JP7549345B2 - Composition for improving malignant tumor disease - Google Patents

Description

The present invention relates to a compound that inhibits NF-κB-inducing kinase (also known as NIK-MAP3K14), which is useful for treating and/or preventing malignant tumors such as malignant lymphoma and multiple myeloma, etc. The present invention also relates to a composition, pharmaceutical composition, and processed food for preventing and treating malignant tumors such as malignant lymphoma and multiple myeloma, etc., using the compound.

NF-κB (Nuclear factor kappa B) is a transcription factor that regulates the expression of various genes involved in immune response, cell proliferation, apoptosis, and carcinogenesis. NF-κB is composed of five members: NF-κBp65 (p65), RelB, c-Rel, NF-κB1 (which exists as both precursor p105 and truncated p50), and NF-κB2 (which exists as both precursor p100 and truncated p52). Mainly, NF-κB1 (truncated p50; NF-κBp50) and p65 form a heterodimer, and NF-κB2 (truncated p52; NF-κBp52) and RelB form a heterodimer.

Furthermore, activation of these NF-κB heterodimers is a signaling pathway that is tightly controlled by a series of events including phosphorylation and protein degradation, and is classified into two pathways, the canonical pathway and the non-canonical pathway.

NIK is a serine/threonine kinase that plays a role in both pathways, but is essential in the non-canonical signaling pathway, where it phosphorylates IKKα, partially degrading NF-κB p100 and liberating NF-κB p52.

NF-κBp52 forms a heterodimer with RelB, which then migrates into the nucleus and controls gene expression. In the classical pathway, it activates the IKKα, IKKβ and IKKγ complex to form a heterodimer with p65 and NF-κBp50, which then migrates into the nucleus and controls gene expression.

NIK is activated by ligands such as B cell activating factor (BAFF), CD40 ligand, and tumor necrosis factor α (TNFα), and it has been shown that NIK is important for the activation of signaling pathways by these ligands. Due to its important role, the expression of NIK is tightly regulated.

Under normal non-stimulated conditions, NIK is degraded by interaction with TNF receptor-associated factors (TRAFs), which are ubiquitin ligases, resulting in low levels of NIK protein in cells. It is believed that when the non-classical pathway is stimulated by a ligand, the activated receptor dissociates the TRAF-NIK complex, thereby increasing the concentration of NIK (Thu and Richmond, Cytokine Growth FR 2010, 21, 213-226: Non-Patent Document 1).

BAFF is produced and secreted by T cells, monocytes/macrophages, dendritic cells, etc., and is known to control the differentiation, activation, survival, etc. of B cells via three types of receptors on B cells (Moore, et al., Science. 1999, 285, 260-263: Non-Patent Document 2).

Known receptors for BAFF include BAFF-R (BAFF-Receptor), TACI (Taransubranesbrane activator, calcium modulator and cyclophilin ligand and interactor), and BMCA (B cell maturation antigen).

BAFF-R and BMCA are primarily expressed in B cells, whereas TACI is expressed in B cells and activated T cells. Interaction of BAFF with BAFF-R activates the non-classical NF-κB signaling pathway via NIK.

It has been shown that the NF-κB pathway is constitutively activated in multiple myeloma (Annuziata, et al. Cancer Cell. 2007, 12, 115-130: Non-Patent Document 3 and Demchenko, et al. Blood. 2010, 115, 3541-3552: Non-Patent Document 4). It has also been shown that multiple myeloma patients have NIK gene amplification, TRAF gene deletion, and point mutations in the TRAF gene, which increase the amount of NIK protein expression and constitutively activate NIK, which is a factor in activating the NF-κB pathway. It has also been shown that inhibition of NIK with shRNA (NIK shRNA) suppresses NF-κB activation and induces cell death in multiple myeloma cell lines (Annuziata, et al. Cancer Cell. 2007, 12, 115-130: Non-Patent Document 3).

It has also been shown that serum BAFF concentrations are elevated in patients with multiple myeloma, and it has been reported that BAFF is secreted not only from monocytes and macrophages but also from multiple myeloma cells, and that the proliferation of multiple myeloma cells is enhanced by autocrine BAFF (Sutherland, et al., Pharmacol. Ther. 2006, 112, 774-786: Non-Patent Document 5 and Novak, et al., Blood. 2004, 103, 689-694: Non-Patent Document 6).

It has also been shown that point mutations in the TRAF gene and increased expression of NIK protein are observed in Hodgkin's lymphoma patients, and it has been reported that NIK shRNA also induces cell death in these patients (Ranuncolo, et al., Blood. 2012, 120, 3756-3763: Non-Patent Document 7).

Furthermore, it has been shown that the cellular mass of NIK protein is increased in adult T-cell leukemia cells, and that NIK shRNA treatment suppresses tumor growth of adult T-cell leukemia in vivo (Saitoh, et al., Blood. 2008, 111, 5118-5129: Non-Patent Document 8).

In addition, it has been shown that the API2-MALT1 fusion protein produced by chromosomal translocation (t(11;18)(q21;q21)) in mucosa associated lymphoid tissue (MALT) lymphoma induces constitutive activation of NIK by cleaving the protein at the 325th arginine position of NIK. This constitutive activation of NIK activates the non-classical NF-κB pathway, which is involved in cell adhesion and apoptosis resistance (Rosebeck, et al., Science. 2011, 331, 468-472; Non-Patent Document 9).

In diffuse large B-cell lymphoma (DLBCL) cells, NIK is highly expressed in the cytoplasm upon stimulation with BAFF. Activation of NIK by this high expression is an important signal transduction mechanism involved in lymphoma proliferation, and it has been shown that NIK shRNA suppresses NIK-induced NF-κB activation in vitro and inhibits proliferation of DLBCL cell lines (Pham, et al. Blood. 2011, 117, 200-210: Non-Patent Document 10).

In addition, BAFF expression has been observed in B lymphoma cells collected from patients with chronic B lymphoma, and it has been shown that this BAFF reduces drug-induced apoptosis (Kern, et al., Blood. 2004, 103, 679-688: Non-Patent Document 11). It has also been reported that BAFF overexpression induces the development of B lymphoma in mice (Sutherland, et al., Pharmacol. Ther. 2006, 112, 774-786: Non-Patent Document 5).

Furthermore, BAFF expression has been observed in the serum and lymphoma cells of patients with MALT lymphoma, DLBCL, Hodgkin's lymphoma, and Burkitt's lymphoma, and it has been shown that this induces lymphoma cell proliferation and apoptosis resistance, and that patients with lymphomas with high BAFF expression have a poorer prognosis than those with low BAFF expression (He, et al., J. Immunol., 2004, 172, 3268-3279: Non-Patent Document 12; Novak, et al., Blood. 2004, 104, 2247-2253: Non-Patent Document 13; and Oki, et al., Haematologica. 2007, 92, 269-270: Non-Patent Document 14).

It has been shown that NIK expression is higher in T lymphoma cells collected from peripheral T lymphoma patients than in T cells collected from healthy individuals, activating the downstream non-classical NF-κB pathway, and that patients with high nuclear NF-κB expression have a poorer prognosis than patients with low nuclear NF-κB expression. It has also been shown that cell death can be induced in peripheral T lymphoma cells by NIK siRNA treatment (Odqvist, et al., Clin. Cancer Res. 2013, 19, 2319-2330: Non-Patent Document 15).

The role of NIK in tumor cell proliferation is not limited to hematopoietic tumors. It has been shown that NIK is highly expressed in certain pancreatic cancer cell lines, and that the cell proliferation is suppressed by NIK siRNA treatment (Nishina, et al., Biochem. Biophys. Res. Commun. 2009, 388, 96-101: Non-Patent Document 16).

It has also been shown that the BAFF concentration in the serum of pancreatic cancer patients is higher than that of healthy individuals, and that the BAFF concentration correlates with tumor growth and progression of the disease. Furthermore, it has been reported that BAFF-R is expressed in pancreatic cancer tissue, that NF-κBp52 and RelB are highly expressed, and that B lymphocytes present around pancreatic cancer tissue produce BAFF (Koizumi, et al., PLoS One. 2013, 8, e71367: Non-Patent Document 17).

It has been reported that high expression of NIK induces constitutive activation of NF-κB in basal-like breast cancer cell lines (Yamamoto, et al., Cancer Sci. 2010.101, 2391-2397: Non-Patent Document 18).

Furthermore, tissue microarray analysis has shown that NIK expression is significantly higher in malignant melanoma than in benign tissue, and NIK shRNA has been shown to suppress tumor growth, induce apoptosis, and arrest the cell cycle in vivo (Thu, et al., Oncogene. 2012, 31, 2580-2592: Non-Patent Document 19).

Furthermore, it has been shown that NF-κB is activated in non-small cell lung cancer tissues and cell lines, and NIK siRNA treatment has been shown to induce apoptosis and inhibit anchorage-independent cell proliferation (Saitoh, et al., Lung Cancer. 2010, 70, 263-270: Non-Patent Document 20).

It has been shown that NIK is highly expressed in liver cancer patients and liver cancer cell lines, and that NIK siRNA treatment and miR-520e, which reduces NIK expression, suppress cell proliferation in vitro and tumor proliferation in vivo (Zhang, et al., Oncogene, 2012, 31, 3607-3620: Non-Patent Document 21).

Helicobacter pylori is known to be deeply involved in the development of gastric cancer, and it has been shown that in patients infected with this bacterium, NIK is constitutively activated at the site of gastric infection, activating the non-classical NF-κB pathway. It has also been reported that gastric cancer cells into which a mutant that suppresses NIK activation has been introduced suppress NF-κB activation by Helicobacter pylori (Feige, et al., Biochim. Biophys. Acta Mol. Cell Res. 2018, 1865, 545-550: Non-Patent Document 22 and Maeda, et al., Gastroenterology. 2000, 119, 97-108: Non-Patent Document 23).

Activation of the non-classical NF-κB pathway is associated with the development of colitis and colon cancer, and in mice lacking NLRP12, which negatively regulates NIK, it has been shown that the non-classical NF-κB pathway is activated via NIK activation, inducing colitis and colon cancer. It has also been reported that in colon cancer patients, expression of OLFM1, which negatively regulates NIK, is higher in non-cancerous areas compared to cancerous areas, and that NIK siRNA treatment in colon cancer cells suppresses cell proliferation and motility (Allen, et al. Immunity 2012, 36, 742-754: Non-Patent Document 24 and Shi, et al., J. Pathol. 2016, 240, 352-365: Non-Patent Document 25).

