Use of myt1 inhibitor

Abstract

The present invention provides a pharmaceutical composition comprising, as an active ingredient, a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or the salt thereof.　

Description

Uses of MYT1 inhibitors

[0001] The present invention relates to a therapeutic agent and method for treating cancer in patients with decreased RB1 function, which uses a MYT1 inhibitor in combination with a chemotherapeutic agent or a molecular targeted agent.

[0002] In recent years, rapid advances in genome sequencing technology have made it possible to decipher genomic information, including specific gene mutations, in cancer cells. Examples of specific gene mutations in cancer cells include EGFR gene mutations, BRAF gene mutations, and gain-of-function genes such as ALK fusion genes and ROS1 fusion genes. For example, an ALK fusion gene is a gain-of-function gene formed by the fusion of an ALK gene encoding a receptor tyrosine kinase with a gene encoding a protein with multimerization function, such as EML4, through chromosomal inversion or translocation. In the development of anticancer drugs, drug discovery research has been conducted with the aim of selectively inhibiting the function of specific gene mutations in cancer cells (see Non-Patent Documents 1 to 3). Treatment methods targeting cancer cells with these gene mutations are expected to be highly selective for cancer cells and highly effective treatment methods.

[0003] On the other hand, gene mutations found in human cancer cells include not only gain-of-function but also loss-of-function mutations. Loss-of-function gene mutations are difficult to target with drugs, and require a different treatment strategy from cancers with gain-of-function gene mutations.

[0004] One of the few successful examples of specifically targeting cancer cells with loss-of-function mutations is the treatment of BRCA1 / 2-deficient tumors with PARP inhibitors (see Non-Patent Document 4). However, no therapeutic strategy has yet been developed to specifically target cancer cells with other loss-of-function mutations.

[0005] Loss of function of the RB1 gene has been considered a cause or promoting factor of retinoblastoma, but in recent years, reports have shown that functional impairments of the RB1 gene, such as loss-of-function mutations and expression suppression, are observed in many human cancers, and as a result, attention has been focused on its involvement in the development and progression of tumors in a wide range of cancers. The RB1 protein is thought to be involved in various cancer biologies, such as cell cycle, inflammation, metabolism, autophagy, apoptosis, differentiation, aging, DNA repair, and genome stability, and it is speculated that there is an important relationship between such functional suppression and the development and progression of cancer, and research is being conducted based on this assumption (see Non-Patent Document 5).

[0006] In fact, it has been reported that in small cell lung cancer and triple-negative breast cancer (a type in which the estrogen receptor, progesterone receptor, and HER2 protein are all negative), RB1 protein deficiency and Aurora kinase A are synthetically lethal (see Non-Patent Document 6), and that in triple-negative breast cancer, RB1 protein deficiency and CHK1 or PLK1 are synthetically lethal (see Non-Patent Document 7).

[0007] It has also been reported that the combined use of RP-6306 and gemcitabine is expected to have an in vivo antitumor effect in NIH:OVCAR-3 cells in which CCNE1 is amplified (see Patent Document 1 and Non-Patent Document 8).

[0008] However, there are still no sufficient therapeutic strategies that specifically target cancer cells in which RB1 function is impaired.

[0009] International Publication No. 2021 / 195781

[0010] Makoto Maemondo et al. NEJM2010Jun 24; 362(25):2380-2388.Paul B. Chapman et al. NEJM2011Jun 30; 364(26):2507-2516.D. Ross Camidge et al. J. Thorac.Oncol. 2019 Jul; 14(7):1233-1243.Kathleen Moore et al. NEJM2018Dec 27; 379(26):2495-2505.Letian Zhang et al. Annu Rev CancerBiol. 2022 April Vol.6:201-221.Xueqian Gong et al. Cancer Discov.2019 Feb;9(2):248-263.Agnieszka K. et al. Cell Rep.2018Jan 30;22(5):1185-1199.David Gallo et al. Nature 2022 Apr;604(7907):749-756.Patricia Jaaks et al. Nature 2022Mar;603(7899):166-173.Alina Malyutina et al. PLoS ComputBiol. 2019 May 20;15(5):e1006752.Sarah E. Taylor et al.CancerRes. 2019 Aug; 79(16):4242-4257.Dorien H. et al. Cancer Res.2016Oct;76(19):5719-5731Paola Indovina et al. Oncotarget2015 Jul;6(20): 17873-17890.Kenta Kurayoshi et al.DOI:10.5772 / intechopen.72125.Jasone E McDermott et al. Cell RepMed. 2020 Apr;1(1):100004Suhas Vasaikar et al. Cell 2019 May;177(4):1035-1049.e19.

[0011] Therefore, an object of the present invention is to provide a novel method for treating or preventing cancer caused by RB1 hypofunction. Another object of the present invention is to provide a novel method for treating or preventing cancer caused by CCNE1 hyperfunction.

[0012] The present invention provides the following: [A-1] A pharmaceutical composition comprising, as an active ingredient, a compound represented by the following formula (1) (also referred to as "Compound 1") or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt: [A-2] A pharmaceutical composition comprising a chemotherapeutic agent or a molecularly targeted agent as an active ingredient, in combination with Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, for treating or preventing cancer. [A-3] A method for treating or preventing cancer in a cancer patient, comprising administering Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt to said cancer patient. [A-4] A method for inhibiting the proliferation of cancer cells, comprising contacting said cancer cells with Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt. [A-5] A method for improving the responsiveness of cancer treatment with a chemotherapeutic agent or a molecularly targeted agent, comprising administering Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt to said cancer patient together with a chemotherapeutic agent or a molecularly targeted agent. [A-6] A method for predicting responsiveness to cancer treatment with a combination of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, and a chemotherapeutic agent or a molecularly targeted agent, comprising: determining, or having a third party determine, whether a cancer patient is a cancer patient in whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected; and determining that the cancer patient is responsive to cancer treatment with a combination of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, and a chemotherapeutic agent or a molecularly targeted agent, if the cancer patient is a cancer patient in whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected.[A-6.1] A method for predicting responsiveness to cancer treatment with a combination of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, and a chemotherapeutic agent or a molecularly targeted agent, comprising: determining, or having a third party determine, whether a cancer patient is a cancer patient in whom positive expression of CCNE1 protein, overexpression of CCNE1 protein, or an amplification of the copy number of CCNE1 gene has been detected; and, if the cancer patient is a cancer patient in whom positive expression of CCNE1 protein, overexpression of CCNE1 protein, or an amplification of the copy number of CCNE1 gene has been detected, determining that the cancer patient will be responsive to cancer treatment with a combination of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, and a chemotherapeutic agent or a molecularly targeted agent. [A-7] A method for selecting a cancer patient for whom administration of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, in combination with a chemotherapeutic agent or a molecularly targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecularly targeted agent alone, the method comprising: determining whether or not the cancer patient is a cancer patient for whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, or having a third party determine this; and, if the cancer patient is a cancer patient for whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, determining the cancer patient as a cancer patient for whom administration of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, in combination with a chemotherapeutic agent or a molecularly targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecularly targeted agent alone.[A-7.1] A method for selecting a cancer patient for whom administration of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, in combination with a chemotherapeutic agent or a molecularly targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecularly targeted agent alone, the method comprising: determining, or having a third party determine, whether the cancer patient is a cancer patient for whom positive expression of CCNE1 protein, overexpression of CCNE1 protein, or amplification of the copy number of the CCNE1 gene has been detected; and, if the cancer patient is a cancer patient for whom positive RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, determining the cancer patient as a cancer patient for whom administration of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, in combination with a chemotherapeutic agent or a molecularly targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecularly targeted agent alone. [A-8] Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt for use in the treatment or prevention of cancer. [A-9] A chemotherapeutic agent or molecular targeted agent for use in combination with Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt for the treatment or prevention of cancer. [A-10] Use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt for the manufacture of a pharmaceutical composition for the treatment or prevention of cancer. [A-11] Use of a chemotherapeutic agent or molecular targeted agent for administration in combination with Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt for the manufacture of a pharmaceutical composition for the treatment or prevention of cancer.

[0013] [A-12] The pharmaceutical composition according to [A-1] for treating cancer. [A-13] The pharmaceutical composition according to any one of [A-1], [A-8], and [A-10], used in combination with a chemotherapeutic agent or a molecular targeted agent, Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, or use. [A-14] The method according to [A-3], further comprising administering a chemotherapeutic agent or a molecular targeted agent to the cancer patient. [A-15] The method according to [A-4], further comprising contacting a chemotherapeutic agent or a molecular targeted agent with the cancer cells. [A-16] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-2] to [A-4] and [A-8] to [A-15], wherein the cancer is a cancer in a cancer patient in which RB1 gene mutation is positive, RB1 gene or protein expression is reduced, or hyperphosphorylated RB1 protein expression is positive. [A-16.1] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-2] to [A-4] and [A-8] to [A-15], wherein the cancer is a cancer in a cancer patient in which CCNE1 protein expression is positive, CCNE1 protein is overexpressed, or the copy number of the CCNE1 gene is amplified. [A-17] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecularly targeted agent for use, or use according to any one of [A-2] to [A-4] and [A-8] to [A-15], wherein the cancer is a cancer in a patient in which positive RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected.[A-17.1] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent for use, or use according to any one of [A-2] to [A-4] and [A-8] to [A-15], wherein the cancer is a cancer in a patient in which positive expression of CCNE1 protein, overexpression of CCNE1 protein, or amplification of the copy number of the CCNE1 gene has been detected. [A-18] The method according to any one of [A-3] to [A-5], comprising detecting, or having a third party detect, a positive RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein. [A-18.1] The method according to any one of [A-3] to [A-5], comprising detecting, or having a third party detect, that the cancer has positive expression of CCNE1 protein, overexpression of CCNE1 protein, or an amplification of the copy number of the CCNE1 gene. [A-19] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof for use, chemotherapeutic agent or molecular targeted agent for use, or use according to any one of [A-1] to [A-18], wherein compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof is compound 1. [A-20] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof, use of a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt is a pharmaceutically acceptable salt of Compound 1.[A-21] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, use of a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein the pharmaceutically acceptable salt is the hydrochloride salt of Compound 1. [A-22] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein the compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt is a pharmaceutically acceptable solvate of said compound 1 or a salt thereof. [A-23] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof, a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt is a pharmaceutically acceptable solvate of a salt of Compound 1. [A-24] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt, a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt is a hydrate of Compound 1 or a pharmaceutically acceptable salt thereof, or a hydrate of the compound or salt. [A-25] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, use of a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof is a pharmaceutically acceptable hydrate of a salt of Compound 1.[A-25.1] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is a solvate of the hydrochloride of Compound 1. [A-25.2] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is a hydrate of the hydrochloride of Compound 1. [A-26] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, use of a chemotherapeutic agent or molecular targeted agent, or use according to any one of [A-1] to [A-18], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is hydrochloride monohydrate of Compound 1.

[0014] [A-27] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecularly targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecularly targeted agent is gemcitabine. [A-28] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecularly targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecularly targeted agent is carboplatin. [A-29] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecularly targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecularly targeted agent is trastuzumab deruxtecan. [A-30] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecularly targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecularly targeted agent is sacituzumab govitecan. [A-31] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof for use, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent for use, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of compound 1 or a salt thereof is hydrochloride monohydrate of compound 1, and the chemotherapeutic agent or molecular targeted agent is pemetrexed.[A-32] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecularly targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecularly targeted agent is etoposide. [A-33] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecularly targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecularly targeted agent is lurbinectedin. [A-34] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent is fluorouracil. [A-35] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecular targeted agent is trifluridine. [A-36] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, the chemotherapeutic agent or molecular targeted agent for use, or the use according to [A-26], wherein the chemotherapeutic agent or molecular targeted agent is trifluridine, which is further used in combination with tipiracil.[A-37] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or the use according to [A-26], wherein the pharmaceutically acceptable solvate of Compound 1 or a salt thereof is the hydrochloride monohydrate of Compound 1, and the chemotherapeutic agent or molecular targeted agent is a combination of fluorouracil and irinotecan. [A-38] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, a chemotherapeutic agent or molecular targeted agent, or the use according to [A-26], wherein the chemotherapeutic agent or molecular targeted agent is a combination of fluorouracil and irinotecan, further combined with levofolinate. [A-39] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or its salt, the chemotherapeutic agent or molecular targeted agent for use, or the use according to [A-26], wherein the chemotherapeutic agent or molecular targeted agent is a combination of fluorouracil and irinotecan, which is further used in combination with levofolinate and bevacizumab.

[0015] [B-1] A pharmaceutical composition for treating or preventing cancer in a cancer patient in whom a positive RB1 gene mutation, decreased expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, comprising, as an active ingredient, a compound represented by the following formula (2) (also referred to as "Compound 2") or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or the salt thereof: [B-2] A pharmaceutical composition comprising as an active ingredient a chemotherapeutic agent or a molecular targeted agent in combination with compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, for treating or preventing cancer in a cancer patient in whom a positive RB1 gene mutation, a reduced expression of the RB1 gene or protein, or a positive expression of hyperphosphorylated RB1 protein has been detected. [B-3] A method for treating or preventing cancer in a cancer patient in whom a positive RB1 gene mutation, a reduced expression of the RB1 gene or protein, or a positive expression of hyperphosphorylated RB1 protein has been detected, the method comprising administering to the cancer patient compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt. [B-3.5] A method for treating or preventing cancer, comprising detecting or having a third party detect that the cancer is positive for an RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein, and administering compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or its salt to the cancer patient. [B-4] A method for suppressing the proliferation of cancer cells in a cancer patient in which a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, comprising contacting the cancer cells with compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or its salt. [B-4.5] A method for suppressing the proliferation of cancer cells, comprising: detecting or having a third party detect that the cancer is positive for an RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein; and contacting the cancer cells with Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or its salt.[B-5] A method for improving responsiveness to cancer treatment with a chemotherapeutic agent or a molecularly targeted agent, wherein the cancer is a cancer in a patient for which a positive RB1 gene mutation, a reduced expression of the RB1 gene or protein, or a positive expression of hyperphosphorylated RB1 protein has been detected, and the method comprises administering to the cancer patient compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, together with a chemotherapeutic agent or a molecularly targeted agent. [B-5.5] A method for improving responsiveness to cancer treatment with a chemotherapeutic agent or a molecularly targeted agent, comprising detecting or having a third party detect that the cancer is a positive RB1 gene mutation, a reduced expression of the RB1 gene or protein, or a positive expression of hyperphosphorylated RB1 protein, and administering to the cancer patient compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, together with a chemotherapeutic agent or a molecularly targeted agent. [B-6] A method for predicting responsiveness to cancer treatment with a combination of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, and a chemotherapeutic agent or a molecularly targeted agent, comprising: determining, or having a third party determine, whether a cancer patient is a cancer patient in whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected; and determining that the cancer patient is responsive to cancer treatment with a combination of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, and a chemotherapeutic agent or a molecularly targeted agent, if the cancer patient is a cancer patient in whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected.[B-7] A method for selecting a cancer patient for whom administration of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, in combination with a chemotherapeutic agent or a molecularly targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecularly targeted agent alone, the method comprising: determining whether the cancer patient is a cancer patient for whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, or having a third party determine this; and if the cancer patient is a cancer patient for whom RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, determining the cancer patient as a cancer patient for whom administration of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, in combination with a chemotherapeutic agent or a molecularly targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecularly targeted agent alone. [B-8] Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, for use in treating or preventing cancer in cancer patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected. [B-9] A chemotherapeutic agent or molecularly targeted agent for use in combination with Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, for treating or preventing cancer in cancer patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected. [B-10] Use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or its salt, for the manufacture of a pharmaceutical composition for treating or preventing cancer in cancer patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected.[B-11] Use of a chemotherapeutic agent or a molecularly targeted agent for the manufacture of a pharmaceutical composition for treating or preventing cancer in a cancer patient in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, wherein the pharmaceutical composition is administered in combination with compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof.

[0016] [B-12] Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use in combination with a chemotherapeutic agent or a molecular targeted agent, or use thereof, according to any one of [B-1] to [B-11]. [B-13] Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use in combination with a chemotherapeutic agent or a molecular targeted agent, according to any one of [B-1] to [B-11]. [B-14] Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use in combination with a chemotherapeutic agent or a molecular targeted agent, according to any one of [B-1] to [B-11]. [B-15] The pharmaceutical composition, method, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof, a chemotherapeutic agent or molecular targeted agent, or use according to any one of [B-1] to [B-11], wherein compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof, is compound 2. [B-16] The pharmaceutical composition, method, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof, a chemotherapeutic agent, or use according to any one of [B-1] to [B-12], wherein compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or salt thereof, is a pharmaceutically acceptable salt of compound 2. [B-17] The pharmaceutical composition, method, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, a chemotherapeutic agent for use, or use according to any one of [B-1] to [B-13], wherein Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof is a pharmaceutically acceptable solvate of Compound 2 or a salt thereof.

[0017] [C-1] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent or molecular targeted agent for use, or use according to any one of [A-1] to [A-36] or [B-1] to [B-17], wherein the cancer patient in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected is a cancer patient in whom a positive RB1 gene mutation or reduced expression of the RB1 gene or protein has been detected. [C-2] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, chemotherapeutic agent for use, or use according to [C-1], wherein the cancer patient in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein is detected is a cancer patient in whom a positive RB1 gene mutation has been detected. [C-3] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent for use, or use according to [C-1] or [C-2], wherein the cancer patient in whom a positive RB1 gene mutation, decreased expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein is detected is a cancer patient in whom decreased expression of the RB1 gene or protein is detected.[C-4] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent for use, or use according to any one of [C-1] to [C-3], wherein the cancer patient in whom positive for RB1 gene mutation, reduced expression of RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein is detected is a cancer patient in whom positive expression of hyperphosphorylated RB1 protein is detected. [C-5] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to any one of [C-1] to [C-4], wherein the RB1 gene mutation comprises a mutation resulting in the insertion, substitution, deletion, and / or addition of at least one amino acid residue relative to the wild-type RB1 protein. [C-6] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to any one of [C-1] to [C-5], wherein the RB1 gene mutation is a nonsense mutation, a frameshift mutation, a splice site mutation, or a homozygous or heterozygous deletion. [C-7] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [C-1] to [C-6], wherein the RB1 gene mutation is a mutation that reduces the function of RB1.[C-8] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [C-1] to [C-7], wherein the RB1 gene mutation is a human RB1 gene mutation. [C-9] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, chemotherapeutic agent, or use according to any one of [C-1] to [C-8], wherein the human RB1 gene mutation is at least one of the following (1) to (6): (1) the codon corresponding to the serine residue (S) at position 82 of the amino acid sequence of SEQ ID NO: 2 is substituted with a stop codon, (2) the codon corresponding to the tyrosine residue (Y) at position 325 of the amino acid sequence of SEQ ID NO: 2 is substituted with a stop codon, (3) the serine residue (S) at position 350 of the amino acid sequence of SEQ ID NO: 2 is substituted with an isoleucine residue (I), and a portion of the RB1 gene is deleted, (4) The glutamic acid residue (E) at position 837 in the amino acid sequence of SEQ ID NO: 2 is replaced with a lysine residue (K), and a portion of the RB1 gene is homozygously deleted; (5) The codon corresponding to the arginine residue (R) at position 467 in the amino acid sequence of SEQ ID NO: 2 is replaced with a stop codon; (6) At least one base is inserted in the codon corresponding to the serine residue (S) at position 182 in the amino acid sequence of SEQ ID NO: 2, forming a new reading frame starting with an isoleucine residue (I), and the third reading frame therefrom is a stop codon.[C-10] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, chemotherapeutic agent, or use according to any one of [C-1] to [C-9], wherein the human RB1 gene mutation is a mutation that results in at least one of the following (1) to (4): (1) a codon corresponding to the serine residue (S) at position 82 of the amino acid sequence of SEQ ID NO: 2 is substituted with a stop codon, (2) a codon corresponding to the tyrosine residue (Y) at position 325 of the amino acid sequence of SEQ ID NO: 2 is substituted with a stop codon, or (3) a serine residue (S) at position 350 of the amino acid sequence of SEQ ID NO: 2 is substituted with an isoleucine residue (I), and a portion of the RB1 gene is deleted, (4) The glutamic acid residue (E) at position 837 in the amino acid sequence of SEQ ID NO: 2 is substituted with a lysine residue (K), and a portion of the RB1 gene is homozygously deleted. [C-11] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof for use, chemotherapeutic agent for use, or use according to any one of [C-1] to [C-10], wherein the decreased expression of the RB1 gene or protein is due to methylation of the RB1 gene or decreased gene expression mediated by microRNA. [C-12] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 3 or a pharmaceutically acceptable salt thereof, compound 4 or a pharmaceutically acceptable salt thereof, compound 5 or a pharmaceutically acceptable solvate of said compound or salt, compound 6 or a pharmaceutically acceptable salt thereof, compound 7 or a pharmaceutically acceptable salt thereof, compound 8 or a pharmaceutically acceptable salt thereof, compound 9 or a pharmaceutically acceptable salt thereof, compound 10 or a pharmaceutically acceptable salt thereof, compound 11 or a pharmaceutically acceptable salt thereof, compound 12 or a pharmaceutically acceptable salt thereof, compound 13 or a pharmaceutically acceptable salt thereof, compound 14 or a pharmaceutically acceptable salt thereof, compound 15 or a pharmaceutically acceptable salt thereof, compound 16 or a pharmaceutically acceptable salt thereof, compound 17 or a pharmaceutically acceptable salt thereof, compound 18 or a pharmaceutically acceptable salt thereof, compound 19 ...[C-13] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, chemotherapeutic agent, or use according to any one of [C-1] to [C-12], wherein the hyperphosphorylated RB1 protein is an RB1 protein having four or more phosphorylated amino acid residues in the amino acid sequence of the RB1 protein. [C-14] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 3 or a pharmaceutically acceptable salt thereof, compound 4 or a pharmaceutically acceptable salt thereof, compound 5 or a pharmaceutically acceptable solvate of said compound or salt, compound 6 or a pharmaceutically acceptable salt thereof, compound 7 or a pharmaceutically acceptable salt thereof, compound 8 or a pharmaceutically acceptable salt thereof, compound 9 or a pharmaceutically acceptable salt thereof, compound 10 or a pharmaceutically acceptable salt thereof, compound 11 or a pharmaceutically acceptable salt thereof, compound 12 or a pharmaceutically acceptable salt thereof, compound 13 or a pharmaceutically acceptable salt thereof, compound 14 or a pharmaceutically acceptable salt thereof, compound 15 or a pharmaceutically acceptable salt thereof, compound 16 or a pharmaceutically acceptable salt thereof, compound 17 or a pharmaceutically acceptable salt thereof, compound 18 or a pharmaceutically acceptable salt thereof, compound 19 ... [C-15] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, or use according to any one of [C-1] to [C-14], wherein the hyperphosphorylated RB1 protein is an RB1 protein having 15 or more phosphorylated amino acid residues in the amino acid sequence of the RB1 protein. [C-16] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, or use according to any one of [C-1] to [C-15], wherein the hyperphosphorylated RB1 protein is human hyperphosphorylated RB1 protein.[C-17] The hyperphosphorylated human RB1 protein is selected from the group consisting of a threonine residue (T) at position 826, a threonine residue (T) at position 823, a threonine residue (T) at position 821, a serine residue (S) at position 816, a tyrosine residue (Y) at position 813, a serine residue (S) at position 811, a serine residue (S) at position 807, a tyrosine residue (Y) at position 805, a serine residue (S) at position 780, a threonine residue (T) at position 625, a threonine residue (T) at position 601, a threonine residue (T) at position 373, a serine residue (S) at position 360, a threonine residue (T) at position 356, and a serine residue (S) at position 249 in SEQ ID NO: 2. The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, a chemotherapeutic agent for use, or use according to any one of [C-1] to [C-16], wherein the RB1 protein is an RB1 protein in which at least one amino acid residue selected from the group consisting of a phosphodiesterase (S) residue at position 37 and a serine residue (S) at position 37 is phosphorylated. [C-18] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, for use, or use according to any one of [C-1] to [C-17], wherein the hyperphosphorylated human RB1 protein is an RB1 protein in which at least one amino acid residue selected from the group consisting of threonine residue (T) at position 826, threonine residue (T) at position 821, serine residue (S) at position 811, serine residue (S) at position 807, serine residue (S) at position 780, threonine residue (T) at position 373, and threonine residue (T) at position 356 of SEQ ID NO: 2 is phosphorylated.[C-19] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, for use, or use according to any one of [C-1] to [C-18], wherein the hyperphosphorylated human RB1 protein is an RB1 protein in which at least one amino acid residue selected from the group consisting of threonine residue (T) at position 826, threonine residue (T) at position 821, serine residue (S) at position 811, and serine residue (S) at position 807 of SEQ ID NO: 2 is phosphorylated. [C-20] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, for use, or use according to any one of [C-1] to [C-19], wherein the hyperphosphorylated human RB1 protein is an RB1 protein in which at least two amino acid residues selected from the group consisting of the threonine residue (T) at position 826, the threonine residue (T) at position 821, and the serine residue (S) at position 811 and / or the serine residue (S) at position 807 of SEQ ID NO: 2 are phosphorylated. [C-21] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, for use, or use according to any one of [C-1] to [C-20], wherein the hyperphosphorylated human RB1 protein is an RB1 protein in which the threonine residues (T) at positions 826 and 821 of SEQ ID NO: 2 are phosphorylated, and the serine residues (S) at positions 811 and / or 807 are phosphorylated.[C-21.1] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent or molecular targeted agent for use, or use described in any one of [A-1] to [A-36] or [B-1] to [B-17], wherein the cancer patient in whom positive expression of CCNE1 protein, overexpression of CCNE1 protein, or amplification of the copy number of the CCNE1 gene has been detected is a cancer patient in whom positive expression of CCNE1 protein has been detected. [C-21.2] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent or molecular targeted agent for use, or use according to any one of [A-1] to [A-36] or [B-1] to [B-17], wherein the cancer patient in whom positive expression of CCNE1 protein, overexpression of CCNE1 protein, or amplification of the copy number of the CCNE1 gene has been detected is a cancer patient in whom overexpression of CCNE1 protein has been detected. [C-21.3] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent or molecular targeted agent for use, or use according to any one of [A-1] to [A-36] or [B-1] to [B-17], wherein the cancer patient in whom positive expression of CCNE1 protein, overexpression of CCNE1 protein, or amplification of the copy number of the CCNE1 gene has been detected is a cancer patient in whom amplification of the copy number of the CCNE1 gene has been detected.[C-22] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-21], wherein the chemotherapeutic agent or molecular targeted agent is a chemotherapeutic agent. [C-23] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-22], wherein the chemotherapeutic agent is at least one selected from the group consisting of antimetabolites, anticancer antibiotics, mitotic inhibitors, topoisomerase inhibitors, platinum preparations, alkylating agents, and antibody-drug conjugates. [C-24] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is an antimetabolite. [C-25] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the antimetabolite is at least one selected from the group consisting of purine antimetabolites, pyrimidine antimetabolites, antifolates, and ribonucleotide reductase inhibitors.[C-26] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the antimetabolite is a purine antimetabolite. [C-27] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-26], wherein the purine antimetabolite is at least one selected from the group consisting of 6-thioguanine, 6-mercaptopurine, azathioprine, fludarabine, pentostatin, cladribine, clofarabine, and nelarabine. [C-28] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the antimetabolite is a pyrimidine antimetabolite. [C-29] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-28], wherein the pyrimidine antimetabolite is at least one selected from the group consisting of gemcitabine, cytarabine, fluorouracil, capecitabine, tegafur, azacitidine, trifluridine, and floxuridine.[C-30] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-28], wherein the pyrimidine antimetabolite is gemcitabine or fluorouracil. [C-31] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the antimetabolite is a folate anti-metabolite. [C-32] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-31], wherein the antifolate is at least one selected from the group consisting of pemetrexed and methotrexate. [C-33] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-31], wherein the antifolate is pemetrexed. [C-34] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to [C-23], wherein the antimetabolite is a ribonucleotide reductase inhibitor.[C-35] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-34], wherein the ribonucleotide reductase inhibitor is hydroxyurea. [C-36] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is an anticancer antibiotic. [C-37] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-36], wherein the anticancer antibiotic is at least one selected from the group consisting of bleomycin, actinomycin D, doxorubicin, daunorubicin, idarubicin, mitomycin, mitoxantrone, epirubicin, aclarubicin, and valrubicin. [C-38] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is a mitotic inhibitor.[C-39] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-38], wherein the mitosis inhibitor is at least one selected from the group consisting of vinca alkaloids and microtubule inhibitors. [C-40] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-38], wherein the mitosis inhibitor is a vinca alkaloid. [C-41] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-39], wherein the vinca alkaloid is at least one selected from the group consisting of vincristine, vinblastine, and vinorelbine. [C-42] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-39], wherein the mitosis inhibitor is a microtubule inhibitor.[C-43] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-39], wherein the microtubule inhibitor is at least one selected from the group consisting of docetaxel, paclitaxel, eribulin, ixabepilone, and epothilone. [C-44] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is a topoisomerase inhibitor. [C-45] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-44], wherein the topoisomerase inhibitor is at least one selected from the group consisting of topotecan, irinotecan, DXd, etoposide, and teniposide. [C-46] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-44], wherein the topoisomerase inhibitor is at least one selected from the group consisting of irinotecan, DXd, and etoposide.[C-47] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is a platinum preparation. [C-48] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-47], wherein the platinum preparation is at least one selected from the group consisting of cisplatin, carboplatin, and oxaliplatin. [C-49] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-47], wherein the platinum drug is carboplatin. [C-50] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is an alkylating agent.[C-51] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to [C-50], wherein the alkylating agent is at least one selected from the group consisting of cyclophosphamide, ifosfamide, chlorambucil, melphalan, temozolomide, carmustine, lomustine, streptozocin, busulfan, procarbazine, dacarbazine, nimustine, ranimustine, bendamustine, altretamine, thiotepa, mechlorethamine, and lurbinectedin. [C-52] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-50], wherein the alkylating agent is lurbinectedin. [C-53] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is an antibody-drug conjugate.[C-54] The antibody-drug conjugate is selected from the group consisting of trastuzumab deruxtecan, ifinatamab deruxtecan, datopotamab deruxtecan, patritumab deruxtecan, DS-6000 (an antibody-drug conjugate of DXd and an anti-CDH6 antibody), sacituzumab govitecan, tisotumab vedotin, enfortumab vedotin, trastuzumab emtansine, loncastuximab tesirine, moxetumomab pasudotox, belantamab mafodotin, polatuzumab vedotin, and inotuzumab ozoga [C-54] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, a chemotherapeutic agent for use, or a use according to [C-53], wherein the antibody-drug conjugate is at least one selected from the group consisting of trastuzumab deruxtecan and sacituzumab govitecan. [C-55] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, a chemotherapeutic agent for use, or a use according to [C-53], wherein the antibody-drug conjugate is at least one selected from the group consisting of trastuzumab deruxtecan and sacituzumab govitecan. [C-55.1] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, chemotherapeutic agent, or use according to [C-23], wherein said chemotherapeutic agent is a combination of fluorouracil and irinotecan.[C-55.2] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is a combination of fluorouracil and irinotecan, and further in combination with levofolinate. [C-55.3] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, or use according to [C-23], wherein the chemotherapeutic agent is a combination of fluorouracil and irinotecan, and further in combination with levofolinate and bevacizumab. [C-56] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-21], wherein the chemotherapeutic agent or molecular targeted agent is a molecular targeted agent.[C-57] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, for use, chemotherapeutic agent for use, or use according to [C-56], wherein the molecular targeted agent is at least one selected from the group consisting of a WEE1 inhibitor, a FEN1 inhibitor, an RRM1 inhibitor, an RRM2 inhibitor, an AURKB inhibitor, an ATR inhibitor, a TTK inhibitor, a SOD1 inhibitor, an SOD2 inhibitor, a BUB1 inhibitor, a CDC7 inhibitor, an SAE1 inhibitor, a PLK1 inhibitor, a UBA2 inhibitor, a DUT inhibitor, an HDAC3 inhibitor, a CHEK1 inhibitor, an AURKA inhibitor, an MEN1 inhibitor, a DOT1L inhibitor, a CREBBP inhibitor, an EZH2 inhibitor, a PLK4 inhibitor, a HASPIN inhibitor, and a METTL3 inhibitor. [C-58] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, chemotherapeutic agent, or use according to [C-56], wherein the molecular targeted agent is at least one selected from the group consisting of a WEE1 inhibitor, an ATR inhibitor, and a CHEK1 inhibitor. [C-59] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt thereof for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt thereof for use, chemotherapeutic agent for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], and [C-1] to [C-58], wherein compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, or compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof for use, chemotherapeutic agent for use, or use.[C-60] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt thereof for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt thereof for use, chemotherapeutic agent for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], and [C-1] to [C-58], wherein compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, or compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof for use, chemotherapeutic agent for use, or use. [C-61] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-60], wherein the cancer is a solid cancer or a blood cancer. [C-62] The cancer is at least one selected from the group consisting of lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, uterine cancer, ovarian cancer, particularly high-grade serous ovarian cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, bladder cancer, thyroid cancer, skin cancer, head and neck cancer, kidney cancer, liver cancer, prostate cancer, biliary tract cancer, gastrointestinal stromal tumor, adenoid cystic carcinoma, retinoblastoma, brain tumor, neuroendocrine cancer, osteosarcoma, soft tissue sarcoma, leukemia, malignant lymphoma, and multiple myeloma. The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, a chemotherapeutic agent, for use, or use, according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-60].[C-62.1] The pharmaceutical composition, method, use of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], and [C-1] to [C-60], wherein the cancer is at least one selected from the group consisting of lung cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, uterine cancer, ovarian cancer, pancreatic cancer, bladder cancer, thyroid cancer, skin cancer, head and neck cancer, kidney cancer, liver cancer, prostate cancer, adenoid cystic carcinoma, retinoblastoma, brain tumor, neuroendocrine cancer, osteosarcoma, soft tissue sarcoma, leukemia, malignant lymphoma, and multiple myeloma. [C-63] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], and [C-1] to [C-60], wherein the cancer is at least one type selected from the group consisting of ovarian cancer, bladder cancer, breast cancer, lung cancer, and colorectal cancer. [C-64] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-60], wherein the cancer is ovarian cancer, colorectal cancer, or breast cancer.[C-65] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-60], wherein the cancer is ovarian cancer or breast cancer. [C-66] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-65], wherein the positive RB1 gene mutation or decreased expression of the RB1 gene or protein has been detected in a biological sample derived from a cancer patient. [C-66.1] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-65], wherein positive expression of CCNE1 protein, overexpression of CCNE1 protein, or amplification of the copy number of CCNE1 gene is detected in a biological sample derived from a cancer patient.[C-67] ​​The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-66], wherein positive expression of the hyperphosphorylated RB1 protein is detected in a biological sample derived from a cancer patient. [C-68] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-67], wherein the biological sample derived from a cancer patient is cancer cells. [C-69] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, for use, chemotherapeutic agent, for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], and [C-1] to [C-68], wherein the decrease in expression of the RB1 gene or protein is a decrease based on the expression level in a biological sample derived from a healthy subject or in a non-cancerous tissue derived from the cancer patient.[C-69.1] A pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, compound 2 or a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate of said compound or salt for use, chemotherapeutic agent for use, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-68], wherein the positive expression of CCNE1 protein, overexpression of CCNE1 protein, or amplification of the copy number of the CCNE1 gene is decreased relative to the expression level in a biological sample from a healthy subject or in a non-cancerous tissue from the cancer patient. [C-70] The pharmaceutical composition, method, compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], and [C-1] to [C-69], wherein the positive expression of the hyperphosphorylated RB1 protein is determined based on an increase in expression of the hyperphosphorylated RB1 protein, relative to the expression level in a biological sample derived from a healthy subject or in a non-cancerous tissue derived from the cancer patient. [C-71] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-70], wherein the patient is a patient in whom amplification of the copy number of the CCNE1 gene has not been detected.[C-72] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-71], wherein the patient is not a mouse into which OVCAR3 has been transplanted. [C-73] The pharmaceutical composition, method, use of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt, use of a chemotherapeutic agent, or use according to any one of [A-1] to [A-36], [B-1] to [B-17], or [C-1] to [C-72], wherein the patient is a human.

