WO2020246488A1 - COMPOUND STABILIZING COMPLEX CONTAINING NuMA1 AND CKAP5 - Google Patents

COMPOUND STABILIZING COMPLEX CONTAINING NuMA1 AND CKAP5 Download PDF

Info

Publication number
WO2020246488A1
WO2020246488A1 PCT/JP2020/021850 JP2020021850W WO2020246488A1 WO 2020246488 A1 WO2020246488 A1 WO 2020246488A1 JP 2020021850 W JP2020021850 W JP 2020021850W WO 2020246488 A1 WO2020246488 A1 WO 2020246488A1
Authority
WO
WIPO (PCT)
Prior art keywords
numa1
ckap5
compound
complex containing
cancer
Prior art date
Application number
PCT/JP2020/021850
Other languages
French (fr)
Japanese (ja)
Inventor
知樹 石川
靖浩 林
真菜 畠中
大雅 加藤
明日香 合田
清水　弘樹
裕次 小倉
伊藤　雅夫
Original Assignee
第一三共株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 第一三共株式会社 filed Critical 第一三共株式会社
Publication of WO2020246488A1 publication Critical patent/WO2020246488A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/499—Spiro-condensed pyrazines or piperazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/20—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to a compound that stabilizes a complex containing NuMA1 and CKAP5, a method for stabilizing a complex containing NuMA1 and CKAP5, and a method for screening a compound that stabilizes a complex containing NuMA1 and CKAP5.
  • Non-Patent Document 1 Since the 1940s, many anticancer agents such as alkylating agents have been developed (Non-Patent Document 1). However, many of the anticancer drugs currently in use are cytotoxic and exert their effects on all proliferating cells, thus affecting both cancer cells and normal cells. (Non-Patent Document 2).
  • Taxanes, vinca alkaloids, and the like are known and widely used as agents that suppress the growth of cancer cells by inhibiting cell division, but these agents are all used for cell polymerization and depolymerization of microtubules. Since it targets the mechanisms essential for division, it also affects normal cells as described above. In addition, microtubule formation also plays an important role in nerve cells, which are non-dividing cells, and neurotoxicity of these drugs has become a clinical problem (Non-Patent Document 3). However, since suppressing the growth of cancer cells by inhibiting cell division is considered to be an extremely effective treatment method for cancer, there is an anti-cancer drug that has high selectivity for cancer cells while inhibiting cell division. Antineoplastic agents are required.
  • NuMA1 is a protein that is a constituent of the nuclear substrate and at the same time plays an important role in spindle formation and placement during mitosis (Non-Patent Document 4). NuMA1 is known to be highly expressed in some cancer cells. CKAP5 is a protein that binds to microtubules and plays an important role in stabilizing microtubules. Furthermore, it is known that CKAP5 forms a complex with TACC3 and clathrin and acts on cross-linking between microtubules and spindle formation (Non-Patent Document 5).
  • NuMA1 and CKAP5 are both involved in the formation of microtubules and mitotic spindles, but it has not been known how NuMA1 and CKAP5 are related. Furthermore, it was not known how controlling these molecules would affect cancer.
  • the subject of the present invention is a compound capable of hindering the growth of cancer cells highly selectively, a method of hindering the growth of cells highly selectively of cancer cells, and a method of highly selectively hindering the growth of cells of cancer cells. It is to provide a method of screening a compound that can be prevented.
  • NuMA1 a nuclear protein highly expressed in cancer cells
  • NuMA1 contains a complex containing NuMA1 and CKAP5 during cell division (hereinafter referred to as "NuMA1"). It was discovered that it forms a “complex”) and that the NuMA1 complex controls microtubule formation.
  • the present inventors stabilized the binding between the constituent proteins of the NuMA1 complex and suppressed the dissociation or degradation of the NuMA1 complex to form microtubules. It was found that the production of mitotic spindles was suppressed, cell division was inhibited, and cell proliferation was suppressed. Since NuMA1 is known to be highly expressed in cancer cells, targeting the NuMA1 complex can prevent the proliferation of cancer cells in which NuMA1 is highly expressed in a highly selective manner. It is possible. Therefore, the present invention provides a compound that stabilizes the NuMA1 complex, a method for stabilizing the NuMA1 complex, and a method for screening the compound that stabilizes the NuMA1 complex.
  • the present invention relates to the following (1) to (16).
  • the pharmaceutical composition according to (5) which contains a compound that stabilizes a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • a method for stabilizing a complex containing NuMA1 and CKAP5. (10) The method according to (9), wherein the complex containing NuMA1, CKAP5 and ⁇ -tubulin is stabilized.
  • (11) The method according to (9), wherein the complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3 is stabilized.
  • (12) The method according to any one of (9) to (11) used for treating cancer.
  • a method for suppressing the growth of cancer cells which comprises stabilizing a complex containing NuMA1 and CKAP5.
  • the method according to (13), which suppresses the growth of cancer cells which comprises stabilizing a complex containing NuMA1, CKAP5 and ⁇ -tubulin.
  • the method according to (13), which suppresses the growth of cancer cells which comprises stabilizing a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • Another aspect of the present invention relates to the following (17) to (25).
  • Another aspect of the present invention relates to the following (1A) to (52A).
  • (11A) The method according to (9A), wherein the complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3 is stabilized.
  • (12A) The method according to any one of (9A) to (11A) used for treating cancer.
  • (13A) A method for suppressing the growth of cancer cells, which comprises stabilizing a complex containing NuMA1 and CKAP5.
  • (14A) The method according to (13A), which suppresses the growth of cancer cells, which comprises stabilizing a complex containing NuMA1, CKAP5 and ⁇ -tubulin.
  • (15A) The method according to (13A), which suppresses the growth of cancer cells, which comprises stabilizing a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • (21A) A compound that stabilizes a complex containing NuMA1 and CKAP5 for use in the treatment of cancer.
  • (22A) A compound according to (21A) that stabilizes a complex containing NuMA1, CKAP5 and ⁇ -tubulin for use in the treatment of cancer.
  • (23A) A compound according to (21A) that stabilizes a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3 for use in the treatment of cancer.
  • (24A) Use of compounds that stabilize complexes containing NuMA1 and CKAP5 in the manufacture of pharmaceuticals for the treatment of cancer.
  • (25A) The use of a compound that stabilizes a complex containing NuMA1, CKAP5 and ⁇ -tubulin in the manufacture of a medicament for the treatment of cancer, according to the use according to (24A).
  • (26A) The use of a compound that stabilizes a complex, including NuMA1, CKAP5, ⁇ -tubulin and TACC3, in the manufacture of a medicament for the treatment of cancer, according to the use according to (24A).
  • (27A) A compound that regulates the function of a complex containing NuMA1 and CKAP5.
  • (28A) A compound according to (27A) that regulates the function of a complex containing NuMA1, CKAP5 and ⁇ -tubulin.
  • (29A) A compound according to (27A) that regulates the function of a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • (30A) The compound according to any one of (27A) to (29A) used for the treatment of cancer.
  • (31A) A pharmaceutical composition containing a compound that regulates the function of a complex containing NuMA1 and CKAP5.
  • (32A) The pharmaceutical composition according to (31A), which contains a compound that regulates the function of a complex containing NuMA1, CKAP5 and ⁇ -tubulin.
  • (33A) The pharmaceutical composition according to (31A), which contains a compound that regulates the function of a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • 34A The pharmaceutical composition according to any one of (31A) to (33A) used for treating cancer.
  • (35A) A method of coordinating the function of a complex containing NuMA1 and CKAP5.
  • 38A The method according to any one of (35A) to (37A) used for the treatment of cancer.
  • 39A A method for suppressing the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1 and CKAP5.
  • (40A) (39A) the method of suppressing the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1, CKAP5 and ⁇ -tubulin.
  • (41A) (39A), the method of suppressing the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • (42A) The following steps: (A) A step of contacting the compound with the complex containing NuMA1 and CKAP5 (b) A step of confirming whether or not the function of the complex containing NuMA1 and CKAP5 is adjusted by the compound is included. A method for screening a compound that regulates the function of a complex containing.
  • (43A) The following steps: (A) Step of contacting the compound with each of the cells having a high NuMA1 expression level and the cell having a low NuMA1 expression level (b) A step of measuring the cell growth inhibitory action and / or the cell division inhibitory action of the compound in each cell. (C) When the cell growth inhibitory effect in cells with high NuMA1 expression is higher than the cell growth inhibitory effect in cells with low NuMA1 expression, the compound adjusts the function of the complex containing NuMA1 and CKAP5. A method for screening a compound that regulates the function of a complex containing NuMA1 and CKAP5, which comprises the step of identifying the compound to be used.
  • (44A) A method of treating cancer, which comprises regulating the function of a complex containing NuMA1 and CKAP5.
  • (45A) (44A), a method for treating cancer, which comprises adjusting the function of a complex containing NuMA1, CKAP5 and ⁇ -tubulin.
  • (46A) The method of treating cancer according to (44A), which regulates the function of a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • (47A) A compound that regulates the function of a complex containing NuMA1 and CKAP5 for use in the treatment of cancer.
  • (48A) A compound according to (47A) that regulates the function of a complex containing NuMA1, CKAP5 and ⁇ -tubulin for use in the treatment of cancer.
  • (49A) A compound according to (47A) that regulates the function of a complex comprising NuMA1, CKAP5, ⁇ -tubulin and TACC3 for use in the treatment of cancer.
  • (50A) Use of compounds that regulate the function of complexes containing NuMA1 and CKAP5 in the manufacture of pharmaceuticals for the treatment of cancer.
  • 51A The use of a compound according to (50A) that modifies the function of a complex containing NuMA1, CKAP5 and ⁇ -tubulin in the manufacture of a medicament for the treatment of cancer.
  • (52A) Use of a compound according to (50A) that regulates the function of a complex comprising NuMA1, CKAP5, ⁇ -tubulin and TACC3 in the manufacture of a medicament for the treatment of cancer.
  • a compound capable of hindering the growth of cancer cells highly selectively a method of hindering the growth of cancer cells highly selectively, and a method of hindering the growth of cells highly selectively of cancer cells.
  • the vertical axis shows the rate of increase in binding with NuMA1 by Ispinesib treatment
  • the horizontal axis shows the rate of increase in binding with NuMA1 by Compound C treatment. It is a figure which showed the binding between NuMA1, CKAP5, and TACC3 by Western blotting when treated with Compound B or Eg5 inhibitor Ispinesib. It is a figure showing the intracellular localization of NuMA1 and CKAP5 when treated with Compound B or the Eg5 inhibitor Ispinesib. Monopolar spindle formation was observed in both Compound B and Ispine sib treatment, but NuMA1 was localized in a ring shape around the centrosome and CKAP5 was not clearly localized in the Ispine sib treatment.
  • NuMA1 and CKAP5 were strongly co-localized near the centrosome by Compound B treatment. It is a figure which showed the influence which the knockdown of NuMA1, TACC3, and CKAP5 has on the binding of Compound A and ⁇ -tubulin.
  • the vertical axis shows the binding ratio of Compound A and ⁇ -tubulin to the UV non-irradiated sample, and the horizontal axis shows the processing content of each sample. It is the figure which showed the elongation of the microtubule when treated with Compound B or the Eg5 inhibitor Ispinesib by immunostaining of EB1.
  • Compound B treatment markedly reduced the signal of EB1 and suppressed microtubule elongation from the centrosome.
  • the NuMA1 complex is formed near the centrosome during cell division by experiments using various compounds (experimental results are shown in FIGS. 4, 6, 7 and 9), and the NuMA1 complex. It was found that microtubule formation was regulated by (experimental results are shown in FIGS. 8 and 10), and that stabilization of the NuMA1 complex suppressed cell proliferation.
  • cell proliferation can be suppressed by stabilizing the NuMA1 complex.
  • NuMA1 is a protein coded by the NUMA1 gene and is also called nuclear mitotic apparatus protein 1.
  • NUMA1 is registered in NCBI as Gene ID: 4926, RefSeq; NM_001286561.1 and NM_006185.3 (Protein: RefSeq; NP_001273490.1 and NP_006176.2).
  • RefSeq As a function of NuMA1, it is known that it binds to microtubules during cell division and is involved in spindle formation and chromosome sequence distribution.
  • CKAP5 is a protein coded by the CKAP5 gene and is also called cytoskeleton associated protein5.
  • CKAP5 is registered with NCBI as Gene ID: 9973, RefSeq; NM_001008938.3 and NM_014756.3 (protein: RefSeq; NP_001008938.1 and NP_055571.2).
  • Gene ID 9973, RefSeq; NM_001008938.3 and NM_014756.3
  • protein RefSeq; NP_001008938.1 and NP_055571.2
  • TACC3 is a protein coded by the TACC3 gene, and is also called transforming acidic coiled-coil containing protein 3.
  • TACC3 is registered in NCBI as Gene ID: 10460, RefSeq; NM_006342.3 (protein: RefSeq; NP_006333.1).
  • ⁇ -tubulin refers to proteins of the tubulin beta family in general, specifically, TUBB, TUBA1B, TUBB1, TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB5, TUBB6, TUBB8, etc. It is a protein coded by the gene of. As a function of ⁇ -tubulin, it is known that it forms microtubules and controls cell division and intracellular transport.
  • clathrin is a protein coded by the CLTC gene and is also called clathrin heavy chain 1.
  • CLTC is registered in NCBI as Gene ID: 1213, RefSeq; NM_004859.4 (protein: RefSeq; NP_004850.1).
  • clathrin it is known that it forms a complex with CKAP5 and TACC3 and stabilizes the mitotic spindle by cross-linking between microtubules.
  • the "NuMA1 complex” means a complex containing NuMA1 and CKAP5.
  • complexes containing other proteins are also included in the NuMA1 complex of the present invention.
  • it is a complex containing NuMA1, CKAP5 and ⁇ -tubulin. More preferably, it is a complex containing NuMA1, CKAP5, ⁇ -tubulin and TACC3.
  • stabilization of the NuMA1 complex means suppressing dissociation or decomposition of the NuMA1 complex.
  • abnormal localization of CKAP5 is caused, inhibiting the elongation of microtubules extending from the centrosome and suppressing cell division.
  • Whether or not the NuMA1 complex is stabilized can be confirmed by, for example, whether or not formation of monopolar spindle microtubules is observed, and whether or not NuMA1 and CKAP5 are co-localized.
  • the compound binds to the complex after NuMA1 and CKAP5 form a complex to stabilize the NuMA1 complex, and CAKP5 combines with NuMA1 after the compound binds to NuMA1. It includes both forming a body and stabilizing the NuMA1 complex.
  • adjusting the function of the NuMA1 complex means reducing the function of elongating microtubules, which is the function of the NuMA1 complex.
  • regulating the function of the NuMa1 complex cell division is suppressed. In other words, it controls the function of the NuMA1 complex.
  • NuMA1 is highly expressed in cancer cells, for example, prostate cancer, neuroblastoma, small cell lung cancer, adrenal cancer, colon cancer, gastric cancer, head and neck cancer, esophageal cancer, non-small cell lung cancer. , Cervical cancer, uterine body cancer, anal cancer, bladder cancer, breast cancer, brain tumor, ovarian cancer, pancreatic cancer, melanoma, thyroid cancer, soft sarcoma, leukemia, malignant lymphoma, etc. It is known that It is also known that NuMA1 is highly expressed in cancers with gene amplification at the chromosomal site of 11q13 where the NuMA1 gene is present.
  • NuMA1 can be confirmed by a known method, for example, immunohistochemical staining or RNA-Seq analysis by next-generation sequence.
  • gene amplification can be confirmed by a method such as the FISH method.
  • Compound A means (-)-11- (4-ethynyl-2,6-difluorobenzoyl)-14-[(4-methoxyphenyl) methyl] -1,2,7,11. , 14-Pentaazatrispyro [2.2.2.4 9 . 26 . A 2 3] Heputadeku 1-ene -8,15- dione.
  • Compound B refers to 5-[(4-chloro-3-fluorophenyl) methyl] -2- [7- (1,1-difluoroethyl) quinazoline-4-yl] -10, 10-Difluoro-2,5,13-Triazadispiro [3.2.5 7 . It is a 2 4] tetradecane -6,14- dione.
  • Compound C means (-)-2- (4-chloro-2,6-difluorobenzoyl) -6-[(4-chloro-3-fluorophenyl) methyl] -11,11. -Difluoro-2,6,14-Triazadispiro [4.2.5 8 . 25 ] Pentadecane-7,15-dione.
  • Compound D refers to 2- (4-chloro-2,6-difluorobenzoyl) -11,11-difluoro-6-[(4-methoxyphenyl) methyl] -2,6,14. -Triazadispiro [4.2.5 8 . 25 ] Pentadecane-7,15-dione.
  • Compound E means (5R, 8R) -2- (2-amino-6-fluoro-4-methylbenzoyl) -8-cyclohexyl-6-[(4-methoxyphenyl) methyl]. -2,6,9-Triazaspiro [4.5] Decane-7,10-dione.
  • Ispinesib means N- (3-aminopropyl) -N-[(1R) -1- [7-chloro-4-oxo-3- (phenylmethyl) -2-quinazolinyl]-. 2-Methylpropyl] -4-methylbenzamide. Ispinesib can be produced by the method described in Bioorganic & Medicinal Chemistry 2013, 21, 496-507. Ispinesib is a drug that is classified as an Eg5 inhibitor.
  • Volasertib means N- [trans-4- [4- (cyclopropylmethyl) -1-piperazinyl] cyclohexyl] -4-[[(7R) -7-ethyl-5,6). 7,8-Tetrahydro-5-methyl-8- (1-methylethyl) -6-oxo-2-pteridinyl] amino] -3-methoxybenzamide.
  • Volasertib can be produced, for example, by the method described in WO2007090844.
  • Volasertib is a drug classified as a Plk1 inhibitor.
  • Alisertib refers to 4-[[9-chloro-7- (2-fluoro-6-methoxyphenyl) -5H-pyrimid [5,4-d] [2] benzazepin-2-yl. ] Amino] -2-methoxybenzoic acid.
  • Alisertib can be produced, for example, by the method described in ACS Medicinal Chemistry Letters 2015, 6, 630-634. Alisertib is a drug classified as an Aurora A kinase inhibitor.
  • the "Eg5 inhibitor” is a compound that inhibits the ATPase motor activity of Eg5, which is a protein coded by the KIF11 (kinesin family member 11) gene. Eg5 inhibitors inhibit centrosome separation and arrest the cell cycle in the M phase, but do not inhibit microtubule function.
  • the "Plk1 inhibitor” is a compound that inhibits the kinase activity of Plk1, which is a protein coded by the PLK1 (pololike kinase 1) gene. Plk1 inhibitors have the effect of arresting the cell cycle in the M phase.
  • Aurora A kinase inhibitor is a compound that inhibits the kinase activity of Aurora A, which is a protein coded by the AURKA (aurora kinase A) gene. Aurora A kinase inhibitors have the effect of arresting the cell cycle in the M phase.
  • Vincristine is an anticancer agent having an inhibitory effect on microtubule polymerization. It has the effect of stopping the cell cycle in the M phase.
  • Paclitaxel is an anticancer agent having an inhibitory effect on microtubule depolymerization. It has the effect of stopping the cell cycle in the M phase.
  • cancer means all malignant tumors.
  • Cancer can be classified into “solid cancer” and "blood cancer”. Solid tumors can be classified into “epithelial cell carcinoma” and “non-epithelial cell carcinoma”.
  • Epithelial cell carcinoma is a cancer that begins in epithelial cells, such as lung cancer, stomach cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, colon cancer, breast cancer, neuroblastoma, and adrenal cancer. , Head and neck cancer, esophageal cancer, cervical cancer, uterine body cancer, anal cancer, bladder cancer, brain tumor, ovarian cancer, melanoma, thyroid cancer and the like.
  • Non-epithelial cell carcinoma is a cancer that develops from non-epithelial cells such as bone and muscle, and examples thereof include soft tissue sarcoma, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma.
  • Hematological cancer is a cancer that develops from a hematopoietic organ and can be classified into, for example, malignant lymphoma, leukemia, multiple myeloma and the like.
  • a preferred embodiment of the present invention is a method of stabilizing the NuMA1 complex. By stabilizing the NuMA1 complex, cell division is suppressed and cell proliferation is suppressed.
  • the method for stabilizing the NuMA1 complex is not particularly limited, and for example, low molecular weight compounds, peptides and the like can be used.
  • the method for stabilizing the NuMA1 complex in addition to NuMA1 and CKAP5, it may also act on other proteins forming the complex to stabilize the NuMA1 complex.
  • the method of the present invention is preferably used for cells in which NuMA1 is highly expressed, and particularly preferably for cancer cells.
  • Cancers with high expression of NuMA1 include prostate cancer, neuroblastoma, small cell lung cancer, adrenal cancer, colon cancer, gastric cancer, head and neck cancer, esophageal cancer, non-small cell lung cancer, and cervix.
  • Cancer, uterine body cancer, anal cancer, bladder cancer, breast cancer, brain tumor, ovarian cancer, pancreatic cancer, melanoma, thyroid cancer, soft sarcoma, leukemia, malignant lymphoma and the like are known.
  • Another preferred embodiment of the present invention is a compound that stabilizes the NuMA1 complex.