NIK and RelB are highly expressed in head and neck tumor cells, and it has been shown that siRNA treatment of NIK and RelB suppresses the motility and invasion of tumor cells (Das, et al., Mol. Carcinog. 2018, In press: Non-Patent Document 26).

Overexpression of NIK and RelB has been observed in patients with renal cancer, and it has been shown that this reduces the 10-year survival rate compared to patients with low expression, and it has been reported that the expression status of NIK is a prognostic factor (Lua, et al., Urol. Int. 2018, 101, 190-196: Non-Patent Document 27).

It has been shown that overexpression of NIK in glioma cells promotes tumor formation, and that activation of the non-classical NF-κB pathway by NIK activation enhances the motility and invasion of glioma cells (Cherry, et al., Mol. Cancer. 2015, 14, 9: Non-Patent Document 28).

It has been shown that NIK mRNA expression is higher in ovarian cancer patient tissues than in normal ovarian tissues, that the NIK/NF-κB p52 (non-classical) pathway is activated in ovarian cancer cells, that NIK shRNA treatment suppresses anchorage-dependent and non-anchorage-dependent cell proliferation, and that NIK shRNA suppresses tumor proliferation in vivo (Uno, et al., PLoS One. 2014, 9, e88347: Non-Patent Document 29).

It has been shown that in patients with endometrial cancer, the degree of differentiation of cancer tissue decreases and the activation of NIK increases as the disease progresses, and it has been reported that activation of NIK suppresses apoptosis of cancer cells (Zhou, et al., Int. J. Gynecol. Cancer. 2015, 25, 770-778: Non-Patent Document 30).

As described above, it is now scientifically common knowledge that in malignant tumors such as multiple myeloma, malignant lymphoma (MALT lymphoma, DLBCL, Burkitt lymphoma, Hodgkin lymphoma, adult T-cell leukemia, peripheral T lymphoma, etc.), pancreatic cancer, breast cancer, malignant melanoma, lung cancer, liver cancer, gastric cancer, colon cancer, head and neck tumors, glioma, renal cancer, ovarian cancer, and uterine cancer, overexpression of BAFF and overexpression of NIK occur, and activation of NIK activates NF-κB, resulting in the disease.

Therefore, pharmaceuticals and the like that can inhibit NIK activation by BAFF and NIK activation associated with NIK overexpression and suppress the non-classical NF-κB signaling pathway have a therapeutic effect against malignant tumors in which BAFF overexpression and excessive activation of NIK and non-classical NF-κB signaling are observed.

In addition, multiple myeloma is known to be accompanied by characteristic symptoms called CRAB (hypercalcemia, renal dysfunction, anemia, and bone lesions).

It has been reported that osteoclast activating factors such as monoclonal immunoglobulin (M protein), immunoglobulin free light chain, interleukin 6 (IL-6) and macrophage inflammatory protein 1α (MIP-1α) expressed by multiple myeloma cells are involved in the development of CRAB (Rajkumar, et al., Lancet Oncol. 2014, 15, e538-548: Non-Patent Document 31; Harmer, et al., Front. Endocrinol. 2019, 9, 788: Non-Patent Document 32; Roussou, et al., Leukemia. 2009, 23, 2177-2181: Non-Patent Document 33).

It is known that M proteins and free immunoglobulin light chains caused by multiple myeloma are deposited in the kidney and cause kidney damage, and are also deposited in organs throughout the body, causing amyloidosis in various organs and resulting in a variety of symptoms such as neuropathy and arrhythmia.Furthermore, in the blood, they cause hyperviscosity syndrome.

It has been shown that IL-6 secretion in multiple myeloma promotes differentiation into osteoclasts and activates osteoclasts, which has been reported to progress bone lesions (Harmer, et al., Front. Endocrinol. 2019, 9, 788: Non-Patent Document 32). In addition, MIP-1α secreted by multiple myeloma has also been shown to promote differentiation and activation of osteoclasts and promote bone lesions (Roussou, et al., Leukemia. 2009, 23, 2177-2181: Non-Patent Document 33; Tsubaki, et al., J. Cell. Biochem. 2010, 111, 1661-1672: Non-Patent Document 34). It has also been reported that the progression of bone lesions caused by these factors leads to the development of hypercalcemia (Lee, et al., Intern. Med. J. 2017, 47, 938-951: Non-Patent Document 35). That is, osteoclast activating factors cause bone lesions, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms.

It has been reported that NF-κB2 mutant mice develop myeloma and have increased expression of M protein (McCarhty, et al., BMC Cancer. 2012, 12, 203: Non-Patent Document 36). Furthermore, it has been reported that activation of the NF-κB pathway increases the expression of osteoclast activating factors such as IL-6 and MIP-1α (Vrabel, et al., Blood Rev. 2019, 34, 56-66: Non-Patent Document 37).

As described above, it is now scientifically common knowledge that in multiple myeloma, activation of the NF-κB pathway leads to the expression of M protein and bone lesions caused by osteoclast activating factor.

Therefore, pharmaceuticals and the like that can inhibit NIK activation and NIK activation associated with NIK overexpression and suppress the non-classical NF-κB signaling pathway have therapeutic effects on nephropathy due to increased expression of M protein or free immunoglobulin light chain caused by multiple myeloma, amyloidosis, bone lesions (bone destruction) due to hyperviscosity syndrome or increased expression of osteoclast activating factor, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms, i.e., have therapeutic effects on CRAB.

Patent Document 1 (JP Patent Publication No. 2016-531858 A) discloses a 3-(1H-pyrazol-4-yl)-1H-pyrrolo[2,3-c]pyridine derivative as a NIK inhibitor. It is shown that the compound is an inhibitor of NF-κB-inducing kinase, also known as NIK-MAP3K14, and the patent right recognizes that the compound is a compound for use in the prevention or treatment of cancer.

In addition, Patent Document 1 only lists the EC50 of the compound against three types of cancer cells (all of which are multiple myeloma cell lines), JJN-3, L-363, and LP-1, as examples. From the above, it is generally recognized as common general knowledge that NIK inhibitors have a wide range of therapeutic effects against malignant cancer cells.

Furthermore, Patent Document 2 (JP Patent Publication No. 7-082263) discloses that a xanthone compound having a structure of formula (10) has anticancer activity.

なお、（Ｒ^１ ～Ｒ^７ ：Ｈ、－ＯＨ、Ｃ１－６ アルキル、Ｃ１－６ アルコキシ、エポキシプロポキシ）、およびベンゾフェノン化合物を表す。

In addition, (R ¹ to R ⁷ : H, —OH, C1-6 alkyl, C1-6 alkoxy, epoxypropoxy) and benzophenone compounds.

In addition, Patent Document 3 (JP Patent Publication 2017-031146 A) describes that mangiferin inhibits NIK and is effective against multiple myeloma and malignant melanoma. As described above, compounds having a xanthone skeleton have been found to have anticancer activity.

Special table 2016-531858 publication Japanese Patent Application Publication No. 7-082263 JP 2017-031146 A