[0018] In the above numbering scheme, unless otherwise specified, the numbers cited in the dependent claims include numbers with different decimal points. For example, [B-5] cited in the dependent claims indicates that it includes not only [B-5] but also [B-5.5], etc. The same applies to other numbering schemes.

[0019] According to one aspect of the present invention, there is provided a method for treating or preventing cancer in a patient in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected. According to another aspect of the present invention, there is provided a method for treating or preventing cancer in a patient in whom a positive CCNE1 protein expression, overexpression of the CCNE1 protein, or an amplification of the copy number of the CCNE1 gene has been detected.

[0020] The results of simultaneous measurements of thermogravimetry, differential scanning calorimetry, and mass spectrometry for Sample A are shown. The horizontal axis is temperature (°C). The left vertical axis is the weight change (%) of the sample in thermogravimetry. The right vertical axis is the heat flow (mW / mg) observed in differential thermal analysis (left) and the peak intensity observed in mass analysis (right). The results of simultaneous measurements of thermogravimetry, thermogravimetry, differential scanning calorimetry, and mass spectrometry for Sample A-1 are shown. The horizontal axis is temperature (°C). The left vertical axis is the weight change (%) of the sample in thermogravimetry (these results are for reference only, as the sample was directly measured after filtering and drying through a mesh). The right vertical axis is the heat flow (mW / mg) observed in differential thermal analysis (left) and the peak intensity observed in mass analysis (right). The results of simultaneous measurements of thermogravimetry and differential thermal analysis for Sample A-2 are shown. The horizontal axis is temperature (°C). The right vertical axis is the weight change (%) of the sample in thermogravimetry. The left vertical axis represents heat flow (μV) observed in differential thermal analysis. (A) is a graph showing the maximum Bliss score when pemetrexed and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion strain (RB1 deficient). (B) is a graph showing the maximum HSA score when pemetrexed and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion strain (RB1 deficient). The results of Student's t-test are shown. (A) is a graph showing the maximum Bliss score when gemcitabine and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion strain (RB1 deficient). (B) is a graph showing the maximum HSA score when gemcitabine and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient). The results of Student's t-test are shown. (A) is a graph showing the maximum Bliss score when carboplatin and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient).(B) is a graph showing the maximum HSA score when carboplatin and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient). The results of a Student's t-test are shown. (A) is a graph showing the maximum Bliss score when SN-38 and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient). (B) is a graph showing the maximum HSA score when SN-38 and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient). The results of a Student's t-test are shown. 1A is a graph showing the maximum Bliss score when sacituzumab govitecan (SG) and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion strain (RB1 deficient). 1B is a graph showing the maximum HSA score when SG and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion strain (RB1 deficient). The results of Student's t-test are shown. 1A is a graph showing the maximum Bliss score when DXd and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion strain (RB1 deficient). (B) is a graph showing the maximum HSA score when DXd and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient). The results of Student's t-test are shown. (A) is a graph showing the maximum Bliss score when trastuzumab deruxtecan (T-DXd) and Compound 2 were added to an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient).(B) is a graph showing the maximum HSA score when T-DXd and Compound 2 were added in an RB1 gene wild-type cell line (RB1 proficient) and an RB1 gene mutant or deletion cell line (RB1 deficient). The results of Student's t-test are shown. The expression levels of RB1 protein and phosphorylated RB1 protein in an RB1 gene wild-type cell line were evaluated by Western blotting. The results of Student's t-test are shown. The maximum Bliss score and maximum HSA score are plotted for cells positive for expression of hypophosphorylated RB1 protein and cells positive for expression of hyperphosphorylated RB1 protein. (A) is a graph comparing the maximum Bliss score when gemcitabine and Compound 2 are administered concomitantly. 1B is a graph comparing the maximum HSA score in the combined administration of gemcitabine and compound 2. The results of Student's t-test are shown. The results are from Western blotting evaluation of the expression levels of RB1 protein and phosphorylated RB1 protein in RB1-genetic wild-type cell lines. The graph shows the cytotoxic activity of compound 1 and gemcitabine treatment in cell lines (RB1 gene WT or Amp and hypophosphorylated RB1-expressing cell lines) in which the RB1 gene is wild-type or amplified and which has been determined to be positive for the expression of hypophosphorylated RB1 protein. The graph shows the cytotoxic activity of compound 1 and gemcitabine treatment in cell lines (RB1 gene mt or Loss cell lines) in which the RB1 gene is deleted or has a deletion mutation or has a mutation in the splicing site of the RB1 gene. 1 is a graph showing the cytotoxic activity of Compound 1 and gemcitabine treatment in cell lines (RB1 gene WT or Amp and hyperphosphorylated RB1-expressing cell lines) in which the RB1 gene is wild-type or amplified and which have been determined to be positive for the expression of hyperphosphorylated RB1 protein.

[0023] Figure 1 is a graph plotting the maximum Bliss score and the maximum HSA score for cell lines in which the RB1 gene is wild-type or amplified and determined to be positive for the expression of hypophosphorylated RB1 protein (cell lines expressing WT or Amp RB1 genes and hypophosphorylated RB1), cell lines in which the RB1 gene is deleted or has a deletion mutation or a mutation at a splicing site of the RB1 gene (cell lines expressing mt or Loss RB1 genes), and cell lines in which the RB1 gene is wild-type or amplified and determined to be positive for the expression of hyperphosphorylated RB1 protein (cell lines expressing WT or Amp RB1 genes and hyperphosphorylated RB1). The results of a Tukey test are shown.

[0024] Figure 1 is a graph showing the cytotoxic activity of compound 1 and carboplatin treatment in RB1-proficient cell lines.

[0025] Figure 1 is a graph showing the cytotoxic activity of compound 1 and carboplatin treatment in each RB1-deficient cell line.

[0023] Figure 1 is a graph showing the cytotoxic activity of RB1 proficient cell lines treated with compound 1 and SN-38. Figure 2 is a graph showing the cytotoxic activity of RB1 deficient cell lines treated with compound 1 and SN-38. Figure 3 is a graph showing the cytotoxic activity of RB1 deficient cell lines treated with compound 1 and sacituzumab govitecan. Figure 4 is a graph showing the cytotoxic activity of RB1 proficient cell lines treated with compound 1 and DXd. Figure 5 is a graph showing the cytotoxic activity of RB1 deficient cell lines treated with compound 1 and DXd. Figure 6 is a graph showing the cytotoxic activity of RB1 deficient cell lines treated with compound 1 and pemetrexed. Figure 7 is a graph showing the cytotoxic activity of RB1 deficient cell lines treated with compound 1 or 2 and etoposide. Figure 8 is a graph showing the cytotoxic activity of RB1 deficient cell lines treated with compound 1 or 2 and lurbinectedin. 1 is a graph showing the cytotoxic activity of RB1-deficient cell lines treated with Compound 1 or 2 and 5-fluorouracil (5-FU), 1 is a graph showing the cytotoxic activity of RB1-deficient cell lines treated with Compound 1 or 2, trifluridine, and tipiracil hydrochloride.Graph A shows the change in tumor volume over time after transplantation of DU4475 in the non-drug administration group, the compound 1 alone administration group, the gemcitabine alone administration group, and the combination administration group. Graph B shows the change in mouse body weight over time after transplantation of DU4475 in the non-drug administration group, the compound 1 alone administration group, the gemcitabine alone administration group, and the combination administration group. Graph A shows the change in tumor volume over time after transplantation of NIH:OVCAR-3 in the non-drug administration group, the compound 1 alone administration group, the gemcitabine alone administration group, and the combination administration group. Graph B shows the change in mouse body weight over time after transplantation of NIH:OVCAR-3 in the non-drug administration group, the compound 1 alone administration group, the gemcitabine alone administration group, and the combination administration group. Graph A shows the change in tumor volume over time after transplantation of ES-2 in the non-drug administration group, the compound 1 alone administration group, the gemcitabine alone administration group, and the combination administration group. Graph B shows the change in mouse body weight over time after ES-2 transplantation in the non-drug administration group, the compound 1 alone administration group, the gemcitabine alone administration group, and the combination administration group. Graph A shows the change in tumor volume over time after ES-2 transplantation in the non-drug administration group, the compound 2 alone administration group, the gemcitabine alone administration group, and the combination administration group. Graph A shows the change in tumor volume over time after Colo-824 transplantation in the non-drug administration group, the compound 1 alone administration group, the carboplatin alone administration group, and the combination administration group. Graph B shows the change in mouse body weight over time after Colo-824 transplantation in the non-drug administration group, the compound 1 alone administration group, the carboplatin alone administration group, and the combination administration group. Graph A shows the change in tumor volume over time after Colo-824 transplantation in the non-drug administration group, the compound 1 alone administration group, the carboplatin alone administration group, and the combination administration group. (B) is a graph showing the change in mouse body weight over time after transplantation of Colo-824 in the non-administered group, the group administered with Compound 1 alone, the group administered with trastuzumab deruxtecan alone, and the combination group. (C) is a graph showing the change in tumor volume over time and the change in mouse body weight over time after transplantation of COLO-205 in the non-administered group, the group administered with Compound 1 alone, the group administered with FOLFIRI alone, and the combination group (N=4, mean±SD).1 is a graph showing the time course of tumor volume and mouse body weight after transplantation of COLO-205 in the drug-untreated group, the compound 1 alone treated group, the FOLFIRI and bevacizumab treated group, and the FOLFIRI, bevacizumab, and compound 1 combined treated group (N=5, mean±SD). 2 is a graph showing the cell viability of CCNE1 gene-amplified cell lines ((A): SNU-8 line, (B): HCC1569 line) treated with compound 1 and / or gemcitabine (N=10, mean±SD). 3 is a graph showing the cell viability of CCNE1 gene-amplified cell lines ((A): SNU-8 line, (B): HCC1569 line) treated with compound 1 and / or SN-38 (N=10, mean±SD).

[0021] The following describes embodiments of the present invention, but the present invention is not limited to the following embodiments. The therapeutic or preventive medicament and therapeutic or preventive method of the present invention may be administered or applied to humans.

[0022] As used herein, "one or more" means one or more than one. When "one or more" is used in the context of substituents on a group, the term means a number from one to the maximum number of substituents permitted by the group.

[0023] As used herein, the term "to" indicating a range includes both ends of the range, for example, "A to B" means a range equal to or greater than A and equal to or less than B. As used herein, the term "about," when used in combination with a numerical value, means a range of +10% and -10% of that numerical value. In the present invention, the meaning of the term "and / or" includes any combination of "and" and "or" as appropriate. Specifically, for example, "A, B, and / or C" includes the following seven variations: (i) A, (ii) B, (iii) C, (iv) A and B, (v) A and C, (vi) B and C, and (vii) A, B, and C.

[0024] The term "combination" refers to the use of two or more components in combination. For example, a combination of a MYT1 inhibitor (hereinafter sometimes referred to as the "first component") and a chemotherapeutic agent or molecularly targeted agent (hereinafter sometimes referred to as the "second component") includes "administration as a single formulation containing the first component and the second component" (i.e., administration of the first component and the second component as a combined drug) and "administration of the first component and the second component as separate formulations, either simultaneously or separately." In the latter embodiment, the formulation containing the first component may be administered first, or the formulation containing the second component may be administered first. The latter embodiment may be any of "an embodiment in which the first component and the second component are formulated separately and administered simultaneously via the same administration route," "an embodiment in which the first component and the second component are formulated separately and administered separately via the same administration route at different times," "an embodiment in which the first component and the second component are formulated separately and administered simultaneously via different administration routes (administered from different sites in the same patient)," and "an embodiment in which the first component and the second component are formulated separately and administered separately via different administration routes at different times." In the case of "an embodiment in which the first component and the second component are formulated separately and administered simultaneously via the same administration route," the two formulations may be mixed immediately before administration. "Separately" means that one formulation is administered before or after the other formulation.

[0025] In other words, "combined use" can also be said to be a method of use in which one component is present in the patient's body while the other component is present in the patient's body. That is, a preferred embodiment is one in which the first component and the second component are administered so that they are simultaneously present in the patient's body, for example, in the blood, and a preferred embodiment is one in which one formulation is administered to the patient simultaneously, or one formulation is administered to the patient within 48 hours of the other formulation being administered.

[0026] "Cancer treatment" in the present invention means a reduction in the number of cancer cells in an individual, inhibition of cancer cell proliferation, reduction in tumor volume, reduction in tumor weight, inhibition of cancer cell metastasis, or amelioration of various symptoms caused by cancer, or a combination thereof. Furthermore, "cancer prevention" in the present invention means preventing the development of new cancer cells, preventing an increase in the number of cancer cells due to the re-proliferation of reduced cancer cells, preventing the regrowth of cancer cells whose proliferation has been inhibited, preventing a re-increase in the volume or weight of reduced tumors, or a combination thereof.

[0027] [First Embodiment] One embodiment of the present invention is a pharmaceutical composition comprising, as an active ingredient, a compound represented by the following formula (1) (also referred to as "Compound 1") or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, or a compound represented by the following formula (2) (also referred to as "Compound 2") or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof:

[0028] Compound 2 is a compound described in WO 2021 / 195781, and may produce atropisomers at the bond between the nitrogen atom constituting the pyrrole ring and the phenyl group.

[0029] As used herein, the term "compound or a salt thereof, or a solvate thereof" includes the compound, a salt of the compound, a solvate of the compound, and a solvate of a salt of the compound. In a specific embodiment, Compound 1 or a pharmaceutically acceptable salt thereof, or a solvate thereof is preferably a solvate of the hydrochloride salt, more preferably a hydrate of the hydrochloride salt, and even more preferably a hydrochloride monohydrate of Compound 1.

[0030] The compounds described herein can be in the form of salts, preferably pharmaceutically acceptable salts. The compounds described herein or their salts can be in the form of solvates, preferably pharmaceutically acceptable solvates. Examples of salts of the compounds include hydrochlorides; hydrobromides; hydroiodides; phosphates; phosphonates; sulfates; sulfonates such as methanesulfonate and p-toluenesulfonate; carboxylates such as acetate, citrate, malate, tartrate, succinate, and salicylate; alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as magnesium salt and calcium salt; and ammonium salts such as ammonium salt, alkylammonium salt, dialkylammonium salt, trialkylammonium salt, and tetraalkylammonium salt. These salts are produced, for example, by contacting the compound with an acid or a base. As used herein, the term "solvate" refers to a compound that forms a molecular group together with a solvent, and is not particularly limited as long as it is a solvate formed with a solvent that is acceptable for ingestion accompanying pharmaceutical administration. Examples of solvates include hydrates, alcoholates (ethanol solvates, methanol solvates, 1-propanol solvates, 2-propanol solvates, etc.), and solvates with a single solvent such as dimethyl sulfoxide, as well as solvates formed with multiple solvents per molecule of the compound, or solvates formed with multiple types of solvents per molecule of the compound. When the solvent is water, the solvate is called a hydrate. As the solvate of the compound of the present invention, hydrates are preferred, and specific examples of such hydrates include mono- to decahydrates, preferably mono- to pentahydrates, and more preferably mono- to trihydrates.

[0031] When the compound according to the present invention is obtained in a free form, the compound can be converted into a salt thereof or a hydrate or solvate thereof by a conventional method. Examples include hydrates and ethanolates of Compound 1 or its salts. Specific examples include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nodahydrate, decahydrate, or monoethanolate of Compound 1, or hemihydrate, monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nodahydrate, decahydrate, or monoethanolate of the sodium salt of Compound 1, or a hydrate or ethanolate of the hydrochloride of Compound 1. The hydrate or solvate may be produced in a crystalline or amorphous form, and in the case of a crystalline form, it may take the form of a crystalline polymorph. The hydrate or solvate can be produced by a conventional method, for example, by adding a solvent such as ethanol and / or water to Compound 1, followed by stirring, cooling, concentrating, and / or drying.

[0032] Furthermore, when the compound according to the present invention is obtained as a salt, hydrate, or solvate of the compound, the compound can be converted into its free form by a conventional method.

[0033] The compounds described herein may contain unnatural proportions of isotope atoms in one or more atoms constituting such compounds. The present invention also includes compounds in which any atom in a compound is substituted with another isotope atom having the same atomic number (number of protons) but a different mass number (sum of the number of protons and neutrons), thereby replacing the isotope with an abundance ratio different from the abundance ratio of the isotopes in nature, i.e., compounds labeled with isotope atoms. Examples of isotope elements contained in the compounds of the present specification include hydrogen atoms, carbon atoms, nitrogen atoms, oxygen atoms, phosphorus atoms, sulfur atoms, fluorine atoms, chlorine atoms, etc., and each of these isotopes is 2 H, 3 H, 13 C. 14 C. 15 N, 17 O. 18 O. 32 P, 35S, 18 F, 36 Isotopically labeled compounds include Cl and the like. Compounds labeled with isotope atoms are useful as therapeutic or preventive agents, research reagents (e.g., assay reagents), and diagnostic agents (e.g., in vivo imaging diagnostic agents). Compounds herein containing radioactive or non-radioactive isotopes in all proportions are encompassed within the scope of the present invention. Compounds labeled with isotope atoms can be produced using reagents and solvents containing the corresponding isotope atoms in the same manner as for producing unlabeled compounds.

[0034] The compounds described herein, their salts, or solvates thereof include all stereoisomers thereof (e.g., enantiomers, diastereomers (including cis- and trans-geometric isomers)), racemates of said isomers, and other mixtures thereof. For example, the compounds of the present invention may have one or more asymmetric centers, and the compounds of the present invention include racemic mixtures, diastereomeric mixtures, and enantiomers of such compounds. Furthermore, for example, the compounds of the present invention may have axial chirality, and the compounds of the present invention include each stereoisomer (atropisomer) of such compounds and mixtures thereof.

[0035] <Pharmaceutical Composition> The present invention provides a pharmaceutical composition (hereinafter also referred to as the "pharmaceutical composition of the present invention") containing Compound 1 or a salt thereof or a solvate thereof, or Compound 2 or a salt thereof or a solvate thereof (hereinafter collectively referred to as the "first ingredient").

[0036] The pharmaceutical composition of the present invention can be formulated by a known method by incorporating a pharmaceutically acceptable carrier in addition to the first component. For formulation, commonly used excipients, binders, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. can be used, and the composition can be formulated by a conventional method by blending components generally used as raw materials for pharmaceutical preparations.

[0037] In the formulation, active ingredients used in pharmaceuticals can be processed by known methods into the optimal shape or properties, i.e., dosage form, suited to the method and purpose of use. Examples of commonly used dosage forms include liquid pharmaceutical preparations (liquids) such as injections, suspensions, emulsions, and eye drops, and solid pharmaceutical preparations (solid preparations) such as tablets, powders, fine granules, granules, coated tablets, capsules, dry syrups, lozenges, and suppositories, but are not limited to these.

[0038] For example, to prepare a liquid formulation, a pharmaceutically acceptable carrier or vehicle and, if necessary, pharmaceutically acceptable additives commonly used in the pharmaceutical formulation field are added to the first component in appropriate combination, and then blended into a unit dosage form required for generally accepted pharmaceutical practice. Alternatively, a composition prepared for liquid formulation may be dried to form a solid formulation. Such a solid formulation can be dissolved as needed by adding an appropriate solvent (e.g., sterile water or physiological saline) before administration and then administered. Such a liquid formulation can also be used parenterally (e.g., an injection) in the form of a sterile solution or suspension in water or another pharmaceutically acceptable carrier or vehicle. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using a vehicle such as distilled water for injection. The prepared composition for injection is usually filled into an appropriate ampule. Furthermore, when preparing liquid formulations such as syrups and injectable preparations, the first component is formulated according to conventional methods by adding a pH adjuster, solubilizer, isotonicity adjuster, etc., and, if necessary, a solubilizer, stabilizer, etc.