  • the compound that stabilizes the NuMA1 complex of the present invention is not particularly limited as long as it is a substance that can stabilize the NuMA1 complex, and is, for example, a low molecular weight compound, a peptide, or the like.
  • the substance when the substance stabilizes the NuMA1 complex, it may act on NuMA1 and CKAP5 as well as other proteins forming the complex at the same time to stabilize the NuMA1 complex.
  • the compound that stabilizes the NuMA1 complex of the present invention is a substance that induces a monopolar spindle.
  • the compound that stabilizes the NuMA1 complex of the present invention is preferably used for the treatment of diseases in which NuMA1 is highly expressed, and particularly preferably for the treatment of cancers in which NuMA1 is highly expressed.
  • the compound that stabilizes the NuMA1 complex of the present invention can be administered to a patient either orally or parenterally.
  • Parenteral administration includes, for example, intravenous administration, arterial administration, intramuscular administration, intrathoracic administration, intraperitoneal administration, direct administration to a target site (for example, cancer), and the like.
  • the dose is not particularly limited as long as it is an effective amount for treating the target disease, and may be appropriately selected depending on the age, weight, symptom, health condition, progress of the disease, and the like of the patient.
  • the frequency of administration is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the daily dose may be administered once a day or divided into a plurality of doses. You may.
  • the dose range of the active ingredient is usually from about 0.01 mg / kg body weight to about 500 mg / kg body weight, preferably from about 0.1 mg / kg body weight per day. It is about 100 mg / kg body weight.
  • it is preferably administered once a day or in 2 to 4 divided doses, and repeated at appropriate intervals.
  • Another preferred embodiment of the present invention is a method for screening a compound that stabilizes the NuMA1 complex.
  • Examples of the method for screening the compound that stabilizes the NuMA1 complex include a screening method consisting of the following two steps. (A) A step of contacting the test substance with the complex containing NuMA1 and CKAP5, and (b) a step of confirming whether or not the complex containing NuMA1 and CKAP5 is stabilized by the test substance.
  • the NuMA1 protein and CKAP5 protein used in the above screening method can be either full length or partial length. In addition to NuMA1 and CKAP5, it is preferable to mix tubulin dimers or microtubules at the same time.
  • Another embodiment of the method for screening a compound that stabilizes the NuMA1 complex includes, for example, a screening method consisting of the following three steps.
  • a step of contacting a test substance with cells having a high NuMA1 expression level and a cell having a low NuMA1 expression level and (b) measuring the cell growth inhibitory effect and / or the cell division inhibitory effect of the compound in each cell.
  • Another preferred embodiment of the present invention is a method for treating cancer in which NuMA1 is highly expressed, using a compound that stabilizes the NuMA1 complex.
  • the elution in the column chromatography of the example was carried out under observation by TLC (Thin Layer Chromatography, thin layer chromatography).
  • TLC Thin Layer Chromatography, thin layer chromatography
  • TLC pre - UV and solvent used as an elution solvent as a detection method - bets as Merck (Merck) manufactured by silica gel 60F 254 or silica gel 60NH 2 F 254 S
  • column chromatography as a developing solvent A detector was adopted.
  • silica gel for the column silica gel SK-85 (230-400 mesh) also manufactured by Merck & Co. Ltd. or Fuji Silysia Chemical Chromatolex NH (200-350 mesh) was used.
  • Step 1 Benzyl 1-[(tert-butoxycarbonyl) amino] -4-oxocyclohexane-1-carboxylate 1-[(tert-butoxycarbonyl) amino] -4-oxocyclohexane-1-carboxylic acid ( In a solution of 10.0 g, 38.9 mmol) in dichloromethane (100 ml), ice-cooled, under a nitrogen stream, benzyl alcohol (5.20 ml, 50.6 mmol), 1-ethyl-3- (3-dimethylaminopropyl).
  • Step 2 benzyl 6-[(tert-butoxycarbonyl) amino] -1,4-diazaspiro [2.5] octa-1-encarboxylate
  • Hydroxylamine-O-sulfonic acid (5.05 g) was added to a 3 mmol) metanol (225 ml) solution under ice-cooled and nitrogen stream, and a 2 mol / l ammonia-methanol solution (675 ml) was added and stirred for 2 hours.
  • 44.7 mmol) of a methanol solution (45 ml) was added, and the mixture was stirred at room temperature for 16 hours.
  • Step 3 6-[(tert-butoxycarbonyl) amino] -1,4-diazaspiro [2.5] octa-1-encarboxylic acid
  • Lithium hydroxide monohydrate (1.40 g, 33.4 mmol) was added to a mixed solution of tetrahydrofuran (80 ml), methanol (50 ml) and water (10 ml), and the mixture was stirred for 3 days.
  • the reaction mixture was washed with diethyl ether, the aqueous layer was acidified with 1N aqueous hydrochloric acid solution, diluted with dichloromethane, and the organic layer was washed with 10% aqueous citric acid solution and water.
  • the mixture was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure.
  • reaction solvent was distilled off under reduced pressure, and the mixture was dried under reduced pressure.
  • the residue was powdered with a spartel, tetrahydrofuran (50 ml) was added, triethylamine (10.0 ml, 69.3 mmol) was added under ice-cooling, and the mixture was stirred under a nitrogen stream at an outside temperature of 50 ° C. for 4 hours.
  • di-tert-butyl dicarbonate (4.00 g, 18.3 mmol) was added, and the mixture was stirred at room temperature for 16 hours.
  • the reaction mixture was diluted with dichloromethane and washed with 10% aqueous citric acid solution and water.
  • Step 5 14-[(4-Methoxyphenyl) methyl] -1,2,7,11,14-pentaazatrispyro [2.2.2.4 9 . 26 . 2 3] Heputadeku 1-ene -8,15- dione hydrochloride compound obtained in the above Step 4 (712 mg, 1.47 mmol) in tetrahydrofuran (18 ml) was added under ice-cooling, 4 N hydrochloric acid / 1,4 -Dioxane solution (18 ml) was added and stirred at room temperature for 5 hours. Diethyl ether was added to the reaction mixture, and the mixture was stirred.
  • Step 7 4-ethynyl-2,6-difluorobenzoic acid
  • the compound (1.26 g, 4.71 mmol) obtained in the above step 6 was dissolved in methanol (50 ml), and potassium carbonate (846 mg, 846 mg,) was dissolved at room temperature.
  • An aqueous solution (8 ml) of 6.12 mmol) was added, and the mixture was stirred for 2 hours. Water was added to the reaction mixture, and the metalnol was distilled off under reduced pressure. The obtained aqueous layer was washed with dichloromethane, and then cooled with ice to adjust to acidity by adding a 1N hydrochloric acid aqueous solution.
  • Step 8 11- (4-ethynyl-2,6-difluorobenzoyl) -14-[(4-methoxyphenyl) methyl] -1,2,7,11,14-pentaazatrispyro [2.2. 2.4 9 . 26 .
  • Step 2 1- (tert-Butyloxycarbonyl) -3- ⁇ [(4-chloro-3-fluorophenyl) methyl] amino ⁇ azetidine-3-carboxylic acid
  • the compound obtained in the above step 1 (36.3 g, 0.107 mol) was suspended in dimethyl sulfoxide (350 ml), and hydrogen peroxide solution (17.6 ml, 0.213 mol) was added.
  • Potassium carbonate (44.5 g, 0.320 mol) was added under ice-cooling, and the mixture was stirred for 24 hours while raising the temperature to room temperature. Water was slowly added to the reaction mixture, the mixture was extracted with ethyl acetate, and washed with water and saturated brine.
  • the solvent was distilled off under reduced pressure, the residue obtained by drying was suspended in etanol (600 ml), 8N aqueous potassium hydroxide solution (57.5 ml, 0.460 mol) was added, and the temperature was 80 ° C. for 16 hours. Stirred. After distilling off the solvent under reduced pressure, the mixture was diluted with water and neutralized by adding a 2N hydrochloric acid aqueous solution (230 ml, 0.460 mol) under ice-cooling. The resulting solid was collected by filtration to give the title compound (34.6 g, 96.4 mmol, 90%).
  • Step 4 1-Amino-4,4-difluorocyclohexane-1-carboxylic acid
  • the compound (44.3 g, 0.217 mol) obtained in the above step 3 was added to an 8N aqueous sodium hydroxide solution (271 ml, 2.17 mol).
  • the mixture was suspended in, heated to 120 ° C., and stirred for 28 hours.
  • the reaction mixture was ice-cooled, neutralized by adding 5N aqueous hydrochloric acid solution (434 ml, 2.17 mol), and stirred for a while.
  • the resulting solid was collected by filtration to give the title compound (30.8 g, 0.172 mol, 79%).
  • Step 5 4,4-Difluoro-1- (2,2,2-trifluoroacetamide) cyclohexane-1-carboxylic acid
  • the compound (15.0 g, 83.7 mmol) obtained in the above step 4 and potassium methoxyde. (6.46 g, 92.1 mmol) was suspended in methanol (20 ml) and stirred at 50 ° C. for 30 minutes. After allowing to cool to room temperature, ethyl trifluoroacetate (20.0 ml, 167 mmol) was added, and the mixture was stirred again at 50 ° C. for 6 hours. After allowing to cool to room temperature, the solvent was distilled off under reduced pressure.
  • the compound (17.1 g, 47.7 mmol) obtained in the above step 2 was placed in another flask and suspended in N, N-dimethylformamide (500 ml). Under ice-cooling, N, N-diisopropylethylamine (16.3 ml, 95.3 mmol) was added, and then a solution of the previously prepared acid chloride in N, N-dimethylformamide (100 ml) was added dropwise. After stirring at room temperature for 14 hours, 1,1'-carbonyldiimidazole (23.2 g, 143 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 10 minutes and at 80 ° C. for 8 hours.
  • Step 7 5-[(4-Chloro-3-fluorophenyl) methyl] -10,10-difluoro-2,5,13-triazadispiro [3.2.5 7 . 2 4] tetradecane -6,14- dione hydrochloride
  • dichloromethane 250 ml
  • 4 N hydrochloric acid / 1,4 Dioxane solution 125 ml
  • the solvent was evaporated under reduced pressure, diethyl ether was added, and the mixture was stirred for a while.
  • Step 9 4- (1,1-difluoroethyl) -2-nitrobenzonitrile
  • the compound (14.9 g, 67.4 mmol) obtained in the above step 8 was dissolved in N-methyl-2-pyrrolidinone (55 ml).
  • copper (I) cyanide (12.3 g, 135 mmol) was added, and the mixture was heated and stirred at 160 ° C. for 18 hours.
  • the reaction mixture was diluted with ethyl acetate, saturated aqueous ammonium chloride solution was added, and the mixture was stirred.
  • the resulting insoluble material was filtered through Celite, extracted with ethyl acetate, and washed with water and saturated brine.
  • Step 10 4- (1,1-difluoroethyl) -2-nitrobenzamide Peroxidation of the compound (9.43 g, 44.5 mmol) obtained in the above step 9 in a solution of dimethyl sulfoxide (115 ml) while cooling. Hydrogen peroxide water (7.32 ml, 88.9 mmol) was added, and then potassium carbonate (18.4 g, 133 mmol) was added, and the mixture was stirred at room temperature for 18 hours. Water and saturated brine were added to the reaction mixture, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and saturated brine.
  • Step 12 7- (1,1-difluoroethyl) quinazoline-4 (3H) -one
  • the compound (2.67 g, 13.3 mmol) obtained in the above step 11 and form amidine acetate (4.17 g, 40.0 mmol) was dissolved in etanol (80 ml), and the mixture was heated under reflux for 5 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. Water was added to the obtained residue, and after ultrasonic treatment, the mixture was stirred at room temperature for a while. The solid was collected by filtration to give the title compound (2.59 g, 12.3 mmol, 93%).
  • Step 13 4-Chloro-7- (1,1-difluoroethyl) quinazoline
  • the compound (2.59 g, 12.3 mmol) obtained in the above step 12 was suspended in toluene (55 ml), and N , N-diisopropylethylamine (6.02 ml, 34.6 mmol) and phosphorus oxychloride (5.69 g, 37.0 mmol) were added, and the mixture was heated to 100 ° C. and stirred for 4 hours. After returning to room temperature, it was diluted with ethyl acetate.
  • Step 14 5-[(4-Chloro-3-fluorophenyl) methyl] -2- [7- (1,1-difluoroethyl) quinazoline-4-yl] -10,10-difluoro-2,5 13-Triazadispiro [3.2.5 7 .
  • Step 1 4,4-Difluoro-1- ⁇ [(2,2,2-trichloroethoxy) carbonyl] amino ⁇ cyclohexane-1-carboxylic acid
  • the compound obtained by the method of Step 3 of Example 2 (37. (0 g, 181 mmol) was suspended in an 8N aqueous potassium hydroxide solution (200 ml), heated to 120 ° C., and stirred for 18 hours.
  • the reaction mixture was ice-cooled and neutralized by adding a 5N hydrochloric acid aqueous solution (320 ml) to obtain a suspension.
  • Step 4 6-[(4-Chloro-3-fluorophenyl) methyl] -11,11-difluoro-2,6,14-triazadispiro [4.2.5 8 . 25 ]
  • Pentadecane-7,15-dione hydrochloride The compound (5.70 g, 11.0 mmol) obtained in step 3 above was suspended in 1,4-dioxane (50 ml), and under ice-cooling, 4N hydrochloric acid / A 1,4-dioxane solution (25 ml) was added, and the mixture was stirred at room temperature for 14 hours. The solvent was evaporated under reduced pressure, diethyl ether was added, and the mixture was stirred for a while.
  • Zinc (1.46 g, 22.3 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 3 hours.
  • the insoluble material was filtered through Celite and washed with tetrahydrofuran.
  • the reduced pressure filtrate was concentrated, toluene was added, and the mixture was azeotropically heated.
  • the obtained residue was dissolved in toluene (40 ml), acetic acid (2 ml) was added, and the mixture was stirred at 80 ° C. for 1 hour.
  • Step 5 (5R, 8R) -2- (2-amino-6-fluoro-4-methylbenzoyl) -8-cyclohexyl-6-[(4-methoxyphenyl) methyl] -2,6,9-triazaspiro [4.5]
  • Decane-7,10-Zeon (Compound E) The compound (50.0 mg, 0.123 mmol) obtained in the above step 4 was dissolved in N, N-dimethylformamide (3 ml), triethylamine (34.2 ⁇ l, 0.245 mmol) was added, and the mixture was stirred.
  • 2-amino-6-fluoro-4-methylbenzoic acid (27.0 mg, 0.159 mmol), N, N-diisopropylethylamine (42.7 ⁇ l, 0.245 mmol), N- [1- (cyano) -2-ethoxy-2-oxoethylideneaminooxy) dimethylamino (morpholino)] uronium hexafluorophosphate (57.7 mg, 0.135 mmol) was added, and the mixture was stirred at room temperature for 72 hours. Water was added to the reaction mixture, the mixture was extracted with ethyl acetate, and washed with saturated brine.
  • Example 6 Evaluation of growth inhibitory activity of Compound A, B, C, D, and E against cancer cells
  • Human neuroblastoma cell line SH-SY5Y European Collection of Authenticated Cell Cultures
  • MEM and F12 mixed medium (Thermo Fisher Scientific # 11095 and # 11765) containing (final concentration 15%) (hereinafter referred to as “MEM / F12 medium”).
  • MEM / F12 medium MEM and F12 mixed medium
  • the cell line was exfoliated and collected by TrypLE Express (Thermo Fisher Scientific # 12605), and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant.
  • Example 7 Effect of Compounds A, B, and C on the cell cycle Using Compounds A, B, and C, which were found to have cell growth inhibitory activity in Example 6, how these compounds were added to the cell cycle. We examined whether it had a positive effect.
  • the human neuroblastoma cell line SH-SY5Y is prepared by suspending cells in MEM / F12 medium to a cell concentration of 20000 cells / 1 mL, and dispensing 4 mL each into a 60 mm dish (Corning # 430166), 37 The cells were cultured at 5% CO 2 for 2 days.
  • the cells were then stained using the BD Cyclestest Plus DNA Reagent kit (BD Biosciences, # 340242) according to the protocol attached to the kit. Subsequently, the cell cycle was analyzed using BD FACSCanto (BD Biosciences). The obtained data was analyzed using FlowJo 7.6.5. The results are shown in FIG. As a result of the examination, it was found that Compounds A, B, and C show a cell division arresting action.
  • Example 8 Effect of Compounds A, B, and C on spindle formation Human neuroblastoma cell line SH-SY5Y was suspended in MEM / F12 medium, prepared to a cell concentration of 20000 cells / 1 mL, and 8 wells. 250 ⁇ L of each well of the culture slide (Falcon, # 354118) was dispensed and cultured at 37 ° C. under 5% CO 2 for 24 hours. Subsequently, 250 ⁇ L of Compound A, B, and C diluted in MEM / F12 medium to twice the final concentration was added to each well, and the cells were cultured at 37 ° C. under 5% CO 2 for 24 hours.
  • the antibody dilution buffer-1% BSA (SIGMA, # A9205-50ML) / 0.3% Triton X-100 / D-PBS (-) was used to use the primary antibody ⁇ -tubulin rabbit polyclonal antibody (Cell Signaling Technologies, #). 2146) and Pericentrin mouse monoclonal antibody (Abcam, # ab28144) were diluted 80-fold and 1000-fold, respectively, and treated overnight at 4 ° C. The next day, after washing 3 times with D-PBS (-) in the same manner as above, the secondary antibody Goat anti-mouse IgG Alexa 488 antibody (Life technologies, # A11029) and Goat anti using the same antibody dilution buffer as above.
  • Compound A is a compound in which a photoreactive group that covalently binds to a nearby protein by irradiation with ultraviolet rays and an alkyne for chemical modification by a click reaction are introduced.
  • a pull-down test combining photocrosslinking and chemical crosslinking was performed to identify the protein complex that binds to Compound A.
  • Example preparation Human neuroblastoma cell line SK-N-SH was suspended in MEM medium (Thermo Fisher Scientific # 11095), 2 ⁇ 10 7 cells were seeded in a 15 cm dish, and 1 at 37 ° C. under 5% CO 2. After daily culture, the cell cycle was synchronized to the mitotic phase by treatment with the Eg5 inhibitor Ispinesib (100 nM) for 15 hours. Subsequently, Compound D and Compound E diluted to 400 ⁇ M in MEM medium were added in an amount of 1/10 of each dish and cultured for 30 minutes.
  • proteins and impurities were separated by metalnol / chloroform extraction.
  • 200 ⁇ L of methanol, 50 ⁇ L of chloroform, and 150 ⁇ L of distilled water were added to the sample and then suspended.
  • the mixture was centrifuged (14,000 rpm, room temperature, 2 minutes) and the upper layer was removed.
  • 200 ⁇ L of methanol was added to the lower layer, and the mixture was centrifuged (14,000 rpm, room temperature, 2 minutes), the supernatant was removed, and the precipitate was dried.
  • urea / Tris solution 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 8 M urea, 0.005% dodecyl maltoside
  • DTT solution 100 mM
  • 35 ⁇ L of distilled water was added to the sample, and methanol / chloroform extraction was performed again.
  • urea / HEPES solution 8 M urea, 200 mM HEPES (pH 8.0), 0.005% dodecyl maltoside
  • HEPES buffer 200 mM HEPES (pH 8.0), 0.005%
  • Dodecyl maltoside 75 ⁇ L was added to dilute the urea concentration, trypsin (Modified trypsin (Promega # V5111), 250 ng / ⁇ L trypsin dilution buffer) 10 ⁇ L was added, and the mixture was reacted overnight at 37 ° C.
  • the Trypsin digest was subsequently labeled by the TMT system (Thermo Scientific # 90111).
  • each TMT tag was dissolved in 90 ⁇ L of acetonitrile, 41 ⁇ L of TMT tag dissolved in trypsin digest was added, and the mixture was reacted at room temperature for 1 hour. After the reaction, 8 ⁇ L of 5% hydroxylamine was added, and the reaction was carried out at room temperature for 15 minutes to stop the TMT labeling reaction. All samples were mixed, acetonitrile was removed by speedback and 80 ⁇ L of 50% formic acid was added.
  • the TMT-labeled peptide was desalted with StageTip (C18-SCX), fractionated with ammonium formate or ammonia, and further desalted with StageTip (C18).
  • the LC-MS / MS device used was an Orbitrap Fusion lumos (Thermo Fisher Scientific) equipped with an EASY-nLC1200 (Thermo Fisher Scientific).
  • the analytical column used was a chip column (100 ⁇ m ID, length 150 mm) packed with ReproSil-Pur 120 C18-AQ (2.4 ⁇ m, Dr. Maisch GmbH). Separation by LC was performed at a flow rate of 300 nL / min for 159.5 minutes with a linear gradient of 8-34% acetonitrile (0.125% formic acid).
  • LC-MS3 of Orbitrap Fusion Lumos was used, and positive mode, data-dependent top speed acquisition mode, and cycle time of 3 seconds were used.
  • MS / MS was acquired by CID, and HCD (collision energy 65%, resolution 60,000, scan range m / z 120-500) was acquired for the 10 strongest precursor ions in the MS / MS spectrum.
  • the obtained RAW data was converted to mzXML by the modified ReAdW.exe and analyzed using GFY Core 3.7. Comet was used for the database search, and Swiss-prot (human) was used for the database.