Thu YM, Richmond A. : Cytokine Growth Factor Rev. 2010, 21, 213-226. Moore PA, Belvedere O, Orr A, Pieri K, LaFleur DW, Feng P, Soppet D, Charters M, Gentz R, Parmelee D, Li Y, Galperina O, Giri J, Roschke V, Nardelli B, Carrell J, Sosnovtseva S , Greenfield W, Ruben SM, Olsen HS, Fikes J, Hilbert DM. : Science. 1999, 285, 260-263. Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea A, Zhan F, Lenz G, Hanamura I, Wright G, Xiao W, Dave S, Hur t EM, Tan B, Zhao H, Stephens O, Santra M, Williams DR , Dang L, Barlogie B, Shaughnessy JD Jr, Kuehl WM, Staudt LM. : Cancer Cell. 2007, 12, 115-130. Demchenko YN, Glebov OK, Zingone A, Keats JJ, Bergsagel PL, Kuehl WM. : Blood. 2010, 115, 3541-3552. Sutherland AP, Mackay F, Mackay CR. : Pharmacol Ther. 2006, 112, 774-786. Novak AJ, Darce JR, Arendt BK, Harder B, Henderson K, Kindsvogel W, Gross JA, Greipp PR, Jelinek DF. : Blood. 2004, 103, 689-694. Ranuncolo SM, Pittaluga S, Evbuomwan MO, Jaffe ES, Lewis BA. : Blood. 2012, 120, 3756-3763. Saitoh Y, Yamamoto N, Dewan MZ, Sugimoto H, Martinez Bruyn VJ, Iwasaki Y, Matsubara K, Qi X, Saitoh T, Imoto I, awa J, Utsunomiya A, Watanabe T, Masuda T, Yamamoto N, Yamaoka S. : Blood. 2008, 111, 5118-5129. Rosebeck S, Madden L, Jin X, Gu S, Apel IJ, Appert A, Hamoudi RA, Noels H, Sagaert X, Van Loo P, Baens M, Du MQ, Lucas PC, McAllister-Lucas LM. : Science. 2011, 331, 468-472. Pham LV, Fu L, Tamayo AT, Bueso-Ramos C, Drakos E, Vega F, Medeiros LJ, Ford RJ. : Blood. 2011, 117, 200-210. Kern C, Cornuel JF, Billard C, Tang R, Rouillard D, Stenou V, Defrance T, Ajchenbaum-Cymbalista F, Simonin PY, Feldblum S, Kolb JP. : Blood. 2004, 103, 679-688. He B, Chadburn A, Jou E, Schattner EJ, Knowles DM, Cerutti A. : J Immunol. 2004, 172, 3268-3279. Novak AJ, Grote DM, Stenson M, Ziesmer SC, Witzig TE, Habermann TM, Harder B, Ristow KM, Bram RJ, Jelinek DF, Gross JA , Ansell SM. : Blood. 2004, 104, 2247-2253. Oki Y, Georgakis GV, Migone TS, Kwak LW, Younes A. : Haematologica. 2007, 92, 269-270. Odqvist L, Sanchez-Beato M, Montes-Moreno S, Martin-Sanchez E, Pajares R, Sanchez-Verde L, Ortiz-Romero PL, Ro driguez J, Rodriguez-Pinilla SM, Iniesta-Martinez F, Solera-Arroyo JC, Ramos - Asensio R, Flores T, Palanca JM, Bragado FG, Franjo PD, Piris MA. : Clin Cancer Res. 2013, 19, 2319-2330. Nishina T, Yamaguchi N, Gohda J, Semba K, Inoue J. : Biochem Biophys Res Commun. 2009, 388, 96-101. Koizumi M, Hiasa Y, Kumagi T, Yamanishi H, Azemoto N, Kobata T, Matsuura B, Abe M, Onji M. : PLoS One. 2013, 8, e71367. Yamamoto M, Ito T, Shimizu T, Ishida T, Semba K, Watanabe S, Yamaguchi N, Inoue J. : Cancer Sci. 2010, 101, 2391-2397. Thu YM, Su Y, Yang J, Splittgerber R, Na S, Boyd A, Mosse C, Simons C, Richmond A. : Oncogene. 2012, 31, 2580-2592. Saitoh Y, Martinez Bruyn VJ, Uota S, Hasegawa A, Yamamoto N, Imoto I, Inazawa J, Yamaoka S. : Lung Cancer. 2010, 70, 263-270. Zhang S, Shan C, Kong G, Du Y, Ye L, Zhang X. : Oncogene. 2012, 31, 3607-3620. Feige MH, Vieth M, Sokolova O, Tager C, Naumann M. : Biochim Biophys Acta Mol Cell Res. 2018, 1865, 545-550. Maeda S, Yoshida H, Ogura K, Mitsuno Y, Hirata Y, Yamaji Y, Akanuma M, Shiratori Y, Omata M. : Gastroenterology. 2000, 119, 97-108. Allen IC, Wilson JE, Schneider M, Lich JD, Roberts RA, Arthur JC, Woodford RM, Davis BK, Uronis JM, Herfarth HH, Jobin C, Rogers AB, Ting JP. : Immunity. 2012, 36, 742-754. Shi W, Ye Z, Zhuang L, Li Y, Shuai W, Zuo Z, Mao X, Liu R, Wu J, Chen S, Huang W. : J Pathol. 2016, 240, 352-365. Das R, Coupar J, Clavijo PE, Saleh A, Cheng TF, Yang X, Chen J, Van Waes C, Chen Z. : Mol Carcinog. 2018. In press. Lua J, Qayyum T, Edwards J, Roseweir AK. : Urol Int. 2018, 101, 190-196. Cherry EM, Lee DW, Jung JU, Sitcheran R. : Mol Cancer. 2015 Jan 27;14:9. Uno M, Saitoh Y, Mochida K, Tsuruyama E, Kiyono T, Imoto I, Inazawa J, Yuasa Y, Kubota T, Yamaoka S.: PLoS One. 2014, 9, e88347. Zhou X, Li H, Chai Y, Liu Z. : Int J Gynecol Cancer. 2015, 25, 770-778. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos MV, Kumar S, Hillengass J, Kastritis E, Richardson P, Landgren O, Paiva B, Dispenzieri A, Weiss B, LeLeu X, Zweegman S, Lonial S , Rosinol L, Zamagni E, Jagannath S, Sezer O, Kristinsson SY, Caers J, Usmani SZ, Lahuerta JJ, Johnson HE, Beksac M, Cavo M, Goldschmidt H, Terpos E, Kyle RA, Anderson KC, Durie BG, Miguel JF. : Lancet Oncol. 2014, 15, e538-548. Harmer, D. , Falank, C. , Reagan, M. R. Front. Endocrinol. 2019, 9, 788. Roussou, M. , Tasidou, A. , Dimopoulos, M. A. , Kastritis, E. , Migkou, M. , Christoulas, D. , Gavriatopoulou, M. , Zagouri, F. , Matsouka, C. , Anagnostou, D. , Terpos, E. : Leukemia. 2009, 23, 2177-2181. Tsubaki, M. , Kato, C. , Isono, A. , Kaneko, J. , Isozaki, M. , Satou, T. , Itoh, T. , Kidera, Y. , Tanimori, Y. , Yanae, M. , Nishida S. : J. Cell. Biochem. 2010, 111, 1661-1672. Lee, O. L. , Horvath, N. , Lee, C. , Joshua, D. , Ho, J. , Szer, J. , Quach, H. , Spencer, A. , Harrison, S. , Mollee, P. , Roberts, A. W. , Talaulikar, D. , Brown, R. , Augustson, B. , Ling, S. , Jaksic, W. , Gibson, J. , Kalff, A. , Johnston, A. , Kalro, A. , Ward, C. , Prince, H. M. , Zannettino, A. : Intern. Med. J. 2017, 47, 938-951. McCarthy, B. A. , Yang, L. , Ding, J. , Ren, M. , King, W. , ElSalanti, M. , Zakhary, I. , Sharawy, M. , Cui, H. , Ding, HF. : BMC Cancer. 2012, 12,,203. Vrabel, D. , Pour, L. , Sevcikova, S. : Blood Rev. 2019, 34, 56-66. Hu, L. , Wu, W. , Chai, X. , Luo, J. , Wu, Q. : Bioorg. Med. Chem. Lett. 2011, 21, 4013-4015.

As shown in Patent Document 1, chemical substances are used as drugs to suppress NIK, but in most cases, except for mangiferin in Patent Document 3, the drug is administered intravenously, which places a heavy burden on patients. Therefore, the object is to provide a therapeutic drug and an improving composition that inhibits NIK and improves malignant tumors such as malignant lymphoma and multiple myeloma, even when administered orally.

In addition, although the mangiferin of Patent Document 3 is effective when administered orally, the required amount is large, and it is difficult to take it orally. Therefore, there is a demand for a more effective or active therapeutic drug and improving composition.

Furthermore, new or improved forms of existing drugs that inhibit kinases such as NIK are constantly needed to develop more effective pharmaceutical agents for treating malignancies, etc.

In order to solve the above problems, the present inventors have searched for compounds that inhibit NIK, and have found substances having a xanthone skeleton, including those with a hitherto undiscovered structure, that have NIK inhibitory activity, and have confirmed that these substances actually induce cell death in malignant lymphoma and multiple myeloma.

The inventors also confirmed that the compound significantly inhibits the tumor growth of malignant lymphoma and multiple myeloma in vivo. Furthermore, the inventors confirmed that the compound induces a decrease in the expression of CD138, a malignancy marker of multiple myeloma, and an increase in the expression of CD20, a B cell marker, and inhibits the production of monoclonal immunoglobulins, free immunoglobulin light chains, and bone destruction-inducing factors in multiple myeloma, thereby completing the present invention.

That is, the composition for improving malignant tumor diseases according to the present invention is characterized by containing a compound represented by the following formula (1) or (5).

In addition, the composition for improving malignant tumors containing the compound of formula (1) or (5) can be used as a composition for improving malignant tumors associated with NIK overexpression and malignant tumors associated with BAFF overexpression. It can also be used as a therapeutic agent for malignant tumors.

At least one of the compounds of the above formulas (1), (2), (3), (4) and (5) can be used as a drug for inducing dedifferentiation of plasma cells caused by multiple myeloma into B-cell-like cells. Furthermore, the above compounds can also be used as a drug for improving associated symptoms called CRAB (hypercalcemia, renal dysfunction, anemia, bone lesions) characteristic of multiple myeloma.

The therapeutic agent and improving composition for malignant tumors such as malignant lymphoma and multiple myeloma according to the present invention can improve malignant lymphoma and multiple myeloma. For example, it can effectively induce cell death in malignant tumor cells such as malignant lymphoma cells and multiple myeloma cells. In addition, it does not affect normal cells at a concentration that induces cell death in malignant tumor cells.

In addition, it is believed that the above-mentioned malignant tumors promote the development, proliferation and survival of malignant tumor cells by overexpression of NIK protein. Therefore, the composition for improving malignant tumors such as malignant lymphoma and multiple myeloma according to the present invention is believed to have an improving effect not only on the malignant tumors shown in the examples but also on other malignant tumors.

Furthermore, it can inhibit monoclonal immunoglobulin production, free immunoglobulin light chain production, and bone destruction-inducing factor production associated with multiple myeloma. Therefore, it can suppress renal disorders due to the enhanced expression of M protein and free immunoglobulin light chains associated with these productions, amyloidosis, bone lesions (bone destruction) due to hyperviscosity syndrome and enhanced expression of osteoclast activating factor, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms. That is, it can suppress the expression of CRAB.

FIG. 1 shows a synthesis procedure for norathyriol. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using Raji cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using CCRF-SB cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using Namalwa cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using Z138 cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using SU-DHL-5 cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using L363 cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using KMS-28BM cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using ARH77 cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using IM9 cells. 1 is a graph showing the results of examining the cell death-inducing effect of each agent by the trypan blue dye method using RPMI1788 cells. 1 shows photographs depicting the results of immunoblotting for the NIK inhibitory effect of each agent using IM9 cells. The photographs show the results of immunoblotting for the activation kinetics of IKK, NF-κB p65, and NF-κB p52, which are downstream signals of NIK, by each agent, using IM9 cells. This panel shows the results of a flow cytometry study of the inhibition of CD138 expression by each agent using L363 cells. This panel shows the results of a flow cytometry study of the inhibition of CD138 expression by each agent using L363 cells. This panel shows the results of examining the increase in CD20 expression by each agent by flow cytometry using L363 cells. This panel shows the results of examining the increase in CD20 expression by each agent by flow cytometry using L363 cells. 1 is a graph showing the results of an enzyme-linked immunosorbent assay (ELISA) study on the inhibition of IgG secretion by each agent using L363 cells. The graph shows the results of an ELISA study on the inhibition of λ-chain secretion of free immunoglobulin light chains by various drugs using L363 cells. 1 is a graph showing the results of examining the bone destruction-related factor suppressing effect of each drug by ELISA using L363 cells. This panel shows the results of investigating the inhibition of CD138 expression by mangiferin, mangiferin 8a, and norathyriol by flow cytometry using KMS-28BM cells. This panel shows the results of investigating increased CD20 expression by mangiferin, mangiferin 8a, and norathyriol using KMS-28BM cells by flow cytometry. 1 is a graph showing the results of investigating the inhibition of IgG secretion by mangiferin, mangiferin 8a, and norathyriol by ELISA using KMS-28BM cells. 1 is a graph showing the results of investigating the inhibition of λ-chain secretion of immunoglobulin free light chains by mangiferin, mangiferin 8a, and norathyriol by ELISA using KMS-28BM cells. 1 is a graph showing the results of investigating the inhibitory effects of mangiferin, mangiferin 8a, and norathyriol on bone resorption-related factors by ELISA using KMS-28BM cells. This is a graph showing the time course of tumor growth when Raji cells were transplanted into NOD/ShiJic-scidJcl mice and norathyriol was orally administered. FIG. 26 shows photographs of tumors in untreated mice and in mice administered norathyriol on day 27. 1 is a graph showing the time course of tumor growth when L363 cells were transplanted into NOD/ShiJic-scidJcl mice and norathyriol was orally administered. FIG. 28 shows photographs of tumors in untreated mice and in mice administered norathyriol on day 8.