[0039] Specific examples of pharmaceutically acceptable carriers or vehicles include water (e.g., sterilized water), vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc. Specific examples of pharmaceutically acceptable additives commonly used in the field of pharmaceutical formulations include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts (e.g., sodium chloride), etc. The amount of the active ingredient in these formulations should be an appropriate dose within the indicated range.

[0040] Examples of vehicles for injections include water (e.g., sterile water) and liquid oil. Water as a vehicle for injections may contain, as necessary, adjuvants, solubilizing agents, liquid oil, buffers, soothing agents (also known as local anesthetics), stabilizers, antioxidants, and the like. Examples of adjuvants include components that adjust osmotic pressure, such as sugars (e.g., D-mannose), sugar alcohols (e.g., D-sorbitol, D-mannitol), and salts (e.g., sodium chloride). Suitable solubilizing agents include alcohols (specifically, ethanol, polyalcohols (e.g., propylene glycol, polyethylene glycol)), and nonionic surfactants (e.g., Polysorbate 80 (registered trademark), HCO-50). The aqueous solution for aqueous injections may be physiological saline.

[0041] The liquid oil may be any oil that is liquid at room temperature, such as sesame oil or soybean oil. Suitable solubilizing agents include benzyl benzoate and benzyl alcohol. Furthermore, the liquid oil used as a vehicle for injections may contain, as needed, a buffer (e.g., phosphate buffer solution, sodium acetate buffer solution), a soothing agent (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol, phenol), an antioxidant, etc.

[0042] For example, to produce a solid preparation, an excipient and, if necessary, pharmaceutically acceptable additives commonly used in the field of pharmaceutical preparations, such as a binder, a disintegrant, a lubricant, a colorant, and a flavoring agent, are added to the first component in appropriate combination, and then the mixture is processed into tablets, powders, fine granules, granules, coated tablets, capsules, dry syrups, troches, suppositories, etc., by conventional methods.

[0043] Examples of pharmaceutically acceptable additives used in such solid preparations include animal and vegetable oils such as soybean oil, beef tallow, and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octyldodecyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; silicone resins; silicone oils; surfactants such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene polyoxypropylene block copolymers; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, and methylcellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerin, propylene glycol, dipropylene glycol, and sorbitol; sugars such as lactose, lactose hydrate, fructose, and sucrose; inorganic powders such as silicic anhydride, aluminum magnesium silicate, and aluminum silicate; and purified water.

[0044] Examples of excipients include sugars (e.g., lactose, lactose hydrate, fructose, sucrose, etc.), sugar alcohols (e.g., mannitol, etc.), starches (corn starch, potato starch, wheat starch, rice starch, partially pregelatinized starch, pregelatinized starch, etc.), celluloses (e.g., crystalline cellulose), inorganic salts (e.g., calcium silicate, anhydrous calcium hydrogen phosphate, precipitated calcium carbonate, etc.), etc.

[0045] Examples of binders include polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polypropylene glycol-polyoxyethylene block polymers, and the like.

[0046] Examples of disintegrants include croscarmellose sodium, carmellose sodium, hydroxypropyl cellulose, carmellose, carmellose calcium, methylcellulose, crystalline cellulose, sodium lauryl sulfate, povidone, polysorbate, and the like.

[0047] Examples of lubricants include magnesium stearate, calcium stearate, talc, sucrose fatty acid esters, sodium stearyl fumarate, and hardened oils.

[0048] Coloring agents that are permitted to be added to pharmaceuticals are used, and flavoring agents include cocoa powder, peppermint, aromatic powder, peppermint oil, borneol, cinnamon powder, etc.

[0049] These tablets or granules may be coated with sugar or other necessary coatings.

[0050] In certain embodiments, the preferred content of Compound 1 varies depending on the dosage form, but is typically 0.01 to 100% by weight based on the total weight of the pharmaceutical composition. In certain embodiments, the preferred content of Compound 2 varies depending on the dosage form, but is typically 0.01 to 100% by weight based on the total weight of the pharmaceutical composition.

[0051] In certain embodiments, the pharmaceutical composition is particularly suitable for treating or preventing cancer in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, or in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has occurred. The pharmaceutical composition, in combination with a chemotherapeutic agent or a molecular targeted agent, is particularly suitable for treating or preventing cancer in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, or in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has occurred. The positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein may be detected by a third party.

[0052] <Chemotherapy Agents> Chemotherapeutic agents are chemically synthesized substances with anticancer activity (also referred to as antitumor activity) (hereinafter also referred to as anticancer agents). Chemotherapeutic agents may be selected from the group consisting of, for example, antimetabolites, anticancer antibiotics, mitotic inhibitors, topoisomerase inhibitors, platinum preparations, alkylating agents, and antibody-drug conjugates. Chemotherapeutic agents may be used in combination with components other than chemotherapeutic agents. For example, trifluridine may be used in combination with tipiracil. A combination of trifluridine and tipiracil hydrochloride is commercially available as Lonsurf (registered trademark).

[0053] Antimetabolites are substances that have a chemical structure similar to the main chemical structure of a substance that serves as a substrate for a metabolic enzyme (e.g., purine, pyrimidine, folic acid, ribonucleotide), and that antagonize or inhibit the metabolic mechanism of an organism. Examples of antimetabolites include purine antimetabolites, pyrimidine antimetabolites, folic acid antimetabolites, and ribonucleotide reductase inhibitors. More preferred antimetabolites are purine antimetabolites.

[0054] Purine antimetabolites have a chemical structure similar to that of purine nucleobases and inhibit the metabolism of purines. Examples of purine antimetabolites include 6-thioguanine, 6-mercaptopurine, azathioprine, fludarabine, pentostatin, cladribine, clofarabine, and nelarabine.

[0055] Pyrimidine antimetabolites have a chemical structure similar to pyrimidine nucleobases and inhibit pyrimidine metabolism. Examples of pyrimidine antimetabolites include gemcitabine, cytarabine, fluorouracil, capecitabine, tegafur, azacitidine, trifluridine, and floxuridine. More preferred pyrimidine antimetabolites are gemcitabine or fluorouracil (5-FU). A treatment in which 5-fluorouracil is administered in combination with levofolinate is also known. Levofolinate (l-leucovorin) enhances the activity of 5-fluorouracil. Levofolinate (dl-leucovorin) may also be used instead of levofolinate. A treatment in which 5-fluorouracil is administered in combination with levofolinate and irinotecan is known as FOLFIRI therapy. A quadruple therapy is known that combines FOLFIRI therapy with bevacizumab, cetuximab, or panitumumab. In one example of quadruple therapy, a molecular targeted drug (bevacizumab, cetuximab, or panitumumab) is first administered, followed by levofolinate infusion over 120 minutes, irinotecan infusion over 90 minutes, a short injection of fluorouracil, and then fluorouracil infusion over 46 hours. This administration schedule is repeated for 14 days (one cycle).

[0056] Antifolates are substances that inhibit DNA biosynthesis by suppressing or inhibiting the activity of enzymes that reduce folic acid to active folic acid, which is essential for nucleic acid synthesis. Examples of antifolates include methotrexate and pemetrexed. A more preferred antifolate is pemetrexed.

[0057] Ribonucleotide reductase inhibitors are substances that inhibit DNA biosynthesis by suppressing or inhibiting the enzyme that reduces ribonucleotides to deoxyribonucleotides. Examples of ribonucleotide reductase inhibitors include hydroxyurea.

[0058] Anticancer antibiotics are drugs synthesized based on chemical substances (also called antibiotics) produced by microorganisms, and are substances that exhibit antitumor effects by destroying the cell membranes of cancer cells or inhibiting DNA or RNA synthesis. Examples of anticancer antibiotics include bleomycin, actinomycin D, doxorubicin, daunorubicin, idarubicin, mitomycin, mitoxantrone, epirubicin, aclarubicin, and valrubicin.

[0059] Mitotic inhibitors are substances that inhibit the division of cancer cells by disrupting the cell cycle. They are classified into vinca alkaloids and microtubule inhibitors (including taxane compounds). Vinca alkaloids are compounds derived from the chemical structure of compounds extracted from Catharanthus roseus. They act on the tubulin protein of microtubules that form the mitotic spindle during cell division, causing the cell cycle to stop midway through division. Examples of vinca alkaloids include vincristine, vinblastine, and vinorelbine. Microtubule inhibitors include taxane compounds derived from the chemical structure of compounds extracted from yew trees. They inhibit cell division by promoting microtubule polymerization or inhibiting microtubule depolymerization during cell division. Examples of microtubule inhibitors include taxane compounds (e.g., docetaxel, paclitaxel), eribulin, ixabepilone, and epothilone.

[0060] Topoisomerase inhibitors are substances that inhibit topoisomerase I or II, which are necessary for DNA replication in actively proliferating cells such as cancer cells. Topoisomerase I is an enzyme that cleaves one strand of the double helix structure of DNA and is involved in recombination, while topoisomerase II is an enzyme that cleaves both strands of the double helix structure and is involved in recombination. Examples of topoisomerase inhibitors include topotecan, irinotecan, DXd (also known as "N-[(1S,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10,13-dioxo-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]-2-hydroxyacetamide"), etoposide, and teniposide. More preferred topoisomerase inhibitors are irinotecan, DXd, and etoposide.

[0061] Platinum agents are compounds that bind to the DNA of cancer cells, inhibiting DNA replication and inducing apoptosis in cancer cells. Examples of platinum agents include cisplatin, carboplatin, and oxaliplatin. A more preferred platinum agent is carboplatin.

[0062] Alkylating agents are compounds that bind to DNA in cancer cells and inhibit DNA replication. Examples of alkylating agents include cyclophosphamide, ifosfamide, chlorambucil, melphalan, temozolomide, carmustine, lomustine, streptozocin, busulfan, procarbazine, dacarbazine, nimustine, ranimustine, bendamustine, altretamine, thiotepa, mechlorethamine, and lurbinectedin. A more preferred alkylating agent is lurbinectedin.

[0063] Antibody-drug conjugates (ADCs) are substances in which an anticancer compound is bound to an antibody that recognizes a specific molecule present in cancer cells. Generally, the anticancer compound is bound to the antibody via a linker. The antibody's specific recognition of cancer cells allows the compound bound to the antibody to be selectively delivered to the cancer cells. Examples of antibody-drug conjugates include trastuzumab deruxtecan (T-DXd), ifinatamab deruxtecan, datopotamab deruxtecan, patritumab deruxtecan, DS-6000 (an antibody-drug conjugate of DXd and an anti-CDH6 antibody), sacituzumab govitecan (SG), tisotumab vedotin, enfortumab vedotin, trastuzumab emtansine, loncastuximab tesillin, moxetumomab pasudotox, belantamab mafodotin, polatuzumab vedotin, inotuzumab ozogamicin, brentuximab vedotin, and gemtuzumab ozogamicin. More preferred antibody-drug conjugates are trastuzumab deruxtecan or sacituzumab govitecan.

[0064] In this embodiment, preferred combinations of the first component and the second component include, for example, a combination of the hydrochloride monohydrate of Compound 1 with at least one chemotherapeutic agent selected from the group consisting of gemcitabine, carboplatin, trastuzumab deruxtecan, sacituzumab govitecan, pemetrexed, etoposide, lurbinectedin, fluorouracil, and trifluridine.

[0065] <Molecularly targeted agent> As used herein, the term "molecularly targeted agent" refers to a drug having an antitumor effect and specifically inhibiting a specific protein. It should be noted that, as used herein, the term "molecularly targeted agent" does not include Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, Compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, or a chemotherapeutic agent. Examples of molecularly targeted agents include WEE1 inhibitors, FEN1 inhibitors, RRM1 inhibitors, RRM2 inhibitors, AURKB inhibitors, ATR inhibitors, TTK inhibitors, SOD1 inhibitors, SOD2 inhibitors, BUB1 inhibitors, CDC7 inhibitors, SAE1 inhibitors, PLK1 inhibitors, UBA2 inhibitors, DUT inhibitors, HDAC3 inhibitors, CHEK1 inhibitors, AURKA inhibitors, MEN1 inhibitors, DOT1L inhibitors, CREBBP inhibitors, EZH2 inhibitors, PLK4 inhibitors, HASPIN inhibitors, and METTL3 inhibitors. The molecular targeted agent may be at least one selected from the group consisting of a WEE1 inhibitor, a FEN1 inhibitor, an RRM1 inhibitor, an RRM2 inhibitor, an AURKB inhibitor, an ATR inhibitor, a TTK inhibitor, a SOD1 inhibitor, an SOD2 inhibitor, a BUB1 inhibitor, a CDC7 inhibitor, an SAE1 inhibitor, a PLK1 inhibitor, a UBA2 inhibitor, a DUT inhibitor, an HDAC3 inhibitor, a CHEK1 inhibitor, an AURKA inhibitor, an MEN1 inhibitor, a DOT1L inhibitor, a CREBBP inhibitor, an EZH2 inhibitor, a PLK4 inhibitor, a HASPIN inhibitor, and a METTL3 inhibitor, or may be a WEE1 inhibitor, an ATR inhibitor, or a CHEK1 inhibitor. Specific examples of each molecular targeted agent include the inhibitors described in WO2022 / 213204. Examples of WEE1 inhibitors include AZD-1775 (adavosertib) and Zn-C3 (azenosertib). ATR inhibitors include RP-3500 (camonsertib), beruzosertib, selalasertib, and BAY-1895344 (elimsertib). CHEK1 inhibitors include prexasertib, GDC-0575, ravusertib, and SRA737.

[0066] The pharmaceutical composition of this embodiment is particularly suitable for treating or preventing cancer in patients with cancer who have a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein.Furthermore, the pharmaceutical composition of this embodiment is particularly suitable for treating or preventing cancer in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected.

[0067] The cancer may be a solid cancer or a blood cancer. In a specific embodiment, the cancer is lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, uterine cancer, or ovarian cancer, particularly high-grade serous ovarian cancer, fallopian tube cancer, peritoneal cancer, pancreatic cancer, bladder cancer, thyroid cancer, skin cancer, head and neck cancer, kidney cancer, liver cancer, prostate cancer, adenoid cystic carcinoma, retinoblastoma, brain tumor, biliary tract cancer, gastrointestinal stromal tumor, neuroendocrine cancer, osteosarcoma, soft tissue sarcoma, leukemia, malignant lymphoma, or multiple myeloma. The pharmaceutical composition is suitable for the treatment or prevention of at least one cancer selected from the group consisting of ovarian cancer, bladder cancer, breast cancer, lung cancer, and colon cancer, preferably for the treatment or prevention of ovarian cancer, colon cancer, or breast cancer, and more preferably for the treatment or prevention of ovarian cancer or breast cancer.

[0068] RB1 (retinoblastoma gene, also known as Rb or RB) is a gene encoding the RB1 protein, a representative cell cycle regulator, and is involved in the G1 / S checkpoint. The RB1 protein (also known as RB1 or pRb) forms a complex with E2F, a transcription factor responsible for inducing the expression of genes involved in the transition from the G1 phase to the S phase of the cell cycle, thereby suppressing the activity of E2F. When E2F activity is suppressed, the transition from the G1 phase to the S phase is inhibited.

[0069] During the cell cycle, cyclin D is generally synthesized in response to cell proliferation stimuli and binds to CDK4 / CDK6 to form a complex. This complex phosphorylates the RB protein. When the RB protein is phosphorylated, the transcription factor E2F bound to the RB protein is released, inducing the expression of a group of genes required for S phase progression or DNA replication. Among the induced genes is cyclin E, which forms a complex with CDK2 and further phosphorylates RB1, thereby further inactivating the RB1 protein. If there is a mutation in the RB1 gene (particularly a mutation that reduces the function of the RB1 protein), if the expression level of the RB1 gene or protein is reduced, or if the RB1 protein is hyperphosphorylated, the cell will proceed to the next stage of the cell cycle. Similarly, CCNE1 gene amplification, increased CCNE1 protein expression, CCNE1 gene gain-of-function mutations, FBXW7 gene deletion or mutation, decreased FBXW7 protein expression, CDK4 gene amplification, increased CDK4 protein expression, CDK4 gene gain-of-function mutations, CDK6 gene amplification, increased CDK6 protein expression, CDK6 gene gain-of-function mutations, E2F3 gene amplification, increased E2F3 protein expression, E2F3 gene gain-of-function mutations, PPP2R1A gene deletion or mutation, and / or decreased PPP2R1A protein expression causes cells to progress to the next stage of the cell cycle. Therefore, in the treatment of cancers in which such gene amplification, gene mutation or deletion, and / or protein expression changes have occurred, it is believed that the therapeutic efficacy can be further enhanced by using a MYT1 inhibitor in combination with a chemotherapeutic agent or a molecularly targeted agent.

[0070] As used herein, "positive for RB1 gene mutation" means that, when the nucleotide sequence corresponding to the RB1 gene of interest is analyzed, some mutation (e.g., a mutation resulting in the insertion, substitution, deletion, and / or addition of at least one amino acid residue relative to the wild-type RB1 protein) is found in the nucleotide sequence compared to the nucleotide sequence of the wild-type RB1 gene, or, if a mutation in the nucleotide sequence of the RB1 gene is reflected in a base change in a transcription product or an amino acid change in a translation product (e.g., an insertion, substitution, deletion, and / or addition of at least one amino acid residue compared to the wild-type RB1 protein), the change is detected in the transcription product or translation product. In certain embodiments, a positive RB1 gene mutation is detected in a biological sample (e.g., cancer cells) derived from a cancer patient. As used herein, "detecting a mutation" generally means detecting a mutation in genomic DNA; however, if the mutation in the genomic DNA is reflected in a base change in a transcription product or an amino acid change in a translation product, it also includes detecting the change in the transcription product or translation product (i.e., indirect detection). A preferred embodiment of the method of the present invention is a method for detecting a mutation by directly determining the base sequence of the RB1 gene region of a cancer cell. There are no particular limitations on the method for detecting a positive RB1 gene mutation, and confirmation and determination can be performed, for example, by NGS (next-generation sequencer).

[0071] In the present invention, the term "RB1 gene region" refers to a certain region on genomic DNA that contains the RB1 gene. This region independently includes, in addition to the translated region, untranslated regions such as expression control regions of each gene (e.g., promoter regions and enhancer regions) and the 3'-terminal untranslated region of each gene. In this method, a DNA sample is first prepared from a biological sample. Examples of DNA samples include genomic DNA samples and cDNA samples prepared by reverse transcription from RNA.

[0072] There are no particular limitations on the method for extracting genomic DNA or RNA from a biological sample, and any known method can be appropriately selected and used. For example, methods for extracting genomic DNA include the SDS phenol method (a method in which tissue stored in a solution containing urea or ethanol is denatured with a protease (proteinase K), a surfactant (SDS), and phenol to denature the proteins in the tissue, and then DNA is precipitated from the tissue with ethanol to extract it), and DNA extraction methods using Clean Columns (registered trademark, manufactured by NexTec), AquaPure (registered trademark, manufactured by Bio-Rad), ZR Plant / Seed DNA Kit (manufactured by Zymo Research), AquaGenomicSolution (registered trademark, manufactured by MoBiTec), prepGEM (registered trademark, manufactured by ZyGEM), and BuccalQuick (registered trademark, manufactured by TrimGen).

[0073] Furthermore, the method for extracting RNA from a biological sample and the method for preparing cDNA from the extracted RNA are not particularly limited, and known methods can be appropriately selected and used. Examples include extraction methods using phenol and a chaotropic salt (more specifically, extraction methods using commercially available kits such as Trizol (Invitrogen) and Isogen (Wako Pure Chemical Industries, Ltd.)), and methods using other commercially available kits (RNAPrep Total RNA Extraction Kit (Beckman Coulter), RNeasy Mini (QIAGEN), RNA Extraction Kit (Pharmacia Biotech), etc.). Furthermore, the reverse transcriptase used to prepare cDNA from the extracted RNA is not particularly limited, and examples include reverse transcriptases derived from retroviruses such as RAV (Rous associated virus) and AMV (Avian myeloblastosis virus), and reverse transcriptases derived from mouse retroviruses such as MMLV (Moloney murine leukemia virus).

[0074] In this embodiment, DNA containing the RB1 gene region is then isolated, and the nucleotide sequence of the isolated DNA is determined. The DNA can be isolated, for example, by PCR using genomic DNA or RNA as a template, using a pair of oligonucleotide primers designed to flank all or part of the RB1 gene region. The nucleotide sequence of the isolated DNA can be determined by methods known to those skilled in the art, such as the Maxam-Gilbert method or the Sanger method, and a next-generation sequencer or the like, which is capable of performing analysis to rapidly and comprehensively read out the nucleotide sequence of genes, can also be used.

[0075] By comparing the determined DNA or cDNA base sequence with a control (for example, if the biological sample is derived from a cancer patient, with the DNA or cDNA base sequence derived from non-cancerous tissue of the same patient or with a publicly known database), it is possible to determine whether or not there is a mutation in the RB1 gene region in the cancer cells of the biological sample.

[0076] Mutations in the RB1 gene region can be detected by various methods that allow detection of mutations, in addition to direct determination of the base sequence of DNA or cDNA.

[0077] For example, mutation detection in the present invention can also be performed by the following method. First, a DNA or cDNA sample is prepared from a biological sample. Next, an oligonucleotide probe is prepared, which has a base sequence complementary to a base sequence containing the mutation site in the RB1 gene region and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. The oligonucleotide probe is then hybridized to the DNA or cDNA sample, and the base sequence containing the mutation site in the RB1 gene region is amplified using the DNA or cDNA sample hybridized with the oligonucleotide probe as a template. Fluorescence emitted by the reporter fluorescent dye due to degradation of the oligonucleotide probe accompanying the amplification is then detected, and the detected fluorescence is then compared with that of a control. Examples of such methods include the double-dye probe method, also known as the TaqMan® probe method.

[0078] In yet another method, a DNA or cDNA sample is prepared from a biological sample. Next, a base sequence containing a mutation site in the RB1 gene region is amplified using the DNA or cDNA sample as a template in a reaction system containing an intercalator that emits fluorescence when inserted between DNA double strands. The temperature of the reaction system is then changed, and fluctuations in the intensity of the fluorescence emitted by the intercalator are detected. The detected fluctuations in the intensity of the fluorescence associated with the temperature change are compared with a control. Examples of such methods include high-resolution melting (HRM) analysis.

[0079] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Then, DNA containing all or part of the RB1 gene region is amplified. The amplified DNA is then cleaved with a restriction enzyme. The DNA fragments are then separated according to size. The sizes of the detected DNA fragments are then compared with a control. Examples of such methods include methods using restriction fragment length polymorphism (RFLP) and PCR-RFLP.

[0080] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Then, DNA containing all or part of the RB1 gene region is amplified. The amplified DNA is then dissociated into single-stranded DNA. The dissociated single-stranded DNA is then separated on a non-denaturing gel. The mobility of the separated single-stranded DNA on the gel is compared with that of a control. Such a method includes, for example, PCR-SSCP (single-strand conformation polymorphism) method.

[0081] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. DNA containing all or part of the RB1 gene region is then amplified. The amplified DNA is then separated on a gel containing increasing concentrations of DNA denaturing agent. The mobility of the separated DNA on the gel is then compared with that of a control. Such a method includes, for example, denaturant gradient gel electrophoresis (DGGE).

[0082] Yet another method uses DNA containing a mutation site in the RB1 gene region prepared from a biological sample and a substrate on which an oligonucleotide probe that hybridizes to the DNA is immobilized, such as a DNA array method.

[0083] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. An oligonucleotide primer is then prepared, which has a base 3' to all or part of the bases in the RB1 gene region and a base sequence complementary to the base sequence 3' to that base. A ddNTP primer extension reaction is then carried out using the DNA as a template and the primer. The primer extension reaction product is then subjected to a mass spectrometer to measure the mass. The genotype is then determined based on the mass measurement results. The determined genotype is then compared with a control. Examples of such methods include MALDI-TOF / MS.

[0084] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. An oligonucleotide probe is then prepared, consisting of 5'-"a base sequence complementary to all or a portion of the RB1 gene region and the base sequence on its 5' side"-"a base sequence that does not hybridize to all or a portion of the RB1 gene region's 5' side base sequence and the base sequence on its 3' side"-3' (flap). Also, an "oligonucleotide probe having a base sequence complementary to all or a portion of the RB1 gene region's 5' side base sequence and the base sequence on its 3' side" is prepared. The two oligonucleotide probes are then hybridized to the prepared DNA or cDNA sample. The hybridized DNA is then cleaved with a single-stranded DNA cleaving enzyme to release the flap. The single-stranded DNA cleaving enzyme is not particularly limited, and examples include cleavase. In this method, an oligonucleotide probe having a sequence complementary to the flap and labeled with a reporter fluorescent compound and a quencher fluorescent compound is then hybridized to the flap. The intensity of the emitted fluorescence is then measured. The measured fluorescence intensity is then compared with a control. Examples of such methods include the Invader method.

[0085] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Next, DNA containing all or part of the RB1 gene region is amplified. The amplified DNA is then dissociated into single strands, and only one strand of the dissociated single-stranded DNA is separated. Next, an extension reaction is carried out one base at a time, starting from the vicinity of all or part of the bases in the RB1 gene region. The pyrophosphate produced during this reaction is enzymatically induced to emit light, and the intensity of the emitted light is measured. The measured fluorescence intensity is then compared with a control. Examples of such methods include pyrosequencing.

[0086] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Then, DNA containing all or part of the RB1 gene region is amplified. Next, an "oligonucleotide primer having a base sequence complementary to the 5' base and the 3' base sequence of all or part of the RB1 gene region" is prepared. Next, a single-base extension reaction is carried out using the prepared primer and the amplified DNA as a template in the presence of fluorescently labeled nucleotides. The degree of fluorescence polarization is then measured. The measured degree of fluorescence polarization is then compared with a control. Examples of such methods include the AcycloPrime method.

[0087] In yet another method, a DNA or cDNA sample is first prepared from a biological sample. Then, DNA containing all or part of the RB1 gene region is amplified. Next, an "oligonucleotide primer having a base sequence complementary to the 5'-side base and the 3'-side base sequence of all or part of the RB1 gene region" is prepared. Next, a single-base extension reaction is carried out using the prepared primer and the amplified DNA as a template in the presence of fluorescently labeled nucleotides. Next, the base type used in the single-base extension reaction is determined. The determined base type is then compared with a control. An example of such a method is the SNuPE method.

[0088] If the mutation involves an amino acid change in the RB1 protein, the sample prepared from the biological sample may be a protein. In such cases, the mutation can be detected by a method using a molecule (e.g., an antibody) that specifically binds to the site where the amino acid change occurs due to the mutation, peptide mass fingerprinting (PMF), protein sequencer (Edman degradation), or the like.

[0089] As used herein, "decreased expression of the RB1 gene or protein" means that, when the RB1 gene or protein of a subject is analyzed, the expression level of the RB1 gene or protein is lower than that of a control (e.g., expression level in a healthy subject or in non-cancerous tissue of the same patient). In a specific embodiment, the decreased expression level of the RB1 gene or protein is detected in a biological sample (e.g., cancer cells) derived from a cancer patient.

[0090] The method for detecting decreased expression of the RB1 gene is not particularly limited, and examples include a method in which the expression level of RB1 is detected at the transcription level or translation level and compared with the control. In a method for detecting the expression level of the RB1 gene at the transcription level, RNA or cDNA is first prepared from a biological sample. The method for extracting RNA from a biological sample and the method for preparing cDNA from the extracted RNA are not particularly limited, and known methods can be appropriately selected and used. Examples include extraction methods using phenol and chaotropic salts (more specifically, extraction methods using commercially available kits such as Trizol (Invitrogen) and Isogen (Wako Pure Chemical Industries)), and methods using other commercially available kits (RNAPrep Total RNA Extraction Kit (Beckman Coulter), RNeasy Mini (QIAGEN), RNA Extraction Kit (Pharmacia Biotech), etc.). Furthermore, the reverse transcriptase used to prepare cDNA from the extracted RNA is not particularly limited, and examples thereof include reverse transcriptases derived from retroviruses such as RAV (Rous associated virus) and AMV (Avian myeloblastosis virus), and reverse transcriptases derived from mouse retroviruses such as MMLV (Moloney murine leukemia virus).