  • search conditions up to 2 uncleaved peptides were allowed, carbamoid methylation of cysteine residue was selected for fixed modification, TMT tag at the N-terminal of peptide, TMT tag for lysine, and oxidation of methionine was selected for variable modification.
  • the allowable range with the calculated mass of the peptide was set to 50 ppm, the allowable range with the calculated value of the product ion was set to 0.8 Da, and the monoisotopic was selected as the mass value.
  • the FDR of peptides and proteins was set to 1% or less, and filtering was performed using LDA (Core).
  • LDA Core
  • the values obtained by Core were normalized by data using R if necessary, and the p-value was calculated.
  • ALDH1B1, EXOC5, NUMA1, TUBB2B, TUBB4A, TUBB3, TUBB, TUBB2A, and CKAP5 were identified as activity-dependent binding proteins to Compound A that are competing with Compound D. The results are shown in FIG.
  • Example 10 Effect of NuMA1 knockdown due to RNA interference on suppression of cell proliferation of Compound B and Ispinesib (Eg5 inhibitor)
  • Human lung cancer cell line LK-2 (Health Science Research Resources Bank) has fetal bovine serum (final concentration 10). %)
  • RPMI1640 medium Thermo Fisher Scientific # 11875
  • the human neuroblastoma cell line SH-SY5Y was subcultured in MEM / F12 medium. First, each cell line was prepared to a cell concentration of 300,000 cells / 1 mL.
  • Negative control siRNA (Dharmacon, ON-TARGET plus Non-targeting Pool, # D-001810-10-50) and NuMA1 siRNA (Dharmacon, ON-TARGET plus NUMA1 siRNA, # J-005272-05-0002) have final concentrations. It was mixed with Lipofectamine RNAiMAX (Thermo Fisher Scientific, # 13778-150) using Opti-MEM medium (Thermo Fisher Scientific, # 11058-021) to 10 nM. Dispense 1 mL of cell suspension into each well of 6-well plate (IWAKI, # 3810-006), 1 mL of the above medium for each cell line, and 0.5 mL of siRNA-RNAiMAX reagent mixture at 37 ° C.
  • Example 11 Search for Compound C-dependent NuMA1-binding protein [Sample preparation]
  • the human neuroblastoma cell line SH-SY5Y was seeded on a 10 cm dish (Corning # 430293) and cultured at 37 ° C. under 5% CO 2 using MEM / F12 medium until the next day. Subsequently, Compound C and Ispinesib diluted with DMSO to a concentration 1000 times the final concentration were added to each dish and cultured overnight at 37 ° C. and 5% CO 2 . The next day, after washing 3 times with D-PBS (-), 0.5% formalin (20% neutral buffered formalin solution (Wako Pure Chemical Industries, Ltd.
  • the cells were lysed by adding cOmplete Tablets, Mini EASYpack (Roche # 04 693 124 001) and PhosSTOP EASYpack (Roche # 04 906 837 001) to the mixture in proportions. After incubating on ice for about 20 minutes, the cells were crushed by passing the needle 10 times with a syringe 27G ⁇ 1/2 (Terumo Corporation # SS-10M2713) with a Myjector injection needle. Centrifuge at 15,000 x g at 4 ° C for 10 minutes. Add 0.1 mL of 1% SDS to the precipitate, crush it with an ultrasonic crusher model UR-20P (Tomy Seiko Co.
  • Example 12 Enhancement of binding between NuMA1 and TACC3 and CKAP5 by Compound B treatment It was confirmed by Western blotting after immunoprecipitation of NuMA1 that the binding between NuMA1 and TACC3 or CKAP5 was enhanced by Compound B treatment.
  • the sample preparation method the method described in Example 11 except that Compound B was used instead of Compound C and the dish used in the experiment was changed from a 10 cm dish to a 15 cm dish (IWAKI # 3030-150).
  • Example 11 In the immunoprecipitation experiment, the method of Example 11 was followed, but each SDS extract fraction was 0.85 mL, 85 ⁇ L of washed Dynabeads was used during pre-clearing and immunoprecipitation, and 8.5 ⁇ L of anti-NUMA1 antibody was used during immunoprecipitation. (1.7 ⁇ g) was added, and only the point where 60 ⁇ L of the sample buffer was added was changed. [Electrophoresis] Samples were electrophoresed at 10 ⁇ L / lane using Perfect NT Gel 5-20% (DRC, # NTH-576HP10CD).
  • anti-NUMA1 antibody 5000-fold dilution
  • anti-CKAP5 antibody Cho-TOG (D2Z8J) Rabbit mAb (Cell Signaling Technology # 67774S), 500-fold dilution
  • anti-TACC3 antibody TACC3 (C-5)) (Santa Cruz Biotechnology # sc-271165)
  • anti- ⁇ -tubulin antibody Monoclonal Anti- ⁇ -tubulin antibody (Sigma-Aldrich # T8328), 1000-fold dilution
  • Anti- Incubation was performed using ⁇ -Actin antibody and Mouse monoclonal (Sigma-Aldrich # A1978) 40,000-fold diluted) at 4 ° C.
  • ECL Anti-Rabbit IgG Horseradish Peroxidase linked whole antibody from donky (GE Healthcare, # NA934V)
  • ECL Anti-Mouse IgG Horseradish Peroxidase linked whole antibody from sheep
  • Can Get Signal Solution 1 and Solution 2 were used to dilute the primary antibody and the secondary antibody, respectively. It was also washed 5 times with TBS-Tween between each step.
  • 3 ⁇ 10 5 human lung cancer cell lines LK-2 were seeded on a 3.5 cm glass bottom dish, cultured for 1 day at 37 ° C. under 5% CO 2 , and then in 20 nM Compound B or 1 nM Ispine sib for 16 hours. Processed. Cells were fixed with 100% metanol for 5 minutes (for CKAP5 staining) and washed 3 times with D-PBS (-). After membrane permeation treatment with D-PBS (-) containing 0.1% TritonX-100 for 5 minutes, washing 3 times with D-PBS (-) and 30 minutes with D-PBS (-) containing 3% bovine serum albumin. Blocked.
  • the primary antibody was diluted with a blocking solution and allowed to stand at room temperature for 30 minutes for reaction. This was washed 3 times with D-PBS (-), and the secondary antibody was also reacted and washed in the same manner, and encapsulated with ProLong Diamond Antifade Mountant (ThermoFisherScientific, # P36965). Observations were analyzed with LASX software using a Leica TSC SP8 STED microscope. The results are shown in FIG. Similar monopolar spindle formation was observed with Compound B and Ispine sib treatment, but NuMA1 was localized around the centrosome in a ring shape and CKAP5 was not clearly localized with Ispine sib treatment. NuMA1 and CKAP5 were strongly co-localized near the centrosome by Compound B treatment.
  • Negative control siRNA (Dharmacon, ON-TARGET plus Non-targeting Pool, # D-001810-10-50), NuMA1 siRNA (Dharmacon, ON-TARGET plus NUMA1 siRNA, # J-005272-05-0002), TACC3 siRNA ( Dharmacon, ON-TARGET plus TACC3 siRNA, # J-004155-07-0002, # J-004155-08-0002), CKAP5 siRNA (Dharmacon, ON-TARGET plus CKAP5 siRNA, # J-006847-07-0002, # J-006847-08-0002) was mixed with Lipofectamine RNAiMAX using Opti-MEM medium to a final concentration of 10 nM.
  • the cells were then irradiated with ultraviolet light (365 nm) at 4 ° C for 10 minutes.
  • the cells were then scraped with a scraper, centrifuged at 1500 rpm for 10 minutes at 4 ° C. to remove the supernatant, washed with 1 mL of PBS, and the pellet was suspended in 100 ⁇ L of D-PBS (-).
  • the cells were crushed on ice using an ultrasonic crusher, centrifuged at 4 ° C. and 15000 rpm for 30 minutes, and the supernatant was used as a cell lysate and stored at -80 ° C until use.
  • the protein concentration of each sample was measured with the SpectraMax Plus assayer (molecular device) using the DC Protein Assay (BIO-RAD) and with D-PBS (-) at a concentration of 2 mg / mL. Prepared. 45 ⁇ L of 2 mg / mL cell lysate and 5 ⁇ L of Click reagent mix (50 mM CuSO4 (Nacalai Tesque, # 09604-85), 1.25 mM TAMRA-Azide (Click chemistry tools, # AZ10905), 50 mM TCEP (Bond-) Breaker TCEP Solution, Neutral pH (PCC), Thermo Fisher Scientific, # 77720), 10 mM THPTA (Tris (3-hydroxypropyltriazolylmethyl) amine, SIGMA, # 762342-500MG), 10% SDS (SIGMA, # L6026-250G)) Was mixed and reacted at room temperature for 2 hours.
  • Click reagent mix 50
  • sample buffer (4 ⁇ LDS sample buffer (Life technologies, # NP0008) and 2-Mercaptoethanol (SIGMA, # 6250) mixed at 10: 1) was added to each sample, and the mixture was treated at 70 ° C. for 10 minutes. did.
  • Samples were electrophoresed in triplicate at 10 ⁇ L / lane using a gel (Perfect NT Gel, 5-20%) (DRC, # NTH-576HP10CD) and TAMRA fluorescence was measured with Typhoon FLA9500 (GE Healthcare).
  • ⁇ -Tubulin (50 kDa) band density was quantified using ImageQuant TL (GE Healthcare). The results are shown in FIG.
  • Knockdown of NuMA1 and CKAP5 markedly reduced the binding of Compound A to ⁇ -tubulin.
  • knockdown of TACC3 showed moderate attenuation of binding.
  • no effect was observed in the knockdown of CLTC.
  • Example 15 Inhibition of microtubule elongation by Compound B treatment It is known that CKAP5 and TACC3 form a complex and play an important role in the stability of microtubules. Since it was found that the abnormal localization of CKAP5 was caused by Example 13, it is a growth end marker of microtubules whether or not the elongation of microtubules extending from the centrosome is suppressed by Compound B treatment. It was examined by immunostaining of EB1.
  • Anti-EB1 antibody (Abcam, # ab53358), anti-rat IgG-Alexa 647-labeled antibody (Abcam, # ab150155), CellMask Green (Thermo Fisher Scientific, C37608), DAPI Solution (4', 6-Diamidino-2-phenylindole Dihydrochloride Solution) ) (Dojin Chemical Research Institute, D523) was used. 6 ⁇ 10 4 human lung cancer cell lines LK-2 were seeded on a clear bottom 96-well plate, cultured daily under 5% CO 2 at 37 ° C, and then in 100 nM Compound B or 10 nM Ispine sib for 16 hours. Processed.
  • the cells were fixed in 100% metanol for 5 minutes and washed twice with D-PBS (-). After membrane permeation treatment with D-PBS (-) containing 0.1% TritonX-100 for 5 minutes, washing 3 times with D-PBS (-) and 30 minutes with D-PBS (-) containing 3% bovine serum albumin. Blocked.
  • the primary antibody was diluted with a blocking solution and allowed to stand at room temperature for 30 minutes for reaction. This was washed 3 times with D-PBS (-), and was similarly reacted and washed with a mixed solution of the secondary antibody and CellMask Green. Measured using Cell Voyager 7000S manufactured by Yokogawa Electric Co., Ltd.
  • the human prostate cancer cell line LNCaP clone FGC (American Type Culture Collection) is an RPMI1640 medium (Fujifilm Wako Junyaku # 187-02705) containing bovine fetal serum (final concentration 10%), and the human esophalized cell line TE10 (physical chemistry).
  • the laboratory bioimmortalized cell line is the RPMI1640 cell line containing bovine fetal serum (final concentration 10%) (Thermo Fisher Scientific # 11875), and the human esophalized cell line TE14 (physical and chemical research institute biomortalized cell line) is the bovine immortalized cell line.
  • RPMI 1640 medium (Thermo Fisher Scientific # 11875) containing 10% final concentration
  • Detroit 562 American Type Culture Collection
  • a human head and neck cancer cell line is an EMEM medium (Fuji Film Japanese) containing bovine fetal serum (10% final concentration).
  • human head and neck cancer cell line HSC3 (Bio-mortalized cell line of the Institute of Physical and Chemical Research) is an EMEM medium containing bovine fetal serum (final concentration 10%) (Fujifilm Wako Junyaku # 051-07615) , Human immortalized cell line AML193 (American Type Culture Collection) is available in bovine fetal cell line (finalized 5%) and ITS Liquid Media Supplement (SIGMA I3146) and finalized 5 ng / mL human GM-CSF (Miltenyi Biotec # 130-095-).
  • IMDM cell line containing 372) (Thermo Fisher Scientific # 12440)
  • human lung cancer cell line NCI-H1395 (American Type Culture Collection) is an RPMI1640 cell line containing bovine fetal cell line (final concentration 10%) (Fujifilm Wako Pure Cell Line # 187-). 02705)
  • human lung cancer cell line NCI-H23 (American Type Culture Collection) is an RPMI1640 medium (Fujifilm Wako Pure Drug # 187-02705) containing bovine fetal serum (final concentration 10%), human keratomortalized cell line A375 (American).
  • Type Culture Collection is a DMEM medium (Fujifilm Wako Junyaku # 043-30085) containing bovine fetal serum (final concentration 10%), human pancreatic cancer cell line.
  • BxPC-3 American Type Culture Collection
  • RPMI1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd. # 187-02705) containing fetal bovine serum (final concentration 10%), and human lung cancer cell line LK-2 contains fetal bovine serum (final concentration 10%).
  • RPMI 1640 medium (Thermo Fisher Scientific # 11875).
  • Each cell line was exfoliated and collected by TrypLE Express (Thermo Fisher Scientific # 12605), and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant. Suspend the cells in a similar medium, prepare each cell to a cell concentration of 20000 cells / 1 mL, dispense 100 ⁇ L into each well of a 96-well plate (Corning # 3904), 37 ° C., 5% CO Incubated under 2 for 24 hours. Subsequently, 50 ⁇ L of the compound solution of the example diluted in each medium to a predetermined concentration was added to each well (day 1), and the cells were cultured at 37 ° C. under 5% CO 2 for 3 days.
  • the concentration (GI50 value) that inhibits the growth of each cell of the compound by 50% was calculated by semi-logarithm plotting the cell growth rate and the compound concentration at each concentration. The results are shown in Table 1. Both Compound B and Compound C showed a cell growth inhibitory effect for each cancer type.
  • Example 17 Screening of a compound that stabilizes a complex containing NuMA1 and CKAP5 using cells having different NuMA1 expression levels as an index Human lung cancer cell line LK-2 (Health Science Research Resources Bank) is a cow. Subculture was maintained in RPMI 1640 medium (Thermo Fisher Scientific # 11875) containing fetal bovine serum (final concentration 10%). First, each cell line was prepared to a cell concentration of 300,000 cells / 1 mL. Negative control siRNA (Dharmacon, ON-TARGET plus Non-targeting Pool, # D-001810-10-50) and NuMA1 siRNA (Dharmacon, ON-TARGET plus NUMA1 siRNA, # J-005272-05-0002) have final concentrations.
  • Negative control siRNA Dharmacon, ON-TARGET plus Non-targeting Pool, # D-001810-10-50
  • NuMA1 siRNA Dharmacon, ON-TARGET plus NUMA1 siRNA, # J-005272-05-0002
  • RNAiMAX Lipofectamine RNAiMAX (Thermo Fisher Scientific, # 13778-150) using Opti-MEM medium (Thermo Fisher Scientific, # 11058-021) to 10 nM.
  • the culture supernatant was removed, 2.5 mL of the above medium was added to each cell line, and 0.5 mL of the siRNA-RNAiMAX reagent mixture prepared in the same manner as above was added.
  • the cells were peeled and recovered by TrypLE Express, and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant.
  • the cells in the above medium for each cell line prepare to a cell concentration of 20000 cells / 1 mL, dispense 100 ⁇ L into each well of 96-well plates (Corning # 3904), 37 ° C, 5% CO 2 Incubated underneath for 24 hours.
  • Compound B Ispinesib, Volasertib, Alisertib, and other microtubule polymerization inhibitors Vincristine (for injection of Japanese chemicals and oncobin), which are microtubule polymerization inhibitors, and microtubules, which were diluted to a predetermined concentration in the above medium for each cell line, were used.
  • Paclitaxel Flujifilm Wako Pure Chemical Industries, Ltd., # 167-28166
  • a depolymerization inhibitor was added to each well in an amount of 50 ⁇ L (day 1) and cultured at 37 ° C. under 5% CO 2 for 3 days.
  • CellTiter-Glo 2.0 Assay which is a reagent for ATP measurement, was added to each well at a rate of 30 ⁇ L / well, and the luminescence amount of each well was measured by EnVision. From the luminescence amount of the compound-free group (C4) and the compound-added group (T4) after culturing for 3 days, the cell suppression rate was calculated based on the following formula.
  • IC50 value For the concentration that suppresses each cell of the compound by 50% (IC50 value), calculate by semi-log plotting the cell proliferation rate and compound concentration at each concentration, and the ratio of IC50 in the siContorl treatment group to IC50 in the siNuMA1 treatment group is shown in Table 2. Shown in. Compound B suppresses the expression of NuMA1 by siRNA treatment, which increases the IC50 value by 10 times or more (the pharmacological activity drops to 1/10 or less), whereas other cell division inhibitors suppress the expression of NuMA1. No change in IC50 value was observed. In other words, by using cells with different expression levels of NuMA1 and selecting compounds with significantly different cell growth inhibitory effects among the cells, it is possible to screen for compounds that stabilize the complex containing NuMA1 and CKAP5. I found it.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention addresses the problem of providing: a compound that is capable of inhibiting cell proliferation with high selectivity for cancer cells; a method for inhibiting cell proliferation with high selectivity for cancer cells; and a method for screening a compound that is capable of inhibiting cell proliferation with high selectivity for cancer cells.　The present invention has been completed on the basis of a finding that cell proliferation can be inhibited with high selectivity for cancer cells by stabilizing a complex containing NuMA1 and CKAP5 (hereinafter called "NuMA1 complex"). The present invention provides: a compound that stabilizes the NuMA1 complex; a method for stabilizing the NuMA1 complex; and a method for screening a compound that stabilizes the NuMA1 complex.

Description

ＮｕＭＡ１およびＣＫＡＰ５を含む複合体を安定化する化合物Compounds that stabilize the complex containing NuMA1 and CKAP5
　本発明は、NuMA1およびCKAP5を含む複合体を安定化する化合物、NuMA1およびCKAP5を含む複合体を安定化させる方法、およびNuMA1およびCKAP5を含む複合体を安定化する化合物のスクリ－ニング方法に関する。 The present invention relates to a compound that stabilizes a complex containing NuMA1 and CKAP5, a method for stabilizing a complex containing NuMA1 and CKAP5, and a method for screening a compound that stabilizes a complex containing NuMA1 and CKAP5.
　1940年代以降、アルキル化剤をはじめとして多くの抗がん剤が開発されてきた（非特許文献１）。しかし、現在使われている抗がん剤の多くは、細胞毒性を有しており、すべての増殖細胞に作用を発揮するため、がん細胞および正常細胞のいずれにも影響を及ぼしてしまう。（非特許文献２）。 Since the 1940s, many anticancer agents such as alkylating agents have been developed (Non-Patent Document 1). However, many of the anticancer drugs currently in use are cytotoxic and exert their effects on all proliferating cells, thus affecting both cancer cells and normal cells. (Non-Patent Document 2).
　細胞分裂を阻害することによりがん細胞の増殖を抑制する薬剤として、タキサンやビンカアルカロイド等が知られ、広く使われているが、これら薬剤は、微小管の重合および脱重合等、すべて細胞の分裂に必須のメカニズムを標的としていることから、上述のように正常細胞にも影響を及ぼしてしまう。また、微小管形成は非分裂細胞である神経細胞においても重要な役割を果たしており、これら薬剤は神経毒性が臨床上問題となっている（非特許文献３）。しかしながら、細胞分裂を阻害することによるがん細胞の増殖抑制は極めて有効ながんの治療方法と考えられるため、細胞分裂を阻害しつつも、がん細胞に対して高い選択性を有する抗がん剤が求められている。 Taxanes, vinca alkaloids, and the like are known and widely used as agents that suppress the growth of cancer cells by inhibiting cell division, but these agents are all used for cell polymerization and depolymerization of microtubules. Since it targets the mechanisms essential for division, it also affects normal cells as described above. In addition, microtubule formation also plays an important role in nerve cells, which are non-dividing cells, and neurotoxicity of these drugs has become a clinical problem (Non-Patent Document 3). However, since suppressing the growth of cancer cells by inhibiting cell division is considered to be an extremely effective treatment method for cancer, there is an anti-cancer drug that has high selectivity for cancer cells while inhibiting cell division. Antineoplastic agents are required.
　NuMA1は、核基質の構成因子であると同時に、分裂期においては紡錘体形成および配置に重要な役割を果たしているタンパク質である（非特許文献４）。NuMA1は一部のがん細胞に高発現していることが知られている。
CKAP5は、微小管と結合するタンパク質であり、微小管の安定化に重要な役割を担っている。さらに、CKAP5は、TACC3およびclathrinと複合体を形成し微小管間の架橋および紡錘体形成に働いていることが知られている（非特許文献５）。 NuMA1 is a protein that is a constituent of the nuclear substrate and at the same time plays an important role in spindle formation and placement during mitosis (Non-Patent Document 4). NuMA1 is known to be highly expressed in some cancer cells.