The composition for improving malignant tumors such as malignant lymphoma and multiple myeloma according to the present invention will be described below. Note that the following description is an example of one embodiment and one example of the present invention, and the present invention is not limited to the following description. The following embodiment can be modified without departing from the spirit of the present invention.

The therapeutic agent for malignant tumors such as malignant lymphoma and multiple myeloma according to the present invention contains, as an active ingredient, a substance having a xanthone skeleton and represented by formula (1), (2), (3), (4), (5), (6) or (7). Formula (1) is 1,2',3',4',6-penta-O-propionylmangiferin, hereinafter referred to as "mangiferin 8a". Mangiferin is represented by formula (8).

Formula (2) is 1,3,6,7-tetrahydroxyxanthone, hereinafter referred to as "noratiriol". Formula (3) is 1,3,5,6-tetrahydroxyxanthone, hereinafter referred to as "tetrahydroxyxanthone". Formula (4) is 1,3,6-trihydroxy-methoxyxanthone, hereinafter referred to as "methoxyxanthone". Formula (5) is 9-hydroxyxanthene, hereinafter referred to as "xanthohydrol". Formula (6) is 1,3,6-trihydroxy-7-methoxy-2,8-bis(3-methyl-2-buten-1-yl)-9H-xanthen-9-one, hereinafter referred to as "α-mangostin". Formula (7) is 1,3,6,7-tetrahydroxy-2,8-bis(3-methyl-2-buten-1-yl)-9H-xanthen-9-one, hereinafter referred to as "γ-mangostin".

As can be seen from formula (1), mangiferin 8a is a compound in which a propionyl group is ether-bonded to a hydroxyl group of mangiferin (see formula (8)). As shown in the following examples, this compound exhibits superior NIK and IKK activity inhibition to mangiferin, and exhibits a high cell death induction effect against malignant tumors such as malignant lymphoma and multiple myeloma.

Norathyriol (CAS number 3542-72-1) is a compound in which a hydroxy group has been added to a portion of xanthone (CAS number: 90-47-1), and has a xanthone skeleton similar to mangiferin 8a. Similarly to mangiferin 8a, norathyriol exhibits greater inhibition of NIK and IKK activity than mangiferin, and exhibits a high cell death induction effect against malignant tumors such as malignant lymphoma and multiple myeloma.

Tetrahydroxyxanthone (CAS number 5084-31-1) is a compound in which a hydroxy group has been added to a portion of xanthone, and has a xanthone skeleton similar to mangiferin 8a. Similarly to mangiferin 8a, it exhibits greater inhibition of NIK and IKK activity than mangiferin, and exhibits a high cell death induction effect against malignant tumors such as malignant lymphoma and multiple myeloma.

Methoxyxanthone (CAS number 41357-84-0) is a compound in which a hydroxy group is bonded to a portion of xanthone and a methyl group is bonded to the hydroxy group via an ether bond, and has a xanthone skeleton similar to mangiferin 8a. Similarly to mangiferin 8a, it exhibits greater inhibition of NIK and IKK activity than mangiferin, and exhibits a high cell death induction effect against malignant tumors such as malignant lymphoma and multiple myeloma.

Xanthohydrol (CAS number 96-46-0) is a compound in which the ketone position of xanthone is replaced by a hydroxyl group and has a xanthone skeleton. Like mangiferin 8a, it exhibits greater inhibition of NIK and IKK activity than mangiferin and exhibits a high cell death induction effect against malignant tumors such as malignant lymphoma and multiple myeloma.

α-Mangostin (CAS number 6147-11-1) is a compound in which a bismethylbutenyl group and a hydroxyl group are bound to a portion of xanthone, and has a xanthone skeleton. As with mangiferin 8a, α-Mangostin shows a higher inhibition of NIK and IKK activity than mangiferin, and shows a higher cell death induction effect against malignant tumors such as malignant lymphoma and multiple myeloma.

γ-mangostin (CAS number 96-46-0) is a compound in which a bismethylbutenyl group, a hydroxyl group, and a methyl group are bonded to a portion of the hydroxyl group, and has a xanthone skeleton. As with mangiferin 8a, it exhibits greater inhibition of NIK and IKK activity than mangiferin, and exhibits a high cell death induction effect against malignant tumors such as malignant lymphoma and multiple myeloma.

These compounds, like mangiferin, are relatively low molecular weight compounds, are highly soluble in water, and can be orally ingested. The composition for improving malignant tumor diseases according to the present invention contains at least one of these seven compounds as an active ingredient. It may also contain other medicament-acceptable ingredients.

The composition for improving malignant tumor disease according to the present invention can be provided as a malignant tumor disease therapeutic drug (a pharmaceutical composition including a dedifferentiation inducer into B-cell-like cells or a CARB improver). As a pharmaceutical composition, the composition according to the present invention can be effective when administered orally. Therefore, it can be provided as an internal medicine. For example, the powdered composition for improving malignant tumor disease can be formulated and provided as a capsule, granule, powder, tablet, or the like. When preparing an oral preparation, additives such as a binder, lubricant, disintegrant, colorant, flavoring agent, preservative, antioxidant, and stabilizer can be added to produce a capsule, granule, powder, or tablet by a conventional method.

Furthermore, the pharmaceutical composition according to the present invention may be formulated into an external preparation such as a liquid, ointment, cream, gelling agent, patch, or aerosol, and administered parenterally. When preparing an external preparation, water, lower alcohol, solubilizer, surfactant, emulsion stabilizer, gelling agent, adhesive, and other necessary base components may be blended. In addition, additives such as vascular dilating agents, adrenal cortical hormones, keratolytic agents, moisturizers, bactericides, antioxidants, refreshing agents, fragrances, and colorants may be appropriately blended.

In addition, the composition for improving malignant tumor disease according to the present invention can be provided as a processed food. That is, even if the composition for improving malignant tumor disease according to the present invention is ingested as a processed food, it has the same effect as the composition for improvement according to the present invention.

The processed foods according to the present invention include not only general processed foods including luxury foods and health foods such as candy, gum, jelly, biscuits, cookies, rice crackers, bread, noodles, fish and meat paste products, tea, soft drinks, coffee drinks, milk drinks, whey drinks, lactic acid bacteria drinks, yogurt, ice cream, pudding, etc., but also health functional foods such as foods for specified health uses and foods with nutrient functions as specified by the Health Functional Foods System of the Ministry of Health, Labor and Welfare, and further include nutritional supplements, feed, food additives, etc.

The processed foods according to the present invention can be prepared by adding the composition for improving malignant tumors to the raw materials of these processed foods. Note that when norathyriol, α-mangostin and γ-mangostin are added to these processed foods, the application is excluded when materials containing norathyriol, α-mangostin and γ-mangostin are used as raw materials. The composition for improving malignant tumors according to the present invention will be described below based on the examples.

Example 1: Synthesis of mangiferin 8a
Mangiferin 8a was obtained in two steps by synthesizing the raw material 1,3,2',3',4',6,6',7-octa-O-propionylmangiferin and then synthesizing it further. (1) Synthesis of 1,3,2',3',4',6,6',7-octa-O-propionylmangiferin

Mangiferin (2.1 g, 4.98 mmol), propionic anhydride (12.8 mL, 99.4 mmol) and dry pyridine (60 mL) were heated at 80°C for 5 hours. The reaction solution was poured into ice water (400 mL) and extracted with ethyl acetate. The ethyl acetate layer was washed with ice-cold 10% sulfuric acid, saturated aqueous sodium bicarbonate solution and saturated saline solution in this order, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified using column chromatography (n-hexane/ethyl acetate = 1/1) to obtain 1,3,2',3',4',6,6',7-octa-O-propionylmangiferin (3.71 g, 86%) of formula (9) as a colorless solid.

¹ H-NMR (800 MHz, DMSO-d ₆ ) δ: 0.70-1.26 (24H, m, COCH ₂ CH ₃ ), 1.92-2.89 (16H, m, COCH ₂ CH ₃ ), 3.83-3.91 (1H, m, H-6'a), 4.05-4.15 (2H, m, H-5' and H-6'b), 4.98-5.05 (1H, m, H-4'), 5.09-5.21 (1H, m, H-1'), 5.43-5.51 (2H, m, H-3' and H-2'), 7.50-7.54 (1H, m, H-4), 7.68-7.71 (1H, m, H-5), 7.96-7.98 (1H, m, H-8).
^13C -NMR (200MHz, DMSO- _d6 ) δ: 8.68/8.77/8.79/8.92/8.95/8.98/9.00/9.01/9.04/ 9.06/9.17/9.19/9.28 (COCH ₂ CH ₃ ), 26.6/26.7/26.9/26.8/26.93/26.97/27.00/ 27.09/27.35/27.37 (COCH ₂ CH ₃ ), 62.1/62.2 (C6'), 68.20/68.23 (C4'), 69.6/70.1 ( C2'), 70.9/71.3 (C1'), 73.6/73.7 (C3'), 75.06/75.10 (C5'), 110.3/111.8 (C4), 111.7/112.6 (C9a), 113.3/ 113.4 (C5), 118.9/119.0 (C2), 119.7/119.7 (C8a), 120.36/120.40 (C8), 139.57/139.59 (C7' ), 147.9 (C6), 149.1/150.9 (C1), 152.5/152.6 (C8b), 153.4/154.8 (C4a), 156.6/156.8 (C3), 171.09/171.13/171.15/171.20/171.72/171.79/171.82/171.93/172.37/172.44 /172.78/172.87/173.16/173.23/173.26 (COCH ₂ CH ₃ ), 173.56/173.60 (C9).
HRMS (FAB) m/z: [M+Na] ⁺ _Calcd for _{C43H50O19Na 893.2844} ; Found _893.2883 .

(2) Synthesis of mangiferin 8a (1,2',3',4',6-penta-O-propionyl mangiferin) A mixture of 1,3,2',3',4',6,6',7-octa-O-propionyl mangiferin (3.7 g, 4.25 mmol), ammonium acetate (3.8 g, 49.4 mmol), methanol (80 mL) and water (40 mL) was stirred at room temperature for 5.5 hours. After removing methanol from the reaction solution by vacuum concentration, the residue was diluted with ethyl acetate (100 mL). The mixture was washed with water and saturated saline solution in this order, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography [CHCl ₃ /CH ₃ OH (20:1)] to obtain a pale yellow solid (2.54 g). The solid was dissolved in methanol (80 mL) and decolorized with activated carbon to obtain mangiferin 8a (2.16 g, 73%) of formula (1) as a colorless solid.