[0091] The oligonucleotide primers or oligonucleotide probes are then used in an amplification reaction or hybridization reaction, respectively, to detect the amplified product or hybrid product. Examples of such methods include RT-PCR, Northern blotting, dot blotting, DNA array analysis, in situ hybridization, RNase protection assay, and mRNA-seq. Those skilled in the art can routinely design oligonucleotide primers or oligonucleotide probes suitable for each method based on the nucleotide sequence of RB1 cDNA.

[0092] It is known in the art that promoter hypermethylation is one of the factors behind decreased gene expression. Therefore, in detecting whether or not RB1 function is inhibited, it is conceivable to detect methylation of the RB1 gene promoter as an indicator. Promoter methylation can be detected by known methods, such as a method of directly detecting changes in the base sequence after bisulfite treatment, which has the activity of converting methylated cytosine to uracil, by base sequencing, or a method of indirectly detecting changes using a restriction endonuclease that can recognize (cleave) the base sequence before bisulfite treatment but cannot recognize (cleave) the base sequence after bisulfite treatment.

[0093] The method for detecting decreased expression of RB1 protein is not particularly limited, and can be confirmed and determined, for example, by IHC (immunohistochemical staining) using an antibody specific to RB1 protein. In antibody-based protein detection methods, a protein sample is first prepared from a biological sample. Then, RB1 protein is detected by an antigen-antibody reaction using an antibody specific to RB1 protein. When the antibody specific to RB1 protein is labeled, RB1 protein can be detected directly. However, when the antibody is unlabeled, RB1 protein can be detected indirectly by further reacting it with a labeled molecule that recognizes the antibody (e.g., a secondary antibody or protein A), utilizing the label of the molecule. Examples of such methods include immunohistochemistry (immunostaining), Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, and analysis using an antibody array. This method also has the advantage of simultaneously obtaining additional information, such as the morphology and distribution of cancer cells in tissue, through immunohistochemistry.

[0094] There are no particular limitations on the type or origin of the antibody used, but a monoclonal antibody is preferred. As long as it can detect the RB1 protein with sufficient specificity, an oligoclonal antibody (a mixture of several to several dozen types of antibodies) or a polyclonal antibody can also be used. Fab, Fab', F(ab') ２ , Fv, scFv, sc(Fv) ２ Functional fragments of antibodies and multimers thereof (for example, dimers, trimers, tetramers, polymers) such as dsFv, diabodies, etc. can also be used. The anti-RB1 protein antibody may be a commercially available product.

[0095] RB1 protein can also be detected using mass spectrometry (MS). Analysis using a mass spectrometer coupled to liquid chromatography (LC / MS) is particularly advantageous due to its sensitivity. Detection by mass spectrometry can be performed, for example, by labeling the protein in the protein sample, fractionating the labeled protein, subjecting the fractionated protein to mass spectrometry, and identifying the RB1 protein from the mass spectrometry values. Isotopic labeling reagents known in the art can be used as the label, and suitable labeling reagents are commercially available. Fractionation can also be performed by methods known in the art, for example, using a commercially available ion exchange column.

[0096] As used herein, the amplification of the CCNE1 gene copy number can be determined during diagnostic or prognostic assays by assessing the copy number of the CCNE1 gene using a biological sample derived from a cancer patient (e.g., using a next-generation sequencer, digital PCR, array CGH, or FISH). "Amplified copy number of the CCNE1 gene" may refer to a state in which the copy number is higher than that obtained by measuring a biological sample derived from a healthy individual (also referred to as a healthy adult), or may refer to a state in which the copy number is higher than that obtained by measuring a biological sample collected from the cancer patient before the onset of the disease. Positive expression of CCNE1 protein (referring to the protein encoded by the CCNE1 gene, also referred to as cyclin E1) and overexpression of CCNE1 protein can be detected or determined, for example, by immunohistochemistry (IHC) using an antibody specific to the CCNE1 protein, without particular limitation. "Overexpression of CCNE1 protein" may refer to a state in which the CCNE1 protein is expressed at a level greater than that obtained by measuring a biological sample derived from a healthy individual, or may refer to a state in which the CCNE1 protein is expressed at a level greater than that obtained by measuring a biological sample collected from the cancer patient before the patient became ill.

[0097] As used herein, a "patient" may be a mouse, rat, guinea pig, monkey, dog, sheep, horse, or human. In the present invention, a "cancer patient" may refer not only to a person currently suffering from cancer, but also to a person suspected of suffering from cancer. In a specific embodiment, the subject to be treated or prevented is a cancer patient in whom an increase in the CCNE1 gene expression has not been detected compared to a control. Furthermore, in a specific embodiment, the subject to be treated or prevented is not a mouse transplanted with OVCAR3. The pharmaceutical composition can be suitably used in humans.

[0098] As used herein, the term "biological sample derived from a cancer patient" is not particularly limited as long as it is a biological sample capable of detecting the presence or absence of an RB1 gene mutation, the presence or absence of reduced expression of the RB1 gene or protein, or the presence or absence of expression of hyperphosphorylated RB1 protein. Preferably, the biological sample is a cancer biopsy specimen, blood, urine, body cavity fluid, tumor cell-derived circulating DNA (ctDNA), or other specimen. Protein extracts or nucleic acid extracts (e.g., mRNA extracts, cDNA preparations or cRNA preparations prepared from mRNA extracts) obtained from the specimens may also be used. As used herein, "biological sample" includes samples derived from cancer patients and samples derived from cancer cell cultures.

[0099] The RB1 gene mutation may include a mutation that results in the insertion, deletion, or addition of at least one amino acid residue, or the substitution of an existing amino acid residue, relative to the wild-type RB1 protein. The RB1 gene mutation may also be a nonsense mutation, a frameshift mutation, a splice site mutation, or a homozygous or heterozygous deletion. Preferably, the RB1 gene mutation is a mutation that reduces the function of RB1. "Mutations that reduce the function of RB1" can be confirmed, for example, via the Internet at: https: / / www.oncokb.org / gene / RB1 [searched February 20, 2023].

[0100] A typical DNA (cDNA) nucleotide sequence of a human wild-type RB1 gene is shown in SEQ ID NO: 1 (NCBI Reference No.: NM_000321.3), and a typical amino acid sequence of a human wild-type RB1 protein is shown in SEQ ID NO: 2 (NCBI Reference No.: NP_000312.2). In the case of a human RB1 gene, an RB1 gene mutation refers to a nucleotide sequence that is different from the human RB1 genomic sequence set forth at positions 48,303,751 to 48,481,890 in NCBI Reference No. NC_13.11, a nucleotide sequence that is different from the human RB1 genomic sequence set forth at positions 4921 to 5161 in NCBI Reference No. NG_9009.1, or a mutation that results in an amino acid sequence that is different from the amino acid sequence of the human RB1 protein set forth in SEQ ID NO: 2, and may result in at least one of the following (1) to (6): Even in RB1 without mutation, individual differences in the sequence may occur due to polymorphism, etc. (1) The codon corresponding to the serine residue (S) at position 82 in the amino acid sequence of SEQ ID NO: 2 is replaced with a stop codon, (2) The codon corresponding to the tyrosine residue (Y) at position 325 in the amino acid sequence of SEQ ID NO: 2 is replaced with a stop codon, (3) The serine residue (S) at position 350 in the amino acid sequence of SEQ ID NO: 2 is replaced with an isoleucine residue (I), and a portion of the RB1 gene is deleted, (4) The glutamic acid residue (E) at position 837 in the amino acid sequence of SEQ ID NO: 2 is replaced with a lysine residue (K), and a portion of the RB1 gene is homozygously deleted, (5) The codon corresponding to the arginine residue (R) at position 467 in the amino acid sequence of SEQ ID NO: 2 is replaced with a stop codon, (6) At least one base is inserted in the codon corresponding to the serine residue (S) at position 182 of the amino acid sequence of SEQ ID NO: 2, forming a new reading frame starting from the isoleucine residue (I), and the third reading frame therefrom is a stop codon.

[0101] As used herein, decreased expression of the RB1 gene or protein includes decreased gene expression due to methylation of the RB1 gene or via microRNA.

[0102] As used herein, "hyperphosphorylated RB1 protein" refers to an RB1 protein that has two or more phosphorylated amino acid residues (also called phosphorylation sites) in the amino acid sequence of the RB1 protein, preferably an RB1 protein that has three or more or four or more phosphorylation sites, more preferably eight or more, and most preferably 15 or more phosphorylation sites.

[0103] In the case of human RB1 protein, "positive expression of hyperphosphorylated RB1 protein" may be determined, for example, by phosphorylation of at least one amino acid residue selected from the group consisting of the threonine residue (T) at position 826, the threonine residue (T) at position 823, the threonine residue (T) at position 821, the serine residue (S) at position 816, the tyrosine residue (Y) at position 813, the serine residue (S) at position 811, the serine residue (S) at position 807, the tyrosine residue (Y) at position 805, the serine residue (S) at position 780, the threonine residue (T) at position 625, the threonine residue (T) at position 601, the threonine residue (T) at position 373, the serine residue (S) at position 360, the threonine residue (T) at position 356, the serine residue (S) at position 249, and the serine residue (S) at position 37 in SEQ ID NO: 2 (NCBI reference number: NP_000312.2), which is a typical amino acid sequence of human wild-type RB1 protein.

[0104] In the case of human RB1 protein, "positive expression of hyperphosphorylated RB1 protein" is preferably determined by phosphorylation of at least one amino acid residue selected from the group consisting of the threonine residue (T) at position 826, the threonine residue (T) at position 821, the serine residue (S) at position 811, the serine residue (S) at position 807, the serine residue (S) at position 780, the threonine residue (T) at position 373, and the threonine residue (T) at position 356 of SEQ ID NO: 2.

[0105] In the case of human RB1 protein, "positive expression of hyperphosphorylated RB1 protein" is more preferably determined by phosphorylation of at least one amino acid residue selected from the group consisting of the threonine residue (T) at position 826, the threonine residue (T) at position 821, the serine residue (S) at position 811, and the serine residue (S) at position 807 of SEQ ID NO: 2.

[0106] It is more preferable that, in the case of human RB1 protein, "positive expression of hyperphosphorylated RB1 protein" is determined by phosphorylation of at least two amino acid residues selected from the group consisting of the threonine residue (T) at position 826, the threonine residue (T) at position 821, and the serine residue (S) at position 811 and / or the serine residue (S) at position 807 of SEQ ID NO: 2.

[0107] In the case of human RB1 protein, "positive expression of hyperphosphorylated RB1 protein" is most preferably determined by phosphorylation of the threonine residues (T) at positions 826 and 821 of SEQ ID NO: 2, and phosphorylation of the serine residues (S) at positions 811 and / or 807 of SEQ ID NO: 2.

[0108] In certain embodiments, hyperphosphorylated RB1 protein is detected in a biological sample (eg, cancer cells) from a cancer patient.

[0109] Positive expression of hyperphosphorylated RB1 protein can be detected or assessed, for example, by immunohistochemical staining (IHC) using an antibody specific to phosphorylated RB1 protein. It is also possible to combine antibodies specific to RB1 protein in which multiple amino acid residues are phosphorylated. In antibody-based protein detection methods, a protein sample is first prepared from a biological sample. Then, phosphorylated RB1 protein is detected by an antigen-antibody reaction using an antibody specific to phosphorylated RB1 protein. When the antibody specific to phosphorylated RB1 protein is labeled, phosphorylated RB1 protein can be detected directly. However, when the antibody is unlabeled, phosphorylated RB1 protein can be detected indirectly by further reacting it with a labeled molecule (e.g., a secondary antibody or protein A) that recognizes the antibody, utilizing the label of the molecule. Examples of such methods include immunohistochemistry (immunostaining), Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, and analysis using an antibody array. This method also has the advantage that additional information such as the morphology and distribution of cancer cells in tissues can be simultaneously obtained by immunohistochemistry.

[0110] In certain embodiments, the pharmaceutical composition comprises a first component, such as Compound 1 or Compound 2, as an active ingredient. Compound 1 is a type of substance (also called a MYT1 inhibitor) that can directly or indirectly neutralize, block, inhibit, reduce, or interfere with MYT1 activity in vitro, in a cell culture system, or in an animal. MYT1 inhibitory activity IC ５０ For measuring, the method described in Non-Patent Document 8 can be referred to.

[0111] MYT1 protein, also known as membrane-bound tyrosine-threonine-specific cdc2 inhibitory kinase, is encoded by the PKMYT1 gene. MYT1 protein is primarily localized in the endoplasmic reticulum and Golgi apparatus. It is a member of the Wee kinase family, which includes Wee1 and Wee1b proteins, and negatively regulates the CDK1-cyclin B complex, which promotes progression from the G2 phase to the M phase. Normally, MYT1 protein is inactivated at the onset of the M phase of the cell cycle and negatively acts at the DNA replication checkpoint (G2 / M phase). Overexpression of MYT1 has been observed in various cancers, including hepatocellular carcinoma and clear cell renal cell carcinoma. Examples of MYT1 protein activity include threonine kinase activity and tyrosine kinase activity. The inhibition of MYT1 threonine kinase activity or tyrosine kinase activity by a compound can be confirmed, for example, by treating a purified MYT1 protein preparation with a test compound, adding ATP, and measuring the degree of ATP hydrolysis. The degree of ATP hydrolysis can be evaluated using Kinase-Glo (Promega) or ADP-Glo ​​(Promega) (Non-Patent Document 8). When the threonine kinase activity or tyrosine kinase activity of MYT1 is inhibited by the test compound in comparison with a control (e.g., the degree of ATP hydrolysis by MYT1 not treated with the test compound), attenuation of the ATP hydrolysis is confirmed.

[0112] The inhibition of the threonine kinase activity of the MYT1 protein by a compound can be confirmed, for example, by treating a purified MYT1 protein preparation with a test compound, adding CDK1, a substrate of MYT1, and using the degree of phosphorylation of the Thr14 site of CDK1 as an indicator. The degree of phosphorylation of this site can be evaluated by Western blotting using an antibody (manufactured by Abcam) that specifically recognizes phosphorylation of the Thr14 site of CDK1, ELISA using an antibody that specifically recognizes CDK1 and an antibody that specifically recognizes phosphorylation of the Thr14 site of CDK1, or AlphaLISA (manufactured by PerkinElmer) using an antibody that specifically recognizes CDK1 and an antibody that specifically recognizes phosphorylation of the Thr14 site of CDK1. When the threonine kinase activity of MYT1 is inhibited by the test compound in comparison with a control (e.g., the degree of phosphorylation of the Thr14 site of CDK1 by MYT1 not treated with the test compound), attenuation of the phosphorylation is confirmed.

[0113] Furthermore, whether a compound inhibits the threonine kinase activity of MYT1 can be confirmed, for example, by measuring the degree of phosphorylation of the Thr14 site of CDK1, a substrate protein for the threonine kinase activity of MYT1, in a lysate of cells treated with the test compound. The degree of phosphorylation of this site can be evaluated by Western blotting using an antibody (manufactured by Abcam) that specifically recognizes phosphorylation of the Thr14 site of CDK1, ELISA using an antibody that specifically recognizes CDK1 and an antibody that specifically recognizes phosphorylation of the Thr14 site of CDK1, or AlphaLISA (manufactured by PerkinElmer) (Non-Patent Document 8) using an antibody that specifically recognizes CDK1 and an antibody that specifically recognizes phosphorylation of the Thr14 site of CDK1. When the test compound inhibits the threonine kinase activity of MYT1 in comparison with a control (e.g., the degree of phosphorylation of the Thr14 site of CDK1 in a lysate of cells not treated with the test compound), attenuation of the phosphorylation is confirmed.

[0114] Furthermore, inhibition of MYT1 expression by a compound can be confirmed, for example, by detecting decreased MYT1 expression in cells treated with the test compound. A typical method for detecting decreased MYT1 expression is to detect the expression level of MYT1 at the transcriptional or translational level and confirm that the expression level is lower than a control (e.g., the expression level in cells not treated with the test compound).

[0115] In a method for detecting the expression level of MYT1 at the transcription level, RNA or cDNA is first prepared from cells treated with a test compound. The method for extracting RNA from the cells is not particularly limited, and any known method can be appropriately selected and used. Examples include extraction methods using phenol and a chaotropic salt (more specifically, extraction methods using commercially available kits such as Trizol (Invitrogen) and Isogen (Wako Pure Chemical Industries, Ltd.)), and methods using other commercially available kits (RNAPrep Total RNA Extraction Kit (Beckman Coulter), RNeasy Mini (QIAGEN), RNA Extraction Kit (Pharmacia Biotech), etc.). Furthermore, the reverse transcriptase used to prepare cDNA from the extracted RNA is not particularly limited, and examples include reverse transcriptases derived from retroviruses such as RAV (Rous associated virus) and AMV (Avian myeloblastosis virus), and reverse transcriptases derived from mouse retroviruses such as MMLV (Moloney murine leukemia virus).

[0116] The oligonucleotide primers or oligonucleotide probes are then used in an amplification reaction or hybridization reaction, respectively, to detect the amplified product or hybrid product. Examples of such methods include RT-PCR, Northern blotting, dot blotting, DNA arrays, in situ hybridization, RNase protection assays, and mRNA-seq. Those skilled in the art can routinely design oligonucleotide primers or oligonucleotide probes suitable for each method based on the nucleotide sequence of the MYT1 cDNA.

[0117] In a method for detecting the expression level of MYT1 at the translational level, a protein sample is first prepared from cells treated with a test compound. An antigen-antibody reaction is then carried out using an antibody specific to the MYT1 protein to detect the MYT1 protein. In such a protein detection method using an antibody, for example, an antibody specific to the MYT1 protein is added to the protein sample to carry out an antigen-antibody reaction, and binding of the antibody to the MYT1 protein is detected. When the antibody specific to the MYT1 protein is labeled, the MYT1 protein can be detected directly. However, when the antibody is unlabeled, the MYT1 protein can be detected indirectly by further reacting it with a labeled molecule that recognizes the antibody (e.g., a secondary antibody or protein A), utilizing the label of the molecule. Examples of such methods include immunohistochemistry (immunostaining), Western blotting, ELISA, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, and analysis using an antibody array.

[0118] There are no particular limitations on the type or origin of the antibody used, but a monoclonal antibody is preferred. Oligoclonal antibodies (a mixture of several to several dozen types of antibodies) or polyclonal antibodies can also be used, as long as they are capable of detecting the MYT1 protein with sufficient specificity. Functional fragments of antibodies and multimers thereof (e.g., dimers, trimers, tetramers, polymers), such as Fab, Fab', F(ab')2, Fv, scFv, sc(Fv)2, dsFv, and diabodies, can also be used. Such anti-MYT1 protein antibodies may be commercially available.

[0119] MYT1 protein can also be detected using mass spectrometry (MS). Analysis using a mass spectrometer coupled to liquid chromatography (LC / MS) is particularly advantageous due to its sensitivity. Detection by mass spectrometry can be performed, for example, by labeling the protein in the protein sample, fractionating the labeled protein, subjecting the fractionated protein to mass spectrometry, and identifying the MYT1 protein from the mass spectrometry values. Isotopic labeling reagents known in the art can be used as the label, and suitable labeling reagents are commercially available. Fractionation can also be performed by methods known in the art, for example, using a commercially available ion exchange column.

[0120] As used herein, "having MYT1 inhibitory activity" means reducing MYT1 activity in vitro, in a cell culture system, or in an animal, and is, for example, measured as an inhibitory activity of MYT1, IC ５０ is preferably 10 μM or less, 5 μM or less, or 1 μM or less. More preferably, the MYT1 inhibitory activity (IC ５０ ) is 100 nM or less, 10 nM or less, 3 nM or less, 100 pM or less, or 10 pM or less. ５０ Particularly preferred are compounds having a MYT1 inhibitory activity (IC ５０ ) is less than 20 nM, or 1 nM to 20 nM.５０ The smaller the RB1 gene mutation, the lower the RB1 gene or protein expression, or the more effective the compound can be in the treatment or prevention of cancer in patients in whom a positive RB1 gene mutation, a decreased RB1 gene or protein expression, or a positive hyperphosphorylated RB1 protein expression has been detected.

[0121] The compound selected by the method of this embodiment has enhanced therapeutic efficacy when used in combination with a chemotherapeutic agent in the treatment of cancer in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, or in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has occurred.

[0122] In certain embodiments, the pharmaceutical composition may further contain, in addition to the first component, other MYT1 inhibitors known to those skilled in the art. For example, the MYT1 inhibitor may be at least one selected from the group consisting of a small molecule compound, a polypeptide, and a polynucleotide. When the MYT1 inhibitor is a small molecule compound, the MYT1 inhibitor is preferably a compound with a molecular weight of 2000 g / mol or less, more preferably a compound with a molecular weight of 1000 g / mol or less. When the MYT1 inhibitor is a polypeptide, an antibody (e.g., an anti-MYT1 antibody) may be used as the MYT1 inhibitor. When the MYT1 inhibitor is a polynucleotide, the MYT1 inhibitor may be selected from the group consisting of a ribozyme, an antisense molecule, an inhibitor oligonucleotide, an aptamer, a microRNA, and a small interfering RNA (siRNA), preferably an antisense nucleic acid, a microRNA, or a small interfering RNA (siRNA).

[0123] In the present disclosure, a solvate of a compound refers to a compound that forms a single molecular group together with a solvent, and when the solvent is water, it is called a hydrate. As the solvate of the compound of the present disclosure, a hydrate is preferred, and specific examples of such hydrates include mono- to decahydrates, preferably mono- to pentahydrates, and more preferably mono- to trihydrates. The solvates of the compound of the present disclosure include solvates with a single solvent such as water, alcohols (e.g., methanol, ethanol, 1-propanol, 2-propanol, etc.), and dimethylformamide, as well as solvates with multiple solvents.

[0124] In a specific embodiment, the preferred content of Compound 2 varies depending on the dosage form, but is usually 0.01 to 100% by weight based on the mass of the entire pharmaceutical composition.

[0125] In certain embodiments, a preferred combination of a MYT1 inhibitor and a second component is a combination in which the MYT1 inhibitor comprises compound 1 hydrochloride monohydrate, and the second component is at least one selected from the group consisting of gemcitabine, pemetrexed, irinotecan (a prodrug of SN-38), carboplatin, and an antibody-drug conjugate comprising DXd as a payload (e.g., trastuzumab deruxtecan (used as Enhertz (trade name, Daiichi Sankyo Co., Ltd.))), an antibody-drug conjugate comprising SN-38 as a payload (e.g., sacituzumab govitecan), etoposide, lurbinectedin, fluorouracil, and trifluridine.

[0126] In certain embodiments, a preferred combination of a MYT1 inhibitor and a second component is a combination in which the MYT1 inhibitor comprises compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, and the second component is at least one selected from the group consisting of gemcitabine, pemetrexed, irinotecan (a prodrug of SN-38), carboplatin, and an antibody-drug conjugate comprising DXd as a payload (e.g., trastuzumab deruxtecan (used as Enhertz (trade name, Daiichi Sankyo Co., Ltd.))), an antibody-drug conjugate comprising SN-38 as a payload (e.g., sacituzumab govitecan), etoposide, lurbinectedin, fluorouracil, and trifluridine.

[0127] Pharmaceutical compositions according to certain embodiments may contain a pharmaceutically acceptable additive in addition to a MYT1 inhibitor (first component) such as Compound 1. The pharmaceutically acceptable additive may be appropriately selected from additives well known to those skilled in the art, and multiple additives may be appropriately used as needed. The additive is appropriately selected depending on whether the preparation is oral or injectable, and may be a mixture with, for example, water, ethanol, saline, liquid paraffin, a surfactant, sucrose, or the like, and the resulting mixture may be encapsulated.

[0128] A pharmaceutical composition comprising a MYT1 inhibitor (first component) such as Compound 1 as an active ingredient is used in combination with a chemotherapeutic agent or molecularly targeted agent (second component). In one embodiment, the pharmaceutical composition comprising the MYT1 inhibitor is administered simultaneously with or separately from the second component. In another embodiment, the pharmaceutical composition comprising the MYT1 inhibitor is administered as a combination drug with the second component.

[0129] The dosage of a MYT1 inhibitor such as Compound 1 is preferably 0.001 to 50 mg per kg of body weight of the subject per day (0.001 to 50 mg / kg / day), more preferably 0.001 to 20 mg / kg / day, and even more preferably 0.002 to 10 mg / kg / day. When the dosage of the MYT1 inhibitor is within these ranges, the cancer treatment or prevention effect is further enhanced. The frequency of administration of the MYT1 inhibitor can be, for example, once or more per week, twice per week, once per day, or twice per day.

[0130] The dosage of the second component is preferably 0.005 to 300 mg per kg of body weight of the subject per day (0.005 to 300 mg / kg / day), more preferably 0.01 to 250 mg / kg / day, and even more preferably 0.02 to 200 mg / kg / day. When the dosage of the second component is within these ranges, the cancer treatment or prevention effect is further enhanced. The frequency of administration of the second component can be, for example, once or more per week, twice per week, once per day, or twice per day.

[0131] The dosage of a MYT1 inhibitor (first component) such as Compound 1 and a chemotherapeutic agent or molecular targeted agent (second component) is preferably 0.0001 to 50 mg / kg / day for the first component and 0.005 to 300 mg / kg / day for the second component per kg of body weight of the subject to be administered, more preferably 0.001 to 20 mg / kg / day for the first component and 0.01 to 250 mg / kg / day for the second component, and even more preferably 0.002 to 10 mg / kg / day for the first component and 0.02 to 200 mg / kg / day for the second component. When the dosages of the first component and the second component are within these ranges, the therapeutic or preventive effect on cancer is further enhanced.

[0132] In the present invention, administration methods include oral, rectal, parenteral (intravenous, intramuscular, subcutaneous, transdermal), intracisternal, intravaginal, intraperitoneal, intravesical, or topical (injection, infusion, powder, ointment, gel, or cream) administration, and inhalation (buccal or nasal spray). Dosage forms include, for example, tablets, capsules, granules, powders, pills, aqueous and non-aqueous oral solutions and suspensions, and parenteral solutions packaged in containers adapted for individual dosages. Dosage forms can also be adapted for various administration methods, including controlled-release formulations such as subcutaneous implants.

[0133] The first component can be administered by, for example, any of the administration methods described above, and the second component can be administered by, for example, the same or different administration method as the administration method of the first component. The interval between the administration of the first component and the second component can be, for example, 0 to 14 days, 0 to 10 days, or 0 to 7 days. The administration interval can be determined using, for example, AUC, Cmax, Tmax, elimination half-life, or the subject's health status as indicators. For example, the first component can be administered orally (tablets, capsules, etc.), and the second component can be administered by infusion. For example, both the first component and the second component can be administered orally (tablets, capsules, etc.). For example, the first component can be administered by infusion, and the second component can be administered orally (tablets, capsules, etc.). For example, both the first component and the second component can be administered by infusion. In such cases, the first component and the second component can be administered at any interval, such as three times a day, twice a day, once a day, once a week, or once every two weeks. More specifically, both the first component and the second component may be administered once a day, and in this case, they may be administered before, between, or after meals. "Before, between, or after meals" may refer to before, between, or after breakfast, lunch, dinner, a late-night snack, or a snack. If the administration interval between the first component and the second component is within 24 hours, both components can be administered once a day. If necessary, the first component and the second component may be administered twice a day and once a day, once a day and once a day, once a day and twice a day, once a day and once every two days, once a day and once every three days, once a day and once every seven days, three times a day and once every seven days, or twice a day and once every seven days. If necessary, the administration interval of the second component alone or the administration interval of the first component alone can be increased or decreased. The administration of the second component may be initiated on the same day as the administration of the first component, or the administration of the first component and the administration of the second component may be initiated on different days. The administration period is 7 days per course, and the total number of courses may be one or more courses, or may be two or more courses. Furthermore, each course may be administered consecutively, or a drug holiday may be provided. A drug holiday may also be provided midway between courses.If necessary, during the course of treatment, only the first component can be continuously administered and the second component can be discontinued, or only the first component can be discontinued and the second component can be continuously administered. The first and second components can also be contained in the same tablet, capsule, etc.