CKAP5 is a protein that binds to microtubules and plays an important role in stabilizing microtubules. Furthermore, it is known that CKAP5 forms a complex with TACC3 and clathrin and acts on cross-linking between microtubules and spindle formation (Non-Patent Document 5).
　NuMA1およびCKAP5は、いずれも微小管および紡錘体の形成に関わっているが、これまでNuMA1とCKAP5がどのように関係しているのかは知られていない。さらに、これら分子を制御することががんにどのような影響を及ぼすのかも知られていなかった。 NuMA1 and CKAP5 are both involved in the formation of microtubules and mitotic spindles, but it has not been known how NuMA1 and CKAP5 are related. Furthermore, it was not known how controlling these molecules would affect cancer.
本発明の課題は、がん細胞高選択的に細胞の増殖を妨げることが可能な化合物、がん細胞高選択的に細胞の増殖を妨げる方法、およびがん細胞高選択的に細胞の増殖を妨げることが可能な化合物のスクリ－ニング方法を提供することである。 The subject of the present invention is a compound capable of hindering the growth of cancer cells highly selectively, a method of hindering the growth of cells highly selectively of cancer cells, and a method of highly selectively hindering the growth of cells of cancer cells. It is to provide a method of screening a compound that can be prevented.
本発明者らは、がん細胞の細胞分裂について研究を行った結果、がん細胞に高発現している核内タンパク質であるNuMA1が、細胞分裂時にNuMA1およびCKAP5を含む複合体（以下「NuMA1複合体」という。）を形成すること、および当該NuMA1複合体が微小管形成を制御していることを発見した。 As a result of research on cell division of cancer cells, the present inventors have found that NuMA1, a nuclear protein highly expressed in cancer cells, contains a complex containing NuMA1 and CKAP5 during cell division (hereinafter referred to as "NuMA1"). It was discovered that it forms a "complex") and that the NuMA1 complex controls microtubule formation.
　そこで本発明者らは、当該NuMA1複合体について更なる研究を行った結果、NuMA1複合体の構成タンパク同士の結合を安定化させてNuMA1複合体の解離もしくは分解を抑制することで、微小管形成とそれに続く紡錘体の生成が抑制されて細胞分裂を阻害し、細胞の増殖が抑制されることを見出した。NuMA1はがん細胞に高発現していることが知られているため、NuMA1複合体を標的とすることで、NuMA1が高発現しているがん細胞高選択的に細胞の増殖を妨げることが可能である。したがって、本発明は、NuMA1複合体を安定化する化合物、NuMA1複合体の安定化方法、およびNuMA1複合体を安定化する化合物のスクリ－ニング方法を提供するものである。 Therefore, as a result of further research on the NuMA1 complex, the present inventors stabilized the binding between the constituent proteins of the NuMA1 complex and suppressed the dissociation or degradation of the NuMA1 complex to form microtubules. It was found that the production of mitotic spindles was suppressed, cell division was inhibited, and cell proliferation was suppressed. Since NuMA1 is known to be highly expressed in cancer cells, targeting the NuMA1 complex can prevent the proliferation of cancer cells in which NuMA1 is highly expressed in a highly selective manner. It is possible. Therefore, the present invention provides a compound that stabilizes the NuMA1 complex, a method for stabilizing the NuMA1 complex, and a method for screening the compound that stabilizes the NuMA1 complex.
　本発明は、下記（１）～（１６）に関する。
（１）NuMA1およびCKAP5を含む複合体を安定化する化合物。
（２）（１）に記載の化合物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化する化合物。
（３）（１）に記載の化合物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化する化合物。
（４）がんの治療に用いられる（１）～（３）のいずれか１つに記載の化合物。
（５）NuMA1およびCKAP5を含む複合体を安定化する化合物を含有する医薬組成物。
（６）（５）に記載の医薬組成物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化する化合物を含有する医薬組成物。
（７）（５）に記載の医薬組成物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化する化合物を含有する医薬組成物。
（８）がんの治療に用いられる（５）～（７）のいずれか１つに記載の医薬組成物。
（９）NuMA1およびCKAP5を含む複合体を安定化させる方法。
（１０）（９）に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化させる方法。
（１１）（９）に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化させる方法。
（１２）がんの治療に用いられる（９）～（１１）のいずれか１つに記載の方法。
（１３）NuMA1およびCKAP5を含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。
（１４）（１３）に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。
（１５）（１３）に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。
（１６）以下の工程：
（a）化合物をNuMA1およびCKAP5を含む複合体と接触させる工程
（b）化合物によりNuMA1およびCKAP5を含む複合体が安定化されているか否かを確認する工程
を含むことを特徴とするNuMA1およびCKAP5を含む複合体を安定化する化合物のスクリ－ニング方法。 The present invention relates to the following (1) to (16).
(1) A compound that stabilizes a complex containing NuMA1 and CKAP5.
(2) The compound according to (1) that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin.
(3) The compound according to (1), which stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(4) The compound according to any one of (1) to (3) used for the treatment of cancer.
(5) A pharmaceutical composition containing a compound that stabilizes a complex containing NuMA1 and CKAP5.
(6) The pharmaceutical composition according to (5), which contains a compound that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin.
(7) The pharmaceutical composition according to (5), which contains a compound that stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(8) The pharmaceutical composition according to any one of (5) to (7) used for treating cancer.
(9) A method for stabilizing a complex containing NuMA1 and CKAP5.
(10) The method according to (9), wherein the complex containing NuMA1, CKAP5 and β-tubulin is stabilized.
(11) The method according to (9), wherein the complex containing NuMA1, CKAP5, β-tubulin and TACC3 is stabilized.
(12) The method according to any one of (9) to (11) used for treating cancer.
(13) A method for suppressing the growth of cancer cells, which comprises stabilizing a complex containing NuMA1 and CKAP5.
(14) The method according to (13), which suppresses the growth of cancer cells, which comprises stabilizing a complex containing NuMA1, CKAP5 and β-tubulin.
(15) The method according to (13), which suppresses the growth of cancer cells, which comprises stabilizing a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(16) The following steps:
(A) A step of contacting the compound with the complex containing NuMA1 and CKAP5 (b) A step of confirming whether or not the complex containing NuMA1 and CKAP5 is stabilized by the compound NuMA1 and CKAP5. A method for screening a compound that stabilizes a complex containing.
　本発明の別の態様としては、下記（１７）～（２５）に関する。
（１７）NuMA1およびCKAP5を含む複合体を安定化させることを特徴とするがんの治療方法。
（１８）（１７）に記載の方法であって、NuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化させることを特徴とするがんの治療方法。
（１９）（１７）に記載の方法であって、NuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化させることを特徴とするがんの治療方法。
（２０）がんの治療に使用するためのNuMA1およびCKAP5を含む複合体を安定化する化合物。
（２１）（２０）に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化する化合物。
（２２）（２０）に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化する化合物。
（２３）がんの治療のための医薬の製造におけるNuMA1およびCKAP5を含む複合体を安定化する化合物の使用。
（２４）（２３）に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化する化合物の使用。
（２５）（２３）に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化する化合物の使用。 Another aspect of the present invention relates to the following (17) to (25).
(17) A method for treating cancer, which comprises stabilizing a complex containing NuMA1 and CKAP5.
(18) The method according to (17), which is a method for treating cancer, which comprises stabilizing a complex containing NuMA1, CKAP5 and β-tubulin.
(19) The method according to (17), which is a method for treating cancer, which comprises stabilizing a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(20) A compound that stabilizes a complex containing NuMA1 and CKAP5 for use in the treatment of cancer.
(21) The compound according to (20), which stabilizes a complex containing NuMA1, CKAP5 and β-tubulin for use in the treatment of cancer.
(22) The compound according to (20), which stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3 for use in the treatment of cancer.
(23) Use of compounds that stabilize complexes containing NuMA1 and CKAP5 in the manufacture of pharmaceuticals for the treatment of cancer.
(24) Use of a compound according to (23) that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin in the manufacture of a medicament for the treatment of cancer.
(25) Use of a compound according to (23) that stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3 in the manufacture of a medicament for the treatment of cancer.
さらに本発明の別の態様としては、下記（１A）～（５２A）に関する。
（１A）NuMA1およびCKAP5を含む複合体を安定化する化合物。
（２A）（１A）に記載の化合物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化する化合物。
（３A）（１A）に記載の化合物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化する化合物。
（４A）がんの治療に用いられる（１A）～（３A）のいずれか１つに記載の化合物。
（５A）NuMA1およびCKAP5を含む複合体を安定化する化合物を含有する医薬組成物。
（６A）（５A）に記載の医薬組成物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化する化合物を含有する医薬組成物。
（７A）（５A）に記載の医薬組成物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化する化合物を含有する医薬組成物。
（８A）がんの治療に用いられる（５A）～（７A）のいずれか１つに記載の医薬組成物。
（９A）NuMA1およびCKAP5を含む複合体を安定化させる方法。
（１０A）（９A）に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化させる方法。 Further, another aspect of the present invention relates to the following (1A) to (52A).
(1A) A compound that stabilizes a complex containing NuMA1 and CKAP5.
(2A) The compound according to (1A) that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin.
(3A) A compound according to (1A) that stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(4A) The compound according to any one of (1A) to (3A) used for the treatment of cancer.
(5A) A pharmaceutical composition containing a compound that stabilizes a complex containing NuMA1 and CKAP5.
(6A) The pharmaceutical composition according to (5A), which contains a compound that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin.
(7A) The pharmaceutical composition according to (5A), which contains a compound that stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(8A) The pharmaceutical composition according to any one of (5A) to (7A) used for treating cancer.
(9A) A method for stabilizing a complex containing NuMA1 and CKAP5.
(10A) The method according to (9A), wherein the complex containing NuMA1, CKAP5 and β-tubulin is stabilized.
（１１A）（９A）に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化させる方法。
（１２A）がんの治療に用いられる（９A）～（１１A）のいずれか１つに記載の方法。
（１３A）NuMA1およびCKAP5を含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。
（１４A）（１３A）に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。
（１５A）（１３A）に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。
（１６A）以下の工程：
（a）化合物をNuMA1およびCKAP5を含む複合体と接触させる工程
（b）化合物によりNuMA1およびCKAP5を含む複合体が安定化されているか否かを確認する工程
を含むことを特徴とするNuMA1およびCKAP5を含む複合体を安定化する化合物のスクリ－ニング方法。
（１７A）以下の工程：
　（a）NuMA1の発現量が高い細胞およびNuMA1の発現量が低い細胞のそれぞれに化合物を接触させる工程
　（b）それぞれの細胞における化合物の細胞増殖抑制作用および／または細胞分裂阻害作用を測定する工程
　（c）NuMA1の発現量の高い細胞における細胞増殖抑制作用の方がNuMA1の発現量の低い細胞における細胞増殖抑制作用よりも高い場合に、当該化合物をNuMA1およびCKAP5を含む複合体を安定化する化合物であると同定する工程
を含むことを特徴とするNuMA1およびCKAP5を含む複合体を安定化する化合物のスクリ－ニング方法。
（１８A）NuMA1およびCKAP5を含む複合体を安定化させることを特徴とするがんの治療方法。
（１９A）（１８A）に記載の方法であって、NuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化させることを特徴とするがんの治療方法。
（２０A）（１８A）に記載の方法であって、NuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化させることを特徴とするがんの治療方法。 (11A) The method according to (9A), wherein the complex containing NuMA1, CKAP5, β-tubulin and TACC3 is stabilized.
(12A) The method according to any one of (9A) to (11A) used for treating cancer.
(13A) A method for suppressing the growth of cancer cells, which comprises stabilizing a complex containing NuMA1 and CKAP5.
(14A) The method according to (13A), which suppresses the growth of cancer cells, which comprises stabilizing a complex containing NuMA1, CKAP5 and β-tubulin.
(15A) The method according to (13A), which suppresses the growth of cancer cells, which comprises stabilizing a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(16A) The following steps:
(A) A step of contacting the compound with the complex containing NuMA1 and CKAP5 (b) A step of confirming whether or not the complex containing NuMA1 and CKAP5 is stabilized by the compound NuMA1 and CKAP5. A method for screening a compound that stabilizes a complex containing.
(17A) The following steps:
(A) Step of contacting the compound with cells having a high expression level of NuMA1 and cells having a low expression level of NuMA1 (b) Step of measuring the cell growth inhibitory action and / or cell division inhibitory action of the compound in each cell (C) Stabilize the complex containing NuMA1 and CKAP5 when the cell growth inhibitory effect in cells with high NuMA1 expression is higher than the cell growth inhibitory effect in cells with low NuMA1 expression. A method for screening a compound that stabilizes a complex containing NuMA1 and CKAP5, which comprises the step of identifying it as a compound.
(18A) A method for treating cancer, which comprises stabilizing a complex containing NuMA1 and CKAP5.
(19A) The method of treating cancer according to (18A), which is characterized by stabilizing a complex containing NuMA1, CKAP5 and β-tubulin.
(20A) The method according to (18A), which is a method for treating cancer, which comprises stabilizing a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
（２１A）がんの治療に使用するためのNuMA1およびCKAP5を含む複合体を安定化する化合物。
（２２A）（２１A）に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化する化合物。
（２３A）（２１A）に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化する化合物。
（２４A）がんの治療のための医薬の製造におけるNuMA1およびCKAP5を含む複合体を安定化する化合物の使用。
（２５A）（２４A）に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化する化合物の使用。
（２６A）（２４A）に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化する化合物の使用。
（２７A）NuMA1およびCKAP5を含む複合体の機能を調整する化合物。
（２８A）（２７A）に記載の化合物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整する化合物。
（２９A）（２７A）に記載の化合物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整する化合物。
（３０A）がんの治療に用いられる（２７A）～（２９A）のいずれか１つに記載の化合物。 (21A) A compound that stabilizes a complex containing NuMA1 and CKAP5 for use in the treatment of cancer.
(22A) A compound according to (21A) that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin for use in the treatment of cancer.
(23A) A compound according to (21A) that stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3 for use in the treatment of cancer.
(24A) Use of compounds that stabilize complexes containing NuMA1 and CKAP5 in the manufacture of pharmaceuticals for the treatment of cancer.
(25A) The use of a compound that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin in the manufacture of a medicament for the treatment of cancer, according to the use according to (24A).
(26A) The use of a compound that stabilizes a complex, including NuMA1, CKAP5, β-tubulin and TACC3, in the manufacture of a medicament for the treatment of cancer, according to the use according to (24A).
(27A) A compound that regulates the function of a complex containing NuMA1 and CKAP5.
(28A) A compound according to (27A) that regulates the function of a complex containing NuMA1, CKAP5 and β-tubulin.
(29A) A compound according to (27A) that regulates the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(30A) The compound according to any one of (27A) to (29A) used for the treatment of cancer.
（３１A）NuMA1およびCKAP5を含む複合体の機能を調整する化合物を含有する医薬組成物。
（３２A）（３１A）に記載の医薬組成物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整する化合物を含有する医薬組成物。
（３３A）（３１A）に記載の医薬組成物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整する化合物を含有する医薬組成物。
（３４A）がんの治療に用いられる（３１A）～（３３A）のいずれか１つに記載の医薬組成物。
（３５A）NuMA1およびCKAP5を含む複合体の機能を調整する方法。
（３６A）（３５A）に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整する方法。
（３７A）（３５A）に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整する方法。
（３８A）がんの治療に用いられる（３５A）～（３７A）のいずれか１つに記載の方法。
（３９A）NuMA1およびCKAP5を含む複合体の機能を調整することを特徴とするがん細胞の増殖を抑制する方法。
（４０A）（３９A）に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整することを特徴とするがん細胞の増殖を抑制する方法。 (31A) A pharmaceutical composition containing a compound that regulates the function of a complex containing NuMA1 and CKAP5.
(32A) The pharmaceutical composition according to (31A), which contains a compound that regulates the function of a complex containing NuMA1, CKAP5 and β-tubulin.
(33A) The pharmaceutical composition according to (31A), which contains a compound that regulates the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(34A) The pharmaceutical composition according to any one of (31A) to (33A) used for treating cancer.
(35A) A method of coordinating the function of a complex containing NuMA1 and CKAP5.
(36A) (35A), the method of adjusting the function of a complex comprising NuMA1, CKAP5 and β-tubulin.
(37A) (35A), the method of adjusting the function of a complex comprising NuMA1, CKAP5, β-tubulin and TACC3.
(38A) The method according to any one of (35A) to (37A) used for the treatment of cancer.
(39A) A method for suppressing the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1 and CKAP5.
(40A) (39A), the method of suppressing the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1, CKAP5 and β-tubulin.
（４１A）（３９A）に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整することを特徴とするがん細胞の増殖を抑制する方法。
（４２A）以下の工程：
（a）化合物をNuMA1およびCKAP5を含む複合体と接触させる工程
（b）化合物によりNuMA1およびCKAP5を含む複合体の機能が調整されているか否か
確認する工程
を含むことを特徴とするNuMA1およびCKAP5を含む複合体の機能を調整する化合物のスクリ－ニング方法。
（４３A）以下の工程：
（a）NuMA1の発現量が高い細胞およびNuMA1の発現量が低い細胞のそれぞれに化合物を接触させる工程
（b）それぞれの細胞における化合物の細胞増殖抑制作用および／または細胞分裂阻害作用を測定する工程
（c）NuMA1の発現量の高い細胞における細胞増殖抑制作用の方がNuMA1の発現量の低い細胞における細胞増殖抑制作用よりも高い場合に、当該化合物をNuMA1およびCKAP5を含む複合体の機能を調整する化合物であると同定する工程
を含むことを特徴とするNuMA1およびCKAP5を含む複合体の機能を調整する化合物のスクリ－ニング方法。
（４４A）NuMA1およびCKAP5を含む複合体の機能を調整することを特徴とするがんの治療方法。
（４５A）（４４A）に記載の方法であって、NuMA1、CKAP5およびβ－チュ－ブリンを含む複合体の機能を調整することを特徴とするがんの治療方法。
（４６A）（４４A）に記載の方法であって、NuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体の機能を調整することを特徴とするがんの治療方法。
（４７A）がんの治療に使用するためのNuMA1およびCKAP5を含む複合体の機能を調整する化合物。
（４８A）（４７A）に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体の機能を調整する化合物。
（４９A）（４７A）に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体の機能を調整する化合物。
（５０A）がんの治療のための医薬の製造におけるNuMA1およびCKAP5を含む複合体の機能を調整する化合物の使用。
（５１A）（５０A）に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体の機能を調整する化合物の使用。
（５２A）（５０A）に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体の機能を調整する化合物の使用。 (41A) (39A), the method of suppressing the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(42A) The following steps:
(A) A step of contacting the compound with the complex containing NuMA1 and CKAP5 (b) A step of confirming whether or not the function of the complex containing NuMA1 and CKAP5 is adjusted by the compound is included. A method for screening a compound that regulates the function of a complex containing.
(43A) The following steps:
(A) Step of contacting the compound with each of the cells having a high NuMA1 expression level and the cell having a low NuMA1 expression level (b) A step of measuring the cell growth inhibitory action and / or the cell division inhibitory action of the compound in each cell. (C) When the cell growth inhibitory effect in cells with high NuMA1 expression is higher than the cell growth inhibitory effect in cells with low NuMA1 expression, the compound adjusts the function of the complex containing NuMA1 and CKAP5. A method for screening a compound that regulates the function of a complex containing NuMA1 and CKAP5, which comprises the step of identifying the compound to be used.
(44A) A method of treating cancer, which comprises regulating the function of a complex containing NuMA1 and CKAP5.
(45A) (44A), a method for treating cancer, which comprises adjusting the function of a complex containing NuMA1, CKAP5 and β-tubulin.
(46A) The method of treating cancer according to (44A), which regulates the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
(47A) A compound that regulates the function of a complex containing NuMA1 and CKAP5 for use in the treatment of cancer.
(48A) A compound according to (47A) that regulates the function of a complex containing NuMA1, CKAP5 and β-tubulin for use in the treatment of cancer.
(49A) A compound according to (47A) that regulates the function of a complex comprising NuMA1, CKAP5, β-tubulin and TACC3 for use in the treatment of cancer.
(50A) Use of compounds that regulate the function of complexes containing NuMA1 and CKAP5 in the manufacture of pharmaceuticals for the treatment of cancer.
(51A) The use of a compound according to (50A) that modifies the function of a complex containing NuMA1, CKAP5 and β-tubulin in the manufacture of a medicament for the treatment of cancer.
(52A) Use of a compound according to (50A) that regulates the function of a complex comprising NuMA1, CKAP5, β-tubulin and TACC3 in the manufacture of a medicament for the treatment of cancer.
　本発明により、がん細胞高選択的に細胞の増殖を妨げることが可能な化合物、がん細胞高選択的に細胞の増殖を妨げる方法、およびがん細胞高選択的に細胞の増殖を妨げることが可能な化合物のスクリ－ニング方法を提供することができる。 According to the present invention, a compound capable of hindering the growth of cancer cells highly selectively, a method of hindering the growth of cancer cells highly selectively, and a method of hindering the growth of cells highly selectively of cancer cells. Can provide a method for screening a compound capable of.