^1H -NMR (800MHz, DMSO-d ₆ ) δ:: 0.92-1.24 (15H, m, COCH ₂ CH ₃ ), 1.92-2.32 (10H, m, COCH ₂ CH ₃ ) , 3.85 (0.5H, dd-like, J=ca. 12.5, 1.5, H-6a'), 4.02-4.13 (2H, m, H-5', H-6a', H-6b'), 4.21 (0.5H, dd, J = 12.5, 4.7, H-6b'), 4.95 (0.5H, d, J = 10.0, H-1'), 5.00 (0.5H, dd, J = 9.6, 9.6, H-1'), 5.01 (0.5H, dd, J = 9.6, 9.6, H-4 '), 5.15 (0.5H, d, J=10.0, H-1'), 5.31 (0.5H, dd, J=9.6, 9.6, H-3') , 5.39 (0.5H, dd, J = 9.6, 9.6, H-3'), 5.48 (0.5H, dd, J = 10.0, 9.6, H-2 '), 5.84 (0.5H, dd, J=10.0, 9.6, H-2'), 6.71/6.73 (each 0.5H, s, H-4), 6.79/6.80 (each 0.5H, s, H-5), 7.278/7.279 (each 0.5H, s, H-8) , 9.00-11.5 (3H, br s, OH x 3).
^13C -NMR (200MHz, DMSO- _d6 ) δ::8.84/8.89/9.00/9.05/9.10/9.14/9.15/9.21/9.23 (COCH ₂ CH ₃ ), 26.7/26.92/26.96/26.98/27.04/27.08/27.12/27.4 (COCH ₂ CH ₃ ), 62.1/62 .4 (C-6'), 68.2/68.4 (C-4'), 68.8/70.5 (C-2'), 71.0/71.1 (C-1') , 73.8/74.2 (C-3'), 74.7/75.3 (C-5'), 99.6/100.8 (C-4), 102.50/102.52 (C-5), 106.6/107.7 (C-9a), 109.1 ( C-8), 113.4 (C-2), 114.0/114.1 (C-8a), 143.87/143.88 (C-7), 149.83/149.89 (C- 6), 151.3 (C-1), 153.4 (C-8b), 157.6/157.7 (C-4a), 161.0/162.4 (C-3), 171.2/172.0/172.3/172.9/173.2/173.5/173.6 (COCH ₂ CH ₃ ) 172.7/172.8 (C-9).
HRMS (FAB) m/z: [M+Na] ⁺ Calcd for C ₃₄ H ₃₈ NaO ₁₆ 725.2058; Found. 725.2074.

Example 2: Synthesis of norathyriol
Norathyriol was synthesized according to the method of Non-Patent Document 38.
The synthesis method shown in this example can be briefly explained as follows. That is, according to the method described in the literature (Non-Patent Document 38), 2,4,5-trimethoxybenzoic acid (Compound II) was treated with thionyl chloride to obtain 2,4,5-trimethoxybenzoyl chloride (Compound III). Next, the obtained compound (Compound III) was subjected to a Friedel-Crafts reaction with 1,3,5-trimethoxybenzene (Compound IV) to obtain 2-hydroxy-2',4,4',5,6'-pentamethoxybenzophenone (Compound V). Furthermore, this compound (V) was treated with tetrabutylammonium hydroxide to obtain 1,3,6,7-tetramethoxyxanthone (Compound VI), which was then demethylated to obtain norathyriol (Compound I) in a yield of 39%.

The synthesis route of this example will be described in detail below with reference to FIG.
(1) Method for producing 2,4,5-trimethoxybenzoic acid chloride (Compound III) Thionyl chloride (5 mL) was gradually added to 2,4,5-trimethoxybenzoic acid (Compound II, 8.49 g, 0.040 mol) at room temperature under an argon atmosphere to dissolve the acid, and the mixture was heated under reflux for 6 hours. After completion of the reaction, the reaction mixture was distilled under reduced pressure to obtain 2,4,5-trimethoxybenzoic acid chloride (Compound III, 8.30 g, 90%). The obtained compound (III) was immediately used in the next reaction.

(2) Method for producing 2-hydroxy-2',4,4',5,6'-pentamethoxybenzophenone (Compound V) To a mixture suspension of 2,4,5-trimethoxybenzoyl chloride (Compound III, 8.07 g, 0.035 mol) obtained as described above, 1,3,5-trimethoxybenzene (Compound IV, 6.48 g, 0.0385 mol) and anhydrous diethyl ether (500 mL) was gradually added aluminum chloride (16 g) at room temperature under an argon atmosphere, and the reaction mixture was stirred at room temperature for 48 hours. The solvent in the reaction solution was distilled off under reduced pressure, and water was added to the residue, which was then extracted with ethyl acetate. The extract was washed with saturated saline and dried over anhydrous sodium sulfate. The desiccant was filtered off using pleated filter paper, and the solvent in the filtrate was distilled off under reduced pressure to obtain a crude product. The resulting crude product was purified by silica gel column chromatography (n-hexane:ethyl acetate=1:1, v/v) to obtain 2-hydroxy-2',4,4',5,6'-pentamethoxybenzophenone (Compound V, 8.43 g, 69%).

(3) Method for Producing 1,3,6,7-Tetramethoxyxanthone (Compound VI) The 2-hydroxy-2',4,4',5,6'-pentamethoxybenzophenone (Compound V, 6.97 g, 0.020 mol) obtained as described above was dissolved in a mixed solvent of pyridine (10 mL) and water (10 mL), and 40% aqueous tetrabutylammonium hydroxide solution (5 mL) was added and heated under reflux for 6 hours. The resulting reaction mixture was poured into 5% hydrochloric acid and then extracted with ethyl acetate. The extract was washed with saturated saline, dried over anhydrous sodium sulfate, and the desiccant was filtered off using pleated filter paper. The filtrate was distilled under reduced pressure to remove the solvent and obtain a crude product. The resulting crude product was purified by silica gel column chromatography (n-hexane:ethyl acetate=1:1, v/v) to obtain 1,3,6,7-tetramethoxyxanthone (Compound VI, 5.82 g, 92%).

(4) Method for Producing Norathyriol (Compound I) A mixture of 1,3,6,7-tetramethoxyxanthone (Compound VI, 4.74 g, 0.015 mol) obtained as described above and pyridine hydrochloride (5.00 g) was heated and stirred at 200° C. for 6 hours. The reaction mixture obtained was allowed to cool to room temperature, poured into 5% hydrochloric acid, and then extracted with ethyl acetate. The extract was washed with saturated saline and dried over anhydrous sodium sulfate. The desiccant was filtered off using pleated filter paper, and the filtrate was distilled under reduced pressure to remove the solvent, thereby obtaining a crude product. The crude product obtained was purified by silica gel column chromatography (chloroform:methanol=7:1, v/v) to obtain norathyriol (Compound I, 2.65 g, 68%) of formula (2).

Example 3: Examination of cell death-inducing effect of each drug on Raji cells
Raji cells (malignant lymphoma cell line) were cultured under conditions of 5% CO ₂ and 37° C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin, and fetal bovine serum.
After seeding on the plate, drugs were added at various concentrations, and cell viability was measured by the trypan blue dye method. The results are shown in FIG.

In the graph, the mangiferin 8a-administered group is represented as "Mangiferin 8a," the norathyriol-administered group as "Norathyriol," the tetrahydroxanthone-administered group as "1,3,5,6-tetrahydroxyxanthone," the methoxyxanthone-administered group as "1,3,6-trihydroxy-5-methoxyxanthone," the xanthohydrol-administered group as "Xanthydrol," the α-mangostin-administered group as "α-mangostin," and the γ-mangostin-administered group as "γ-mangostin" (same below).

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for all drugs. Table 1 shows the results of calculating IC50 (half maximal inhibitory concentration: the same applies below). It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group.

Example 4: Examination of cell death-inducing effect of each drug on CCRF-SB cells
CCRF-SB cells (malignant lymphoma cell line) were cultured under conditions of 5% CO ₂ and 37° C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding CCRF-SB cells in a 96-well plate, drugs were added at various concentrations and the cell viability was measured by the trypan blue dye method. The results are shown in FIG. 3.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for all drugs. Table 2 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group.

Example 5: Examination of cell death-inducing effect of each drug on Namalwa cells
Namalwa cells (malignant lymphoma cell line) were cultured under conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding Namalwa cells in a 96-well plate, drugs were added at various concentrations and cell viability was measured by the trypan blue dye method. The results are shown in FIG. 4.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for all drugs. Table 3 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group.

Example 6: Examination of cell death-inducing effect of each drug on Z138 cells
Z138 cells (malignant lymphoma cell line) were cultured under the conditions of 5% CO ₂ and 37° C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin, and fetal bovine serum.
After seeding on the plate, drugs were added at various concentrations, and cell viability was measured by the trypan blue dye method. The results are shown in FIG.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for all drugs. Table 4 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group.

Example 7: Examination of cell death-inducing effect of each drug on SU-DHL-5 cells
SU-DHL-5 cells (malignant lymphoma cell line) were cultured under conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding SU-DHL-5 cells in a 96-well plate, drugs were added at various concentrations and the cell viability was measured by the trypan blue dye method. The results are shown in Figure 6.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for all drugs. Table 5 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group.

Example 8: Examination of cell death-inducing effect of each drug on L363 cells
L363 cells (multiple myeloma cell line) were cultured under conditions of 5% CO ₂ and 37° C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin, and fetal bovine serum.
After seeding on the plate, drugs were added at various concentrations, and cell viability was measured by the trypan blue dye method. The results are shown in FIG.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for each drug. Table 6 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, and γ-mangostin administration group.

Example 9: Examination of cell death-inducing effect of each drug on KMS-28BM cells
KMS-28BM cells (multiple myeloma cell line) were cultured under conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding KMS-28BM cells in a 96-well plate, drugs were added at various concentrations and cell viability was measured by the trypan blue dye method. The results are shown in FIG. 8.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for each drug. Table 7 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, and γ-mangostin administration group.