[0134] The pharmaceutical composition according to this embodiment can be administered in combination with, for example, a pharmaceutical composition containing a chemotherapeutic agent or a molecular targeted agent described as the second embodiment (combined administration).

[0135] One aspect of this embodiment is a pharmaceutical composition containing Compound 1 for use in combination with a chemotherapeutic agent or a molecularly targeted agent in the treatment or prevention of cancer in a cancer patient who has a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein. Another aspect of this embodiment is a pharmaceutical composition containing Compound 1 for use in combination with a chemotherapeutic agent or a molecularly targeted agent in the treatment or prevention of cancer in a patient in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected.

[0136] Another aspect of this embodiment is use of Compound 1 for the manufacture of a medicament for treating or preventing cancer in a patient with cancer who has been detected to have a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein. Also, another aspect of this embodiment is use of Compound 1 for the manufacture of a medicament for treating or preventing cancer in a patient with cancer who has been detected to have a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein, the medicament being administered in combination with a chemotherapeutic agent or a molecular targeted agent.

[0137] [Second Embodiment] The second embodiment of the present invention is a pharmaceutical composition comprising, as an active ingredient, a chemotherapeutic agent for treating or preventing cancer in combination with a first component (i.e., compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, or compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof). The pharmaceutical composition of this embodiment is particularly suitable for treating or preventing cancer in cancer patients who have a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein. Furthermore, the pharmaceutical composition of this embodiment is particularly suitable for treating or preventing cancer in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected.

[0138] For each term in this specification (e.g., first ingredient, compound 1, compound 2, chemotherapeutic agent, molecularly targeted agent, MYT1 inhibitor, RB1 gene mutation, administration method, type of cancer, etc.), reference can be made to the definition in the first embodiment. The pharmaceutical composition according to this embodiment can be administered in combination with, for example, the pharmaceutical composition containing the first ingredient described as the first embodiment (combined administration).

[0139] In the pharmaceutical composition according to this embodiment, the preferred content of the chemotherapeutic agent or molecular targeted agent varies depending on the dosage form, but is usually 0.01 to 100% by weight based on the total mass of the pharmaceutical composition.

[0140] In certain embodiments, the pharmaceutical composition comprises a chemotherapeutic agent or molecularly targeted agent (second component) as an active ingredient. The pharmaceutical composition comprising the second component as an active ingredient is used in combination with the first component. In some embodiments, the pharmaceutical composition comprising the second component is administered simultaneously with or separately from the first component. In other embodiments, the pharmaceutical composition comprising the second component is administered as a combination drug with the first component.

[0141] In certain embodiments, a preferred combination of the first component and the second component is a combination in which the first component comprises the hydrochloride monohydrate of Compound 1, and the second component is at least one selected from the group consisting of gemcitabine, pemetrexed, irinotecan (a prodrug of SN-38), carboplatin, and an antibody-drug conjugate comprising DXd as a payload (e.g., trastuzumab deruxtecan (used as Enherz)), an antibody-drug conjugate comprising SN-38 as a payload (e.g., sacituzumab govitecan), etoposide, lurbinectedin, fluorouracil, and trifluridine.

[0142] In certain embodiments, a preferred combination of the first component and the second component is a combination in which the first component comprises compound 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or a salt thereof, and the second component is at least one selected from the group consisting of gemcitabine, pemetrexed, irinotecan (a prodrug of SN-38), carboplatin, and an antibody-drug conjugate comprising DXd as a payload (e.g., trastuzumab deruxtecan (used as Enherz)), an antibody-drug conjugate comprising SN-38 as a payload (e.g., sacituzumab govitecan), etoposide, lurbinectedin, fluorouracil, and trifluridine.

[0143] The dosages of the first component and the second component are preferably 0.0001 to 50 mg / kg / day of the first component and 0.005 to 300 mg / kg / day of the second component per kg of body weight of the subject, more preferably 0.001 to 20 mg / kg / day of the first component and 0.01 to 250 mg / kg / day of the chemotherapeutic agent, and even more preferably 0.002 to 10 mg / kg / day of the first component and 0.02 to 200 mg / kg / day of the chemotherapeutic agent. When the dosages of the first component and the chemotherapeutic agent are within these ranges, the therapeutic or preventive effect on cancer is further enhanced.

[0144] The pharmaceutical composition according to this embodiment may contain a pharmaceutically acceptable additive in addition to the second component. The pharmaceutically acceptable additive may be appropriately selected from additives well known to those skilled in the art, and multiple additives may be appropriately used as needed. The additive may be appropriately selected depending on whether the formulation is oral or injectable, and may be a mixture with, for example, water, ethanol, physiological saline, liquid paraffin, a surfactant, sucrose, etc., and the resulting mixture may be encapsulated.

[0145] The pharmaceutical composition of this embodiment may also contain a pharmaceutically acceptable additive, such as a component that inhibits the metabolism of the second component. The second component is metabolized and excreted primarily by enzymes via various metabolic pathways depending on the type of compound. By using a component that inhibits the metabolic enzyme in combination, the blood concentration of the second component can be maintained high or its half-life can be extended, thereby enhancing the antitumor effect of the second component. For example, tipiracil is known as a component that inhibits the metabolism of trifluridine. Trifluridine and tipiracil hydrochloride are commercially available under the trademark Lonsurf (registered trademark). The pharmaceutical composition of this embodiment may also be used in combination with other drugs. For example, when the pharmaceutical composition of this embodiment contains trifluridine, it can be used in combination with tipiracil. When the pharmaceutical composition of this embodiment contains fluorouracil and irinotecan, it can be used in combination with levofolinate and / or bevacizumab.

[0146] An aspect of this embodiment is also a chemotherapeutic or molecularly targeted agent for use in combination with the first component in treating or preventing cancer.

[0147] Another aspect of this embodiment is the use of a chemotherapeutic agent or a molecularly targeted agent for the manufacture of a medicament for treating or preventing cancer, administered in combination with a first component.

[0148] Third Embodiment A third embodiment of the present invention is a method for treating or preventing cancer in a cancer patient, the method comprising administering a first component to the cancer patient.

[0149] The dosages of the first component and the second component are preferably 0.0001 to 50 mg / kg / day of the first component and 0.005 to 300 mg / kg / day of the chemotherapeutic agent per kg of body weight of the subject, more preferably 0.001 to 20 mg / kg / day of the first component and 0.01 to 250 mg / kg / day of the chemotherapeutic agent, and even more preferably 0.002 to 10 mg / kg / day of the first component and 0.02 to 200 mg / kg / day of the chemotherapeutic agent. When the dosages of the first component and the chemotherapeutic agent are within these ranges, the therapeutic or preventive effect on cancer is further enhanced.

[0150] When administering the second component and the first component in combination, the first component and the second component may be administered simultaneously or separately at a fixed interval. The administration routes of the first component and the second component may be the same or different. The first component and the second component may also be administered in the form of a combination drug containing the first component and the second component. That is, the pharmaceutical composition may contain both the first component and the second component.

[0151] [Fourth embodiment] A fourth embodiment of the present invention is a method for inhibiting the proliferation of cancer cells, the method comprising contacting a first component with the cancer cells.

[0152] The method according to this embodiment encompasses in vivo, in vitro, and ex vivo methods. For example, a subject in which a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected may be any animal in which a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected. The animal species may be a mouse, rat, guinea pig, monkey, dog, sheep, horse, or human. In the in vivo case, cancer cell proliferation can be suppressed by administering the first and second components to the subject. In the in vitro case, cancer cell proliferation can be suppressed by adding the first and second components to a system containing cancer cells collected from the subject. In the ex vivo case, cancer cell proliferation can be suppressed by collecting an organ containing cancer cells from the subject (e.g., lungs in the case of lung cancer) and administering the first and second components to the organ. The dosage of the first and second components can be determined appropriately depending on the amount of cancer cells and the types of the first and second components used.

[0153] [Fifth Embodiment] A fifth embodiment of the present invention is a method for improving responsiveness to cancer treatment with a chemotherapeutic agent or a molecularly targeted agent, the method comprising administering a first component to a cancer patient together with the chemotherapeutic agent or the molecularly targeted agent. In a specific embodiment, the cancer is cancer in a cancer patient in which RB1 gene mutation is positive, RB1 gene or protein expression is reduced, or hyperphosphorylated RB1 protein expression is positive.

[0154] The method according to this embodiment involves administering a chemotherapeutic agent or a molecular targeted agent to a patient in whom a positive RB1 gene mutation, decreased expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, in combination with the first component. By administering the chemotherapeutic agent or the molecular targeted agent in combination with the first component, the efficacy of cancer treatment with the chemotherapeutic agent or the molecular targeted agent can be improved.

[0155] When administering the first component to a cancer patient together with a chemotherapeutic agent or a molecular targeted agent, both the first component and the chemotherapeutic agent or the molecular targeted agent may be administered simultaneously, or may be administered separately at a fixed interval. The administration routes of Compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or its salt, and the chemotherapeutic agent or the molecular targeted agent may be the same or different. The first component may also be administered in the form of a combination drug containing the chemotherapeutic agent or the molecular targeted agent. Even for patients for whom existing chemotherapeutic agents or molecular targeted agents are insufficient in terms of therapeutic efficacy, sufficient therapeutic efficacy can be achieved by combining the first component with the first component.

[0156] The dosages of the first and second components are preferably 0.0001 to 50 mg / kg / day of the first component and 0.005 to 300 mg / kg / day of the second component per kg of body weight of the subject, more preferably 0.001 to 20 mg / kg / day of the first component and 0.01 to 250 mg / kg / day of the chemotherapeutic agent, and even more preferably 0.002 to 10 mg / kg / day of the first component and 0.02 to 200 mg / kg / day of the second component. When the dosages of the first and second components are within these ranges, the therapeutic or preventive effect on cancer is further enhanced.

[0157] Sixth Embodiment A sixth embodiment of the present invention is a method for predicting responsiveness to cancer treatment with a combination of a first component and a chemotherapeutic agent or a molecular targeted agent, the method comprising: determining, or having a third party determine, whether a cancer patient is a cancer patient in whom RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected; and, if the cancer patient is a cancer patient in whom RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, determining that the cancer patient is responsive to cancer treatment with a combination of the first component and a chemotherapeutic agent or a molecular targeted agent.

[0158] In the method of this embodiment, the presence or absence of RB1 gene mutation, the presence or absence of reduced expression of the RB1 gene or protein, or the presence or absence of expression of hyperphosphorylated RB1 protein in cancer cells collected from a cancer patient to be treated is detected, or a third party is made to detect the same, and if the RB1 gene mutation is positive, or the expression of the RB1 gene or protein is reduced, or the expression of hyperphosphorylated RB1 protein is positive, the cancer patient is determined to be responsive (effective) to treatment involving administration of compound 1 or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or its salt in combination with a chemotherapeutic agent or a molecular targeted agent.

[0159] According to the method of this embodiment, even in a patient for whom the therapeutic efficacy of existing chemotherapeutic agents or molecular targeted agents is insufficient, if the cancer patient is positive for RB1 gene mutation, or has reduced expression of RB1 gene or protein, or is positive for expression of hyperphosphorylated RB1 protein, it can be determined that the cancer patient has therapeutic efficacy by administering the chemotherapeutic agent or molecular targeted agent in combination with the first component. This makes it possible to suggest effective chemotherapy in advance to cancer patients for whom existing chemotherapeutic agents or molecular targeted agents alone have not produced sufficient therapeutic effects.

[0160] Detection of the presence or absence of RB1 gene mutations, the presence or absence of reduced expression of the RB1 gene or protein, or the presence or absence of expression of hyperphosphorylated RB1 protein in cancer cells can be carried out by methods well known to those skilled in the art, such as direct sequencing, PCR, TaqMan Genotyping, next-generation sequencing, etc.

[0161] One aspect of this embodiment is a method for selecting a cancer patient for whom administration of a combination of a first component and a chemotherapeutic agent or a molecularly targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor (particularly the first component), a chemotherapeutic agent, or a molecularly targeted agent alone. This method includes determining, or having a third party determine, whether the cancer patient is a cancer patient for whom RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, and, if the cancer patient is a cancer patient for whom RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, determining the cancer patient as a cancer patient for whom administration of a combination of the first component and a chemotherapeutic agent is more effective in treating cancer than administration of a MYT1 inhibitor (particularly the first component), a chemotherapeutic agent, or a molecularly targeted agent alone.

[0162] Another aspect of this embodiment is a method for diagnosing that administration of a combination of the first component and a chemotherapeutic agent or a molecularly targeted agent is effective for cancer treatment in a specific cancer patient. This method includes detecting or having a third party detect the presence or absence of an RB1 gene mutation, the presence or absence of reduced expression of the RB1 gene or protein, or the presence or absence of expression of hyperphosphorylated RB1 protein in cancer cells derived from the cancer patient, and determining, based on the presence of the mutation, that the cancer patient is one for whom administration of a combination of the first component and a chemotherapeutic agent or a molecularly targeted agent would be more effective.

[0163] [Seventh Embodiment] The seventh embodiment of the present invention is a method for screening for compounds effective in treating or preventing cancer in patients in whom a positive RB1 gene mutation, reduced expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, the method comprising measuring the MYT1 inhibitory activity of candidate compounds and selecting candidate compounds having MYT1 inhibitory activity as compounds effective in treating cancer.

[0164] The method according to this embodiment includes measuring the MYT1 inhibitory activity of a candidate compound, and selecting the candidate compound as a compound effective in treating cancer if the candidate compound has MYT1 inhibitory activity.

[0165] In the present invention, a MYT1 inhibitor means a substance capable of directly or indirectly neutralizing, blocking, inhibiting, reducing or preventing the activity of MYT1.

[0166] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety, including the following:

[0167] The present disclosure will be described in more detail below based on examples, but the present disclosure is not limited to the following examples.

[0168] Examples of abbreviations used in this specification are listed below along with their meanings. DCM: Dichloromethane DIPEA: N,N-Diisopropyl-N-ethylamine DMA: N,N-Dimethylacetamide DMSO: Dimethyl sulfoxide EtOH: Ethanol TFA: Trifluoroacetic acid THF: Tetrahydrofuran TBME: tert-Butyl methyl ether HATU: O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate XPhos Pd G4: (2-Dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)(2-(2'-(N-methyl)amino-1,1'-biphenyl))palladium(II) Methanesulfonic acid (CAS number: 1599466-81-5) TFE: 2,2,2-Trifluoroethanol

[0169] Mass spectral data were obtained using a Shimadzu Corporation ultra-high performance liquid chromatograph (product name: Nexera UC) equipped with a single quadrupole mass spectrometer (product name: LCMS-2020) or a Waters Corporation Acquity ultra-high performance liquid chromatograph (UPLC or UPLC I-Class) equipped with a single quadrupole mass spectrometer (SQD or SQD2).

[0170] The high performance liquid chromatography conditions used were Conditions A, X, or Y in Tables 1 and 2 below. In Tables 7 and 8 below, "TFA" means trifluoroacetic acid, "FA" means formic acid, "AA" means ammonium acetate, and "AC" means ammonium bicarbonate.

[0171] Commercially available reagents were used without further purification. All non-aqueous reactions were carried out using commercially available anhydrous solvents. Concentration under reduced pressure or solvent evaporation was carried out using a rotary evaporator.

[0172] As used herein, "room temperature" means a temperature of about 20°C to about 25°C.

[0173] Example 1: Preparation of Compound 1 3-amino-4-(6,7-difluoro-1H-indazol-4-yl)-6-methyl-1H-1,7-phenanthrolin-2-one (Compound 1) was prepared according to the following method.

[0174] Compound c-266 6,7-difluoro-1H-indazole-4-carboxylic acid 4-Bromo-6,7-difluoro-1H-indazole (10 g, 42.92 mmol), oxalic acid dihydrate (8.12 g, 64.37 mmol), palladium acetate (0.48 g, 2.15 mmol), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (1.24 g, 2.15 mmol) were added to a reaction vessel, and the reaction vessel was degassed under reduced pressure and purged with nitrogen. To this mixture, acetic anhydride (6.09 mL, 64.37 mmol) and DMF (90 mL) were added, and the reaction vessel was purged with nitrogen. The reaction mixture was heated to 80°C, and DIPEA (11.99 mL, 68.66 mmol) was added dropwise over 1 hour. The reaction mixture was stirred at 80°C for 1 hour, after which oxalic acid dihydrate (5.41 g, 42.92 mmol) and acetic anhydride (4.06 mL, 42.92 mmol) were added, and DIPEA (7.5 mL, 42.9 mmol) was added dropwise over 1 hour. The reaction mixture was stirred at 80°C for 1 hour. After cooling the reaction mixture to 0°C, 5 M aqueous potassium hydroxide solution (103 mL, 0.51 mol) was added dropwise. The reaction mixture was stirred at 0°C for 30 minutes and then stirred at room temperature for 1 hour. Isopropyl acetate (100 mL) was added to the reaction mixture, and the insoluble matter was filtered off. The organic layer was discarded, and the aqueous layer was washed with isopropyl acetate (100 mL). Concentrated hydrochloric acid was added to the aqueous layer to adjust the pH to 3-4. The resulting solid was collected by filtration and washed twice with water (20 mL). The resulting solid was dried under reduced pressure at 50°C to give the title compound (8.47 g, yield 96%) as a light brown solid. LCMS: m / z 199 [M+H] ＋ HPLC retention time: 2.03 minutes (Analysis conditions Y)

[0175] Compound c-267 6,7-difluoro-N-methoxy-N-methyl-1H-indazole-4-carboxamide A DMF solution (21 mL) of 6,7-difluoro-1H-indazole-4-carboxylic acid (compound c-266, 3.0 g, 15.14 mmol) in a reaction vessel was cooled to 0°C, and then N,O-dimethylhydroxylamine hydrochloride (1.77 g, 18.17 mmol) and a 50% propylphosphonic anhydride ethyl acetate solution (11.7 mL, 19.68 mmol) were added. DIPEA (7.93 mL, 45.4 mmol) was added dropwise to this mixture over 10 minutes. The reaction mixture was stirred for 30 minutes, and then 20% aqueous ammonium chloride solution (21 mL) was added, followed by extraction with ethyl acetate (21 mL). The aqueous layer was added with ethyl acetate (21 mL), and this procedure was repeated twice. The resulting organic layers were mixed and washed sequentially with 5% aqueous sodium bicarbonate solution (30 mL) and 10% aqueous sodium chloride solution (30 mL), and then the organic layer was concentrated under reduced pressure. The obtained crude product was dissolved in acetone (9 mL) at room temperature, and n-heptane (36 mL) was added. After cooling the suspension to 0°C, it was stirred for 1 hour, filtered, and the solid was washed with heptane. The obtained solid was dried under reduced pressure at 40°C to give the title compound (2.62 g, yield 72%) as a light brown solid. LCMS: m / z 242 [M+H] ＋ HPLC retention time: 1.29 minutes (Analysis conditions Y)

[0176] Compound c-102 6,7-difluoro-N-methoxy-N-methyl-1-(oxan-2-yl)indazole-4-carboxamide A DCM solution (20 mL) of 6,7-difluoro-N-methoxy-N-methyl-1H-indazole-4-carboxamide (Compound c-267, 2 g, 8.29 mmol) in a reaction vessel was cooled to 10° C., and p-toluenesulfonic acid monohydrate (0.158 g, 0.829 mmol) was added thereto, followed by dropwise addition of 3,4-dihydro-2H-pyran (2.27 mL, 24.88 mmol). The reaction mixture was heated to 35° C. and stirred for 30 minutes. The reaction mixture was cooled to 10° C., and a 5% aqueous sodium bicarbonate solution (14 mL) was added thereto. The organic layer was separated and concentrated under reduced pressure, and then toluene (14 mL) was added thereto for azeotropy to obtain the title compound (2.81 g). LCMS: m / z 326 [M+H]+ HPLC retention time: 0.72 minutes (Analysis Condition A)

[0177] Compound c-270 6-iodo-8-methylquinolin-5-amine A suspension of 8-methylquinolin-5-amine (3.0 g, 18.96 mmol) in acetonitrile (21.0 mL) was placed in a reaction vessel and cooled with ice. TFA (1.53 mL, 19.91 mmol), N-iodosuccinimide (4.48 g, 19.91 mmol), and acetonitrile (12.0 mL) were added, and the mixture was stirred under ice-cooling for 30 minutes. A 5% aqueous solution of sodium sulfite (20.0 mL) was added to the reaction mixture, and the mixture was stirred at room temperature. A 5% aqueous solution of sodium sulfite (15.0 mL) and a 5% aqueous solution of sodium carbonate (10.0 mL) were then added and the mixture was stirred. The solid was collected by filtration and washed with water to give the title compound (4.71 g, 87% yield) as a brown solid. LCMS: m / z 285 [M+H] ＋ HPLC retention time: 1.09 minutes (Analysis conditions Y)

[0178] Compound c-271 (E)-N'-(6-iodo-8-methylquinolin-5-yl)-N,N-dimethylmethanimidamide N,N-Dimethylformamide dimethyl acetal (3.77 mL, 28.2 mmol) was added to an ethanol suspension (20.0 mL) of 6-iodo-8-methylquinolin-5-amine (compound c-270, 4.00 g, 14.08 mmol) in a reaction vessel, and the mixture was stirred at 90°C for 2 hours. The reaction mixture was concentrated under reduced pressure, and a 10% aqueous sodium chloride solution (30 mL) was added, followed by extraction with isopropyl acetate (30 mL). The resulting organic layer was concentrated under reduced pressure, and TBME and n-heptane were added to the concentrated residue, followed by stirring. The solid was collected by filtration and washed with n-heptane to obtain the title compound (3.8 g, yield 80%) as a light brown solid. LCMS: m / z 340 [M+H] ＋ HPLC retention time: 0.77 minutes (Analysis conditions Y)

[0179] Compound c-272 (E)-N'-[6-[6,7-difluoro-1-(oxan-2-yl)indazole-4-carbonyl]-8-methylquinolin-5-yl]-N,N-dimethylmethaneimidamide A toluene solution (1.5 mL) of (E)-N'-(6-iodo-8-methylquinolin-5-yl)-N,N-dimethylmethanimidamide (Compound c-271, 146 mg, 0.430 mmol) in a reaction vessel was cooled to -10°C, and a 1.3 M solution of isopropylmagnesium chloride lithium chloride complex in THF (0.355 mL, 0.461 mmol) was added, followed by stirring at -10°C for 1 hour. To the reaction mixture, a toluene solution (0.25 mL) of 6,7-difluoro-N-methoxy-N-methyl-1-(oxan-2-yl)indazole-4-carboxamide (Compound c-102, 100 mg, 0.307 mmol) was added dropwise. The reaction mixture was stirred at -10°C for 10 minutes and then at 0°C for 30 minutes. A 17% aqueous ammonium chloride solution was added to the reaction mixture, which was extracted with ethyl acetate and concentrated under reduced pressure. Acetonitrile (1.0 mL) was added to the residue, and the mixture was dissolved at 70° C. and then cooled to 35° C. The solid was collected by filtration and washed with acetonitrile (2 mL) to give the title compound (36 mg, yield 24%) as a yellow solid. LCMS: m / z 478 [M+H] ＋ HPLC retention time: 1.84 minutes (Analysis conditions Y)

[0180] Compound c-273 (5-amino-8-methylquinolin-6-yl)(6,7-difluoro-2-(tetrahydro-2H-pyran-2-yl)-1H-indazol-4-yl)methanone A DMSO / water (10 / 1 (v / v)) mixed solution (2.2 mL) was added to (E)-N'-[6-[6,7-difluoro-1-(oxan-2-yl)indazole-4-carbonyl]-8-methylquinolin-5-yl]-N,N-dimethylmethanimidamide (Compound c-272, 100 mg, 0.21 mmol) in a reaction vessel and stirred. This mixture was cooled to 10°C, and 50% aqueous potassium hydroxide solution (78 μL, 1.05 mmol) was added, followed by stirring for 2 hours and 30 minutes. 20% aqueous ammonium chloride solution (2 mL) was added dropwise to the reaction mixture, followed by stirring for 30 minutes. The solid was collected by filtration and washed with water (2 mL) to obtain the title compound (82 mg, yield 93%) as a solid. LCMS: m / z 423 [M+H] ＋ HPLC retention time: 4.05 minutes (Analysis conditions X)

[0181] Compound c-274 2-chloro-N-[6-[6,7-difluoro-1-(oxan-2-yl)indazole-4-carbonyl]-8-methylquinolin-5-yl]acetamide Chloroacetyl chloride (0.321 mL, 4.01 mmol) was added to a DMA solution (22.6 mL) of (5-amino-8-methylquinolin-6-yl)-[6,7-difluoro-1-(oxan-2-yl)indazol-4-yl]methanone (compound c-273, 1.13 g, 2.67 mmol) in a reaction vessel, and the mixture was stirred at room temperature for 1 hour to give a DMA solution of the title compound. LCMS: m / z 499 [M+H] ＋ HPLC retention time: 0.99 minutes (Analysis conditions A)

[0182] Compound c-2754-[6,7-difluoro-1-(oxan-2-yl)indazol-4-yl]-6-methyl-3-pyridin-1-ium-1-yl-1H-1,7-phenanthrolin-2-one; chloride Pyridine (11.3 mL) was added to a DMA solution of 2-chloro-N-[6-[6,7-difluoro-1-(oxan-2-yl)indazole-4-carbonyl]-8-methylquinolin-5-yl]acetamide (Compound c-274) in a reaction vessel, and the mixture was stirred at 65° C. for 2 hours. The reaction mixture was cooled to room temperature to give a DMA / pyridine solution of the title compound. LCMS: m / z 524 [M] ＋ HPLC retention time: 0.55 minutes (Analysis conditions A)

[0183] Compound c-276: 3-amino-4-[6,7-difluoro-1-(oxan-2-yl)indazol-4-yl]-6-methyl-1H-1,7-phenanthrolin-2-one A DMA / pyridine solution of 4-[6,7-difluoro-1-(oxan-2-yl)indazol-4-yl]-6-methyl-3-pyridin-1-ium-1-yl-1H-1,7-phenanthrolin-2-one chloride (Compound c-275) in a reaction vessel was cooled to 0°C, and hydrazine monohydrate (1.3 mL, 26.7 mmol) was added, followed by stirring at 65°C for 7 hours. Water (100 mL) was added at room temperature, and the resulting solid was collected by filtration and washed with an acetonitrile / water (1 / 4 (v / v)) mixed solution (50 mL) to obtain the title compound (1.14 g, yield 92%) as a light brown solid. LCMS: m / z 462 [M+H] ＋ HPLC retention time: 0.69 minutes (Analysis conditions A)

[0184] Compound 13-amino-4-(6,7-difluoro-1H-indazol-4-yl)-6-methyl-1H-1,7-phenanthrolin-2-one A reaction vessel was charged with a TFE solution (22.8 mL) of 3-amino-4-[6,7-difluoro-1-(oxan-2-yl)indazol-4-yl]-6-methyl-1H-1,7-phenanthrolin-2-one (compound c-276, 1.14 g, 2.47 mmol) and triethylsilane (3.16 mL, 19.76 mmol), and chlorotrimethylsilane (1.58 mL, 12.35 mmol) was stirred at room temperature for 1 hour. 50 mL of aqueous sodium bicarbonate was added to the reaction mixture to adjust the pH to 8-9. The resulting solid was collected by filtration and washed twice with water (100 mL) and twice with an acetonitrile / water mixture (4 / 1 (v / v), 50 mL). Acetonitrile (8.7 mL) was added to the obtained solid, and the solid was collected by filtration and washed twice with acetonitrile (4 mL) to obtain the title compound (805 mg, 86%) as a light brown solid. LCMS: m / z 378 [M+H] ＋ HPLC retention time: 0.50 minutes (Analysis conditions A)

[0185] (2) Preparation of Compound 1 Hydrochloride Monohydrate DMSO (1.7 mL) was added to 3-amino-4-(6,7-difluoro-1H-indazol-4-yl)-6-methyl-1H-1,7-phenanthrolin-2-one (Compound 1, 342.5 mg) in a reaction vessel, and the mixture was dissolved at room temperature. 2 M hydrochloric acid (2.57 mL) and water (0.86 mL) were added to this solution, and the mixture was stirred at room temperature for 6 minutes. A wet solid of the hydrochloride salt of Compound 1 (Sample A-2 described below) was added to the reaction vessel, and the mixture was stirred at room temperature for 23 hours. The solid was then filtered and washed with 2-propanol (4.3 mL). The resulting solid was dried in vacuo for 4 days to obtain a solid of the title compound (Sample A). The results of simultaneous measurements of thermogravimetry, thermogravimetry, differential scanning calorimetry, and mass spectrometry for the resulting solid are shown in Figure 1. Ion peaks corresponding to the m / z values ​​of water and HCl were detected at around 100°C and 230°C, respectively, and the weight loss value corresponded to one molecule of water and one molecule of HCl per molecule of Compound 1. Furthermore, when the chloride ion content was analyzed by ultra-high performance liquid chromatography, the quantitative value was 7.87±0.19%, which corresponds to hydrochloride monohydrate. From the above, it was confirmed that the obtained solid was the hydrochloride monohydrate of Compound 1.