Compound A, B, C, D, Eの細胞増殖抑制効果を解析した図である。縦軸に細胞増殖率を示し、横軸に化合物の濃度を示す。It is the figure which analyzed the cell growth inhibitory effect of Compound A, B, C, D, E. The vertical axis shows the cell proliferation rate, and the horizontal axis shows the concentration of the compound. Compound A, B, Cの細胞周期に及ぼす影響をPI染色後FACSで解析した図である。縦軸に、細胞数カウントを示し、横軸にPI染色強度を示す。It is the figure which analyzed the influence of Compound A, B, and C on the cell cycle by FACS after PI staining. The vertical axis shows the cell number count, and the horizontal axis shows the PI staining intensity. Compound A, B, Cの紡錘体形成を細胞免疫染色により解析した図である。Compound A、B、Cは、単極性のスピンドル微小管（以下、monopolar spindleということもある。）を誘導している。It is the figure which analyzed the spindle formation of Compound A, B, C by cell immunostaining. Compounds A, B, and C induce unipolar spindle microtubules (hereinafter, also referred to as monopolar spindles). Compound Aのプルダウン実験およびLC－MS/MS解析により結合タンパクの同定結果を示した図である。縦軸に、Compound Dによる競合比率を示し、横軸に同定された各タンパク質を示す。It is the figure which showed the identification result of the binding protein by the pull-down experiment of Compound A and LC-MS / MS analysis. The vertical axis shows the competition ratio by Compound D, and the horizontal axis shows each protein identified. NuMA1ノックダウンがCompound BとEg5阻害剤Ispinesibの細胞増殖抑制効果に及ぼす影響を示した図である。縦軸に、細胞増殖率を示し、横軸に化合物濃度を示す。It is a figure which showed the effect of the NuMA1 knockdown on the cell growth inhibitory effect of Compound B and the Eg5 inhibitor Ispinesib. The vertical axis shows the cell proliferation rate, and the horizontal axis shows the compound concentration. Compound CもしくはEg5阻害剤Ispinesibを処理したとき、NuMA1と結合しているタンパクをLC－MS/MSで解析した結果を示した図である。縦軸に、Ispinesib処理によるNuMA1との結合上昇率を示し、横軸にCompound C処理によるNuMA1との結合上昇率を示す。It is a figure which showed the result of analyzing the protein which binds to NuMA1 by LC-MS / MS when treated with Compound C or Eg5 inhibitor Ispinesib. The vertical axis shows the rate of increase in binding with NuMA1 by Ispinesib treatment, and the horizontal axis shows the rate of increase in binding with NuMA1 by Compound C treatment. Compound BもしくはEg5阻害剤Ispinesibを処理したとき、NuMA1とCKAP5、TACC3間の結合をウエスタンブロットにより示した図である。It is a figure which showed the binding between NuMA1, CKAP5, and TACC3 by Western blotting when treated with Compound B or Eg5 inhibitor Ispinesib. Compound BもしくはEg5阻害剤Ispinesibを処理したとき、NuMA1およびCKAP5の細胞内局在を示した図である。Compound BおよびIspinesib処理のいずれもmonopolar spindleの形成が認められたが、Ispinesib処理によりNuMA1は中心体の周囲にリング状に局在し、CKAP5は明確な局在が認められなかったのに対し、Compound B処理によりNuMA1およびCKAP5は中心体近傍に強く共局在していた。It is a figure showing the intracellular localization of NuMA1 and CKAP5 when treated with Compound B or the Eg5 inhibitor Ispinesib. Monopolar spindle formation was observed in both Compound B and Ispine sib treatment, but NuMA1 was localized in a ring shape around the centrosome and CKAP5 was not clearly localized in the Ispine sib treatment. NuMA1 and CKAP5 were strongly co-localized near the centrosome by Compound B treatment. NuMA1、TACC3、およびCKAP5のノックダウンがCompound Aとβ－チューブリンの結合に及ぼす影響を示した図である。縦軸に、UV非照射サンプルに対するCompound Aとβ－チューブリンの結合比率を示し、横軸に各サンプルの処理内容を示す。It is a figure which showed the influence which the knockdown of NuMA1, TACC3, and CKAP5 has on the binding of Compound A and β-tubulin. The vertical axis shows the binding ratio of Compound A and β-tubulin to the UV non-irradiated sample, and the horizontal axis shows the processing content of each sample. Compound BもしくはEg5阻害剤Ispinesibで処理したときの微小管の伸長をEB1の免疫染色により示した図である。Compound B処理によりEB1のシグナルが顕著に減少し、中心体からの微小管伸長が抑制されている。It is the figure which showed the elongation of the microtubule when treated with Compound B or the Eg5 inhibitor Ispinesib by immunostaining of EB1. Compound B treatment markedly reduced the signal of EB1 and suppressed microtubule elongation from the centrosome. Compound BもしくはEg5阻害剤Ispinesibで処理したときの微小管の伸長をEB1の免疫染色の結果を画像解析してグラフ化した図である。Compound B処理によりEB1のシグナルが顕著に減少し、微小管伸長が抑制されている。It is the figure which image-analyzed the result of the immunostaining of EB1 and graphed the elongation of the microtubule when treated with Compound B or the Eg5 inhibitor Ispinesib. Compound B treatment markedly reduced the signal of EB1 and suppressed microtubule elongation.
以下、本発明の実施の形態の一例について詳細に説明するが、本発明は、これらに限定されない。 Hereinafter, examples of embodiments of the present invention will be described in detail, but the present invention is not limited thereto.
　本発明は、各種化合物を用いた実験により、細胞分裂時に中心体近傍においてNuMA1複合体が形成されること（実験結果は、図４、６、７および９に示される）、および当該NuMA1複合体によって微小管形成が制御されていること（実験結果は、図８および１０に示される）を見出し、NuMA1複合体を安定化することで細胞の増殖が抑制されることを見出したものである。 In the present invention, the NuMA1 complex is formed near the centrosome during cell division by experiments using various compounds (experimental results are shown in FIGS. 4, 6, 7 and 9), and the NuMA1 complex. It was found that microtubule formation was regulated by (experimental results are shown in FIGS. 8 and 10), and that stabilization of the NuMA1 complex suppressed cell proliferation.
より詳細には、細胞分裂時に中心体近傍においてNuMA1複合体を安定化させることにより、CKAP5の局在異常が引き起こされて中心体から伸びる微小管の伸長が阻害されることで中心体分離が阻害されて細胞増殖が抑制されるものである。 More specifically, by stabilizing the NuMA1 complex near the centrosome during cell division, abnormal localization of CKAP5 is caused and the elongation of microtubules extending from the centrosome is inhibited, thereby inhibiting centrosome separation. The cell proliferation is suppressed.
図１、２および３に示すとおり、NuMA1複合体を安定化することによって細胞増殖を抑制することができる。 As shown in FIGS. 1, 2 and 3, cell proliferation can be suppressed by stabilizing the NuMA1 complex.
　本発明において、「NuMA1」とは、NUMA1遺伝子によりコ－ドされるタンパク質でありnuclear mitotic apparatus protein 1とも呼ばれる。NUMA1はNCBIにGene ID:4926、RefSeq; NM_001286561.1およびNM_006185.3（タンパク質：RefSeq; NP_001273490.1およびNP_006176.2） として登録されている。NuMA1の機能としては、細胞分裂期に微小管に結合し紡錘体の形成および染色体の配列分配に関与することが知られている。 In the present invention, "NuMA1" is a protein coded by the NUMA1 gene and is also called nuclear mitotic apparatus protein 1. NUMA1 is registered in NCBI as Gene ID: 4926, RefSeq; NM_001286561.1 and NM_006185.3 (Protein: RefSeq; NP_001273490.1 and NP_006176.2). As a function of NuMA1, it is known that it binds to microtubules during cell division and is involved in spindle formation and chromosome sequence distribution.
　本発明において、「CKAP5」とは、CKAP5遺伝子によりコ－ドされるタンパク質であり、cytoskeleton associated protein 5とも呼ばれる。CKAP5はNCBIにGene ID:9793、RefSeq; NM_001008938.3およびNM_014756.3（タンパク質：RefSeq; NP_001008938.1およびNP_055571.2） として登録されている。CKAP5の機能としては、微小管のプラス端に結合し微小管形成を制御することが知られている。 In the present invention, "CKAP5" is a protein coded by the CKAP5 gene and is also called cytoskeleton associated protein5. CKAP5 is registered with NCBI as Gene ID: 9973, RefSeq; NM_001008938.3 and NM_014756.3 (protein: RefSeq; NP_001008938.1 and NP_055571.2). As a function of CKAP5, it is known to bind to the positive end of microtubules and control microtubule formation.
　本発明において、「TACC3」とは、TACC3遺伝子によりコ－ドされるタンパク質であり、transforming acidic coiled－coil containing protein 3とも呼ばれる。TACC3はNCBIにGene ID:10460、RefSeq; NM_006342.3（タンパク質：RefSeq; NP_006333.1） として登録されている。TACC3の機能としては、CKAP5やclathrinと複合体を形成し、微小管間を架橋することで紡錘体を安定化することが知られている。 In the present invention, "TACC3" is a protein coded by the TACC3 gene, and is also called transforming acidic coiled-coil containing protein 3. TACC3 is registered in NCBI as Gene ID: 10460, RefSeq; NM_006342.3 (protein: RefSeq; NP_006333.1). As a function of TACC3, it is known that it forms a complex with CKAP5 and clathrin and stabilizes the mitotic spindle by cross-linking between microtubules.
　本発明において、「β－チューブリン」とは、tubulin betaファミリ－全般のタンパク質を指し、具体的には、TUBB、TUBA1B、TUBB1、TUBB2A、TUBB2B、TUBB3、TUBB4A、TUBB4B、TUBB5、TUBB6、TUBB8等の遺伝子によりコ－ドされるタンパク質である。β－チューブリンの機能としては、微小管を形成し、細胞分裂および細胞内輸送を制御していることが知られている。 In the present invention, "β-tubulin" refers to proteins of the tubulin beta family in general, specifically, TUBB, TUBA1B, TUBB1, TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB4B, TUBB5, TUBB6, TUBB8, etc. It is a protein coded by the gene of. As a function of β-tubulin, it is known that it forms microtubules and controls cell division and intracellular transport.
　本発明において、「clathrin」とは、CLTC遺伝子によりコ－ドされるタンパク質であり、clathrin heavy chain 1とも呼ばれる。CLTCはNCBIにGene ID:1213、RefSeq; NM_004859.4（タンパク質：RefSeq; NP_004850.1） として登録されている。clathrinの機能としては、CKAP5やTACC3と複合体を形成し、微小管間を架橋することで紡錘体を安定化することが知られている。 In the present invention, "clathrin" is a protein coded by the CLTC gene and is also called clathrin heavy chain 1. CLTC is registered in NCBI as Gene ID: 1213, RefSeq; NM_004859.4 (protein: RefSeq; NP_004850.1). As a function of clathrin, it is known that it forms a complex with CKAP5 and TACC3 and stabilizes the mitotic spindle by cross-linking between microtubules.
　本発明において、「NuMA1複合体」とは、NuMA1およびCKAP5含む複合体を意味する。NuMA1およびCKAP5に加え、その他のタンパクを含む複合体も本発明のNuMA1複合体に含まれる。好ましくは、NuMA1、CKAP5およびβ－チューブリンを含む複合体である。より好ましくは、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体である。 In the present invention, the "NuMA1 complex" means a complex containing NuMA1 and CKAP5. In addition to NuMA1 and CKAP5, complexes containing other proteins are also included in the NuMA1 complex of the present invention. Preferably, it is a complex containing NuMA1, CKAP5 and β-tubulin. More preferably, it is a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
　本発明において、「NuMA1複合体の安定化」とは、NuMA1複合体の解離もしくは分解を抑制することを意味する。NuMA1複合体を安定化した結果、CKAP5の局在異常が引き起こされ、中心体から伸びる微小管の伸長を阻害し、細胞分裂を抑制する。NuMA1複合体が安定化しているかどうかは、例えば、単極性のスピンドル微小管（monopolar spindle）の形成が認められるかどうか、NuMA1およびCKAP5が共局在しているかどうか等によって確認することができる。 In the present invention, "stabilization of the NuMA1 complex" means suppressing dissociation or decomposition of the NuMA1 complex. As a result of stabilizing the NuMA1 complex, abnormal localization of CKAP5 is caused, inhibiting the elongation of microtubules extending from the centrosome and suppressing cell division. Whether or not the NuMA1 complex is stabilized can be confirmed by, for example, whether or not formation of monopolar spindle microtubules is observed, and whether or not NuMA1 and CKAP5 are co-localized.
　NuMA1複合体の安定化には、NuMA1とCKAP5が複合体を形成した後に化合物が複合体に結合してNuMA1複合体を安定化すること、および、NuMA1に化合物が結合した後にCAKP5がNuMA1と複合体を形成し、NuMA1複合体が安定化されることのいずれをも含む。 To stabilize the NuMA1 complex, the compound binds to the complex after NuMA1 and CKAP5 form a complex to stabilize the NuMA1 complex, and CAKP5 combines with NuMA1 after the compound binds to NuMA1. It includes both forming a body and stabilizing the NuMA1 complex.
　本発明において、「NuMA1複合体の機能を調整する」とは、NuMA1複合体の機能である、微小管を伸長させる機能を低下させることを意味する。NuMa1複合体の機能を調整することにより、細胞分裂が抑制される。NuMA1複合体の機能を制御すると言い換えることもできる。 In the present invention, "adjusting the function of the NuMA1 complex" means reducing the function of elongating microtubules, which is the function of the NuMA1 complex. By regulating the function of the NuMa1 complex, cell division is suppressed. In other words, it controls the function of the NuMA1 complex.
　NuMA1は、がん細胞に多く発現しており、例えば、前立腺がん、神経芽腫、小細胞肺がん、副腎がん、大腸がん、胃がん、頭頸部がん、食道がん、非小細胞肺がん、子宮頸がん、子宮体がん、肛門がん、膀胱がん、乳がん、脳腫瘍、卵巣がん、膵臓がん、黒色腫、甲状腺がん、軟部肉腫、白血病、悪性リンパ腫等に多く発現していることが知られている。また、NuMA1遺伝子が存在する11q13の染色体部位に遺伝子増幅があるがんについてもNuMA1が高発現していることが知られている。 NuMA1 is highly expressed in cancer cells, for example, prostate cancer, neuroblastoma, small cell lung cancer, adrenal cancer, colon cancer, gastric cancer, head and neck cancer, esophageal cancer, non-small cell lung cancer. , Cervical cancer, uterine body cancer, anal cancer, bladder cancer, breast cancer, brain tumor, ovarian cancer, pancreatic cancer, melanoma, thyroid cancer, soft sarcoma, leukemia, malignant lymphoma, etc. It is known that It is also known that NuMA1 is highly expressed in cancers with gene amplification at the chromosomal site of 11q13 where the NuMA1 gene is present.
　NuMA1は、公知の方法、例えば、免疫組織染色や次世代シ－クエンスによるRNA－Seq解析のような方法で確認することができる。また、遺伝子増幅に関しては、FISH法のような方法で確認することができる。 NuMA1 can be confirmed by a known method, for example, immunohistochemical staining or RNA-Seq analysis by next-generation sequence. In addition, gene amplification can be confirmed by a method such as the FISH method.
　本明細書において、「Compound A」とは、（－）－１１－（４－エチニル－２，６－ジフルオロベンゾイル）－１４－［（４－メトキシフェニル）メチル］－１，２，７，１１，１４－ペンタアザトリスピロ［２．２．２．４９．２６．２３］ヘプタデク－１－エン－８，１５－ジオンである。 In the present specification, "Compound A" means (-)-11- (4-ethynyl-2,6-difluorobenzoyl)-14-[(4-methoxyphenyl) methyl] -1,2,7,11. , 14-Pentaazatrispyro [2.2.2.4 9 . 26 . A 2 3] Heputadeku 1-ene -8,15- dione.
　本明細書において、「Compound B」とは、５－［（４－クロロ－３－フルオロフェニル）メチル］－２－［７－（１，１－ジフルオロエチル）キナゾリン－４－イル］－１０，１０－ジフルオロ－２，５，１３－トリアザジスピロ［３．２．５７．２４］テトラデカン－６，１４－ジオンである。 As used herein, "Compound B" refers to 5-[(4-chloro-3-fluorophenyl) methyl] -2- [7- (1,1-difluoroethyl) quinazoline-4-yl] -10, 10-Difluoro-2,5,13-Triazadispiro [3.2.5 7 . It is a 2 4] tetradecane -6,14- dione.
　本明細書において、「Compound C」とは、（－）－２－（４－クロロ－２，６－ジフルオロベンゾイル）－６－［（４－クロロ－３－フルオロフェニル）メチル］－１１，１１－ジフルオロ－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－７，１５－ジオンである。 In the present specification, "Compound C" means (-)-2- (4-chloro-2,6-difluorobenzoyl) -6-[(4-chloro-3-fluorophenyl) methyl] -11,11. -Difluoro-2,6,14-Triazadispiro [4.2.5 8 . 25 ] Pentadecane-7,15-dione.
　本明細書において、「Compound D」とは、２－（４－クロロ－２，６－ジフルオロベンゾイル）－１１，１１－ジフルオロ－６－［（４－メトキシフェニル）メチル］－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－７，１５－ジオンである。 As used herein, "Compound D" refers to 2- (4-chloro-2,6-difluorobenzoyl) -11,11-difluoro-6-[(4-methoxyphenyl) methyl] -2,6,14. -Triazadispiro [4.2.5 8 . 25 ] Pentadecane-7,15-dione.
　本明細書において、「Compound E」とは、（５Ｒ，８Ｒ）－２－（２－アミノ－６－フルオロ－４－メチルベンゾイル）－８－シクロヘキシル－６－［（４－メトキシフェニル）メチル］－２，６，９－トリアザスピロ［４．５］デカン－７，１０－ジオンである。 In the present specification, "Compound E" means (5R, 8R) -2- (2-amino-6-fluoro-4-methylbenzoyl) -8-cyclohexyl-6-[(4-methoxyphenyl) methyl]. -2,6,9-Triazaspiro [4.5] Decane-7,10-dione.
　本明細書において、「Ispinesib」とは、N－（３－アミノプロピル）－N－［（１Ｒ）－１－［７－クロロ－４－オキソ－３－（フェニルメチル）－２－キナゾリニル］－２－メチルプロピル］－４－メチルベンズアミドである。Ispinesibは、Bioorganic & Medicinal Chemistry 2013, 21, 496－507に記載の方法で製造することができる。IspinesibはEg5阻害剤に分類される薬剤である。 In the present specification, "Ispinesib" means N- (3-aminopropyl) -N-[(1R) -1- [7-chloro-4-oxo-3- (phenylmethyl) -2-quinazolinyl]-. 2-Methylpropyl] -4-methylbenzamide. Ispinesib can be produced by the method described in Bioorganic & Medicinal Chemistry 2013, 21, 496-507. Ispinesib is a drug that is classified as an Eg5 inhibitor.
　本明細書において、「Volasertib」とは、Ｎ－［トランス－４－［４－（シクロプロピルメチル）－１－ピペラジニル］シクロヘキシル］－４－［［（７Ｒ）－７－エチル－５，６，７，８－テトラヒドロ－５－メチル－８－（１－メチルエチル）－６－オキソ－２－プテリジニル］アミノ］－３－メトキシベンズアミドである。Volasertibは、例えばWO2007090844に記載の方法で製造することができる。VolasertibはPlk1阻害剤に分類される薬剤である。 In the present specification, "Volasertib" means N- [trans-4- [4- (cyclopropylmethyl) -1-piperazinyl] cyclohexyl] -4-[[(7R) -7-ethyl-5,6). 7,8-Tetrahydro-5-methyl-8- (1-methylethyl) -6-oxo-2-pteridinyl] amino] -3-methoxybenzamide. Volasertib can be produced, for example, by the method described in WO2007090844. Volasertib is a drug classified as a Plk1 inhibitor.
　本明細書において、「Alisertib」とは、４－［［９－クロロ－７－（２－フルオロ－６－メトキシフェニル）－５Ｈ－ピリミド［５，４－ｄ］［２］ベンズアゼピン－２－イル］アミノ］－２－メトキシ安息香酸である。Alisertibは、例えばACS Medicinal Chemistry Letters 2015, 6, 630-634に記載の方法で製造することができる。AlisertibはAurora A kinase阻害剤に分類される薬剤である。 As used herein, "Alisertib" refers to 4-[[9-chloro-7- (2-fluoro-6-methoxyphenyl) -5H-pyrimid [5,4-d] [2] benzazepin-2-yl. ] Amino] -2-methoxybenzoic acid. Alisertib can be produced, for example, by the method described in ACS Medicinal Chemistry Letters 2015, 6, 630-634. Alisertib is a drug classified as an Aurora A kinase inhibitor.