Example 10: Examination of cell death-inducing effect of each drug on ARH77 cells
ARH77 cells (multiple myeloma cell line) were cultured under conditions of 5% CO ₂ and 37° C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding the ARH77 cells in a 96-well plate, drugs were added at various concentrations, and the cell viability was measured by the trypan blue dye method. The results are shown in FIG. 9.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for all drugs. Table 8 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group.

Example 11: Examination of cell death-inducing effect of each drug on IM9 cells
IM9 cells (multiple myeloma cell line) were cultured under conditions of 5% CO ₂ and 37°C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding IM9 cells in a 96-well plate, drugs were added at various concentrations and cell viability was measured by the trypan blue dye method. The results are shown in FIG. 10.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates cell viability (%). The control was the addition of only 0.5% DMSO in PBS used to dissolve the reagent. A concentration-dependent decrease in cell viability was confirmed for all drugs. Table 9 shows the results of calculating IC50 values. It was found that cell death was induced at a lower concentration than mangiferin in the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group.

Example 12: Examination of cell death-inducing effect of each drug on RPMI1788 cells
RPMI1788 cells (normal B lymphocyte cell line) were cultured under conditions of 5% CO ₂ and 37° C. The culture medium used was RPMI-1640 medium supplemented with 100 μg/mL penicillin, 100 U/mL streptomycin and fetal bovine serum. After seeding RPMI1788 cells in a 96-well plate, drugs were added at various concentrations, and the cell viability was measured by the trypan blue dye method. The results are shown in FIG. 11.

The horizontal axis indicates the concentration of each drug, and the vertical axis indicates the cell viability (%). The control was the one to which only 0.5% DMSO in PBS used for dissolving the reagent was added. No decrease in cell viability was observed for any drug. Table 10 shows the results of calculating IC50 values. The IC50 values for all drugs were 100 μM or more.

Therefore, it was found that the composition for improving malignant tumor diseases according to the present invention induces cell death in malignant tumor cells at a concentration that does not affect normal cells.

Example 13: Examination of NIK activation inhibition by administration of each drug
The NIK inhibitory effect of each drug administered was examined by immunoblotting using IM9 cells, and an inhibitory effect on NIK activation was observed (FIG. 12).

IM9 cells were seeded in a 150 ^cm2 flask and cultured for 72 hours under conditions of 37°C and 5% _CO2 (Control), IM9 cells were seeded in a 150 ^cm2 flask and precultured for 24 hours, and then added to a final concentration of 100 μM mangiferin, 10 μM mangiferin 8a, 10 μM norathyriol, 10 μM methoxyxanthone, 50 μM tetrahydroxyxanthone, 10 μM xanthohydrol, 50 μM α-mangostin and 50 μM γ-mangostin, and cultured for 72 hours under conditions of 37°C and 5% _CO2 . Proteins were extracted from these cell suspensions using a cell lysis solution and used as samples. 50 μM DMF, an NF-κB p65 nuclear migration inhibitor, was used as the target.

Each sample was subjected to SDS-PAGE and then transferred onto a PVDF membrane, and phosphorylation of NIK was examined using an anti-phospho-NIK antibody and an anti-NIK antibody.

The results of immunoblotting are shown in Figure 12. On the horizontal axis of the photograph, control, 50 μM DMF, 100 μM mangiferin, 10 μM mangiferin 8a, 10 μM
The cases where norathyriol, 10 μM methoxyxanthone, 50 μM tetrahydroxyxanthone, control, 10 μM xanthohydrol, 50 μM α-mangostin, and 50 μM γ-mangostin were added are shown side by side.

The antibody type is indicated vertically. Specifically, the case of anti-phospho-NIK antibody (indicated as "Phospho-NIK") and the case of anti-NIK antibody (indicated as "NIK") are shown. In the photograph of anti-NIK antibody, no decrease in the intensity of the NIK band was observed compared to the control. On the other hand, in the case of anti-phospho-NIK antibody, the immunoblotting results of the samples were faint for mangiferin, mangiferin 8a, norathyriol, methoxyxanthone, tetrahydroxyxanthone, xanthohydrol, α-mangostin, and γ-mangostin. The NF-κB p65 nuclear transport inhibitor DMF did not result in faintness compared to the control.

Example 14: Investigation of downstream signals of NIK by administration of each drug
We investigated the activation kinetics of IKK, NF-κB p52, and NF-κB p65, which are downstream signals of NIK, by administration of each drug, and found that it inhibited the activity of IKK and the nuclear translocation of NF-κB p52 and NF-κB p65 (FIG. 13).

Each sample was prepared in the same manner as in Example 13, and after SDS-PAGE, it was transferred to a PVDF membrane and assayed using anti-phospho-IKK antibody, anti-IKK antibody, anti-NF-κB p52 antibody, anti-NF-κB p65 antibody, and anti-Lamin antibody.

The results of immunoblotting are shown in Figure 13. On the horizontal axis of the photograph, control, 50 μM DMF, 100 μM mangiferin, 10 μM mangiferin 8a, 10 μM
The cases where norathyriol, 10 μM methoxyxanthone, 50 μM tetrahydroxyxanthone, control, 10 μM xanthohydrol, 50 μM α-mangostin, and 50 μM γ-mangostin were added are shown side by side.

The types of antibodies are indicated vertically, specifically, anti-phospho-IKK antibody (denoted as "Phospho-IKK"), anti-IKK antibody (denoted as "IKK"), anti-NF-κBp52 antibody (denoted as "NF-κBp52nuclear"), anti-NF-κBp65 antibody (denoted as "NF-κBp65nuclear"), and anti-Lamin antibody (denoted as "Lamin").

In addition, nuclei and cytoplasmic matrix were separated from the cells by extracting cytoplasmic and nuclear fractions using ProteoExtract (registered trademark) Subcellular Proteome Extraction Kit (Merck).

In the photograph of anti-IKK antibody, the band of IKK was not decreased in intensity compared to the control. On the other hand, in the case of anti-phospho-IKK antibody, the immunoblotting results of the samples were faint in mangiferin, mangiferin 8a, norathyriol, methoxyxanthone, tetrahydroxyxanthone, xanthohydrol, α-mangostin and γ-mangostin. In the case of DMF, an NF-κB p65 nuclear transport inhibitor, the band did not become faint compared to the control.

Next, we refer to the anti-NF-κBp52 antibody (referred to as "NF-κBp52nuclear"), anti-NF-κBp65 antibody (referred to as "NF-κBp65nuclear"), and anti-Lamin antibody (referred to as "Lamin") against intranuclear substances. Lamin was present regardless of all drugs, but it was confirmed that intranuclear NF-κBp52 was faint with mangiferin, mangiferin 8a, norathyriol, methoxyxanthone, tetrahydroxyxanthone, xanthohydrol, α-mangostin, and γ-mangostin, but an increase was observed with DMF compared to the control. In addition, intranuclear NF-κBp65 was decreased (the shadow became fainter) with all drugs.

Lamin is a fibrous protein that maintains the structure and regulates transcription in the cell nucleus. Therefore, in the state where intranuclear substances were detected in all samples, it was shown that NF-κB p65 and NF-κB p52 in the nucleus were not present or were present in very small amounts when each drug was added.

Example 15: Inhibitory effect of administration of each drug on CD138 expression in L363 cells
The inhibitory effect on CD138 expression by administration of each agent was examined by flow cytometry using L363 cells, and the inhibitory effect on CD138 expression was observed.

L363 cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), L363 cells were seeded in a 75 ^cm2 flask and precultured for 4 hours, after which 50 μM mangiferin, 1 μM mangiferin 8a, 1 μM norathyriol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to the final concentrations, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the cells were stained with anti-CD138 antibody, which is a malignancy marker for multiple myeloma, and the expression of CD138 was measured using BD LSRFortessa.

The results are shown in Figures 14 and 15. The horizontal axis represents the expression level of CD138, and the vertical axis represents the number of cells. In addition, the solid line in the panel represents a negative control that was not treated with anti-CD138 antibody or drug, the dotted line represents a positive control that was not treated with drug but was treated with anti-CD138 antibody, and the dashed line represents treatment with drug and anti-CD138 antibody.

Compared with the negative control group not treated with drugs or anti-CD138 antibody, the positive control group showed a significant increase in CD138 expression. Compared with the positive control group, the mangiferin administration group (FIG. 14(a)), mangiferin 8a administration group (FIG. 14(b)), norathyriol administration group (FIG. 14(c)), tetrahydroxyxanthone administration group (FIG. 14(d)), methoxyxanthone administration group (FIG. 15(e)), xanthohydrol administration group (FIG. 15(f)), α-mangostin administration group (FIG. 15(g)), and γ-mangostin administration group (FIG. 15(h)) showed a significant decrease in CD138 expression, almost the same as the negative control. That is, it was found that mangiferin, mangiferin 8a, norathyriol, tetrahydroxyxanthone, methoxyxanthone, xanthohydrol, α-mangostin, and γ-mangostin suppress the expression of CD138, which is a malignancy marker for multiple myeloma.

Example 16: Effect of administration of each drug on increasing CD20 expression in L363 cells
The increasing effect of CD20 expression by administration of each agent was examined by flow cytometry using L363 cells, and the increasing effect of CD20 expression was observed.

L363 cells were seeded in a 75 cm ² flask and cultured for 10 days under conditions of 37°C and 5% CO ₂ (Control), L363 cells were seeded in a 75 cm ² flask and pre-cultured for 4 hours, and then added to a final concentration of 50 μM mangiferin, 1 μM mangiferin 8a, 1 μM norathyriol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin, and cultured for 10 days under conditions of 37°C and 5% CO _2. After culturing for 10 days, the cells were stained with anti-CD20 antibody, which is a B cell marker, and CD20 expression was measured using BD LSRFortessa. CCRF-SB cells were used as a CD20 positive control.

The results are shown in Figures 16 and 17. The horizontal axis represents the expression level of CD20, and the vertical axis represents the number of cells. In addition, the solid line in the panel represents a negative control in which cells were not treated with an anti-CD20 antibody or drug, the dotted line represents a positive control in which cells were not treated with a drug but were treated with an anti-CD20 antibody, and the dashed line represents cells treated with a drug and an anti-CD20 antibody. The dashed and dotted line represents CCRF-SB cells treated with an anti-CD20 antibody.

Compared to the negative control group not treated with drugs or anti-CD20 antibodies, the positive control showed almost no increase in CD20 expression, and was comparable to that of the negative control. In other words, it is clear that L363 cells do not express CD20. On the other hand, in CCRF-SB cells used as a CD20 positive control, CD20 expression was significantly increased compared to the negative and positive controls of L363 cells. This indicates that CCRF-SB cells express CD20.