[0186] (3) Preparation of Sample A-1 To 3-amino-4-(6,7-difluoro-1H-indazol-4-yl)-6-methyl-1H-1,7-phenanthrolin-2-one (Compound 1, 71.7 mg) in a reaction vessel, 2 M hydrochloric acid (0.10 mL) and DMSO (0.30 mL) were added and stirred at room temperature for approximately 1 minute. DMSO (0.40 mL) was then added and stirred at room temperature for 1 minute. DMSO (0.40 mL) was then added and stirred at room temperature for 1 minute. The resulting suspension (0.045 mL) was lyophilized at −20°C for 3 days. Water (0.015 mL) was added to the resulting lyophilized product, and the mixture was shaken and stirred at room temperature for 7 days to obtain a solid hydrochloride hydrate of Compound 1 (Sample A-1). The results of simultaneous measurements of thermogravimetry, thermogravimetry, differential scanning calorimetry, and mass spectrometry of the resulting solid are shown in FIG. 2. It was confirmed to be the hydrochloride hydrate of Compound 1.

[0187] (4) Preparation of Sample A-2 DMSO (0.15 mL) was added to 3-amino-4-(6,7-difluoro-1H-indazol-4-yl)-6-methyl-1H-1,7-phenanthrolin-2-one (Compound 1, 30.0 mg) in a reaction vessel, and the mixture was dissolved at room temperature. 2 M hydrochloric acid (0.23 mL) and water (0.075 mL) were added to this solution, and the mixture was stirred at room temperature for 3 minutes. A wet solid of the hydrochloride salt of Compound 1 (Sample A-1 described above) was added to the reaction vessel, and the mixture was stirred at room temperature for 23 hours. The solid was then filtered and washed with 2-propanol (0.15 mL). The resulting solid was dried under vacuum overnight to obtain a wet solid of the hydrochloride salt of Compound 1. The same procedure was repeated twice using the resulting solid to obtain a wet solid of the hydrochloride salt of Compound 1 (Sample A-2). The results of simultaneous thermogravimetry and differential thermal analysis of the resulting solid are shown in Figure 3. The solid was confirmed to be the hydrochloride hydrate of Compound 1.

[0188] Thermogravimetry and simultaneous differential thermal analysis (TG-DTA) of sample A-2 were carried out under the following conditions: Measuring device: STA7200RV+AS-3T (Hitachi High-Tech Science) Measurement range: 30 to 350°C Heating rate: 10°C / min Atmosphere: Nitrogen Measurement: The sample was weighed in an open aluminum pan, covered with a mesh, and then the measurement was carried out.

[0189] Thermogravimetry, thermogravimetry, differential scanning calorimetry, and simultaneous mass spectrometry (TG-DSC-MS) of Sample A were performed under the following conditions: Measurement equipment: STA449F1 Jupiter (NETZSCH) + JMS-Q1500GC (JEOL) Measurement range: 50 to 350°C Heating rate: 10°C / min Atmosphere: Helium, 50 mL / min Transfer line temperature: 300°C Ion source temperature: 250°C Ionization method, voltage, current: EI, 70 eV, 20 μA Relative EM voltage: +700 V Measurement mode: scan (m / z 10-400) Measurement: The sample was precisely weighed into an open aluminum pan and measured.

[0190] Thermogravimetry, thermogravimetry, differential scanning calorimetry, and simultaneous mass spectrometry (TG-DSC-MS) of sample A-1 were performed under the following conditions: Measurement equipment: STA449F1 Jupiter (NETZSCH) + JMS-Q1500GC (JEOL) Measurement range: 50 to 350°C Heating rate: 10°C / min Atmosphere: Helium, 50 mL / min Transfer line temperature: 300°C Ion source temperature: 250°C Ionization method, voltage, current: EI, 70 eV, 20 μA Relative EM voltage: +700 V Measurement mode: scan (m / z 10-400) Measurement: The sample was filtered through a sintered filter, vacuum dried overnight, and then placed on an open aluminum pan for measurement.

[0191] The Cl content of Sample A was analyzed by ultra high performance liquid chromatography under the following conditions. HPLC analysis condition 1 Measurement apparatus: Shimadzu Nexera X3 UHPLC (Shimadzu), Corona Veo RS Charged Aerosol Detector (Thermo Fisher Scientific) Column: Acclaim Trinity P1 3 μm, 2.1 × 100 mm (Thermo Fisher Scientific) Detector: Corona Veo RS Charged Aerosol Detector Mobile phase: 20 mM ammonium acetate solution (pH = 6.6) / acetonitrile = 55 / 45 (isocratic conditions) Post-addition solvent: acetonitrile Flow rate: 0.3 mL / min Injection volume: 5 μL Analysis time: 7 min Column temperature: 40°C CAD temperature: 35°C Sample treatment: 1 mg of sample was accurately weighed, 1 mL of 0.1 mg / mL hippuric acid methanol solution and 1 mL of methanol were added, and the mixture was stirred with a vortex mixer to prepare a sample solution (sample concentration 0.5 mg / mL).

[0192] (5) Preparation of Compound 2 (S)-2-amino-1-(3-hydroxy-2,6-dimethylphenyl)-5,6-dimethylpyrrolo[2,3-b]pyridine-3-carboxamide (also referred to as “Compound 2”) was prepared according to the method described in Patent Document 1.

[0193] (6) Evaluation of Compound 1 and Compound 2 (6-1) Binding Test to the ATP Binding Site of MYT1 The binding ability of Compound 1 and Compound 2 to the ATP binding site of MYT1 kinase protein was evaluated. 5X Kinase Buffer A (PV3189, manufactured by Thermo Fisher Scientific) was diluted five-fold with Milli-Q water to prepare 1X Kinase Buffer. MYT1 protein (manufactured by Carnabiosciences) was diluted to a concentration of 0.005 μM, and Eu-Anti-GST Antibody was diluted to a concentration of 1 μM with 1X Kinase Buffer. Kinase Tracer 178 (PV5593, manufactured by Thermo Fisher Scientific) was also diluted to a concentration of 0.1 μM with 1X Kinase Buffer. 2.5 μL of Compound 1 and Compound 2 diluted with DMSO was added to a 96-well plate, followed by 5 μL of a 1:1 mixture of the diluted MYT1 protein and Eu-GST-Antibody. 2.5 μL of the diluted Kinase Tracer 178 was then added, mixed well, and allowed to stand at room temperature for 30 minutes. Fluorescence at 665 nm and 615 nm generated by irradiation with 340 nm excitation light was then detected using Envision (PerkinElmer). The intensity of the 665 nm fluorescence wavelength was divided by the intensity of the 615 nm fluorescence wavelength to calculate the percentage of Tracer binding to MYT1 protein for each condition. The signal without Compound 1 and Compound 2 was set to 100%, and the signal without MYT1 protein was set to 0%, and the inhibition rate was calculated for each concentration of Compound 1 and Compound 2 added, and the IC ５０ The results are shown in Table 3. IC of 10 μM or less ５０ When the above expression is observed, it can be determined that the compound exhibits binding activity to the ATP binding site of the MYT1 protein.

[0194] (6-2) MYT1 Kinase Activity Inhibition Test The MYT1 kinase activity inhibitory ability of Compound 1 and Compound 2 was evaluated by assessing the phosphorylation level of Y15 of CDK1 protein by MYT1 protein using ELISA. The Cyclex Wee1 Kinase Assay / Inhibitor Screening Kit Ver. 3 (MBL, CY-1172V3) was used. 10 μL of 5 nM MYT1 protein (Carnabiosciences) was prepared using Kinase Buffer. 30 μL of 83.3 μM ATP was also prepared using Kinase Buffer. Compounds 1 and 2 were diluted to each concentration in 10 μL amounts using Kinase Buffer. 30 μL of ATP was added to the kit plate, followed by 10 μL of compound. 10 μL of the diluted MYT1 protein solution was added, mixed, and allowed to stand at room temperature for 60 minutes. The reaction solution was removed from each well, and the wells were washed with Wash Buffer. After removing the wash buffer, 100 μL of HRP-conjugated anti-phospho-tyrosine antibody was added and the mixture was left to stand at room temperature for 60 minutes. The reaction solution was then removed from the wells, and the wells were washed with wash buffer. After removing the wash buffer, 100 μL of substrate reagent was added to each well and the mixture was left to stand at room temperature for 8 minutes. Then, 100 μL of stop solution (1N sulfuric acid) was added to each well according to the protocol. The absorbance at 450 nm and 590 nm was measured using Envision (PerkinElmer). The absorbance at 590 nm was subtracted from the absorbance at 450 nm to evaluate the phosphorylation level of CDK1 protein in each well. The inhibition rate when compound 1 and compound 2 were added at each concentration was calculated, and IC ５０ The results are shown in Table 3. IC of 10 μM or less ５０ When the above expression is observed, it can be determined that the kinase activity of MYT1 protein is inhibited.

[0195] (6-3) Results As shown in Table 3, Compound 1 and Compound 2 had an IC value of 10 μM or less in the binding assay to the ATP binding site of MYT1. ５０ This suggests that it binds to the ATP binding site of the MYT1 protein. In addition, in a MYT1 kinase activity inhibition test, it had an IC value of 10 μM or less. ５０ These results suggest that the compound is capable of inhibiting the kinase activity of MYT1 protein, i.e., is a MYT1 inhibitor.

[0196] Example 2: Anticancer activity test of MYT1 inhibitor in combination with various anticancer drugs 1. Experimental materials and methods (1) Cell lines MDA-MB-175VII (assigned by ATCC), HCC38 (assigned by ATCC), MDA-MB-361 (assigned by ATCC), DU4475 (assigned by ATCC), Colo-824 (CLS Cell Lines) Service), MDA-MB-157 (assigned by ATCC), MDA-MB-468 (assigned by ATCC) (all of these are breast cancer lines), HLC-1 (assigned by RIKEN), EKVX (assigned by NCI), NCI-H1395 (assigned by ATCC), DMS114 (assigned by ATCC), SW1271 (assigned by ATCC), NCI-H1155 (assigned by ATCC), Lu65 (assigned by JCRB; JCRB0079, Hirohashi, S. and Simosato, The following clones were used in the evaluation of the present invention: NCI-H2228 (assigned from ATCC), NCI-H596 (assigned from ATCC), NCI-H446 (assigned from ATCC), ChaGo-K-1 (assigned from ATCC) (all of these are lung cancer clones), RT-4 (assigned from ATCC), HT-1197 (assigned from ATCC), TCCSUP (assigned from ATCC), 5637 (assigned from ATCC) (all of these are bladder cancer clones), OVMANA (assigned from JCRB; JCRB1045, established by Gorai, I. et al.), RMG-I (assigned from JCRB; IFO50315, established by Nozawa, S. et al.), and NCI:OVCAR-3 (assigned from ATCC) (all of these are ovarian cancer clones).

[0197] MDA-MB-175VII, HCC38, MDA-MB-361, HLC-1, EKVX, NCI-H1395, DMS114, SW1271, RT-4, HT-1197, OVMANA, and RMG-I are wild-type cancer cell lines that do not have RB1 gene mutations or deletions, and RB1 is functional in these cell lines. DU4475, Colo-824, MDA-MB-157, MDA-MB-468, ChaGo-K-1, TCCSUP, and NIH:OVCAR-3 are cancer cell lines lacking the RB1 gene, NCI-H1155, Lu65, NCI-H596, and 5637 are cancer cell lines with truncating mutations in the RB1 gene, and NCI-H2228 and NCI-H446 are cancer cell lines with mutations in the splicing site of the RB1 gene, resulting in reduced RB1 function. Information on these cancer cell lines and gene mutations is summarized in Table 4. １ Nature. 2019 May;569(7757):503-508. ２ Cell Model Passports (https: / / cellmodelpassports.sanger.ac.uk / , accessed 2022 / 12 / 27) ３ Nature. 2012 Mar 28;483(7391):603-7.

[0198] (2) Cytotoxicity Test of Various Anticancer Drugs with the Addition of a MYT1 Inhibitor Pemetrexed (Tokyo Chemical Industry Co., Ltd.), gemcitabine (Nippon Kayaku Co., Ltd.), carboplatin (Sandoz), SN-38 (active metabolite of irinotecan, a topoisomerase I inhibitor, Tokyo Chemical Industry Co., Ltd.), sacituzumab govitecan (antibody-drug conjugate containing SN-38 as a payload and recognizing Trop2, MedChem Express), DXd (exatecan derivative for antibody-drug conjugate, a topoisomerase I inhibitor, MedChem Express), and trastuzumab deruxtecan (antibody-drug conjugate containing DXd as a payload and recognizing HER2, Daiichi Sankyo Co., Ltd.) were used for evaluation.

[0199] Using an Echo (Beckman Coulter), various concentrations of pemetrexed (0.0195 μM to 5.00 μM, common ratio 2), gemcitabine (0.835 nM to 214 nM, common ratio 2), carboplatin (0.781 μM to 200 μM, common ratio 2), SN-38 (0.610 nM to 156 nM, common ratio 2), sacituzumab govitecan (SG) (0.195 nM to 50.0 nM, common ratio 2), DXd (0.195 nM to 50.0 nM, common ratio 2), or trastuzumab deruxtecan (T-DXd) (1.95 nM to 500 nM, common ratio 2) were added to each well of a cell culture plate. As a control, the solvent used to dilute the anticancer drugs was added to empty wells.

[0200] The MYT1 inhibitor (compound 2) (4.88 nM to 5.00 μM, common ratio 2) was added to cell culture plates containing the above anticancer drugs using Echo (Beckman Coulter) to create combinations of various concentrations of anticancer drugs and various concentrations of MYT1 inhibitors. As a control, only DMSO, which was used to dilute the various MYT1 inhibitors, was added to empty wells.

[0201] Various cells cultured under the conditions summarized in Table 5 were seeded onto the above plates at the cell numbers summarized in Table 6 and incubated at 37°C. After incubation with pemetrexed, gemcitabine, carboplatin, SN-38, sacituzumab govitecan (SG), DXd, and trastuzumab deruxtecan (T-DXd) for 7 days, 4 days, 4 days, 4 days, 4 or 5 days, and 4 days and 5 days, respectively, the CellTiter-Glo 2.0 Cell Viability Assay (Promega) was added, and cell number was monitored by measuring luminescence intensity using a Multimode Plate Reader EnVision® Xcite (PerkinElmer). Cell viability was calculated assuming the signal in the control well as 100%, and cytotoxicity was calculated by subtracting the viability from the 100% value. The following combined effect was evaluated using Compound 2 at 313 nM. When the survival rate under the condition of treatment with Compound 2 alone was below 80%, the MYT1 inhibitor concentration was reduced by a common ratio of 2, and the combined effect was evaluated at a concentration at which the survival rate was 80% or higher.

[0202] Based on the cytotoxic activity of each concentration of each chemotherapeutic agent in wells to which no MYT1 inhibitor was added, and the cytotoxic activity of each concentration of the MYT1 inhibitor in wells to which no chemotherapeutic agent was added, a Bliss score based on Bliss independence, which serves as an index of the combined effect of a chemotherapeutic agent and a MYT1 inhibitor, was calculated (see Non-Patent Documents 9 and 10). Furthermore, an HSA score, which serves as an index of the combined effect different from Bliss independence, was calculated (see Non-Patent Document 10). The maximum Bliss score, maximum HSA score, and the corresponding MYT1 inhibitor concentration are summarized in Table 7. When the maximum value was below 0, each score was assigned a value of 0.00.

[0203] 4 to 10, the maximum Bliss score and maximum HSA score were plotted for cell lines with wild-type RB1 gene, in which RB1 was functional, and for cell lines in which RB1 function was reduced due to mutation or deletion of the RB1 gene. Student's t-test was performed to determine whether there was a difference in the maximum Bliss score and maximum HSA score between cell lines in which RB1 was functional and cell lines in which RB1 function was reduced, and p-values ​​were calculated. Asterisks are indicated in each figure based on the p-values ​​(*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0204] 2. Results (1) Pemetrexed As shown in Table 7 and Figure 4, it was confirmed that the Bliss score and HSA score of the cytotoxic activity of pemetrexed were significantly higher in lung cancer cell lines in which mutation or deletion of the RB1 gene was observed and RB1 function was impaired when treated with Compound 2, compared to cancer cell lines in which RB1 function was not observed and mutation or deletion of the RB1 gene was not observed.

[0205] These results demonstrate that the administration of compound 2 to cancers such as lung cancer, which have a decreased function of RB1 due to mutation or deletion of the RB1 gene, can effectively enhance the cytotoxic activity of chemotherapeutic agents such as pemetrexed.

[0206] (2) Gemcitabine As shown in Table 7 and Figure 5, in lung cancer, breast cancer, bladder cancer, and ovarian cancer cell lines in which mutations or deletions in the RB1 gene were observed and RB1 function was impaired, the Bliss score and HSA score of the cytotoxic activity of gemcitabine were confirmed to be significantly higher when treated with Compound 2 than in cancer cell lines in which RB1 was functional and no mutations or deletions in the RB1 gene were observed.

[0207] These results demonstrate that the administration of Compound 2 to cancers such as lung cancer, breast cancer, bladder cancer, and ovarian cancer, which have reduced RB1 function due to mutations or deletions in the RB1 gene, can effectively enhance the cytotoxic activity of chemotherapeutic agents such as gemcitabine.

[0208] (3) Carboplatin As shown in Table 7 and Figure 6, in lung cancer, breast cancer, bladder cancer, and ovarian cancer cell lines in which mutations or deletions in the RB1 gene were observed and RB1 function was impaired, the Bliss score and HSA score of the cytotoxic activity of carboplatin were confirmed to be significantly higher when treated with Compound 2 than in cancer cell lines in which RB1 was functional and no mutations or deletions in the RB1 gene were observed.

[0209] These results demonstrate that the administration of compound 2 to cancers such as lung cancer, breast cancer, bladder cancer, and ovarian cancer, which have reduced RB1 function due to mutations or deletions in the RB1 gene, can effectively enhance the cytotoxic activity of chemotherapeutic agents such as carboplatin.

[0210] (4) SN-38 As shown in Table 7 and FIG. 7 , in lung cancer, breast cancer, and ovarian cancer cell lines in which mutation or deletion of the RB1 gene was observed and RB1 function was impaired, the Bliss score and HSA score of the cytotoxic activity of SN-38 were confirmed to be significantly higher when treated with Compound 2 than in cancer cell lines in which RB1 was functional and no mutation or deletion of the RB1 gene was observed.

[0211] These results demonstrate that the administration of Compound 2 to cancers such as lung cancer, breast cancer, and ovarian cancer, which have reduced RB1 function due to mutations or deletions in the RB1 gene, can effectively enhance the cytotoxic activity of chemotherapeutic agents such as SN-38.

[0212] (5) Sacituzumab govitecan (SG) As shown in Table 7 and Figure 8, in lung cancer and breast cancer cell lines in which mutations or deletions in the RB1 gene were observed and RB1 function was impaired, the Bliss score and HSA score of the cytotoxic activity of sacituzumab govitecan (SG) were significantly higher when treated with Compound 2 than in cancer cell lines in which RB1 was functional and no mutations or deletions in the RB1 gene were observed.

[0213] These results demonstrate that administration of compound 2 to cancers such as lung cancer and breast cancer, which have reduced RB1 function due to mutations or deletions in the RB1 gene, can effectively enhance the cytotoxic activity of antibody-drug conjugates that use chemotherapeutic agents such as SN-38 as the payload.

[0214] (6) DXd As shown in Table 7 and Figure 9, in lung cancer, breast cancer, and ovarian cancer cell lines in which mutations or deletions in the RB1 gene were observed and RB1 function was impaired, the Bliss score and HSA score of the cytotoxic activity of DXd when treated with compound 2 were significantly higher than in cancer cell lines in which RB1 function was not observed and mutations or deletions in the RB1 gene were not observed.

[0215] These results demonstrate that administration of compound 2 to cancers such as lung cancer, breast cancer, and ovarian cancer, which have reduced RB1 function due to mutations or deletions in the RB1 gene, can effectively enhance the cytotoxic activity of chemotherapeutic agents such as DXd.

[0216] (7) Trastuzumab deruxtecan (T-DXd) As shown in Table 7 and FIG. 10, in breast cancer cell lines in which mutation or deletion of the RB1 gene was observed and RB1 function was impaired, the Bliss score and HSA score of the cytotoxic activity of trastuzumab deruxtecan (T-DXd) were significantly higher when treated with Compound 2 than in cancer cell lines in which RB1 function was not observed and mutation or deletion of the RB1 gene was not observed.

[0217] These results demonstrate that administration of compound 2 to cancers such as breast cancer, which have reduced RB1 function due to mutations or deletions in the RB1 gene, can effectively enhance the cytotoxic activity of antibody-drug conjugates that use DXd or other chemotherapeutic agents as payloads.

[0218] Example 3: Cytotoxicity test of gemcitabine with the addition of a MYT1 inhibitor in cancer cell lines positive for the expression of hypophosphorylated RB1 protein and cancer cell lines positive for the expression of hyperphosphorylated RB1 protein 1. Experimental Materials and Methods (1) Cell Lines The following cell lines were used for evaluation: RMG-I (ovarian cancer line, provided by JCRB; IFO50315, established by Nozawa, S. et al.), OVTOKO (ovarian cancer line, provided by JCRB; JCRB1048, established by Gorai, I. et al.), OVMANA (ovarian cancer line, provided by JCRB; JCRB1045, established by Gorai, I. et al.), ES-2 (ovarian cancer line, provided by ATCC), JHOC-5 (ovarian cancer line, provided by RIKEN), PA-1 (ovarian cancer line, provided by ATCC), MDA-MB-175VII (breast cancer line, provided by ATCC), NCI-H1395 (lung cancer line, provided by ATCC), and RT-4 (bladder cancer line, provided by ATCC).

[0219] RMG-I, MDA-MB-175VII, NCI-H1395, OVTOKO, OVMANA, RT-4, ES-2, JHOC-5, and PA-1 are RB1 wild-type cancer cell lines that do not have mutations or deletions in the RB1 gene, and RB1 is expressed in these cell lines.

[0220] RMG-I is a CCNE1-amplified cell line, and CCNE1 function is enhanced. MDA-MB-175VII, NCI-H1395, OVTOKO, OVMANA, RT-4, ES-2, JHOC-5, and PA-1 are RB1 wild-type cancer cell lines that do not have CCNE1 gene mutations or amplifications, and CCNE1 function is not enhanced in these cell lines.

[0221] RMG-I, MDA-MB-175VII, NCI-H1395, OVTOKO, OVMANA, RT-4, ES-2, JHOC-5, and PA-1 are wild-type cancer cell lines that do not have mutations or deletions in the FBXW7 gene, and FBXW7 is functional in these cell lines.

[0222] OVTOKO is a cell line that has the R183G mutation in the PPP2R1A gene, and is a cancer cell line that has reduced PPP2R1A function according to Non-Patent Documents 11 and 12. RMG-I, MDA-MB-175VII, NCI-H1395, OVMANA, RT-4, ES-2, JHOC-5, and PA-1 are PPP2R1A wild-type cancer cell lines that do not have mutations or deletions in the PPP2R1A gene, and PPP2R1A is functional in these cell lines.

[0223] The information on these cancer cell lines and gene mutations is summarized in Table 8.

[0224] (2) Evaluation of the phosphorylation level of RB1 protein in each cancer cell line PhosSTOP TM (Roche, 4906845001) One tablet was dissolved in 1 mL of MilliQ water to prepare a 10x Phosphatase inhibitor solution. TM A 25x protease inhibitor solution was prepared by dissolving one tablet of protease inhibitor cocktail (Roche, 11697498001) in 1 mL of MilliQ water. Cell lysis buffer (1x cell lysis buffer, 1x phosphatase inhibitor solution, 1x protease inhibitor solution) was prepared using 10x cell lysis buffer (Cell Singaling Technology, #9803), 10x phosphatase inhibitor solution, 25x protease inhibitor solution, and MilliQ water, and cooled on ice.

[0225] Each cancer cell line was cultured under the conditions listed in Table 9. The cell surface was washed with PBS (phosphate-buffered saline, manufactured by Nacalai Tesque, 14249-24), and the cells were then collected using a scraper. The cells were then suspended in cell lysis buffer and incubated on ice for 15 minutes. The cells were then centrifuged at 15,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The collected supernatant was analyzed using Direct Detector. TMProtein concentrations were quantified using a spectrometer (Merck). Western blotting samples (1 μg / μL or 0.5 μg / μL final protein concentration, 1x sample buffer) were prepared for each cell line using protein extracts from each cell line, sample buffer (SDS-PAGE grade, 6x concentrated, containing a reducing agent) (Nacalai tesque, 09499-14), and MilliQ water.

[0226] Each sample was diluted with SuperSep™ to ensure equal amounts of protein. ＴＭ The resultant solution was applied to a 17-well plate containing 5-20% Ace (Fujifilm Wako Pure Chemical Industries, Ltd., #194-15021) and subjected to SDS-PAGE using SDS-PAGE buffer, pH 8.5 (Fujifilm Wako Pure Chemical Industries, Ltd., #192-16801). The resultant was then transferred to a Trans-Blot Turbo Transfer Pack PVDF (Mini) (Bio-Rad, #1704156) and blocked for 30 minutes at room temperature using Bullet Blocking One (Nacalai Tesque, 13779-01). The primary antibody was diluted with Signal Enhancer HIKARI for Western Blotting and ELISA Solution A (Nacalai tesque, 02272-74), and the secondary antibody was diluted with Signal Enhancer HIKARI for Western Blotting and ELISA Solution B (Nacalai tesque, 02297-64), and blotting was performed in that order. The antibodies used and their dilutions are shown in Table 10.

[0227] For detection, use Pierce TMECL Plus Western Blotting Substrate (Thermo Fischer Scientific, 32132) was used. Figure 11 shows the results of blotting obtained using a ChemiDoc Touch MP (Bio-Rad). Band intensities detected by Western blotting were quantified using ImageLab (Bio-Rad), and the values ​​(relative values) of various band intensities relative to the quantitative value of GAPDH (housekeeping gene) were calculated. The value of the cell line showing the lowest value was set to 1, and the relative values ​​(phosphorylation levels) of each cell line are shown in Table 17.