　本発明において、「Eg5阻害剤」とは、KIF11（kinesin family member 11）遺伝子がコ－ドするタンパク質であるEg5のATPaseモ－タ－活性を阻害する化合物のことである。Eg5阻害剤は、中心体の分離を阻害し細胞周期をM期で停止させるが、微小管の機能は阻害しない。 In the present invention, the "Eg5 inhibitor" is a compound that inhibits the ATPase motor activity of Eg5, which is a protein coded by the KIF11 (kinesin family member 11) gene. Eg5 inhibitors inhibit centrosome separation and arrest the cell cycle in the M phase, but do not inhibit microtubule function.
　本明細書において、「Plk1阻害剤」とは、PLK1（polo like kinase 1）遺伝子がコ－ドするタンパク質であるPlk1のキナーゼ活性を阻害する化合物のことである。Plk1阻害剤は、細胞周期をM期で停止させる作用を持つ。 In the present specification, the "Plk1 inhibitor" is a compound that inhibits the kinase activity of Plk1, which is a protein coded by the PLK1 (pololike kinase 1) gene. Plk1 inhibitors have the effect of arresting the cell cycle in the M phase.
　本明細書において、「Aurora A kinase阻害剤」とは、AURKA（aurora kinase A）遺伝子がコ－ドするタンパク質であるAurora Aのキナーゼ活性を阻害する化合物のことである。Aurora A kinase阻害剤は、細胞周期をM期で停止させる作用を持つ。 In the present specification, the "Aurora A kinase inhibitor" is a compound that inhibits the kinase activity of Aurora A, which is a protein coded by the AURKA (aurora kinase A) gene. Aurora A kinase inhibitors have the effect of arresting the cell cycle in the M phase.
　本明細書において、「Vincristine」とは、微小管重合阻害作用を持つ抗がん剤である。細胞周期をM期で停止させる作用を持つ。 In the present specification, "Vincristine" is an anticancer agent having an inhibitory effect on microtubule polymerization. It has the effect of stopping the cell cycle in the M phase.
本明細書において、「Paclitaxel」とは、微小管脱重合阻害作用を持つ抗がん剤である。細胞周期をM期で停止させる作用を持つ。 In the present specification, "Paclitaxel" is an anticancer agent having an inhibitory effect on microtubule depolymerization. It has the effect of stopping the cell cycle in the M phase.
　本明細書において、「がん」とは全ての悪性腫瘍を意味する。 In the present specification, "cancer" means all malignant tumors.
がんは、「固形がん」と「血液がん」に分類することができる。固形がんは、「上皮細胞がん」と「非上皮細胞がん」に分類することができる。上皮細胞がんは、上皮細胞から発生するがんであり、例えば、肺がん、胃がん、肝臓がん、腎臓がん、前立腺がん、膵臓がん、大腸がん、乳がん、神経芽腫、副腎がん、頭頸部がん、食道がん、子宮頸がん、子宮体がん、肛門がん、膀胱がん、脳腫瘍、卵巣がん、黒色腫、および甲状腺がん等が挙げられる。非上皮細胞がんは、骨や筋肉などの非上皮細胞から発生するがんであり、例えば、軟部肉腫、骨肉腫、軟骨肉腫、および横紋筋肉腫等が挙げられる。血液がんは、造血器から発生するがんであり、例えば、悪性リンパ腫、白血病、多発性骨髄腫等に分類することができる。 Cancer can be classified into "solid cancer" and "blood cancer". Solid tumors can be classified into "epithelial cell carcinoma" and "non-epithelial cell carcinoma". Epithelial cell carcinoma is a cancer that begins in epithelial cells, such as lung cancer, stomach cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, colon cancer, breast cancer, neuroblastoma, and adrenal cancer. , Head and neck cancer, esophageal cancer, cervical cancer, uterine body cancer, anal cancer, bladder cancer, brain tumor, ovarian cancer, melanoma, thyroid cancer and the like. Non-epithelial cell carcinoma is a cancer that develops from non-epithelial cells such as bone and muscle, and examples thereof include soft tissue sarcoma, osteosarcoma, chondrosarcoma, and rhabdomyosarcoma. Hematological cancer is a cancer that develops from a hematopoietic organ and can be classified into, for example, malignant lymphoma, leukemia, multiple myeloma and the like.
  次に本発明の好適な態様について説明する。 Next, a preferred embodiment of the present invention will be described.
本発明の好適な一態様としては、NuMA1複合体を安定化させる方法である。NuMA1複合体を安定化させることにより、細胞分裂が抑制されて細胞の増殖が抑制される。 A preferred embodiment of the present invention is a method of stabilizing the NuMA1 complex. By stabilizing the NuMA1 complex, cell division is suppressed and cell proliferation is suppressed.
本発明において、NuMA1複合体を安定化する方法は特に限定されず、例えば、低分子化合物、ペプチド等を用いることができる。また、NuMA1複合体を安定化する際、NuMA1およびCKAP5に加え、同時に複合体を形成するその他のタンパクにも作用してNuMA1複合体を安定化させてもよい。 In the present invention, the method for stabilizing the NuMA1 complex is not particularly limited, and for example, low molecular weight compounds, peptides and the like can be used. In addition, when stabilizing the NuMA1 complex, in addition to NuMA1 and CKAP5, it may also act on other proteins forming the complex to stabilize the NuMA1 complex.
　本発明の方法は、NuMA1が高発現している細胞に用いるのが好ましく、がん細胞に用いるのが特に好ましい。NuMA1が高発現しているがんとしては、前立腺がん、神経芽腫、小細胞肺がん、副腎がん、大腸がん、胃がん、頭頸部がん、食道がん、非小細胞肺がん、子宮頸がん、子宮体がん、肛門がん、膀胱がん、乳がん、脳腫瘍、卵巣がん、膵臓がん、黒色腫、甲状腺がん、軟部肉腫、白血病、悪性リンパ腫等が知られている。 The method of the present invention is preferably used for cells in which NuMA1 is highly expressed, and particularly preferably for cancer cells. Cancers with high expression of NuMA1 include prostate cancer, neuroblastoma, small cell lung cancer, adrenal cancer, colon cancer, gastric cancer, head and neck cancer, esophageal cancer, non-small cell lung cancer, and cervix. Cancer, uterine body cancer, anal cancer, bladder cancer, breast cancer, brain tumor, ovarian cancer, pancreatic cancer, melanoma, thyroid cancer, soft sarcoma, leukemia, malignant lymphoma and the like are known.
　本発明の別な好適な一態様としては、NuMA1複合体を安定化する化合物である。 Another preferred embodiment of the present invention is a compound that stabilizes the NuMA1 complex.
本発明のNuMA1複合体を安定化する化合物は、NuMA1複合体を安定化できる物質であれば特に限定されず、例えば、低分子化合物、ペプチド等である。また、当該物質がNuMA1複合体を安定化する際、NuMA1およびCKAP5に加え、同時に複合体を形成するその他のタンパクにも作用してNuMA1複合体を安定化させてもよい。 The compound that stabilizes the NuMA1 complex of the present invention is not particularly limited as long as it is a substance that can stabilize the NuMA1 complex, and is, for example, a low molecular weight compound, a peptide, or the like. In addition, when the substance stabilizes the NuMA1 complex, it may act on NuMA1 and CKAP5 as well as other proteins forming the complex at the same time to stabilize the NuMA1 complex.
　NuMA1複合体が解離もしくは分解すると、CKAP5の働きにより微小管が伸長して紡錘体を形成する。CAKP5の働きが阻害されるとmonopolar spindleが形成されるため、本発明のNuMA1複合体を安定化する化合物は、monopolar spindleを誘導する物質であるということができる。 When the NuMA1 complex is dissociated or decomposed, microtubules are elongated by the action of CKAP5 to form a spindle. Since a monopolar spindle is formed when the action of CAKP5 is inhibited, it can be said that the compound that stabilizes the NuMA1 complex of the present invention is a substance that induces a monopolar spindle.
　Monopolar spindleの状態に誘導されると、正常な染色体の分配が阻害され、結果として細胞の分裂と増殖が阻害される。 When induced to the state of Monopolar spindle, normal chromosome distribution is inhibited, and as a result, cell division and proliferation are inhibited.
　本発明のNuMA1複合体を安定化する化合物は、NuMA1が高発現している疾患の治療に用いるのが好ましく、NuMA1が高発現しているがんの治療に用いるのが特に好ましい。 The compound that stabilizes the NuMA1 complex of the present invention is preferably used for the treatment of diseases in which NuMA1 is highly expressed, and particularly preferably for the treatment of cancers in which NuMA1 is highly expressed.
　本発明のNuMA1複合体を安定化する化合物の患者への投与は、経口的な投与でも非経口的な投与でも可能である。非経口的な投与としては、例えば、静脈投与、動脈投与、筋肉内投与、胸腔内投与、腹腔内投与、標的部位（例えば、がん）への直接投与などが挙げられる。 The compound that stabilizes the NuMA1 complex of the present invention can be administered to a patient either orally or parenterally. Parenteral administration includes, for example, intravenous administration, arterial administration, intramuscular administration, intrathoracic administration, intraperitoneal administration, direct administration to a target site (for example, cancer), and the like.
投与量は、目的の疾患を治療するのに有効な量であれば特に制限はなく、患者の年齢、体重、症状、健康状態、疾患の進行状況などに応じて、適宜選択すればよい。投与頻度としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、１日あたりの投与量を、１日に１回で投与してもよいし、複数回に分けて投与してもよい。本発明の薬剤をヒトに投与する場合、有効成分の投与量の範囲は、１日当たり、通常、約０．０１ｍｇ／ｋｇ体重～約５００ｍｇ/ｋｇ体重、好ましくは、約０．１ｍｇ／ｋｇ体重～約１００ｍｇ／ｋｇ体重である。ヒトに投与する場合、好ましくは、１日あたり１回、あるいは２～４回に分けて投与され、適当な間隔で繰り返すことが好ましい。 The dose is not particularly limited as long as it is an effective amount for treating the target disease, and may be appropriately selected depending on the age, weight, symptom, health condition, progress of the disease, and the like of the patient. The frequency of administration is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the daily dose may be administered once a day or divided into a plurality of doses. You may. When the agent of the present invention is administered to humans, the dose range of the active ingredient is usually from about 0.01 mg / kg body weight to about 500 mg / kg body weight, preferably from about 0.1 mg / kg body weight per day. It is about 100 mg / kg body weight. When administered to humans, it is preferably administered once a day or in 2 to 4 divided doses, and repeated at appropriate intervals.
　本発明の別の好適な一態様としては、NuMA1複合体を安定化する化合物のスクリ－ニング方法である。 Another preferred embodiment of the present invention is a method for screening a compound that stabilizes the NuMA1 complex.
　NuMA1複合体を安定化する化合物のスクリ－ニング方法としては、例えば、以下の２工程からなるスクリ－ニング方法が挙げられる。
（a）試験物質をNuMA1およびCKAP5を含む複合体と接触させる工程、および（b）試験物質によりNuMA1およびCKAP5を含む複合体が安定化されているか否かを確認する工程。 Examples of the method for screening the compound that stabilizes the NuMA1 complex include a screening method consisting of the following two steps.
(A) A step of contacting the test substance with the complex containing NuMA1 and CKAP5, and (b) a step of confirming whether or not the complex containing NuMA1 and CKAP5 is stabilized by the test substance.
　上記スクリーニング法に用いるNuMA1タンパクおよびCKAP5タンパクは、全長または部分長のいずれも用いることができる。また、NuMA1およびCKAP5に加えて、チューブリンダイマーもしくは微小管を同時に混合することが好ましい。 The NuMA1 protein and CKAP5 protein used in the above screening method can be either full length or partial length. In addition to NuMA1 and CKAP5, it is preferable to mix tubulin dimers or microtubules at the same time.
　NuMA1複合体を安定化する化合物のスクリ－ニング方法の別の態様としては、例えば、以下の３工程からなるスクリ－ニング方法が挙げられる。
（a）NuMA1の発現量が高い細胞およびNuMA1の発現量が低い細胞のそれぞれに試験物質を接触させる工程、（b）それぞれの細胞における化合物の細胞増殖抑制作用および／または細胞分裂阻害作用を測定する工程、および（c）NuMA1の発現量の高い細胞における細胞増殖抑制作用の方がNuMA1の発現量の低い細胞における細胞増殖抑制作用よりも高い場合に、当該化合物をNuMA1およびCKAP5を含む複合体を安定化する試験物質であると同定する工程。 Another embodiment of the method for screening a compound that stabilizes the NuMA1 complex includes, for example, a screening method consisting of the following three steps.
(A) A step of contacting a test substance with cells having a high NuMA1 expression level and a cell having a low NuMA1 expression level, and (b) measuring the cell growth inhibitory effect and / or the cell division inhibitory effect of the compound in each cell. And (c) a complex containing NuMA1 and CKAP5 when the cell growth inhibitory effect in cells with high NuMA1 expression is higher than the cell growth inhibitory effect in cells with low NuMA1 expression. The process of identifying a test substance that stabilizes.
　さらに、本発明の別の好適な一態様としては、NuMA1複合体を安定化する化合物を用いた、NuMA1が高発現しているがんの治療方法である。 Furthermore, another preferred embodiment of the present invention is a method for treating cancer in which NuMA1 is highly expressed, using a compound that stabilizes the NuMA1 complex.
　以下、実施例を挙げて、本発明をさらに詳細に説明するが、本発明の範囲はこれら特定の化合物に限定されるものではなく、これらはいかなる意味においても限定的に解釈されない。また、本明細書において、特に記載のない試薬、溶媒および出発材料は、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ、東京化成工業等の市販の供給源から容易に入手可能である。 Hereinafter, the present invention will be described in more detail with reference to examples, but the scope of the present invention is not limited to these specific compounds, and these are not interpreted in a limited manner in any sense. In addition, reagents, solvents and starting materials not otherwise described herein are readily available from commercially available sources such as Sigma-Aldrich and Tokyo Chemical Industry.
　実施例のカラムクロマトグラフィ－における溶出はＴＬＣ（Ｔｈｉｎ　Ｌａｙｅｒ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ，薄層クロマトグラフィ－）による観察下に行われた。ＴＬＣ観察においては、ＴＬＣプレ－トとしてメルク（Ｍｅｒｃｋ）社製のシリカゲル６０Ｆ２５４またはシリカゲル６０ＮＨ２Ｆ２５４Ｓを、展開溶媒としてはカラムクロマトグラフィ－で溶出溶媒として用いられた溶媒を、検出法としてＵＶ検出器を採用した。カラム用シリカゲルは同じくメルク社製のシリカゲルＳＫ－８５（２３０－４００メッシュ）、もしくは富士シリシア化学Ｃｈｒｏｍａｔｏｒｅｘ　ＮＨ（２００－３５０メッシュ）を用いた。通常のカラムクロマトグラフィ－の他に、昭光サイエンス社の自動精製装置（Ｐｕｒｉｆ）もしくはバイオタ－ジ社の自動精製装置（ＨＯＲＩＺＯＮ，ＳＰ１もしくはＩｓｏｌｅｒａ）、またそのカラムカ－トリッジとして昭光サイエンス社製Ｐｕｒｉｆ－Ｐａｃｋシリ－ズ各種、バイオタ－ジ社製ＳＮＡＰカ－トリッジシリ－ズ各種を適宜使用した。溶出溶媒は各参考例および実施例で指定した溶媒を用いた。なお、参考例および実施例で用いる略号は、次のような意義を有する。
ｍｇ：ミリグラム，　ｇ：グラム，　μｌ：マイクロリットル，　ｍｌ：ミリリットル，　ｌ：リットル，　ＭＨｚ：メガヘルツ。
　以下の参考例および実施例において、核磁気共鳴（以下、１Ｈ　ＮＭＲ：４００ＭＨｚ）スペクトルは、テトラメチルシランを標準物質として、ケミカルシフト値をδ値（ｐｐｍ）にて記載した。分裂パタ－ンは一重線をｓ、二重線をｄ、三重線をｔ、多重線をｍ、ブロ－ドをｂｒで示した。
ＣＤＣｌ３：　重クロロホルム、ＣＤ３ＯＤ：　重メタノ－ル、ＤＭＳＯ－ｄ６：　重ジメチルスルホキシド。実施例中、特に断りのない限り、ヘキサンはｎ－ヘキサンを意味する。 The elution in the column chromatography of the example was carried out under observation by TLC (Thin Layer Chromatography, thin layer chromatography). In the TLC observation, TLC pre - UV and solvent used as an elution solvent, as a detection method - bets as Merck (Merck) manufactured by silica gel 60F 254 or silica gel 60NH 2 F 254 S, column chromatography as a developing solvent A detector was adopted. As the silica gel for the column, silica gel SK-85 (230-400 mesh) also manufactured by Merck & Co. Ltd. or Fuji Silysia Chemical Chromatolex NH (200-350 mesh) was used. In addition to normal column chromatography, Shoko Science's automatic purification device (Purif) or Biotage's automatic purification device (HORIZON, SP1 or Isolera), and its column cartridge, Shoko Science's Purif-Pack series. -Various types of SNAP cartridge series manufactured by Biotage Co., Ltd. were used as appropriate. As the elution solvent, the solvent specified in each Reference Example and Example was used. The abbreviations used in Reference Examples and Examples have the following meanings.
mg: milligram, g: gram, μl: microliter, ml: milliliter, l: liter, MHz: megahertz.
In the following Reference Examples and Examples, the nuclear magnetic resonance (hereinafter, 1 H NMR: 400 MHz) spectrum is described with a chemical shift value of δ value (ppm) using tetramethylsilane as a standard substance. The split pattern is indicated by s for the single line, d for the double line, t for the triple line, m for the multiple line, and br for the broad line.
CDCl 3 : Deuterated chloroform, CD 3 OD: Deuterated methanol, DMSO-d 6 : Deuterated dimethyl sulfoxide. In the examples, hexane means n-hexane unless otherwise noted.
（実施例１）Compound Aの製造 (Example 1) Manufacture of Compound A
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
［工程１］　ベンジル　1－［(ｔｅｒｔ－ブトキシカルボニル)アミノ］－４－オキソシクロヘキサン－１－カルボキシレ－ト
　１－［(ｔｅｒｔ－ブトキシカルボニル)アミノ］－４－オキソシクロヘキサン－１－カルボン酸（１０．０ｇ，　３８．９ｍｍｏｌ）のジクロロメタン（１００ｍｌ）溶液に、氷冷、窒素気流下、ベンジルアルコ－ル（５．２０ｍｌ，　５０．６ｍｍｏｌ）、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩（９．７０ｇ，　５０．６ｍｍｏｌ）、４－ジメチルアミノピリジン（４７５ｍｇ，　３．８９ｍｍｏｌ）、およびトリエチルアミン（７．３０ｍｌ，　５０．６ｍｍｏｌ）を加えて、室温で４日間撹拌した。反応液をジクロロメタンで希釈し、水で洗浄した。無水硫酸マグネシウムで乾燥し、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝３０：７０－５０：５０）で精製して、標記化合物（１１．２ｇ，　３２．３ｍｍｏｌ，　８３％）を得た。
1H－NMR (CDCl3) δ : 7.41－7.32 (5H, m), 5.21 (2H, s), 4.95 (1H, br s), 2.55－2.32 (8H, m), 1.44 (9H, s). [Step 1] Benzyl 1-[(tert-butoxycarbonyl) amino] -4-oxocyclohexane-1-carboxylate 1-[(tert-butoxycarbonyl) amino] -4-oxocyclohexane-1-carboxylic acid ( In a solution of 10.0 g, 38.9 mmol) in dichloromethane (100 ml), ice-cooled, under a nitrogen stream, benzyl alcohol (5.20 ml, 50.6 mmol), 1-ethyl-3- (3-dimethylaminopropyl). Carbodiimide hydrochloride (9.70 g, 50.6 mmol), 4-dimethylaminopyridine (475 mg, 3.89 mmol), and triethylamine (7.30 ml, 50.6 mmol) were added and stirred at room temperature for 4 days. The reaction was diluted with dichloromethane and washed with water. The mixture was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 30: 70-50: 50) to obtain the title compound (11.2 g, 32.3 mmol, 83%).