Furthermore, the mangiferin administration group (Figure 16(a)), mangiferin 8a administration group (Figure 16(b)), norathyriol administration group (Figure 16(c)), tetrahydroxyxanthone administration group (Figure 16(d)), methoxyxanthone administration group (Figure 17(e)), xanthohydrol administration group (Figure 17(f)), α-mangostin administration group (Figure 17(g)), and γ-mangostin administration group (Figure 17(h)) showed significantly increased CD20 expression compared to the positive control.

That is, mangiferin, mangiferin 8a, norathyriol, tetrahydroxyxanthone, methoxyxanthone, xanthohydrol, α-mangostin, and γ-mangostin suppressed anti-CD138 antibody, a marker of malignancy of multiple myeloma, and increased the expression of CD20, a B cell marker. As a result, it was found that the above substances convert multiple myeloma cells into B cell-like cells.

In multiple myeloma, CD20 is not usually expressed, and the effect of the anti-CD20 antibody rituximab, which binds to CD20 and exerts an antitumor effect, is not observed. However, the increase in CD20 expression caused by the composition containing mangiferin 8a, norathyriol, tetrahydroxyxanthone, methoxyxanthone, xanthohydrol, α-mangostin, and γ-mangostin according to the present invention allows rituximab to bind to cells and exert an antitumor effect. In other words, by converting multiple myeloma into B-cell-like cells using the above-mentioned substances, the antitumor effect of rituximab can be expected.

In addition, improvement of multiple myeloma using a composition containing mangiferin 8a, norathyriol, tetrahydroxyxanthone, methoxyxanthone, xanthohydrol, α-mangostin, and γ-mangostin and rituximab may also be considered as the present invention.

Example 17: Inhibitory effect of administration of each drug on IgG secretion in L363 cells
The inhibitory effect on IgG secretion by administration of each drug was examined by enzyme-linked immunosorbent assay (ELISA) using L363 cells, and the inhibitory effect on IgG secretion was observed.

L363 cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), L363 cells were seeded in a 75 ^cm2 flask and precultured for 4 hours, after which 50 μM mangiferin, 1 μM mangiferin 8a, 1 μM norathyriol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to the final concentrations, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the culture supernatant was collected and the amount of IgG secretion was measured by ELISA. This measurement was performed using a human anti-IgG antibody ELISA kit (Funakoshi).

The results are shown in Figure 18. The horizontal axis represents the sample group, and the vertical axis represents the amount of IgG secreted (ng/mL). In the control, IgG was 2861ng/mL, whereas in the mangiferin administration group, mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group, IgG was 140ng/mL, 40ng/mL, 59ng/mL, 145ng/mL, 67ng/mL, 106ng/mL, 24ng/mL, and 61ng/mL, respectively.

As described above, the mangiferin administration group was found to reduce IgG secretion, and the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group were found to significantly suppress IgG secretion at lower doses than the mangiferin administration group.

In other words, the control group developed kidney damage, amyloidosis, and hyperviscosity syndrome due to IgG production, while the mangiferin-administered group, mangiferin 8a-administered group, norathyriol-administered group, tetrahydroxyxanthone-administered group, xanthohydrol-administered group, α-mangostin-administered group, and γ-mangostin-administered group suppressed kidney damage, amyloidosis, and hyperviscosity syndrome.

Example 18: Inhibitory effect of administration of each drug on the secretion of λ-chain of free immunoglobulin light chain in L363 cells
The inhibitory effect of administration of each agent on the secretion of the λ-chain of free immunoglobulin light chain was examined by enzyme-linked immunosorbent assay (ELISA) using L363 cells, and the inhibitory effect on the secretion of the λ-chain of free immunoglobulin light chain was observed.

L363 cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), L363 cells were seeded in a 75 ^cm2 flask and precultured for 4 hours, after which 50 μM mangiferin, 1 μM mangiferin 8a, 1 μM norathyriol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin were added to the final concentrations, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the culture supernatant was collected and the amount of λ chain secretion of immunoglobulin free light chain was measured by ELISA. This measurement was performed using a human anti-λ chain antibody ELISA kit (Funakoshi).

The results are shown in Figure 19. The horizontal axis represents the sample group, and the vertical axis represents the amount of λ chain secretion (μg/L). In the control, IgG was 142 μg/L, whereas in the mangiferin administration group, mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group, IgG was 30 μg/L, 23 μg/L, 23 μg/L, 33 μg/L, 31 μg/L, 29 μg/L, 30 μg/L, and 31 μg/L, respectively.

As described above, the mangiferin administration group was found to reduce the secretion of λ chain, and the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group were found to significantly suppress λ chain secretion at lower doses than the mangiferin administration group. In other words, the control group caused renal damage, amyloidosis, and hyperviscosity syndrome due to λ chain production, while the mangiferin administration group, mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group suppressed renal damage, amyloidosis, and hyperviscosity syndrome.

Example 19: Inhibitory effect of administration of each drug on secretion of bone destruction-related factors in L363 cells
The inhibitory effect of each drug on the secretion of bone destruction-related factors was examined by Luminex using L363 cells, and the inhibitory effect on the secretion of bone destruction-related factors was observed.

L363 cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), L363 cells were seeded in a 75 ^cm2 flask and pre-cultured for 4 hours, and then added to a final concentration of 50 μM mangiferin, 1 μM mangiferin 8a, 1 μM norathyriol, 5 μM tetrahydroxyxanthone, 5 μM methoxyxanthone, 1 μM xanthohydrol, 50 μM α-mangostin and 5 μM γ-mangostin, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the culture supernatant was collected and the secretion amount of MIP-1α, a bone destruction-related factor, was measured by Luminex. This measurement was performed using Human Magnetic Luminex Assay (R&D).

The results are shown in Figure 20. The horizontal axis represents the sample group, and the vertical axis represents the amount of MIP-1α secreted (pg/mL). In the control, the amount of MIP-1α secreted was 245 pg/mL. In the mangiferin administration group, mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group, the amounts of MIP-1α secreted were 0 pg/mL, 0 pg/mL, 0 pg/mL, 0 pg/mL, 0.73 pg/mL, 0 pg/mL, 0 pg/mL, and 0 pg/mL, respectively.

As described above, the mangiferin administration group was found to reduce the secretion of MIP-1α, and the mangiferin 8a administration group, norathyriol administration group, tetrahydroxyxanthone administration group, methoxyxanthone administration group, xanthohydrol administration group, α-mangostin administration group, and γ-mangostin administration group were found to significantly suppress MIP-1α secretion at lower doses than the mangiferin administration group. In other words, the control group developed bone lesions (bone destruction), hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms due to the production of MIP-1α, while the mangiferin-administered group, mangiferin 8a-administered group, norathyriol-administered group, tetrahydroxyxanthone-administered group, xanthohydrol-administered group, α-mangostin-administered group, and γ-mangostin-administered group suppressed bone lesions (bone destruction), hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms.

Example 20: Inhibitory effect of administration of each drug on CD138 expression in KMS-28BM cells
The inhibitory effect on CD138 expression by administration of each agent was examined by flow cytometry using KMS-28BM cells, and the inhibitory effect on CD138 expression was observed.

KMS-28BM cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), and KMS-28BM cells were seeded in a 75 ^cm2 flask and precultured for 4 hours, after which 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM norathyriol were added to the final concentrations, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the cells were stained with anti-CD138 antibody, which is a malignancy marker for multiple myeloma, and the expression of CD138 was measured using BD LSRFortessa.

The results are shown in Figure 21. The horizontal axis represents the expression level of CD138, and the vertical axis represents the number of cells. The solid line in the panel represents a negative control not treated with anti-CD138 antibody or drug, the dotted line represents a positive control not treated with drug but treated with anti-CD138 antibody, and the dashed line represents a control treated with drug and anti-CD138 antibody. Compared with the negative control group not treated with drug or anti-CD138 antibody, CD138 expression was significantly increased in the positive control.

In the mangiferin administration group (Figure 21 (a)), mangiferin 8a administration group (Figure 21 (b)), and norathyriol administration group (Figure 21 (c)), the CD138 expression level was significantly lower than in the positive control, and was almost the same as in the negative control. In other words, it was found that mangiferin, mangiferin 8a, and norathyriol suppress the expression of CD138, which is a malignancy marker for multiple myeloma.

Example 21: Effect of administration of each drug on increasing CD20 expression in KMS-28BM cells
The increasing effect of CD20 expression by administration of each drug was examined by flow cytometry using KMS-28BM cells, and the increasing effect of CD20 expression was observed.

KMS-28BM cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control). KMS-28BM cells were seeded in a 75 ^cm2 flask and precultured for 4 hours, after which 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM norathyriol were added to the final concentrations, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the cells were stained with anti-CD20 antibody, which is a B cell marker, and CD20 expression was measured using BD LSRFortessa. CCRF-SB cells were used as a CD20 positive control.

The results are shown in Figure 22. The horizontal axis represents the expression level of CD20, and the vertical axis represents the number of cells. In addition, the solid line in the panel represents a negative control in which cells were not treated with an anti-CD20 antibody or drug, the dotted line represents a positive control in which cells were not treated with a drug but were treated with an anti-CD20 antibody, and the dashed line represents cells treated with a drug and an anti-CD20 antibody. The dashed and dotted line represents CCRF-SB cells treated with an anti-CD20 antibody.

Compared to the negative control group not treated with drugs or anti-CD20 antibodies, the positive control showed almost no increase in CD20 expression, and was at the same level as the negative control. In other words, it is understood that KMS-28BM cells do not express CD20. On the other hand, in CCRF-SB cells used as a CD20 positive control, CD20 expression was significantly increased compared to the negative and positive controls of KMS-28BM cells. This indicates that CCRF-SB cells express CD20. Furthermore, the mangiferin administration group (Figure 22(a)), mangiferin 8a administration group (Figure 22(b)), and norathyriol administration group (Figure 22(c)) showed significantly increased CD20 expression levels compared to the positive control.

That is, mangiferin, mangiferin 8a, and norathyriol suppressed anti-CD138 antibody, a marker of malignancy of multiple myeloma, and increased the expression of CD20, a B cell marker. As a result, it was found that the above substances transformed multiple myeloma cells (KMS-28BM cells) into B cell-like cells.