[0228] Table 11 and Figure 11 show that the phosphorylation levels of RB1 protein at T826, T821, S807 and / or S811, S780, T373, or T356 were higher in the three cell lines, ES-2, JHOC-5, and PA-1, compared to the six cell lines, RMG-I, MDA-MB-175VII, NCI-H1395, OVTOKO, OVMANA, and RT-4. Generally, a high phosphorylation level of an amino acid residue indicates high expression of a protein in which that amino acid residue is phosphorylated. Therefore, the three cell lines, ES-2, JHOC-5, and PA-1, were determined to be cell lines positive for the expression of hyperphosphorylated RB1 protein. Furthermore, it was found that the phosphorylation levels of amino acid residues other than S795, S788, and T252 were lower in the two cell lines, RMG-I and MDA-MB-175VII, than in the three cell lines, ES-2, JHOC-5, and PA-1. Furthermore, it was found that the phosphorylation levels of all measured amino acid residues were lower in the four cell lines, NCI-H1395, OVTOKO, OVMANA, and RT-4, than in the three cell lines, ES-2, JHOOC-5, and PA-1. Hereinafter, these six cell lines, RMG-I, MDA-MB-175VII, NCI-H1395, OVTOKO, OVMANA, and RT-4, were analyzed as a group "positive for expression of hypophosphorylated RB1 protein."

[0229] (3) Analysis of E2F3 gene expression levels in ovarian cancer cell lines. Expression Public 23Q2 was downloaded from the DepMap portal (URL: https: / / depmap.org / portal / [searched July 14, 2023]), and the expression levels of the E2F3 gene for each cell line are shown in Table 12. The expression levels of the E2F3 gene in each cell line determined to be positive for hypophosphorylated RB1 protein or hyperphosphorylated RB1 protein were plotted, and a Student's t-test was performed to determine whether there was a difference in E2F3 gene expression levels between hypophosphorylated RB1 protein expression-positive cell lines and hyperphosphorylated RB1 protein expression-positive cell lines. The p-values ​​were calculated and shown in Figure 12 (*: p<0.05, **: p<0.01).

[0230] (4) Cytotoxicity Test of Gemcitabine with MYT1 Inhibitor Addition Gemcitabine (Nippon Kayaku Co., Ltd.) was used for evaluation. Using Echo (Beckman Coulter), various concentrations of gemcitabine (0.835 nM to 214 nM, common ratio 2) were added to each well of a cell culture plate. As a control, only the solvent used to dilute the various anticancer drugs was added to empty wells. MYT1 inhibitor compound 2 (4.88 nM to 5.00 μM, common ratio 2) was added to the above-mentioned gemcitabine-containing cell culture plate using Echo (Beckman Coulter), creating combinations of various concentrations of anticancer drugs and various concentrations of MYT1 inhibitors. As a control, only DMSO used to dilute the various MYT1 inhibitors was added to empty wells.

[0231] Various cells cultured under the conditions summarized in Table 9 were seeded onto the above plates at the cell numbers summarized in Table 13 and incubated at 37°C. After 4 days of incubation, CellTiter-Glo 2.0 Cell Viability Assay (Promega) was added to the culture medium, and cell number was monitored by measuring luminescence intensity using a Multimode Plate Reader EnVision® Xcite (PerkinElmer). Cell viability was calculated based on the signal from the control well as 100%, and cytotoxicity was expressed by subtracting viability from the 100% value. Compound 2 was used at 313 nM to evaluate the effect of combination therapy as follows. Alternatively, if the viability under single-agent treatment conditions was below 75%, the MYT1 inhibitor concentration was reduced by a common ratio of 2, and the effect of combination therapy was evaluated at a concentration that resulted in a viability of 75% or higher.

[0232] Based on the cytotoxic activity of each concentration of gemcitabine in wells without MYT1 inhibitor and the cytotoxic activity of each concentration of MYT1 inhibitor in wells without gemcitabine, a Bliss score based on Bliss independence was calculated as an index of the combined effect of gemcitabine and a MYT1 inhibitor (see Non-Patent Document 9). Furthermore, an HSA score was calculated as an index of the combined effect different from Bliss independence (see Non-Patent Document 9). The maximum Bliss score, maximum HSA score, and the corresponding MYT1 inhibitor concentration are summarized in Table 14. When the maximum value was below 0, the score was assigned a value of 0.00. Each score was calculated to three significant digits.

[0233] The maximum Bliss score and maximum HSA score for cell lines determined to be positive for hypophosphorylated RB1 protein or hyperphosphorylated RB1 protein were plotted, and a Student's t-test was performed to calculate p-values. The results are shown in Figure 13. Asterisks are indicated in Figure 13 based on p-values ​​(n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0234] 2. Results FIG. 11 suggests that the expression of hyperphosphorylated RB1 protein is independent of gene mutation, amplification, or deletion of RB1, CCNE1, FBXW7, and PPP2R1A.

[0235] The RB1 protein plays a role in the G1 / S checkpoint by suppressing the activity of E2F protein, a transcription factor that promotes the transition from G1 to S phase. However, it is known that hyperphosphorylation reduces the inhibitory function of E2F protein (Non-patent Document 12).

[0236] Target factors of E2F transcription factors include E2F genes, and it is known that a positive feedback loop exists (Non-Patent Document 14). This suggests that the expression level of E2F genes can be used as an indicator of the transition to S phase and the functional decline of RB1 protein.

[0237] As shown in Table 12 and Figure 12, the expression level of the E2F3 gene was significantly higher in ovarian cancer cell lines that were positive for the expression of hyperphosphorylated RB1 protein than in ovarian cancer cell lines that were positive for the expression of hypophosphorylated RB1 protein, suggesting that the function of RB1 protein was impaired in ovarian cancer cell lines that were positive for the expression of hyperphosphorylated RB1 protein. It is also known that hyperphosphorylated RB1 protein is expressed in ovarian cancer and colorectal cancer (Non-Patent Documents 15 and 16).

[0238] As shown in Table 14 and FIG. 13 , it was confirmed that the Bliss score and HSA score of the cytotoxic activity of gemcitabine upon treatment with Compound 2 were significantly higher in cancer cell lines in which hyperphosphorylated RB1 protein was observed to be expressed, compared with cancer cell lines positive for the expression of hypophosphorylated RB1 protein.

[0239] These results revealed that cancers determined to be positive for the expression of hyperphosphorylated RB1 protein have reduced function of RB1 protein, and that administering the MYT1 inhibitor Compound 2 to these cancers can synergistically enhance the cytotoxic activity of chemotherapeutic agents such as gemcitabine.

[0240] Example 4: Anticancer activity test of combined use of MYT1 inhibitor and various anticancer agents in cancer cell lines with reduced or deleted RB1 function 1. Experimental Materials and Methods (1) Cell Lines OVMANA (gift from JCRB; JCRB1045, established by Gorai, I. et al.), RMG-I (gift from JCRB; IFO50315, established by Nozawa, S. et al.), NIH:OVCAR-3 (gift from ATCC), ES-2 (ovarian cancer line, gifted from ATCC), JHOC-5 (ovarian cancer line, gifted from RIKEN), PA-1 (ovarian cancer line, gifted from ATCC) (all of these are ovarian cancer lines), RT-4 (bladder cancer line, gifted from ATCC), TCCSUP (gift from ATCC), 5637 (gift from ATCC) (all of these are bladder cancer lines), DU4475 (gift from ATCC), Colo-824 (CLS Cell Lines) The following strains were used in the evaluation of the present invention: NCI-H1395 (assigned by ATCC), Lu65 (assigned by JCRB; JCRB0079, established by Hirohashi, S. and Simosato, Y.), NCI-H2228 (assigned by ATCC), NCI-H446 (assigned by ATCC), ChaGo-K-1 (assigned by ATCC), NCI-H82 (assigned by ATCC), (all of which are lung cancer strains), and COLO205 (colon cancer strain, assigned by ATCC).

[0241] OVMANA, RMG-I, ES-2, JHOC-5, PA-1, RT-4, and NCI-H1395 are RB1 wild-type cancer cell lines with no RB1 gene mutations or deletions, and these cells express RB1. COLO205 is a cancer cell line with RB1 gene amplification and expresses RB1. DU4475, Colo-824, ChaGo-K-1, TCCSUP, and NIH:OVCAR-3 are cancer cell lines with RB1 gene deletions. 5637 and Lu65 are cancer cell lines with truncated deletion mutations in the RB1 gene. NCI-H446, NCI-H2228, and NCI-H82 are cancer cell lines with mutations in the splicing site of the RB1 gene, resulting in reduced RB1 function. Information on these cancer cell lines and gene mutations is summarized in Table 15. 1: Nature 2019 May 8;569(7757):503-508. 2: Nature 2012 Mar 28;483(7391):603-7. 3: Cell Model Passports (https: / / cellmodelpassports.sanger.ac.uk / ,2022 / 12 / 27access) 4: DepMap portal Copy Number Public 23Q2(https: / / depmap.org / portal / )

[0242] (2) Evaluation of the phosphorylation level of RB1 protein in each cancer cell line. One tablet of PhosSTOP™ (Roche, 4906845001) was dissolved in 1 mL of MilliQ water to prepare a 10x phosphatase inhibitor solution. TMA 25x protease inhibitor solution was prepared by dissolving one tablet of protease inhibitor cocktail (Roche, 11697498001) in 1 mL of MilliQ water. Cell lysis buffer (1x cell lysis buffer, 1x phosphatase inhibitor solution, 1x protease inhibitor solution) was prepared using 10x cell lysis buffer (Cell Singaling Technology, #9803), 10x phosphatase inhibitor solution, 25x protease inhibitor solution, and MilliQ water, and cooled on ice.

[0243] OVMANA, ES-2, and COLO205 cell lines were cultured under the conditions listed in Table 16. The cell surface was washed with PBS (phosphate-buffered saline, manufactured by Nacalai Tesque, 14249-24), and the cells were then collected using a scraper. The cells were then suspended in cell lysis buffer and incubated on ice for 15 minutes. The cells were then centrifuged at 15,000 rpm for 10 minutes at 4°C, and the supernatant was collected. The collected supernatant was analyzed using Direct Detector. TM Protein concentrations were quantified using a spectrometer (Merck). Western blotting samples (1 μg / μL or 0.5 μg / μL final protein concentration, 1x sample buffer) were prepared for each cell line using protein extracts from each cell line, sample buffer (SDS-PAGE grade, 6x concentrated, containing a reducing agent) (Nacalai tesque, 09499-14), and MilliQ water.

[0244] Each sample was diluted with SuperSep™ to ensure equal amounts of protein. ＴＭThe 5-20% Ace gel was applied to a 17-well plate (Fujifilm Wako Pure Chemical Industries, Ltd., #194-15021) and subjected to SDS-PAGE using SDS-PAGE buffer, pH 8.5 (Fujifilm Wako Pure Chemical Industries, Ltd., #192-16801). The gel was then transferred to a Transblot Turbo Transfer Pack PVDF (Mini) (Bio-Rad, #1704156) and blocked with Bullet Blocking One (Nacalai Tesque, 13779-01) at room temperature for 30 minutes. The primary antibody was diluted with Signal Enhancer HIKARI for Western Blotting and ELISA Solution A (Nacalai Tesque, 02272-74), and the secondary antibody was diluted with Signal Enhancer HIKARI for Western Blotting and ELISA Solution B (Nacalai Tesque, 02297-64), followed by blotting. The antibodies and dilutions used are shown in Table 17.

[0245] For detection, use Pierce TM ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, 32132) was used. Figure 19 shows the results of blotting obtained using ChemiDoc Touch MP (Bio-Rad).

[0246] Non-Patent Documents 15 and 16 show that ovarian cancer and colon cancer have high levels of RB1 phosphorylation. Figure 14 reveals that the phosphorylation levels of RB1 protein expressed by the colon cancer cell line COLO205 at T826, T821, S807 and / or S811, S795, S788, S780, T373, T356, or T252 are higher than those of the hypophosphorylated RB1-expressing cell line OVMANA and equivalent to those of the hyperphosphorylated RB1-expressing cell line ES-2. Therefore, COLO205 was determined to be a cell line positive for the expression of hyperphosphorylated RB1 protein, similar to ES-2. Table 15 lists cell lines with wild-type or amplified RB1 genes, and indicates whether they are hyperphosphorylated RB1-expressing or hypophosphorylated RB1-expressing cell lines. Hyperphosphorylated RB1-expressing cell lines are considered to have a loss or reduction of RB1 function.

[0247] (3) Cytotoxicity Test of Various Anticancer Drugs When Compound 1 or Compound 2, an MYT1 Inhibitor, is Added Gemcitabine (Nippon Kayaku Co., Ltd.), carboplatin (Sandoz), SN-38 (active metabolite of irinotecan, topoisomerase I inhibitor, Tokyo Chemical Industry Co., Ltd.), sacituzumab govitecan (antibody-drug conjugate containing SN-38 as a payload and recognizing Trop2, MedChem Express), DXd (exatecan derivative for antibody-drug conjugate, topoisomerase I inhibitor, MedChem Express), pemetrexed (Tokyo Chemical Industry Co., Ltd.), etoposide (Clinigen), lurbinectedin (MedChem Express), fluorouracil (Selleck Biotech), and trifluridine-tipiracil (Selleck Biotech) were used for evaluation.

[0248] Echo (Beckman Using a 100-well platelet count (manufactured by Coulter), various concentrations of gemcitabine (0.86 nM to 220 nM, common ratio 2), carboplatin (0.77 nM to 200 nM, common ratio 2), SN-38 (0.61 nM to 160 nM, common ratio 2), sacituzumab govitecan (0.20 nM to 50 nM, common ratio 2), DXd (0.20 nM to 50 nM, common ratio 2), pemetrexed (20 nM to 5 μM, common ratio 2), etoposide (9.7 nM to 2.5 μM, common ratio 2), lurbinectedin (0.023 nM to 6.0 nM, common ratio 2), fluorouracil (78 nM to 20 μM, common ratio 2), and trifluridine-tipiracil (0.78 μM to 20 μM, common ratio 2) were incubated in a cell culture medium. The MYT1 inhibitors (compound 1, 310 nM, 620 nM, 1200 nM) and (compound 2, 160 nM, 310 nM, 620 nM) were added to each well of the plate. As a control, only the solvent used to dilute the various anticancer drugs was added to empty wells. The MYT1 inhibitors (compound 1, 310 nM, 620 nM) and (compound 2, 160 nM, 310 nM, 620 nM) were also added to the cell culture plates containing the above-mentioned various anticancer drugs using an Echo (Beckman Coulter) to create combinations of various concentrations of the various anticancer drugs and various concentrations of the various MYT1 inhibitors. As a control, only DMSO used to dilute the various MYT1 inhibitors was added to empty wells.

[0249] Various cells cultured under the conditions summarized in Table 16 were seeded onto the above plates at the cell numbers summarized in Table 18 and incubated at 37°C. After incubation for 4 days for combinations with gemcitabine, carboplatin, SN-38, DXd, etoposide, lurbinectedin, fluorouracil, and trifluridine / tipiracil, 5 days for combinations with sacituzumab govitecan, and 7 days for combinations with pemetrexed, a CellTiter-Glo 2.0 Cell Viability Assay (Promega) was added to the culture medium, and cell numbers were monitored by measuring luminescence intensity using a Multimode Plate Reader EnVision® Xcite (PerkinElmer). Cell viability was calculated assuming the signal from the control well as 100%, and cytotoxicity was calculated by subtracting viability from the 100% value. The combination effects were evaluated as follows for each combination of various anticancer drug concentrations and MYT1 inhibitor concentrations. Figures 15 to 30 show graphs plotting the survival rates under each condition of cell lines determined to have a wild-type or amplified RB1 gene and to be positive for the expression of hypophosphorylated RB1 protein (cell lines expressing WT or Amp RB1 gene and hypophosphorylated RB1), cell lines having a deletion or deletion mutation in the RB1 gene or a mutation at a splicing site of the RB1 gene (cell lines expressing mt or Loss RB1 gene), and cell lines determined to have a wild-type or amplified RB1 gene and to be positive for the expression of hyperphosphorylated RB1 protein (cell lines expressing WT or Amp RB1 gene and hyperphosphorylated RB1), respectively.

[0250] Based on the cytotoxic activity of each concentration of each chemotherapeutic agent in wells to which no MYT1 inhibitor was added, and the cytotoxic activity of each concentration of the MYT1 inhibitor in wells to which no chemotherapeutic agent was added, a Bliss score based on Bliss independence, which serves as an index of the combined effect of a chemotherapeutic agent and a MYT1 inhibitor, was calculated (see Non-Patent Document 9). Furthermore, an HSA score, which serves as an index of the combined effect different from Bliss independence, was calculated (see Non-Patent Document 9). The maximum Bliss score and maximum HSA score are summarized in Table 19. If the maximum value was below 0, each score was assigned a value of 0.00. Each score was calculated to two significant digits. A maximum Bliss score of 10 or greater was considered to indicate a synergistic combined effect. Furthermore, a maximum HSA score of 10 or greater was considered to indicate a combined effect.

[0251] Cancer cell lines with a deleted or deleted mutation in the RB1 gene or a mutation in the splicing site of the RB1 gene (RB1 gene mt or Loss cell lines) and cancer cell lines with a wild-type or amplified RB1 gene and determined to be positive for the expression of hyperphosphorylated RB1 protein (RB1 gene WT or Amp and hyperphosphorylated RB1-expressing cell lines) were classified as RB1-deficient cell lines, as these suggested that RB1 function was deleted or impaired. On the other hand, cell lines with a wild-type or amplified RB1 gene and determined to be positive for the expression of hypophosphorylated RB1 protein (RB1 gene WT or Amp and hypophosphorylated RB1-expressing cell lines) were classified as RB1-proficient cell lines, as they were considered to have normal RB1 function.

[0252] The anticancer activity of the combined use of gemcitabine and Compound 1 was evaluated in cell lines in which the RB1 gene was determined to be wild-type or amplified and to be positive for the expression of hypophosphorylated RB1 protein (cell lines in which the RB1 gene is WT or Amp and hypophosphorylated RB1 is expressed), cell lines in which the RB1 gene is deleted or has a deletion mutation or a mutation at a splicing site of the RB1 gene (cell lines in which the RB1 gene is mt or Loss), and cell lines in which the RB1 gene was determined to be wild-type or amplified and to be positive for the expression of hyperphosphorylated RB1 protein (cell lines in which the RB1 gene is WT or Amp and hyperphosphorylated RB1 is expressed). The cytotoxic activity is shown in Figures 15 to 17 . The maximum Bliss score and the maximum HSA score for each cell line were plotted, and a Tukey test was performed to calculate p-values ​​for the maximum Bliss score and the maximum HSA score among cell lines whose RB1 gene was determined to be wild-type or amplified and positive for expression of hypophosphorylated RB1 protein (cell lines expressing WT or Amp RB1 genes and hypophosphorylated RB1), cell lines whose RB1 gene was deleted or had a deletion mutation or a mutation at a splicing site of the RB1 gene (cell lines expressing mt or Loss RB1 genes), and cell lines whose RB1 gene was determined to be wild-type or amplified and positive for expression of hyperphosphorylated RB1 protein (cell lines expressing WT or Amp RB1 genes and hyperphosphorylated RB1). The results are shown in FIG. Asterisks are indicated in Figure 18 based on p-values ​​(n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0253] 2. Results (1) Gemcitabine As shown in FIGS. 15 to 18 and Table 19, it was confirmed that the maximum Bliss score and maximum HSA score of the cytotoxic activity by gemcitabine upon treatment with the MYT1 inhibitor Compound 1 were significantly higher in cancer cell lines having a deletion or deletion mutation in the RB1 gene or a mutation in a splicing site of the RB1 gene (cell lines with mt or Loss RB1 gene) and cancer cell lines having a wild-type or amplified RB1 gene and determined to be positive for the expression of hyperphosphorylated RB1 protein (cell lines with WT or Amp RB1 gene and hyperphosphorylated RB1 expression) than in cancer cell lines having a wild-type or amplified RB1 gene and determined to be positive for the expression of hypophosphorylated RB1 protein (cell lines with WT or Amp RB1 gene and hypophosphorylated RB1 expression). Furthermore, in cancers in which RB1 function is deleted or reduced, the maximum Bliss score and maximum HSA score are 10 or more, so a synergistic combined efficacy is expected.

[0254] Cancer cell lines with a deletion or deletion mutation in the RB1 gene or a mutation in the splicing site of the RB1 gene (RB1 gene mt or Loss cell lines) and cancer cell lines with a wild-type or amplified RB1 gene and determined to be positive for the expression of hyperphosphorylated RB1 protein (RB1 gene WT or Amp and hyperphosphorylated RB1-expressing cell lines) suggest that RB1 function is deleted or reduced (RB1 deficient). It is also known that hyperphosphorylated RB1 is expressed in ovarian cancer and colorectal cancer (Non-Patent Documents 15 and 16).

[0255] These results demonstrate that the administration of compound 1, an MYT1 inhibitor, can effectively and synergistically enhance the cytotoxic activity of gemcitabine in cancers in which RB1 function is absent or reduced (RB1 deficient).

[0256] (2) Carboplatin Figures 19 to 20 and Table 19 show that, with regard to the cytotoxic activity of carboplatin when treated with the MYT1 inhibitor Compound 1, cancers in which RB1 function is deleted or reduced (RB1 deficient) have higher maximum Bliss scores and maximum HSA scores than RB1 proficient lines in which RB1 function is active. Furthermore, since the maximum Bliss scores and maximum HSA scores are 10 or higher in cancers in which RB1 function is deleted or reduced, a synergistic combined efficacy is expected.

[0257] These results demonstrate that the cytotoxic activity of carboplatin can be effectively and synergistically enhanced by administering compound 1, an MYT1 inhibitor, to cancers in which RB1 function is lost or reduced.

[0258] (3) Topoisomerase I inhibitors, such as SN-38, sacituzumab govitecan, or DXd, or antibody-drug conjugates containing a topoisomerase I inhibitor as a payload. Figures 21 to 22 and Table 19 show that, with regard to the cytotoxic activity of SN-38 upon treatment with the MYT1 inhibitor compound 1, cancers in which RB1 function is deleted or reduced (RB1 deficient) have higher maximum Bliss scores and maximum HSA scores than RB1 proficient strains in which RB1 function is active. Furthermore, since the maximum Bliss scores and maximum HSA scores are 10 or greater in cancers in which RB1 function is deleted or reduced, a synergistic combination efficacy is expected.

[0259] These results demonstrate that the cytotoxic activity of SN-38 can be effectively and synergistically enhanced by administering Compound 1, an MYT1 inhibitor, to cancers in which RB1 function is lost or reduced.

[0260] Furthermore, Figure 23 and Table 19 show that with regard to the cytotoxic activity of sacituzumab govitecan, an antibody-drug conjugate that contains SN-38 as a payload during treatment with a MYT1 inhibitor (Compound 1) and recognizes Trop2, the maximum Bliss score and maximum HSA score are 10 or greater in cancers with RB1 function loss or reduction.

[0261] These results demonstrated that the cytotoxic activity of sacituzumab govitecan can be synergistically enhanced by administering compound 1, an MYT1 inhibitor, to cancers in which RB1 function is deleted or reduced.

[0262] 24 to 25 and Table 19 show that, with regard to the cytotoxic activity of DXd upon treatment with the MYT1 inhibitor of Compound 1, cancers in which RB1 function is deleted or reduced (RB1 deficient) have higher maximum Bliss scores and maximum HSA scores than RB1 proficient strains in which RB1 function is active. Furthermore, since the maximum Bliss scores and maximum HSA scores are 10 or higher in cancers in which RB1 function is deleted or reduced, a synergistic combined pharmacological effect is expected.

[0263] These results demonstrated that the cytotoxic activity of DXd can be effectively and synergistically enhanced by administering compound 1, an MYT1 inhibitor, to cancers in which RB1 function is deleted or reduced.

[0264] These results suggest that administration of compound 1, an MYT1 inhibitor, to cancers in which RB1 function is deleted or reduced can effectively and synergistically enhance the cytotoxic activity of topoisomerase I inhibitors and antibody-drug conjugates containing a topoisomerase I inhibitor as a payload.

[0265] (4) Pemetrexed Figure 26 and Table 19 show that, regarding the cytotoxic activity of pemetrexed upon treatment with the MYT1 inhibitor Compound 1, the maximum Bliss score and the maximum HSA score were 10 or more in cancers in which RB1 function was deleted or reduced.

[0266] These results revealed that the cytotoxic activity of pemetrexed can be synergistically enhanced by administering Compound 1, an MYT1 inhibitor, to cancers in which RB1 function is deleted or reduced.

[0267] (5) Etoposide Figure 27 and Table 19 show that, regarding the cytotoxic activity of etoposide when treated with the MYT1 inhibitor Compound 1 or Compound 2, the maximum Bliss score and the maximum HSA score are 10 or more in cancers in which RB1 function is deleted or reduced.

[0268] These results revealed that the cytotoxic activity of etoposide can be synergistically enhanced by administering Compound 1 or Compound 2, which is a MYT1 inhibitor, to cancers in which RB1 function is deleted or reduced.

[0269] (6) Lurbinectedin Figure 28 and Table 19 show that, with regard to the cytotoxic activity of lurbinectedin when treated with the MYT1 inhibitors Compound 1 or Compound 2, the maximum Bliss score and the maximum HSA score were 10 or more in cancers in which RB1 function was deleted or reduced.

[0270] These results demonstrate that the cytotoxic activity of lurbinectedin can be synergistically enhanced by administering compound 1 or compound 2, which is an MYT1 inhibitor, to cancers in which RB1 function is deleted or reduced.

[0271] (7) Fluorouracil Figure 29 and Table 19 show that, regarding the cytotoxic activity of fluorouracil when treated with the MYT1 inhibitor Compound 1 or Compound 2, in cancers in which RB1 function is deleted or reduced, the maximum Bliss score and the maximum HSA score are 10 or more.

[0272] These results revealed that the cytotoxic activity of fluorouracil can be synergistically enhanced by administering Compound 2 or Compound 1, which are MYT1 inhibitors, to cancers in which RB1 function is deleted or reduced.

[0273] (8) Trifluridine-tipiracil Figure 30 and Table 19 show that, regarding the cytotoxic activity of trifluridine-tipiracil when compound 1 or compound 2 inhibits MYT1, the maximum Bliss score and the maximum HSA score are 10 or more in cancers in which RB1 function is deleted or reduced.

[0274] These results demonstrate that the cytotoxic activity of trifluridine / tipiracil can be synergistically enhanced by administering the MYT1 inhibitors Compound 1 or Compound 2 to cancers in which RB1 function is deleted or reduced.

[0275] Example 5: In vivo combined efficacy test of gemcitabine and MYT1 inhibitor in RB1-deficient or -deficient cancer 1. Experimental materials and methods (1) Combined efficacy test of gemcitabine and Compound 1 in DU4475-bearing mice To investigate the combined effect of a MYT1 inhibitor and an anticancer drug on RB1-deficient cancer in vivo, gemcitabine alone, Compound 1 alone, or a combination of these were administered to mice transplanted with DU4475. After culturing DU4475 in vitro, the cells were collected on the day of transplantation into mice, and 2.4 × 10 cells were added to a Matrigel (Corning, 356234) solution (final concentration 50%) diluted with Hanks' Balanced Salt solution (Sigma-Aldrich, H9269). ７ The prepared cell suspension was transferred to BALB / c-nu / nu mice (CAnN.Cg-Foxn1 <nu>0.2 mL of each solution was implanted into the groin of a mouse (Jackson Laboratory Japan, Inc.).

[0276] Next, mice (DU4475 tumor-bearing mice) in which tumor (cancer cell) engraftment was confirmed were orally administered Compound 1 suspended in a mixture of 10% DMSO (Wako Pure Chemical Industries, Ltd., 043-07216), 10% Cremophor (Sigma-Aldrich, C5135), 15% polyethylene glycol 400 (Wako Pure Chemical Industries, Ltd., 161-09065), and 15% hydroxypropyl-β-cyclodextrin (Nihon Shokuhin Kako Co., Ltd., 7585-39-9) once daily at 2.5 mg / kg for 12 days, or gemcitabine (Nippon Kayaku Co., Ltd.) diluted with saline was intraperitoneally administered at 60 mg / kg once every four days for a total of three times, or a combination of these. Four tumor-bearing mice were used for each group.

[0277] Administration of various drugs began on day 7 after transplantation, and tumor volume and body weight were measured until day 19. Tumor volume was calculated by measuring the minor and major axes of the tumor using an electronic caliper (Mitutoyo, CD-15AX) and calculating each by the least squares method. Tumor volume and body weight were also measured in the same way for DU4475 tumor-bearing mice that had not received drug administration (non-drug administration group). The rate of change in body weight from day 7 was calculated. Mice that showed a weight loss rate of more than 20% were euthanized.