1 H-NMR (CDCl 3 ) δ: 7.41-7.32 (5H, m), 5.21 (2H, s), 4.95 (1H, br s), 2.55-2.32 (8H, m), 1.44 (9H, s).
［工程２］　ベンジル　６－［(ｔｅｒｔ－ブトキシカルボニル)アミノ］－１,４－ジアザスピロ［２．５］オクタ－１－エンカルボキシレ－ト
　上記工程１で得られた化合物（１１．２ｇ，　３２．３ｍｍｏｌ）のメタノ－ル（２２５ｍｌ）溶液に、氷冷、窒素気流下、２ｍｏｌ／ｌアンモニア－メタノ－ル溶液（６７５ｍｌ）を加え２時間撹拌後、ヒドロキシアミン－Ｏ－スルホン酸（５．０５ｇ，　４４．７ｍｍｏｌ）のメタノ－ル（４５ｍｌ）溶液を加え、室温にて１６時間撹拌した。減圧下反応液のアンモニアを留去後、氷冷、窒素気流下、トリエチルアミン（１３．５ｍｌ,　９３．６ｍｍｏｌ）、ヨウ素（１１．２ｇ，　４３.８ｍｍｏｌ）を加え１時間撹拌した。反応液をジクロロメタンで希釈し、飽和チオ硫酸ナトリウム水溶液と水で洗浄した。無水硫酸マグネシウムで乾燥し、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝５：９５－４０：６０）で精製して、標記化合物（７．６６ｇ，　２１．３ｍｍｏｌ，　６６％）を得た。
1H－NMR (CDCl3) δ : 7.40－7.35(5H, m), 5.21 (2H, s), 4.79 (1H, bs), 2.32－2.12 (4H, m), 1.87－1.72 (2H, m), 1.44(9H, s), 0.90－0.75 (2H, m). [Step 2] benzyl 6-[(tert-butoxycarbonyl) amino] -1,4-diazaspiro [2.5] octa-1-encarboxylate The compound obtained in the above step 1 (11.2 g, 32). Hydroxylamine-O-sulfonic acid (5.05 g) was added to a 3 mmol) metanol (225 ml) solution under ice-cooled and nitrogen stream, and a 2 mol / l ammonia-methanol solution (675 ml) was added and stirred for 2 hours. , 44.7 mmol) of a methanol solution (45 ml) was added, and the mixture was stirred at room temperature for 16 hours. After distilling off ammonia in the reaction solution under reduced pressure, triethylamine (13.5 ml, 93.6 mmol) and iodine (11.2 g, 43.8 mmol) were added under ice-cooling and a nitrogen stream, and the mixture was stirred for 1 hour. The reaction mixture was diluted with dichloromethane and washed with saturated aqueous sodium thiosulfate solution and water. The mixture was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 5: 95-40: 60) to obtain the title compound (7.66 g, 21.3 mmol, 66%).
1 H-NMR (CDCl 3 ) δ: 7.40-7.35 (5H, m), 5.21 (2H, s), 4.79 (1H, bs), 2.32-2.12 (4H, m), 1.87-1.72 (2H, m) , 1.44 (9H, s), 0.90-0.75 (2H, m).
［工程３］　６－［(ｔｅｒｔ－ブトキシカルボニル)アミノ］－１,４－ジアザスピロ［２．５］オクタ－１－エンカルボン酸
　上記工程２で得られた化合物（７．６６ｇ，　２１．３ｍｍｏｌ）のテトラヒドロフラン（８０ｍｌ）、メタノ－ル（５０ｍｌ）および水（１０ｍｌ）の混合溶液に、水酸化リチウム一水和物（１．４０ｇ，　３３．４ｍｍｏｌ）を加え、３日間撹拌した。反応液をジエチルエ－テルで洗浄し、水層を１規定塩酸水溶液で酸性とし、ジクロロメタンで希釈し、有機層を１０％クエン酸水溶液と水で洗浄した。無水硫酸マグネシウムで乾燥し、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝２５：７５－７０：３０）で精製して、標記化合物（４．１６ｇ，　１５．５ｍｍｏｌ，　７３％）を得た。
1H－NMR (DMSO－d6) δ : 7.20 (1H, br s), 2.18－1.99 (2H, m), 1.93－1.74 (4H, m), 1.39 (9H, s), 0.65－0.52 (2H, m). [Step 3] 6-[(tert-butoxycarbonyl) amino] -1,4-diazaspiro [2.5] octa-1-encarboxylic acid The compound obtained in the above step 2 (7.66 g, 21.3 mmol). Lithium hydroxide monohydrate (1.40 g, 33.4 mmol) was added to a mixed solution of tetrahydrofuran (80 ml), methanol (50 ml) and water (10 ml), and the mixture was stirred for 3 days. The reaction mixture was washed with diethyl ether, the aqueous layer was acidified with 1N aqueous hydrochloric acid solution, diluted with dichloromethane, and the organic layer was washed with 10% aqueous citric acid solution and water. The mixture was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 25: 75-70: 30) to obtain the title compound (4.16 g, 15.5 mmol, 73%).
1 H-NMR (DMSO-d 6 ) δ: 7.20 (1H, br s), 2.18-1.99 (2H, m), 1.93-1.74 (4H, m), 1.39 (9H, s), 0.65-0.52 (2H) , m).
［工程４］　ｔｅｒｔ－ブチル　１４－［(４－メトキシフェニル)メチル］－８，１５－ジオキソ－１，２，７，１１，１４－ペンタアザトリスピロ［２．２．２．４９．２６．２３］ヘプタデク－１－エン－１１－カルボキシレ－ト
　１－（４－メトキシフェニル）メタナミン（１．０３ｇ，　７．５１ｍｍｏｌ）、ｔｅｒｔ－ブチル　３－オキソピロリジン－１－カルボキシレ－ト（１．３８ｇ，　７．４５ｍｍｏｌ）のメタノ－ル（３．５ｍｌ）溶液を窒素気流下、室温で３０分間撹拌した。４－（２－イソシアノエチル）モルホリン（１．０５ｇ，　７．４９ｍｍｏｌ）のメタノ－ル（７．６ｍｌ）溶液、および上記工程３で得られた化合物（２．００ｇ，　７．４４ｍｍｏｌ）を加え、外温５０℃で８時間撹拌した。反応液をメタノ－ル（３２ｍｌ）で希釈し、氷冷下、クロロトリメチルシラン（１０．０ｍｌ,　７９．２ｍｍｏｌ）を加え室温にて１６時間撹拌した。さらに、反応液にクロロトリメチルシラン（５．００ｍｌ，　３９．６ｍｍｏｌ）を加え２時間撹拌した。減圧下反応溶媒を留去し、減圧下乾燥した。残留物をスパ－テルで粉状としテトラヒドロフラン（５０ｍｌ）を加え、氷冷下、トリエチルアミン（１０．０ｍｌ，　６９．３ｍｍｏｌ）を加え、窒素気流下、外温５０℃で４時間撹拌した。室温に戻した後、ジ－ｔｅｒｔ－ブチルジカ－ボネ－ト（４．００ｇ，　１８．３ｍｍｏｌ）を加え、室温で１６時間撹拌した。反応液をジクロロメタンで希釈し、１０％クエン酸水溶液と水で洗浄した。無水硫酸マグネシウムで乾燥し、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝５０：５０－７５：２５）で精製して、標記化合物（６７２ｍｇ，　１．３９ｍｍｏｌ，　１９％）を得た。
1H－NMR (DMSO－d6) δ : 8.80 (1H, s), 7.13－6.85 (4H, m), 4.78－4.38 (4H, m), 3.72 (3H, s), 3.67－3.27 (2H, m), 2.35－2.15 (4H, m), 1.92－1.67 (4H, m), 1.49－1.24 (9H, m), 1.16－1.01 (2H, m).  [Step 4] tert-Butyl 14-[(4-Methoxyphenyl) methyl] -8,15-dioxo-1,2,7,11,14-pentaazatrispyro [2.2.2.4 9 . 26 . 2 3] Heputadeku-1-ene-11-carboxylate - DOO 1- (4-methoxyphenyl) methanamine (1.03 g, 7.51 mmol), tert-butyl 3-oxopyrrolidine-1-carboxylate - DOO (1 A solution of .38 g, 7.45 mmol) of metanol (3.5 ml) was stirred at room temperature for 30 minutes under a nitrogen stream. A solution of 4- (2-isocyanoethyl) morpholine (1.05 g, 7.49 mmol) in metanol (7.6 ml) and the compound obtained in step 3 above (2.00 g, 7.44 mmol) were added. The mixture was stirred at an outside temperature of 50 ° C. for 8 hours. The reaction mixture was diluted with methanol (32 ml), chlorotrimethylsilane (10.0 ml, 79.2 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 16 hours. Further, chlorotrimethylsilane (5.00 ml, 39.6 mmol) was added to the reaction solution, and the mixture was stirred for 2 hours. The reaction solvent was distilled off under reduced pressure, and the mixture was dried under reduced pressure. The residue was powdered with a spartel, tetrahydrofuran (50 ml) was added, triethylamine (10.0 ml, 69.3 mmol) was added under ice-cooling, and the mixture was stirred under a nitrogen stream at an outside temperature of 50 ° C. for 4 hours. After returning to room temperature, di-tert-butyl dicarbonate (4.00 g, 18.3 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with dichloromethane and washed with 10% aqueous citric acid solution and water. The mixture was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 50: 50-75: 25) to give the title compound (672 mg, 1.39 mmol, 19%).
1 H-NMR (DMSO-d 6 ) δ: 8.80 (1H, s), 7.13-6.85 (4H, m), 4.78-4.38 (4H, m), 3.72 (3H, s), 3.67-3.27 (2H, m), 2.35-2.15 (4H, m), 1.92–1.67 (4H, m), 1.49–1.24 (9H, m), 1.16–1.01 (2H, m).
［工程５］　１４－［(４－メトキシフェニル)メチル］－１，２，７，１１，１４－ペンタアザトリスピロ［２．２．２．４９．２６．２３］ヘプタデク－１－エン－８，１５－ジオン　塩酸塩
　上記工程４で得られた化合物（７１２ｍｇ，　１．４７ｍｍｏｌ）のテトラヒドロフラン（１８ｍｌ）溶液に、氷冷下、４規定塩酸／１，４－ジオキサン溶液（１８ｍｌ）を加え、室温で５時間撹拌した。反応液にジエチルエ－テルを加え撹拌した。生じた固体をろ取し、標記化合物（５８０ｍｇ，　１．３８ｍｍｏｌ，　９４％）を得た。
1H－NMR (DMSO－d6) δ : 10.05 (1H, br s), 9.21 (1H, br s), 9.07 (1H,s), 7.17－6.88 (4H, m), 4.83－4.57 (2H, m), 3.73 (3H, s), 3.63－3.24 (4H, m), 2.48－2.08 (4H, m), 1.93－1.67 (4H, m), 1.14－1.01 (2H, m).  [Step 5] 14-[(4-Methoxyphenyl) methyl] -1,2,7,11,14-pentaazatrispyro [2.2.2.4 9 . 26 . 2 3] Heputadeku 1-ene -8,15- dione hydrochloride compound obtained in the above Step 4 (712 mg, 1.47 mmol) in tetrahydrofuran (18 ml) was added under ice-cooling, 4 N hydrochloric acid / 1,4 -Dioxane solution (18 ml) was added and stirred at room temperature for 5 hours. Diethyl ether was added to the reaction mixture, and the mixture was stirred. The resulting solid was collected by filtration to give the title compound (580 mg, 1.38 mmol, 94%).
1 H-NMR (DMSO-d 6 ) δ: 10.05 (1H, br s), 9.21 (1H, br s), 9.07 (1H, s), 7.17-6.88 (4H, m), 4.83-4.57 (2H, m), 3.73 (3H, s), 3.63-3.24 (4H, m), 2.48-2.08 (4H, m), 1.93-1.67 (4H, m), 1.14-1.01 (2H, m).
［工程６］　メチル　２，６－ジフルオロ－４－［（トリメチルシリル）エチニル］ベンゾエ－ト
メチル　４－ブロモ－２，６－ジフルオロベンゾエ－ト（２．００ｇ，　７．７３ｍｍｏｌ）、ヨウ化銅（１４．７ｍｇ，　０．０７７３ｍｍｏｌ）、ビス（トリフェニルホスフィン）パラジウム（ＩＩ）ジクロリド（２１７ｍｇ，　０．３０９ｍｍｏｌ）をガラスチュ－ブに入れ窒素置換した。Ｎ，Ｎ－ジイソプロピルエチルアミン（１．９８ｍｌ，　１１．６ｍｍｏｌ）、Ｎ，Ｎ－ジメチルホルムアミド（１５ｍｌ）、トリメチルシリルアセチレン（１．３４ｍｌ，　９．６６ｍｍｏｌ）を加え、窒素置換後封管して、１００℃で２時間、マイクロウェ－ブ反応装置で処理した。室温に冷却後、セライトで不溶物をろ去し、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝０：１００－５：９５）で精製して、標記化合物（１．２６ｇ，　４．７１ｍｍｏｌ，　６１％）を得た。
1H－NMR (CDCl3) δ : 7.26 (1H, s), 7.05－7.00 (2H, m), 3.95 (3H, s), 0.25 (9H, s). [Step 6] Methyl 2,6-difluoro-4-[(trimethylsilyl) ethynyl] benzoatemethyl 4-bromo-2,6-difluorobenzoate (2.00 g, 7.73 mmol), copper iodide (14) .7 mg, 0.0773 mmol) and bis (triphenylphosphine) palladium (II) dichloride (217 mg, 0.309 mmol) were placed in a glass tube and replaced with nitrogen. N, N-diisopropylethylamine (1.98 ml, 11.6 mmol), N, N-dimethylformamide (15 ml), trimethylsilylacetylene (1.34 ml, 9.66 mmol) were added, and after nitrogen substitution, the tube was sealed and 100 ° C. Was treated with a microwave reactor for 2 hours. After cooling to room temperature, the insoluble material was filtered off with Celite, and the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 0: 100-5: 95) to obtain the title compound (1.26 g, 4.71 mmol, 61%).
1 H-NMR (CDCl 3 ) δ: 7.26 (1H, s), 7.05-7.00 (2H, m), 3.95 (3H, s), 0.25 (9H, s).
［工程７］　４－エチニル－２，６－ジフルオロ安息香酸
　上記工程６で得られた化合物（１．２６ｇ，　４．７１ｍｍｏｌ）をメタノ－ル（５０ｍｌ）に溶解し、室温で炭酸カリウム（８４６ｍｇ，　６．１２ｍｍｏｌ）の水溶液（８ｍｌ）を加えて２時間撹拌した。反応混合物に水を加え、減圧下メタノ－ルを留去した。得られた水層をジクロロメタンで洗浄後、氷冷下、１規定塩酸水溶液を加えて酸性に調整した。酢酸エチルで抽出し、飽和食塩水で洗浄した。無水硫酸ナトリウムで乾燥後、減圧下溶媒を留去して、標記化合物（６７８ｍｇ，　３．７２ｍｍｏｌ，　７９％）を得た。
1H－NMR (CDCl3) δ : 7.13－7.08 (2H, m), 3.31 (1H, s). [Step 7] 4-ethynyl-2,6-difluorobenzoic acid The compound (1.26 g, 4.71 mmol) obtained in the above step 6 was dissolved in methanol (50 ml), and potassium carbonate (846 mg, 846 mg,) was dissolved at room temperature. An aqueous solution (8 ml) of 6.12 mmol) was added, and the mixture was stirred for 2 hours. Water was added to the reaction mixture, and the metalnol was distilled off under reduced pressure. The obtained aqueous layer was washed with dichloromethane, and then cooled with ice to adjust to acidity by adding a 1N hydrochloric acid aqueous solution. It was extracted with ethyl acetate and washed with saturated brine. After drying over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure to give the title compound (678 mg, 3.72 mmol, 79%).
1 1 H-NMR (CDCl 3 ) δ: 7.13-7.08 (2H, m), 3.31 (1H, s).
［工程８］　１１－（４－エチニル－２，６－ジフルオロベンゾイル）－１４－［（４－メトキシフェニル）メチル］－１，２，７，１１，１４－ペンタアザトリスピロ［２．２．２．４９．２６．２３］ヘプタデク－１－エン－８，１５－ジオン
　上記工程５で得られた化合物（１７４ｍｇ，　０．４２０ｍｍｏｌ）と上記工程７で得られた化合物（７５．０ｍｇ，　０．４２０ｍｍｏｌ）のＮ，Ｎ－ジメチルホルムアミド（６ｍｌ）懸濁液に、窒素気流下、ジイソプロピルアミン（３００μｌ，　０．２１０ｍｍｏｌ）とＮ，Ｎ，Ｎ´，Ｎ´－テトラメチル－Ｏ－（７－アザベンゾトリアゾ－ル－１－イル）ウロニウムヘキサフルオロホスファ－ト（２０４ｍｇ，　０．５４０ｍｍｏｌ）を加え、室温で１６時間攪拌した。減圧下反応液溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝６０：４０－９０：１０）で精製して、標記化合物（２１２ｍｇ，　０．３９０ｍｍｏｌ，　９４％）を得た。 [Step 8] 11- (4-ethynyl-2,6-difluorobenzoyl) -14-[(4-methoxyphenyl) methyl] -1,2,7,11,14-pentaazatrispyro [2.2. 2.4 9 . 26 . 2 3] Heputadeku 1-ene -8,15- dione compound obtained in the above Step 5 (174mg, 0.420mmol) and the compound obtained in the above Step 7 (75.0mg, 0.420mmol) N, Diisopropylamine (300 μl, 0.210 mmol) and N, N, N', N'-tetramethyl-O- (7-azabenzotriazol) in a suspension of N-dimethylformamide (6 ml) under a nitrogen stream. -1-yl) Uronium hexafluorophosphate (204 mg, 0.540 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 60: 40-90: 10) to give the title compound (212 mg, 0.390 mmol, 94%).
［工程９］　（－）－１１－（４－エチニル－２，６－ジフルオロベンゾイル）－１４－［（４－メトキシフェニル）メチル］－１，２，７，１１，１４－ペンタアザトリスピロ［２．２．２．４９．２６．２３］ヘプタデク－１－エン－８，１５－ジオン（Compound A）
　上記工程８で得られた化合物を以下の条件にて光学分割を行なった。
カラム：ダイセル　ＣＨＩＲＡＬＰＡＫ　ＩＡ　３０ｍｍＩＤｘ１００ｍｍＬ
溶出溶媒：ヘキサン：２－プロパノ－ル＝９０：１０－８０：２０（Ｖ／Ｖ）
流速：１２．０ｍｌ／ｍｉｎ
温度：２５℃
ピ－ク（保持時間：１３．８分）を分取して、標記化合物（１３１ｍｇ）を得た。
比旋光度［α］Ｄ ２０　＝　－５．８０８　　（ｃ＝１．００７，　クロロホルム）
1H－NMR (CDCl3) δ: 8.17－8.00(1H, m), 7.21－6.78(6H, m), 4.98－3.22(9H, m), 2.68－2.36(4H, m), 1.99－0.87(7H, m). 
MS (m/z) : 548 (M+H)+. [Step 9] (-)-11- (4-ethynyl-2,6-difluorobenzoyl)-14-[(4-methoxyphenyl) methyl] -1,2,7,11,14-pentaazatrispyro [ 2.2.2.4 9 . 26 . 2 3] Heputadeku 1-ene -8,15- dione (Compound A)
The compound obtained in the above step 8 was optically resolved under the following conditions.
Column: Daicel CHIRALPAK IA 30mmIDx100mmL
Elution solvent: Hexane: 2-Propanol = 90: 10-80: 20 (V / V)
Flow velocity: 12.0 ml / min
Temperature: 25 ° C
The peak (retention time: 13.8 minutes) was fractionated to obtain the title compound (131 mg).
Specific rotation [α] D 20 = -5.5808 (c = 1.007, chloroform)
1 H-NMR (CDCl 3 ) δ: 8.17-8.00 (1H, m), 7.21-6.78 (6H, m), 4.98-3.22 (9H, m), 2.68-2.36 (4H, m), 1.99-0.87 ( 7H, m).
MS (m / z): 548 (M + H) + .
（実施例２）Compound Bの製造 (Example 2) Manufacture of Compound B
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
［工程１］　ｔｅｒｔ－ブチル　｛［（４－クロロ－３－フルオロフェニル）メチル］アミノ｝－３－シアノアゼチジン－１－カルボキシレ－ト
　ｔｅｒｔ－ブチル　３－オキソアゼチジン－１－カルボキシレ－ト（２０．０ｇ，　０．１１３ｍｏｌ）をメタノ－ル（４００ｍｌ）に溶解し、４－クロロ－３－フルオロベンジルアミン（１９．０ｇ，　０．１１３ｍｏｌ）、酢酸（３２．４ｍｌ，　０．５６７ｍｏｌ）を加えて室温で１０分間撹拌した。反応液にシアン化カリウム（７．５３ｇ，　０．１１３ｍｏｌ）を加え、６０℃で１６時間撹拌した。室温に放冷後、減圧下溶媒を留去し、得られた残留物に水を加えて撹拌した。生じた固体をろ取して、標記化合物（３６．３ｇ，　０．１０７ｍｏｌ，　９４％）を得た。
1H－NMR (CDCl3) δ : 7.40－7.35 (1H, m), 7.23－7.19 (1H, m), 7.12－7.09 (1H, m), 4.26 (2H, d, J = 9.2 Hz), 3.88 (2H, d, J= 9.2 Hz), 3.83 (2H, d, J = 6.7 Hz), 1.93－1.90 (1H, m), 1.45 (9H, s). [Step 1] tert-Butyl {[(4-chloro-3-fluorophenyl) methyl] amino} -3-cyanoazetidine-1-carboxylate tert-butyl 3-oxoazetidine-1-carboxylate (20. 0 g, 0.113 mol) is dissolved in methanol (400 ml), 4-chloro-3-fluorobenzylamine (19.0 g, 0.113 mol) and acetic acid (32.4 ml, 0.567 mol) are added to room temperature. Was stirred for 10 minutes. Potassium cyanide (7.53 g, 0.113 mol) was added to the reaction mixture, and the mixture was stirred at 60 ° C. for 16 hours. After allowing to cool to room temperature, the solvent was distilled off under reduced pressure, water was added to the obtained residue, and the mixture was stirred. The resulting solid was collected by filtration to give the title compound (36.3 g, 0.107 mol, 94%).