Example 22: Inhibitory effect of administration of each drug on IgG secretion in KMS-28BM cells
The inhibitory effect on IgG secretion by administration of each drug was examined by enzyme-linked immunosorbent assay (ELISA) using KMS-28BM cells, and the inhibitory effect on IgG secretion was observed.

KMS-28BM cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), and KMS-28BM cells were seeded in a 75 ^cm2 flask and precultured for 4 hours, after which 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM norathyriol were added to the final concentrations, and the cells were cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the culture supernatant was collected and the amount of IgG secreted was measured by ELISA. This measurement was performed using a human anti-IgG antibody ELISA kit (Funakoshi).

The results are shown in Figure 23. The horizontal axis represents the sample group, and the vertical axis represents the amount of IgG secreted (ng/mL). In the control, IgG was 2018 ng/mL, whereas in the mangiferin administration group, the mangiferin 8a administration group, and the norathyriol administration group, IgG was 106 ng/mL, 40 ng/mL, and 0 ng/mL, respectively.

As described above, it was found that the mangiferin administration group reduces IgG secretion, and the mangiferin 8a administration group and the norathyriol administration group significantly suppress IgG secretion at a lower dose than the mangiferin administration group.In other words, the control group causes renal damage, amyloidosis, and hyperviscosity syndrome due to IgG production, while the mangiferin administration group, the mangiferin 8a administration group, and the norathyriol administration group suppress renal damage, amyloidosis, and hyperviscosity syndrome.

Example 23: Inhibitory effect of administration of each drug on the secretion of λ-chain of free immunoglobulin light chain in KMS-28BM cells
The inhibitory effect of each drug on the secretion of the λ-chain of free immunoglobulin light chain was examined by enzyme-linked immunosorbent assay (ELISA) using KMS-28BM cells, and the inhibitory effect on the secretion of the λ-chain of free immunoglobulin light chain was observed.

KMS-28BM cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), and KMS-28BM cells were seeded in a 75 ^cm2 flask and precultured for 4 hours, after which 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM norathyriol were added to the final concentrations, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the culture supernatant was collected and the amount of λ chain secretion of immunoglobulin free light chain was measured by ELISA. This measurement was performed using a human anti-λ chain antibody ELISA kit (Funakoshi).

The results are shown in Figure 24. The horizontal axis represents the sample group, and the vertical axis represents the amount of λ chain secretion (μg/L). In the control, IgG was 16 μg/L, whereas in the mangiferin administration group, the mangiferin 8a administration group, and the norathyriol administration group, IgG was 0 μg/L, 0 μg/L, and 6.9 μg/L, respectively.

As described above, it was found that the mangiferin administration group reduces the secretion of λ chain, and the mangiferin 8a administration group and the norathyriol administration group significantly suppress the secretion of λ chain at a lower dose than the mangiferin administration group. In other words, it can be said that the control group caused renal damage, amyloidosis, and hyperviscosity syndrome due to the production of λ chain, and the mangiferin administration group, the mangiferin 8a administration group, and the norathyriol administration group suppressed renal damage, amyloidosis, and hyperviscosity syndrome.

Example 24: Inhibitory effect of administration of each drug on secretion of bone destruction-related factors in KMS-28BM cells
The inhibitory effect of each drug on the secretion of bone destruction-related factors was examined by Luminex using KMS-28BM cells, and the inhibitory effect on the secretion of bone destruction-related factors was observed.

KMS-28BM cells were seeded in a 75 ^cm2 flask and cultured for 10 days under conditions of 37°C and 5% _CO2 (Control), and KMS-28BM cells were seeded in a 75 ^cm2 flask and pre-cultured for 4 hours, after which 1 μM mangiferin, 0.1 μM mangiferin 8a, and 0.05 μM norathyriol were added to the final concentrations, and cultured for 10 days under conditions of 37°C and 5% _CO2 . After culturing for 10 days, the culture supernatant was collected and the secretion amount of IL-6, a bone destruction-related factor, was measured by Luminex. This measurement was performed using Human Magnetic Luminex Assay (R&D).

The results are shown in Figure 25. The horizontal axis represents the sample group, and the vertical axis represents the amount of IL-6 secreted (pg/mL). In the control group, the amount of IL-6 secreted was 153 pg/mL. In the mangiferin administration group, the mangiferin 8a administration group, and the norathyriol administration group, the amount of IL-6 secreted was 36 pg/mL, 28 pg/mL, and 43 pg/mL, respectively.

As described above, the mangiferin administration group was found to reduce IL-6 secretion, and the mangiferin 8a administration group and the norathyriol administration group were found to significantly suppress IL-6 secretion at lower doses than the mangiferin administration group. In other words, the control group caused bone lesions (bone destruction), hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms due to IL-6 production, while the mangiferin administration group, mangiferin 8a administration group, and norathyriol administration group suppressed bone lesions (bone destruction), hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, and neurological symptoms associated with spinal cord compression symptoms.

Example 25: Inhibitory effect of norathyriol administration on tumor growth in Raji cells in vivo
Raji cells were transplanted into NOD/ShiJic-scidJcl mice, and the tumor growth inhibitory effect of orally administering norathyriol was examined. As a result, a significant tumor growth inhibitory effect was observed.

Raji cells were transplanted into NOD/ShiJic-scidJcl mice, and the time when the average tumor volume exceeded ¹⁰⁰ mm3 was set as day 0. Norathyriol was orally administered to the mice at 100 mg/kg every day for 27 days, and the tumor volume was measured.

The group that only received Raji cells was called the control group, and the group that received norathyriol was called the norathyriol-administered group. Each group consisted of 5 mice.

The results are shown in Figure 26. Referring to the figure, the horizontal axis indicates the number of days elapsed (days), and the vertical axis indicates the tumor volume ( ^mm3 ). Open circles indicate the control group (indicated as "Control"). Black diamonds indicate the norathyriol-administered group (indicated as "100 mg/kg norathyriol"). In addition, "*" is indicated for those with significant differences (P<0.01) from the control group.

In the control group (white circle "-O-"), tumor growth was observed over time, reaching 5790 ^mm3 after 27 days. In the norathyriol administration group (black diamond "-◆-"), significant tumor growth inhibition was observed.

In other words, the norathyriol-administered group suppressed tumor growth more significantly than the control group.

Figure 27 shows a photograph of the tumor on the 27th day after the start of administration. Figure 27(a) shows a photograph of the tumor in one mouse in the control group, and Figure 27(b) shows a photograph of the tumor in one mouse in the norathyriol-administered group. In the control group in Figure 27(a), tumor growth is significantly observed. On the other hand, in the norathyriol-administered group in Figure 27(b), a significant decrease in tumor volume was observed compared to the control group.

As described above, it was found that the norathyriol-administered group significantly suppressed tumor growth.

Example 26: Inhibitory effect of administration of norathyriol on tumor growth in L363 cells in vivo
L363 cells were transplanted into NOD/ShiJic-scidJcl mice, and the tumor growth inhibitory effect of oral administration of norathyriol was examined. As a result, a significant tumor growth inhibitory effect was observed.

L363 cells were transplanted into NOD/ShiJic-scidJcl mice, and the time when the average tumor volume exceeded ¹⁰⁰ mm3 was set as day 0. Norathyriol was orally administered to the mice at 100 mg/kg every day for 8 days, and the tumor volume was measured.

The group that only received L363 cells was called the control group, and the group that received norathyriol was called the norathyriol-administered group. Each group consisted of 5 mice.

The results are shown in Figure 28. Referring to the figure, the horizontal axis indicates the number of days passed, and the vertical axis indicates the tumor volume. The open circles indicate the control group (indicated as "Control"). The closed diamonds indicate the norathyriol administration group (indicated as "100 mg/kg norathyriol"). In addition, "*" is indicated for those that were significantly different from the control group (P<0.01).

In the control group (white circle "-O-"), tumor growth was observed over time, reaching 745 ^mm3 after 8 days. In the norathyriol-administered group (black diamond "-◆-"), significant inhibition of tumor growth was observed.

In other words, the norathyriol-administered group suppressed tumor growth more significantly than the control group.

Figure 29 shows a photograph of the tumor 8 days after the start of administration. Figure 29(a) shows a photograph of the tumor in one mouse in the control group, and Figure 29(b) shows a photograph of the tumor in one mouse in the norathyriol-administered group. In the control group in Figure 29(a), tumor growth is significantly observed. On the other hand, in the norathyriol-administered group in Figure 29(b), a significant decrease in volume was observed compared to the control group.

As described above, it was found that the norathyriol-administered group significantly suppressed tumor growth.

The composition for improving malignant tumors according to the present invention can be a pharmaceutical composition useful as a selective NIK inhibitor for malignant tumors caused by activation of NF-κB p52 and NF-κB p65. The composition for improving malignant tumors according to the present invention can be a pharmaceutical composition useful for bone lesions (bone destruction) associated with multiple myeloma, hypercalcemia associated with bone lesions, pathological fractures, spinal cord compression fractures, spinal cord compression symptoms associated with spinal cord compression fractures, neurological symptoms associated with spinal cord compression symptoms, nephropathy, amyloidosis, and hyperviscosity syndrome.

Claims (7)

A composition for improving malignant tumors, comprising as an active ingredient at least one compound of mangiferin 8a and xanthohydrol, both of which have a xanthone skeleton. A drug for the treatment of malignant tumors, the active ingredient of which is at least one compound of mangiferin 8a and xanthohydrol, both of which have a xanthone skeleton. A processed food for improving malignant lymphoma or multiple myeloma, comprising at least one compound having a xanthone skeleton, selected from the group consisting of mangiferin 8a, norathyriol, tetrahydroxyxanthone and methoxyxanthone. An agent for inducing dedifferentiation into B-cell-like cells in multiple myeloma, comprising at least one compound having a xanthone skeleton, namely mangiferin 8a, norathyriol, tetrahydroxyxanthone, methoxyxanthone and xanthohydrol. A drug for improving CRAB associated with multiple myeloma, comprising at least one compound having a xanthone skeleton, namely mangiferin 8a, norathyriol, tetrahydroxyxanthone, methoxyxanthone and xanthohydrol. A composition for improving malignant lymphoma or multiple myeloma, comprising at least one compound having a xanthone skeleton, selected from the group consisting of mangiferin 8a, norathyriol, tetrahydroxyxanthone, methoxyxanthone and xanthohydrol, as an active ingredient. A composition for improving multiple myeloma, comprising at least one compound selected from the group consisting of mangiferin 8a, xanthohydrol, norathyriol, tetrahydroxyxanthone, and methoxyxanthone, and rituximab .

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