[0278] Figure 31 shows the time course of tumor volume and body weight change rate after DU4475 transplantation in the drug-untreated group, compound 1 alone treated group, gemcitabine alone treated group, and combination treated group. In Figure 31, A shows the time course of tumor volume, and B shows the time course of body weight, expressed as relative values. A two-tailed Student's t-test was performed on the tumor volume of each group on day 19 after transplantation, and p values ​​(significance level: 5%) were calculated. First, a comparison was made to see if there was a difference in tumor volume between the compound 1 alone treated group and the combination treated group. If a significant difference was observed, a comparison was also made to see if there was a difference in tumor volume between the gemcitabine alone treated group and the combination treated group. Based on the p values, asterisks are indicated in Figure 31 (n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0279] (2) Efficacy study of combined use of gemcitabine and compound 1 in NIH:OVCAR-3 tumor-bearing mice To investigate the combined effect of a MYT1 inhibitor with an anticancer drug on RB1-deficient cancer in vivo, gemcitabine alone, compound 1 alone, or a combination of these were administered to mice transplanted with NIH:OVCAR-3. After culturing NIH:OVCAR-3 in vitro, the cells were harvested using a trypsin-EDTA solution (Sigma-Aldrich, T4049) on the day of transplantation into mice, and 5 × 10 cells were placed in a Matrigel (Corning, 354234) solution (final concentration 50%) diluted with Hanks' Balanced Salt solution (Sigma-Aldrich, H9269). ７ The prepared cell suspension was transferred to BALB / c-nu / nu mice (CAnN.Cg-Foxn1 <nu>0.2 mL of each solution was implanted into the groin of a mouse (Jackson Laboratory Japan, Inc.).

[0280] Next, mice (NIH:OVCAR-3 tumor-bearing mice) in which tumor (cancer cell) engraftment was confirmed were orally administered Compound 1 suspended in a mixture of 10% DMSO (Wako Pure Chemical Industries, Ltd., 043-07216), 10% Cremophor (Sigma-Aldrich, C5135), 15% polyethylene glycol 400 (Wako Pure Chemical Industries, Ltd., 161-09065), and 15% hydroxypropyl-β-cyclodextrin (Nihon Shokuhin Kako Co., Ltd., 7585-39-9) once daily at 3, 10, 30, or 100 mg / kg for 14 days, or gemcitabine (Nippon Kayaku Co., Ltd.) diluted with saline was intraperitoneally administered at 60 mg / kg once every seven days, for a total of two doses, or a combination of these. Five tumor-bearing mice were used for each group.

[0281] Administration of various drugs began 57 days after transplantation, and tumor volume and body weight were measured for all groups from days 57 to 71. Tumor volume was calculated by measuring the short and long axes of the tumor using an electronic caliper (Mitutoyo, CD-15AX) and calculating the volume using the least squares method. Tumor volume and body weight were also measured in the same way for NIH:OVCAR-3 tumor-bearing mice that did not receive drug treatment (non-drug treatment group). The rate of change in body weight from day 57 was calculated. Mice that showed a weight loss rate of more than 20% were euthanized.

[0282] Figure 32 shows the time course of tumor volume and body weight change rate after NIH:OVCAR-3 transplantation in the non-drug group, the compound 1 monotherapy group, the gemcitabine monotherapy group, and the combination therapy group. In Figure 32, A shows the time course of tumor volume, and B shows the time course of body weight, expressed as relative values. For the tumor volumes of each group on day 71 after transplantation, a two-tailed Student's t-test was performed to compare whether there was a difference in tumor volume between the non-drug group and the gemcitabine monotherapy group. Next, a two-tailed Student's t-test was performed to compare whether there was a difference in tumor volume between the group administered 100 mg / kg of compound 1 monotherapy and the group administered 100 mg / kg of gemcitabine and compound 1 in combination. If a significant difference was observed, a two-tailed Student's t-test was also performed to compare whether there was a difference in tumor volume between the gemcitabine monotherapy group and the group administered 100 mg / kg of gemcitabine and compound 1 in combination. Based on the p-value, asterisks are indicated in Figure 32 (n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). Furthermore, a Williams test was performed to determine whether there was a difference in tumor volume between the group administered gemcitabine alone and the group administered gemcitabine and Compound 1 in combination at each dose, with a significance level of α=0.025. Cases where the combined effect was determined to be significant are indicated by † in Figure 32.

[0283] (3) Efficacy test of combined use of gemcitabine and compound 1 on ES-2 tumor-bearing mice To investigate the combined effect of a MYT1 inhibitor and an anticancer drug on cancers positive for the expression of hyperphosphorylated RB1 protein in vivo (cell lines determined to be positive for the expression of hyperphosphorylated RB1 protein as described above), mice transplanted with ES-2 were administered gemcitabine alone, compound 1 alone, or a combination of gemcitabine and compound 1. After culturing ES-2 in vitro, the cells were collected on the day of transplantation into mice and diluted to 2.5 × 10 cells in Hanks' Balanced Salt solution (Sigma-Aldrich, H9269). ７ The prepared cell suspension was transferred to BALB / c-nu / nu mice (CAnN.Cg-Foxn1 <nu>0.2 mL of each solution was implanted into the groin of a mouse (Jackson Laboratory Japan, Inc.).

[0284] Next, to mice (ES-2 tumor-bearing mice) in which tumor (cancer cell) engraftment was confirmed, Compound 1 suspended in a mixture of 10% DMSO (Wako Pure Chemical Industries, Ltd., 043-07216), 10% Cremophor (Sigma-Aldrich, C5135), 15% polyethylene glycol 400 (Wako Pure Chemical Industries, Ltd., 161-09065), and 15% hydroxypropyl-β-cyclodextrin (Nihon Shokuhin Kako Co., Ltd., 7585-39-9) was orally administered once daily at 3, 10, 30, or 100 mg / kg for 14 days, or gemcitabine solution (Nippon Kayaku Co., Ltd.) diluted with saline was intraperitoneally administered at 60 mg / kg once every 7 days, a total of two times, or a combination of these was administered. In the combined administration group, Compound 1 suspended in the mixed solution was orally administered, and gemcitabine solution diluted with saline was intraperitoneally administered in the same manner as above. Four tumor-bearing mice were used in each group.

[0285] Administration of various drugs began 10 days after transplantation, and tumor volume and body weight were measured until day 24. The short and long diameters of the tumor were measured using an electronic caliper (Mitutoyo, CD-15AX), and tumor volume was calculated by the least squares method. Tumor volume and body weight were also measured in the same way for ES-2 cancer-bearing mice not administered drugs (non-drug administration group). In each group, tumor volumes were measured until tumor volumes reached 2000 mm ３ When any individual with the above condition appeared, administration to the relevant group of mice was discontinued and the mice were euthanized. The rate of change in body weight from day 10 after transplantation was calculated. If the rate of weight loss exceeded 20%, the mice were euthanized.

[0286] The time course of tumor volume after ES-2 transplantation in the drug-untreated group, the compound 1 alone treated group, the gemcitabine alone treated group, and the combination treated group is shown in Figure 33. In Figure 33, A shows the time course of tumor volume, and B shows the time course of body weight, expressed as relative values. On day 21 after transplantation (the final administration day for the compound 1 alone treated group), a two-tailed Student's t-test was performed to determine whether there was a difference in tumor volume between the group administered 100 mg / kg of compound 1 alone and the group administered 100 mg / kg of gemcitabine and compound 1 in combination, and a p-value (significance level: 5%) was calculated. If a significant difference was observed, a two-tailed Student's t-test was performed to determine whether there was a difference in tumor volume between the gemcitabine alone treated group and the group administered 100 mg / kg of gemcitabine and compound 1 in combination on day 24 after transplantation (the day after the final administration day for the gemcitabine alone treated group), and a p-value (significance level: 5%) was calculated. Based on the p-value, asterisks are indicated in Figure 30 (n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). Furthermore, a Williams test was performed to determine whether there was a difference in tumor volume between the group administered gemcitabine alone and the group administered gemcitabine and Compound 1 in combination at each dose, with a significance level of α=0.025. Cases where the combined effect was determined to be significant are indicated by † in Figure 30.

[0287] (4) Efficacy Test of the Combination of Compound 2 and Gemcitabine in ES-2 Tumor-Bearing Mice To investigate the combined effect of a MYT1 inhibitor and an anticancer drug on cancers positive for the expression of hyperphosphorylated RB1 protein in vivo (cell lines determined to be positive for the expression of hyperphosphorylated RB1 protein as described above), mice transplanted with ES-2 cells were administered gemcitabine alone, compound 2 alone, or a combination of gemcitabine and compound 2. After culturing ES-2 cells in vitro, the cells were collected on the day of transplantation into mice and diluted to 2.5 × 10 cells in Hanks' Balanced Salt solution (Sigma-Aldrich, H9269). ７ The prepared cell suspension was transferred to BALB / c-nu / nu mice (CAnN.Cg-Foxn1 <nu>0.2 mL of each solution was implanted into the groin of a mouse (Jackson Laboratory Japan, Inc.).

[0288] Next, to mice (ES-2 tumor-bearing mice) in which tumor (cancer cell) engraftment was confirmed, Compound 2 suspended in a mixture of 10% DMSO (Wako Pure Chemical Industries, Ltd., 043-07216), 10% Cremophor (Sigma-Aldrich, C5135), 15% polyethylene glycol 400 (Wako Pure Chemical Industries, Ltd., 161-09065), and 15% hydroxypropyl-β-cyclodextrin (Nihon Shokuhin Kako Co., Ltd., 7585-39-9) was orally administered once daily at 10 mg / kg for 14 days, or gemcitabine solution (Nippon Kayaku Co., Ltd.) diluted with saline was intraperitoneally administered at 60 mg / kg once every seven days, a total of two times, or a combination of these was administered. In the combined administration group, Compound 1 suspended in the mixed solution was orally administered, and gemcitabine solution diluted with saline was intraperitoneally administered in the same manner as above. Four tumor-bearing mice were used in each group.

[0289] Administration of various drugs began 10 days after transplantation, and tumor volumes were measured until day 24. The short and long diameters of the tumor were measured using an electronic caliper (Mitutoyo, CD-15AX), and the tumor volumes were calculated by the least squares method. Tumor volumes were also measured in the same way for ES-2 tumor-bearing mice that were not administered drugs (non-drug administration group). In each group, tumor volumes were measured until the tumor volume reached 1,400 mm. ３ When any of the above conditions occurred, the administration to the mice in the relevant group was discontinued and the mice were euthanized.

[0290] The time course of tumor volume after ES-2 transplantation in the drug-untreated group, the compound 2 alone treated group, the gemcitabine alone treated group, and the combination treated group is shown in Figure 34. A two-tailed Student's t-test was performed to determine whether there was a difference in tumor volume between the compound 2 alone treated group and the combination treated group on day 17 after transplantation (the final day of administration for the compound 2 alone treated group), and a p-value (significance level: 5%) was calculated. If a significant difference was observed, a two-tailed Student's t-test was performed to determine whether there was a difference in tumor volume between the gemcitabine alone treated group and the combination treated group on day 24 after transplantation (the day after the final day of administration for the gemcitabine alone treated group), and a p-value (significance level: 5%) was calculated. Asterisks are indicated in Figure 34 based on the p-values ​​(n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0291] 2. Results (1) Efficacy test of combined use of gemcitabine and Compound 1 in DU4475 tumor-bearing mice As shown in Figure 31, in vivo efficacy tests using DU4475, which lacks or reduces RB1 function, showed that the DU4475 model does not show significant efficacy when administered with gemcitabine.

[0292] Furthermore, the tumor volume in the combination treatment group was significantly smaller than that in the group administered Compound 1 alone, and was also significantly smaller than that in the group administered gemcitabine alone. Furthermore, no euthanasia procedures were required in either the single-drug or combination-administered groups due to Compound 1 administration.

[0293] These results suggest that even in models in which gemcitabine does not show significant efficacy, the combination of gemcitabine and compound 1 at 2.5 mg / kg exerts a significantly strong synergistic combined antitumor effect. Furthermore, tolerability was confirmed in both the group administered compound 1 alone and the group administered compound 1 in combination with gemcitabine, and no serious weight loss was observed, suggesting that the combination therapy of gemcitabine and compound 1 is a treatment with a therapeutic range for cancers in which RB1 function is deleted or reduced.

[0294] (2) Efficacy test of combined use of gemcitabine and Compound 1 in NIH:OVCAR-3 tumor-bearing mice As shown in Figure 32, in an in vivo efficacy test using NIH:OVCAR-3 mice with RB1 gene mutations and loss or reduced RB1 function, the group administered gemcitabine alone had significantly smaller tumor volumes than the group not administered the drug. This suggests that NIH:OVCAR-3 cells are a model that demonstrates the efficacy of gemcitabine administration.

[0295] Furthermore, the group administered 100 mg / kg of gemcitabine and Compound 1 in combination had a significantly smaller tumor volume than the group administered 100 mg / kg of Compound 1 alone, and the group administered 100 mg / kg of gemcitabine and Compound 1 in combination had a significantly smaller tumor volume than the group administered gemcitabine alone. Furthermore, no euthanasia procedures were required due to administration of Compound 1 in either the single-drug or combined-dose groups.

[0296] Furthermore, a Williams test was performed to determine whether there was a difference in tumor volume between the group administered gemcitabine alone and the groups administered gemcitabine and Compound 1 in combination at each dose, with a significance level of α = 0.025.The group administered gemcitabine and Compound 1 in combination at 3 mg / kg or more had a significantly smaller tumor volume than the group administered gemcitabine alone.

[0297] These findings, along with the lack of significant efficacy in the group administered compound 1 at 100 mg / kg alone, suggest that even in models in which gemcitabine is effective, the combination of gemcitabine and compound 1 at 3 mg / kg exerts a significantly strong synergistic combined antitumor effect. Furthermore, the combined antitumor effect was enhanced in a dose-dependent manner by compound 1, and tolerability was confirmed in both the group administered compound 1 at 100 mg / kg alone and in combination with gemcitabine, without causing serious weight loss. These findings suggest that the combination therapy of gemcitabine and compound 1 is a treatment method with a wide therapeutic range for cancers in which RB1 function is deleted or reduced.

[0298] (3) Efficacy test of combined use of gemcitabine and Compound 1 in ES-2 tumor-bearing mice As shown in Figure 33, in an in vivo efficacy test using ES-2 tumor-bearing mice positive for hyperphosphorylated RB1 expression, the group administered 100 mg / kg of gemcitabine and Compound 1 in combination had significantly smaller tumor volumes than the group administered 100 mg / kg of Compound 1 alone, and the group administered 100 mg / kg of gemcitabine and Compound 1 in combination had significantly smaller tumor volumes than the group administered gemcitabine alone. Furthermore, no euthanasia procedures were performed following administration of Compound 1 in either the single-drug or combined-dose groups.

[0299] Furthermore, a Williams test was performed to determine whether there was a difference in tumor volume between the group administered gemcitabine alone and the groups administered gemcitabine and Compound 1 in combination at various doses, with a significance level of α = 0.025.The group administered gemcitabine and Compound 1 in combination at 10 mg / kg or more had a significantly smaller tumor volume than the group administered gemcitabine alone.

[0300] These findings, along with the lack of significant efficacy in the group administered compound 1 at 100 mg / kg alone, suggest that even in models in which gemcitabine demonstrates efficacy, the combination of gemcitabine and compound 1 at 10 mg / kg exerts a significantly strong synergistic combined antitumor effect. Furthermore, the combined antitumor effect was enhanced in a dose-dependent manner by compound 1, starting with the group administered compound 1 at 3 mg / kg. Compound 1 was well tolerated in both the group administered compound 1 at 100 mg / kg alone and in combination with gemcitabine, and was not accompanied by significant weight loss. This suggests that the combination therapy of gemcitabine and compound 1 is a treatment method with a wide therapeutic window for cancers in which RB1 function is lost or reduced.

[0301] (4) Efficacy test of the combination of Compound 2 and gemcitabine in ES-2 tumor-bearing mice. As shown in Figure 34, in an in vivo antitumor test using ES-2, a cancer cell line positive for the expression of hyperphosphorylated RB1 protein, the combination administration group had significantly smaller tumor volumes than the group administered with Compound 2 alone. Furthermore, the combination administration group had significantly smaller tumor volumes than the group administered with gemcitabine alone. Furthermore, no euthanasia procedures were performed following administration of Compound 2 in either the single-drug administration group or the combination administration group. These findings demonstrate that a synergistic therapeutic effect can be expected by combining Compound 2 and gemcitabine in cancers positive for the expression of hyperphosphorylated RB1 protein.

[0302] The above (1) to (4) suggest that in cancers with RB1 gene deletion or mutation or cancers with RB1 reduced function or deletion that are positive for hyperphosphorylated RB1 expression, the combination therapy of gemcitabine with Compound 1 and Compound 2 is a treatment method with a wide therapeutic range, regardless of the efficacy of gemcitabine against the cancer.

[0303] Example 6: In vivo combined efficacy test of carboplatin and compound 1 in RB1-deficient or -deficient cancer 1. Experimental materials and methods Combined efficacy test of carboplatin and compound 1 in Colo-824 tumor-bearing mice To investigate the combined effect of a MYT1 inhibitor and an anticancer drug on RB1-deficient cancer in vivo, mice transplanted with Colo-824 were administered carboplatin alone, compound 1 alone, or a combination of the two. After culturing Colo-824 in vitro, the cells were collected on the day of transplantation into mice, and 2.5 x 10 cells were placed in a Matrigel (Corning, 356234) solution (final concentration 50%) diluted with Hanks' Balanced Salt solution (Sigma-Aldrich, H9269). ７ The prepared cell suspension was transferred to BALB / c-nu / nu mice (CAnN.Cg-Foxn1 <nu>0.2 mL of each solution was implanted into the groin of a mouse (CrlCrlj, manufactured by Charles River).

[0304] Next, mice (Colo-824 tumor-bearing mice) in which tumor (cancer cell) engraftment was confirmed were orally administered Compound 1 suspended in a mixture of 10% DMSO (Wako Pure Chemical Industries, Ltd., 043-07216), 10% Cremophor (Sigma-Aldrich, C5135), 15% polyethylene glycol 400 (Wako Pure Chemical Industries, Ltd., 161-09065), and 15% hydroxypropyl-β-cyclodextrin (Nihon Shokuhin Kako Co., Ltd., 7585-39-9) once daily at 30 mg / kg for 12 days, or carboplatin (Nippon Kayaku Co., Ltd.) diluted with saline was intraperitoneally administered at 20 mg / kg once every 6 days, for a total of two doses, or a combination of these. Four tumor-bearing mice were used for each group.

[0305] Administration of various drugs began 22 days after transplantation, and tumor volume and body weight were measured until day 34. Tumor volume was calculated by measuring the short and long axes of the tumor using an electronic caliper (Mitutoyo, CD-15AX) and calculating the volume using the least squares method. Tumor volume and body weight were also measured in the same way for Colo-824 tumor-bearing mice that had not received drug administration (non-drug administration group). The rate of change in body weight from day 22 was calculated. Mice that showed a weight loss rate of more than 20% were euthanized.

[0306] Figure 35 shows the time course of tumor volume and body weight change rate after Colo-824 transplantation in the drug-untreated group, the compound 1 monotherapy group, the carboplatin monotherapy group, and the combination therapy group. A two-tailed Student's t-test was performed on the tumor volume of each group 34 days after transplantation, and a p-value (significance level: 5%) was calculated. First, a comparison was made to see if there was a difference in tumor volume between the compound 1 monotherapy group and the combination therapy group. If a significant difference was observed, a comparison was also made to see if there was a difference in tumor volume between the carboplatin monotherapy group and the combination therapy group. Asterisks are indicated in the figures based on the p-value (n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001).

[0307] 2. Results Efficacy study of the combination of carboplatin and Compound 1 in Colo-824 tumor-bearing mice As shown in Figure 35, in an in vivo efficacy study using Colo-824 tumor-bearing mice, in which RB1 function is deleted or reduced, the combination treatment group had significantly smaller tumor volumes than the group treated with Compound 1 alone, and the combination treatment group had significantly smaller tumor volumes than the group treated with carboplatin alone. Furthermore, no euthanasia procedures were required after administration of Compound 1 in either the single-drug or combination-treatment groups.

[0308] These findings suggest that the combination of carboplatin and compound 1 exerts a significantly stronger synergistic antitumor effect. Furthermore, the tolerability of both the compound 1 monotherapy group and the carboplatin-combined therapy group was confirmed, and no serious weight loss was observed, suggesting that the combination therapy of carboplatin and compound 1 is an effective treatment with a therapeutic range for cancers in which RB1 function is lost or reduced.

[0309] Example 7: In vivo combined efficacy test of trastuzumab deruxtecan and compound 1 in RB1-deficient or -deficient cancer 1. Experimental materials and methods Combined efficacy test of trastuzumab deruxtecan and compound 1 in Colo-824 tumor-bearing mice To investigate the combined effect of a MYT1 inhibitor and an anticancer drug on RB1-deficient cancer in vivo, trastuzumab deruxtecan alone, compound 1 alone, or a combination of the two were administered to mice transplanted with Colo-824. After culturing Colo-824 in vitro, the cells were harvested on the day of transplantation into mice and 2.5 x 10 cells were added to a Matrigel (Corning, 356234) solution (final concentration 50%) diluted with Hanks' Balanced Salt solution (Sigma-Aldrich, H9269). ７ The prepared cell suspension was transferred to BALB / c-nu / nu mice (CAnN.Cg-Foxn1 <nu>0.2 mL of each solution was implanted into the groin of a mouse (CrlCrlj, manufactured by Charles River).

[0310] Next, mice (DU4475 tumor-bearing mice) in which tumor (cancer cell) engraftment was confirmed were orally administered Compound 1 suspended in a mixture of 10% DMSO (Wako Pure Chemical Industries, Ltd., 043-07216), 10% Cremophor (Sigma-Aldrich, C5135), 15% polyethylene glycol 400 (Wako Pure Chemical Industries, Ltd., 161-09065), and 15% hydroxypropyl-β-cyclodextrin (Nihon Shokuhin Kako Co., Ltd., 7585-39-9) once daily at 30 mg / kg for 12 days, or trastuzumab deruxtecan (Daiichi Sankyo Co., Ltd.) diluted with saline was intravenously administered at 10 mg / kg once 22 days after tumor engraftment, or a combination of these. Four tumor-bearing mice were used for each group.

[0311] Administration of various drugs began 22 days after transplantation, and tumor volume and body weight were measured until day 34. Tumor volume was calculated by measuring the short and long axes of the tumor using an electronic caliper (Mitutoyo, CD-15AX) and calculating the volume using the least squares method. Tumor volume and body weight were also measured in the same way for Colo-824 tumor-bearing mice that had not received drug administration (non-drug administration group). The rate of change in body weight from day 22 was calculated. Mice that showed a weight loss rate of more than 20% were euthanized.

[0312] Figure 33 shows the time course of tumor volume and body weight change rate after Colo-824 transplantation in the non-drug group, the Compound 1 monotherapy group, the trastuzumab deruxtecan monotherapy group, and the combination therapy group. A two-tailed Student's t-test was performed on the tumor volume of each group 34 days after transplantation, and a p-value (significance level: 5%) was calculated. First, the difference in tumor volume between the Compound 1 monotherapy group and the combination therapy group was compared. If a significant difference was observed, the difference in tumor volume between the trastuzumab deruxtecan monotherapy group and the combination therapy group was also compared. Asterisks are added to each figure based on the p-value (n.s.: p≧0.05, *: p<0.05, **: p<0.01, ***: p<0.001, ***: p<0.0001).

[0313] 2. Results Efficacy study of the combination of trastuzumab deruxtecan and Compound 1 in Colo-824 tumor-bearing mice As shown in Figure 36, in an in vivo efficacy study using Colo-824 mice, which lack or reduce RB1 function, the combination treatment group had significantly smaller tumor volumes than the group treated with Compound 1 alone, and the combination treatment group had significantly smaller tumor volumes than the group treated with trastuzumab deruxtecan alone. Furthermore, no euthanasia procedures were required in either the single-drug or combination treatment groups.

[0314] These findings suggest that the combination of trastuzumab deruxtecan with compound 1 exerts a significant...

Claims

1. A pharmaceutical composition comprising a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the said compound or salt thereof, as an active ingredient. 【Chemistry 1】

2. The pharmaceutical composition according to claim 1, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a solvate of the hydrochloride salt of the compound represented by formula (1).

3. The pharmaceutical composition according to claim 1, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a hydrate of the hydrochloride salt of the compound represented by formula (1).

4. The pharmaceutical composition according to claim 1, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a monohydrate of the hydrochloride salt of the compound represented by formula (1).

5. A pharmaceutical composition comprising a chemotherapeutic agent or molecular targeted agent as an active ingredient for treating or preventing cancer, in combination with a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof. 【Chemistry 2】

6. The pharmaceutical composition according to claim 5, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a solvate of the hydrochloride salt of the compound represented by formula (1).

7. The pharmaceutical composition according to claim 5, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a hydrate of the hydrochloride salt of the compound represented by formula (1).

8. The pharmaceutical composition according to claim 5, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a monohydrate of the hydrochloride salt of the compound represented by formula (1).

9. A method for suppressing the proliferation of cancer cells, A method comprising contacting the cancer cells with a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof. 【Transformation 3】

10. The method according to claim 9, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a solvate of the hydrochloride salt of the compound represented by formula (1).

11. The method according to claim 10, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a hydrate of the hydrochloride salt of the compound represented by formula (1).

12. The method according to claim 11, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a monohydrate of the hydrochloride salt of the compound represented by formula (1).

13. A method for improving the response of cancer treatment with chemotherapeutic agents or molecular targeted agents, A method comprising administering to a cancer patient, together with a chemotherapeutic agent or a molecularly targeted agent, a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the said compound or salt thereof. 【Chemistry 4】

14. The method according to claim 13, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a solvate of the hydrochloride salt of the compound represented by formula (1).

15. The method according to claim 13, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a hydrate of the hydrochloride salt of the compound represented by formula (1).

16. The method according to claim 13, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a monohydrate of the hydrochloride salt of the compound represented by formula (1).

17. A method for predicting the response to cancer treatment by a combination of a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, and a chemotherapeutic agent or molecular targeted agent, Determining, or having a third party determine, whether a cancer patient is a cancer patient in whom a positive RB1 gene mutation, decreased expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, A method comprising determining, if a cancer patient is found to be positive for an RB1 gene mutation, decreased expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein, that the cancer patient is responsive to cancer treatment with a combination of a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, and a chemotherapeutic agent or molecular targeted agent. 【Transformation 5】

18. The method according to claim 17, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a solvate of the hydrochloride salt of the compound represented by formula (1).

19. The method according to claim 17, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a hydrate of the hydrochloride salt of the compound represented by formula (1).

20. The method according to claim 17, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a monohydrate of the hydrochloride salt of the compound represented by formula (1).

21. A method for selecting cancer patients for whom administration of a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, in combination with a chemotherapeutic agent or molecular targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecular targeted agent alone, Determining, or having a third party determine, whether a cancer patient is a cancer patient in whom a positive RB1 gene mutation, decreased expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, A method comprising determining, if a cancer patient is a cancer patient in whom a positive RB1 gene mutation, decreased expression of the RB1 gene or protein, or positive expression of hyperphosphorylated RB1 protein has been detected, that the administration of a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or salt thereof, in combination with a chemotherapeutic agent or molecular targeted agent is more effective in treating cancer than administration of a MYT1 inhibitor, a chemotherapeutic agent, or a molecular targeted agent alone. 【Transformation 6】

22. The method according to claim 21, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a solvate of the hydrochloride salt of the compound represented by formula (1).

23. The method according to claim 21, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a hydrate of the hydrochloride salt of the compound represented by formula (1).

24. The method according to claim 21, wherein the compound represented by formula (1) or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of the compound or a salt thereof, is a monohydrate of the hydrochloride salt of the compound represented by formula (1).

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