benzylidene-L-ascorbic acid, 5,6-O-benzylidene-L-ascorbic acid monosodium salt, 4,6-O-benzylidene-D-glucopyranose, N-benzylideneethylamine, and cyclodextrin-encapsulated benzaldehyde (CDBA), or a pharmaceutically acceptable salt thereof.

3. A cancer metastasis inhibitor for inhibiting epithelial-mesenchymal transition (EMT) of cancer cells, comprising the inhibitor according to claim 1 or 2.

4. An agent for suppressing epithelial-mesenchymal transition (EMT) in cancer cells, comprising the inhibitor according to claim 1 or 2.

5. The cancer metastasis inhibitor of claim 3, wherein the cancer is liver cancer, pancreatic cancer, lung cancer, ovarian cancer, colon adenocarcinoma, chronic myeloid leukemia, pancreatic ductal adenocarcinoma, malignant renal tumor, Wilms' tumor, breast cancer, melanoma, gastric cancer, prostate cancer, cholangiocarcinoma, gallbladder cancer, head and neck cancer, osteosarcoma, diffuse midline glioma, glioblastoma, medulloblastoma, glioma, diffuse large B-cell lymphoma, follicular lymphoma, multiple myeloma, thyroid cancer, uterine cancer, EGFR-TKI therapy-resistant cancer, or radiotherapy-resistant cancer.

6. The EMT inhibitor described in claim 4, wherein the cancer is liver cancer, pancreatic cancer, lung cancer, ovarian cancer, colon adenocarcinoma, chronic myeloid leukemia, pancreatic ductal adenocarcinoma, malignant renal tumor, Wilms' tumor, breast cancer, melanoma, gastric cancer, prostate cancer, cholangiocarcinoma, gallbladder cancer, head and neck cancer, osteosarcoma, diffuse midline glioma, glioblastoma, medulloblastoma, glioma, diffuse large B-cell lymphoma, follicular lymphoma, multiple myeloma, thyroid cancer, uterine cancer, EGFR-TKI therapy-resistant cancer, or radiation therapy-resistant cancer.

7. A combination for cancer treatment and epithelial-mesenchymal transition (EMT) inhibition, comprising a LIN28B inhibitor according to claim 1 or 2 and an EGFR inhibitor, an immune checkpoint inhibitor, a kinase inhibitor, an alkylating agent, an antimetabolite, a microtubule inhibitor, a topoisomerase inhibitor, and / or a cytotoxin.

8. A combination agent for cancer treatment and epithelial-mesenchymal transition (EMT) inhibition, which is used in combination with the LIN28B inhibitor according to claim 1 or 2 and one or more agents selected from the group consisting of: Gefitinib, irinotecan hydrochloride, topotecan hydrochloride, docetaxel, paclitaxel, vinblastine sulfate, vincristine sulfate, vindesine sulfate, etoposide, teniposide, vinorelbine tartrate, busulfan, carboquone, thiotepa, cyclophosphamide, melphalan, estramustine sodium phosphate, mechlorethamine oxide hydrochloride, ifosfamide, ranimustine, nimustine hydrochloride, bleomycin hydrochloride, peplomycin sulfate, zinostatin stimaraate, actinomycin D, aclarubicin hydrochloride, doxorubicin hydrochloride, idarubicin hydrochloride, amrubicin hydrochloride, daunorubicin hydrochloride, pirarubicin, epirubicin hydrochloride, mitomycin, valrubicin, methotrexate, mercaptopurine, fludarabine phosphate, cladribine, fluorouracil, Tegafur, cytarabine, gemcitabine hydrochloride, cytarabine ocfosfate, capecitabine, doxifluridine, carmofur, enocitabine, nedaplatin, carboplatin, cisplatin, fadrozole hydrochloride, anastrozole, exemestane, bicalutamide, flutamide, tamoxifen citrate, toremifene citrate, tretinoin, pentostatin, L-asparagine Nase, dacarbazine, procarbazine hydrochloride, mitoxantrone hydrochloride, sobuzoxane, trastuzumab, rituximab, imatinib mesylate, 5-fluoro-2'-deoxyuridine, ascle, carbocriline, quinolespan, krestin, erlotinib, aquatinib, dacomitinib, osimertinib, sorafenib, sunitinib, bortezomib, oxaliplatin, picibanil, Fulvestrant, letrozole, finasunate, rapamycin, leucovorin, lapatinib, lonafarnib, camptothecin, bryostatin, adezelesin, anthracyclines, carzelesin, bizelesin, dolastatins, auristatins, duocarmycin, eluterobin, taxol, vincristine, prednisone or prednisolone, mechlorethamine, chlorambucil, azathioprine, vinca alkaloids , podophyllotoxin, topoisomerase inhibitors, actinomycin, edrecolomab, plicamycin, and pharmaceutically acceptable salts of the above drugs, cetuximab, bevacizumab, ibritumomab, abagovomab, adecatumumab, afutuzumab, alacizumab, alemtuzumab, anatumomab, apolizumab, bavituximab, belimumab, bivatuzumab mertansine mertansine, blinatumomab, brentuximab vedotin, cantuzumab mertansine, catumaxomab, cetuximab, sitatuzumab bogatox, cixutumumab, clivatuzumab tetraxetan tetraxetan), conatumumab, dacetuzumab, daclizumab, detumomab, ecromeximab, edrecolomab, elotuzumab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, figitumumab, fresolimumab, galiximab, glenbatumumab vedotin, gemtuzumab, ibri Tumomab tiuxetan, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lucatumumab, lumilisimab, mapatumumab, matuzumab, milatuzumab, mitumomab, nacolomab butafenatoxtafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, ofatumumab, olaratumab, oportuzumab monatox, oregovomab, panitumumab, pemtumomab, pertuzumab, pintumomab, pritumumab, ramucirumab, rilotumumab, lobatumumab, sibrotuzumab, tacatuzumab tetraxetan, taplitumomab paptox paptox), tenatumomab, ticilimumab, tigatuzumab, tositumomab, tremelimumab, tuocuzumab, celmoleukin, veltuzumab, visilizumab, volociximab, votumumab, zalutumumab, zanolimumab, anti-PTHrP2C4 antibodies, maytansinoids, calicheamicin.

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