1 H-NMR (CDCl 3 ) δ: 7.40-7.35 (1H, m), 7.23-7.19 (1H, m), 7.12-7.09 (1H, m), 4.26 (2H, d, J = 9.2 Hz), 3.88 (2H, d, J = 9.2 Hz), 3.83 (2H, d, J = 6.7 Hz), 1.93-1.90 (1H, m), 1.45 (9H, s).
［工程２］　１－（ｔｅｒｔ－ブトキシカルボニル）－３－｛［（４－クロロ－３－フルオロフェニル）メチル］アミノ｝アゼチジン－３－カルボン酸
　上記工程１で得られた化合物（３６．３ｇ，　０．１０７ｍｏｌ）をジメチルスルホキシド（３５０ｍｌ）に懸濁し、過酸化水素水（１７．６ｍｌ，　０．２１３ｍｏｌ）を加えた。氷冷下、炭酸カリウム（４４．５ｇ，　０．３２０ｍｏｌ）を加え、室温に昇温させながら２４時間撹拌した。反応液にゆっくりと水を加え、酢酸エチルで抽出し、水、飽和食塩水で洗浄した。減圧下溶媒を留去し、乾燥して得られた残留物をエタノ－ル（６００ｍｌ）に懸濁し、８規定水酸化カリウム水溶液（５７．５ｍｌ，　０．４６０ｍｏｌ）を加えて８０℃で１６時間撹拌した。減圧下溶媒を留去した後に、水で希釈し、氷冷下、２規定塩酸水溶液（２３０ｍｌ，　０．４６０ｍｏｌ）を加えて中和した。生じた固体をろ取して標記化合物（３４．６ｇ，　９６．４ｍｍｏｌ，　９０％）を得た。
1H－NMR (DMSO－d6) δ : 8.24 (1H, br s), 7.56－7.49 (1H, m), 7.44－7.37 (1H, m), 7.25－7.19 (1H, m), 4.09－3.94 (2H, br m), 3.81－3.70 (2H, br m), 3.68 (2H, s), 1.38 (9H, s). [Step 2] 1- (tert-Butyloxycarbonyl) -3-{[(4-chloro-3-fluorophenyl) methyl] amino} azetidine-3-carboxylic acid The compound obtained in the above step 1 (36.3 g, 0.107 mol) was suspended in dimethyl sulfoxide (350 ml), and hydrogen peroxide solution (17.6 ml, 0.213 mol) was added. Potassium carbonate (44.5 g, 0.320 mol) was added under ice-cooling, and the mixture was stirred for 24 hours while raising the temperature to room temperature. Water was slowly added to the reaction mixture, the mixture was extracted with ethyl acetate, and washed with water and saturated brine. The solvent was distilled off under reduced pressure, the residue obtained by drying was suspended in etanol (600 ml), 8N aqueous potassium hydroxide solution (57.5 ml, 0.460 mol) was added, and the temperature was 80 ° C. for 16 hours. Stirred. After distilling off the solvent under reduced pressure, the mixture was diluted with water and neutralized by adding a 2N hydrochloric acid aqueous solution (230 ml, 0.460 mol) under ice-cooling. The resulting solid was collected by filtration to give the title compound (34.6 g, 96.4 mmol, 90%).
1 H-NMR (DMSO-d 6 ) δ: 8.24 (1H, br s), 7.56-7.49 (1H, m), 7.44-7.37 (1H, m), 7.25-7.19 (1H, m), 4.09-3.94 (2H, br m), 3.81-3.70 (2H, br m), 3.68 (2H, s), 1.38 (9H, s).
［工程３］　８，８－ジフルオロ－１，３－ジアザスピロ［４，５］デカン－２，４－ジオン
　炭酸アンモニウム（８５．３ｇ，　８８８ｍｍｏｌ）を水（１Ｌ）に溶解し、シアン化ナトリウム（９．１３ｇ，　１８１ｍｍｏｌ）を加えた後、４，４－ジフルオロシクロヘキサノン（２０．０ｇ，　１４５ｍｍｏｌ）を加え、７０℃で１５時間撹拌した。反応液を氷冷してしばらく撹拌し、生じた固体をろ取して、標記化合物（２８．６ｇ，　１４０ｍｍｏl， ９７％）を得た。
1H－NMR (DMSO－d6) δ : 10.77 (1H, s), 8.53 (1H, s), 1.95－2.15 (4H, m), 1.80－1.90 (2H, m), 1.69－1.77 (2H, m). [Step 3] 8,8-Difluoro-1,3-diazaspiro [4,5] decane-2,4-dione Ammonium carbonate (85.3 g, 888 mmol) was dissolved in water (1 L) and sodium cyanide (9). After adding .13 g, 181 mmol), 4,4-difluorocyclohexanone (20.0 g, 145 mmol) was added, and the mixture was stirred at 70 ° C. for 15 hours. The reaction mixture was ice-cooled and stirred for a while, and the resulting solid was collected by filtration to give the title compound (28.6 g, 140 mmol, 97%).
1 H-NMR (DMSO-d 6 ) δ: 10.77 (1H, s), 8.53 (1H, s), 1.95-2.15 (4H, m), 1.80-1.90 (2H, m), 1.69-1.77 (2H, m).
　［工程４］　１－アミノ－４，４－ジフルオロシクロヘキサン－１－カルボン酸
　上記工程３で得られた化合物（４４．３ｇ，　０．２１７ｍｏｌ）を８規定水酸化ナトリウム水溶液（２７１ｍｌ，　２．１７ｍｏｌ）に懸濁し、１２０℃に加熱して２８時間撹拌した。反応液を氷冷して、５規定塩酸水溶液（４３４ｍｌ，　２．１７ｍｏｌ）を加えて中和してしばらく撹拌した。生じた固体をろ取して、標記化合物（３０．８ｇ，　０．１７２ｍｏｌ，　７９％）を得た。
1H－NMR (DMSO－d6) δ : 7.71－7.53 (2H, br m), 2.52－2.48 (4H, m), 2.28－2.13 (1H, m), 2.08－1.85 (2H, m), 1.71－1.61 (1H, m). [Step 4] 1-Amino-4,4-difluorocyclohexane-1-carboxylic acid The compound (44.3 g, 0.217 mol) obtained in the above step 3 was added to an 8N aqueous sodium hydroxide solution (271 ml, 2.17 mol). The mixture was suspended in, heated to 120 ° C., and stirred for 28 hours. The reaction mixture was ice-cooled, neutralized by adding 5N aqueous hydrochloric acid solution (434 ml, 2.17 mol), and stirred for a while. The resulting solid was collected by filtration to give the title compound (30.8 g, 0.172 mol, 79%).
1 H-NMR (DMSO-d 6 ) δ: 7.71-7.53 (2H, br m), 2.52-2.48 (4H, m), 2.28-2.13 (1H, m), 2.08-1.85 (2H, m), 1.71 -1.61 (1H, m).
［工程５］　４，４－ジフルオロ－１－（２，２，２－トリフルオロアセトアミド）シクロヘキサン－１－カルボン酸
　上記工程４で得られた化合物（１５．０ｇ，　８３．７ｍｍｏｌ）、カリウムメトキシド（６．４６ｇ，　９２．１ｍｍｏｌ）をメタノ－ル（２０ｍｌ）に懸濁し、５０℃で３０分間撹拌した。室温まで放冷後、エチルトリフルオロアセテ－ト（２０．０ｍｌ，　１６７ｍｍｏｌ）を加え、再度５０℃で６時間撹拌した。室温に放冷後、減圧下溶媒を留去した。得られた残留物に１規定塩酸水溶液を加えて酸性とし、酢酸エチルを加えて撹拌した。不溶物をセライトでろ過し、ろ液を酢酸エチルで抽出した。２規定塩酸水溶液、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥した。減圧下溶媒を留去して、標記化合物（１９．０ｇ，　６９．２ｍｍｏｌ，　８３％）を得た。
1H－NMR (DMSO－d6) δ : 13.09 (1H, br s), 9.51 (1H, br s), 2.28－2.19 (2H, m), 2.07－1.85 (6H, m). [Step 5] 4,4-Difluoro-1- (2,2,2-trifluoroacetamide) cyclohexane-1-carboxylic acid The compound (15.0 g, 83.7 mmol) obtained in the above step 4 and potassium methoxyde. (6.46 g, 92.1 mmol) was suspended in methanol (20 ml) and stirred at 50 ° C. for 30 minutes. After allowing to cool to room temperature, ethyl trifluoroacetate (20.0 ml, 167 mmol) was added, and the mixture was stirred again at 50 ° C. for 6 hours. After allowing to cool to room temperature, the solvent was distilled off under reduced pressure. A 1N aqueous hydrochloric acid solution was added to the obtained residue to make it acidic, ethyl acetate was added, and the mixture was stirred. The insoluble material was filtered through Celite, and the filtrate was extracted with ethyl acetate. The product was washed with 2N aqueous hydrochloric acid solution and saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain the title compound (19.0 g, 69.2 mmol, 83%).
1 H-NMR (DMSO-d 6 ) δ: 13.09 (1H, br s), 9.51 (1H, br s), 2.28-2.19 (2H, m), 2.07-1.85 (6H, m).
［工程６］　ｔｅｒｔ－ブチル　５－［（４－クロロ－３－フルオロフェニル）メチル］－１０，１０－ジフルオロ－６，１４－ジオキソ－２，５，１３－トリアザジスピロ［３．２．５７．２４］テトラデカン－２－カルボキシレ－ト
　上記工程５で得られた化合物（１９．７ｇ，　７１．５ｍｍｏｌ）をジクロロメタン（３５０ｍｌ）に懸濁し、室温でオキサリルクロリド（６．１３ｍｌ，　７１．５ｍｍｏｌ）、Ｎ，Ｎ－ジメチルホルムアミド（１ｍｌ）を加え３時間撹拌した。減圧下溶媒を留去し、酸クロリドを得た。別のフラスコに上記工程２で得られた化合物（１７．１ｇ，　４７．７ｍｍｏｌ）を入れ、Ｎ，Ｎ－ジメチルホルムアミド（５００ｍｌ）に懸濁した。氷冷下、Ｎ，Ｎ－ジイソプロピルエチルアミン（１６．３ｍｌ，　９５．３ｍｍｏｌ）を加え、続いて先に調製した酸クロリドのＮ，Ｎ－ジメチルホルムアミド（１００ｍｌ）溶液を滴下した。室温で１４時間撹拌した後に、氷冷下、１，１´－カルボニルジイミダゾ－ル（２３．２ｇ，　１４３ｍｍｏｌ）を加え、室温で１０分間、８０℃で８時間撹拌した。室温に戻した後、１規定塩酸水溶液、水を加え、酢酸エチルで抽出した。有機層を水、飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥した。減圧下溶媒を留去し、得られた残留物にエタノ－ルを加えて超音波処理を行なった。生じた固体をろ取して、標記化合物（１２．０ｇ，　２３．９ｍｍｏｌ，　５０％）を得た。
1H－NMR (CDCl3) δ : 7.40－7.35 (1H, m), 7.07 (1H, br s), 6.99－6.93 (2H, m), 4.91 (2H, br s), 4.56－4.49 (2H, m), 4.00－3.93 (2H, m), 2.39－2.25 (4H, m), 2.12－1.95 (2H, m), 1.81－1.72 (2H, m), 1.45 (9H, s). [Step 6] tert-Butyl 5-[(4-chloro-3-fluorophenyl) methyl] -10,10-difluoro-6,14-dioxo-2,5,13-triazadispiro [3.2.5 7 . 2 4] tetradecane-2 carboxylate - DOO above step 5 in the resulting compound (19.7 g, 71.5 mmol) was suspended in dichloromethane (350 ml), oxalyl at room temperature chloride (6.13 mL, 71.5 mmol) , N, N-dimethylformamide (1 ml) was added, and the mixture was stirred for 3 hours. The solvent was distilled off under reduced pressure to obtain acid chloride. The compound (17.1 g, 47.7 mmol) obtained in the above step 2 was placed in another flask and suspended in N, N-dimethylformamide (500 ml). Under ice-cooling, N, N-diisopropylethylamine (16.3 ml, 95.3 mmol) was added, and then a solution of the previously prepared acid chloride in N, N-dimethylformamide (100 ml) was added dropwise. After stirring at room temperature for 14 hours, 1,1'-carbonyldiimidazole (23.2 g, 143 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 10 minutes and at 80 ° C. for 8 hours. After returning to room temperature, 1N hydrochloric acid aqueous solution and water were added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and ethanol was added to the obtained residue for ultrasonic treatment. The resulting solid was collected by filtration to give the title compound (12.0 g, 23.9 mmol, 50%).
1 H-NMR (CDCl 3 ) δ: 7.40-7.35 (1H, m), 7.07 (1H, br s), 6.99-6.93 (2H, m), 4.91 (2H, br s), 4.56-4.49 (2H, br s) m), 4.00-3.93 (2H, m), 2.39-2.25 (4H, m), 2.12-1.95 (2H, m), 1.81-1.72 (2H, m), 1.45 (9H, s).
［工程７］　５－［（４－クロロ－３－フルオロフェニル）メチル］－１０，１０－ジフルオロ－２，５，１３－トリアザジスピロ［３．２．５７．２４］テトラデカン－６，１４－ジオン　塩酸塩
　上記工程６で得られた化合物（１２．０ｇ，　２３．９ｍｍｏｌ）をジクロロメタン（２５０ｍｌ）に懸濁し、氷冷下、４規定塩酸／１，４－ジオキサン溶液（１２５ｍｌ）を加えて１４時間撹拌した。減圧下溶媒を留去し、ジエチルエ－テルを加えてしばらく撹拌した。生じた固体をろ取して、標記化合物（１０．５ｇ，　２３．９ｍｍｏｌ，　定量的）を得た。
1H－NMR (DMSO－d6) δ : 9.26－9.11 (2H, m), 7.58－7.52 (1H, m), 7.40－7.36 (1H, m), 7.19－7.15 (1H, m), 4.96 (2H, s), 4.42－4.33 (2H, m), 4.24－4.14 (2H, m), 2.27－2.01 (6H, m), 1.93－1.84 (2H, m). [Step 7] 5-[(4-Chloro-3-fluorophenyl) methyl] -10,10-difluoro-2,5,13-triazadispiro [3.2.5 7 . 2 4] tetradecane -6,14- dione hydrochloride The compound obtained in the above step 6 (12.0 g, 23.9 mmol) was suspended in dichloromethane (250 ml), under ice-cooling, 4 N hydrochloric acid / 1,4 Dioxane solution (125 ml) was added and stirred for 14 hours. The solvent was evaporated under reduced pressure, diethyl ether was added, and the mixture was stirred for a while. The resulting solid was collected by filtration to give the title compound (10.5 g, 23.9 mmol, quantitative).
1 H-NMR (DMSO-d 6 ) δ: 9.26-9.11 (2H, m), 7.58-7.52 (1H, m), 7.40-7.36 (1H, m), 7.19-7.15 (1H, m), 4.96 ( 2H, s), 4.42-4.33 (2H, m), 4.24-4.14 (2H, m), 2.27-2.01 (6H, m), 1.93-1.84 (2H, m).
［工程８］　１－クロロ－４－（１，１－ジフルオロエチル）－２－ニトロベンゼン
１－（４－クロロ－３－ニトロフェニル）エタン－１－オン（１４．０ｇ，　７０．１ｍｍｏｌ）のジクロロメタン（２００ｍｌ）溶液に、氷冷下、ビス（２－メトキシエチル）アミノサルファ－トリフルオリド（５０．６ｇ，　２１０ｍｍｏｌ）を加え、室温で４８時間撹拌した。氷冷下、飽和重曹水にゆっくりと注ぎ、ジクロロメタンで抽出した。無水硫酸ナトリウムで乾燥し、減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝０：１００－１０：９０）で精製して、標記化合物（１３．４ｇ，　６０．５ｍｍｏｌ，　８６％）を得た。
1H－NMR (CDCl3) δ : 8.02 (1H, s), 7.68－7.62 (2H, m), 1.95 (3H, t, J = 18.1 Hz). [Step 8] Dichloromethane of 1-chloro-4- (1,1-difluoroethyl) -2-nitrobenzene1- (4-chloro-3-nitrophenyl) ethane-1-one (14.0 g, 70.1 mmol) Bis (2-methoxyethyl) aminosulfa-trifluoride (50.6 g, 210 mmol) was added to the (200 ml) solution under ice-cooling, and the mixture was stirred at room temperature for 48 hours. It was slowly poured into saturated aqueous sodium hydrogen carbonate under ice-cooling and extracted with dichloromethane. The residue obtained by drying over anhydrous sodium sulfate and distilling off the solvent under reduced pressure was purified by silica gel column chromatography (ethyl acetate-hexane = 0: 100-10: 90) to obtain the title compound (13.4 g). , 60.5 mmol, 86%).
1 H-NMR (CDCl 3 ) δ: 8.02 (1H, s), 7.68-7.62 (2H, m), 1.95 (3H, t, J = 18.1 Hz).
［工程９］　４－（１，１－ジフルオロエチル）－２－ニトロベンゾニトリル
　上記工程８で得られた化合物（１４．９ｇ，　６７．４ｍｍｏｌ）をＮ－メチル－２－ピロリジノン（５５ｍｌ）に溶解し、シアン化銅（Ｉ）（１２．３ｇ，　１３５ｍｍｏｌ）を加えて１６０℃で１８時間加熱撹拌した。反応液を酢酸エチルで希釈し、飽和塩化アンモニウム水溶液を加えて撹拌した。生じた不溶物をセライトろ過し、酢酸エチルで抽出し、水、飽和食塩水で洗浄した。無水硫酸ナトリウムで乾燥し、減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝０：１００－２５：７５）で精製して、標記化合物（１０．６ｇ，　４９．９ｍｍｏｌ，　７４％）を得た。
1H－NMR (CDCl3) δ : 8.47 (1H, s), 8.02 (1H, d, J = 8.0 Hz), 7.96 (1H, d, J= 8.0 Hz), 2.00 (3H, t, J = 18.1 Hz). [Step 9] 4- (1,1-difluoroethyl) -2-nitrobenzonitrile The compound (14.9 g, 67.4 mmol) obtained in the above step 8 was dissolved in N-methyl-2-pyrrolidinone (55 ml). Then, copper (I) cyanide (12.3 g, 135 mmol) was added, and the mixture was heated and stirred at 160 ° C. for 18 hours. The reaction mixture was diluted with ethyl acetate, saturated aqueous ammonium chloride solution was added, and the mixture was stirred. The resulting insoluble material was filtered through Celite, extracted with ethyl acetate, and washed with water and saturated brine. The residue obtained by drying over anhydrous sodium sulfate and distilling off the solvent under reduced pressure was purified by silica gel column chromatography (ethyl acetate-hexane = 0: 100-25: 75) to obtain the title compound (10.6 g). , 49.9 mmol, 74%).
1 H-NMR (CDCl 3 ) δ: 8.47 (1H, s), 8.02 (1H, d, J = 8.0 Hz), 7.96 (1H, d, J = 8.0 Hz), 2.00 (3H, t, J = 18.1) Hz).
［工程１０］　４－（１，１－ジフルオロエチル）－２－ニトロベンズアミド
　上記工程９で得られた化合物（９．４３ｇ，　４４．５ｍｍｏｌ）のジメチルスルホキシド（１１５ｍｌ）溶液に、冷却しながら過酸化水素水（７．３２ｍｌ，　８８．９ｍｍｏｌ）を加え、続けて炭酸カリウム（１８．４ｇ，　１３３ｍｍｏｌ）を加えて室温で１８時間撹拌した。反応液に水、飽和食塩水を加え、酢酸エチルで抽出し、有機層を水、飽和食塩水で洗浄した。無水硫酸ナトリウムで乾燥し、減圧下溶媒を留去した。得られた残留物を少量のジクロロメタンに懸濁し、ヘキサンを加えて超音波処理後、室温でしばらく撹拌した。生じた固体をろ取して、標記化合物（９．４３ｇ，　４１．０ｍｍｏｌ，　９２％）を得た。
1H－NMR (CDCl3) δ : 8.22 (1H, s), 7.86－7.81 (1H, m), 7.67 (1H, d, J = 8.0 Hz), 5.88－5.76 (2H, br m), 1.97 (3H, t, J = 18.1 Hz). [Step 10] 4- (1,1-difluoroethyl) -2-nitrobenzamide Peroxidation of the compound (9.43 g, 44.5 mmol) obtained in the above step 9 in a solution of dimethyl sulfoxide (115 ml) while cooling. Hydrogen peroxide water (7.32 ml, 88.9 mmol) was added, and then potassium carbonate (18.4 g, 133 mmol) was added, and the mixture was stirred at room temperature for 18 hours. Water and saturated brine were added to the reaction mixture, the mixture was extracted with ethyl acetate, and the organic layer was washed with water and saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was suspended in a small amount of dichloromethane, hexane was added, the mixture was sonicated, and the mixture was stirred at room temperature for a while. The resulting solid was collected by filtration to give the title compound (9.43 g, 41.0 mmol, 92%).
1 H-NMR (CDCl 3 ) δ: 8.22 (1H, s), 7.86-7.81 (1H, m), 7.67 (1H, d, J = 8.0 Hz), 5.88-5.76 (2H, br m), 1.97 ( 3H, t, J = 18.1 Hz).
［工程１１］　２－アミノ－４－（１，１－ジフルオロエチル）ベンズアミド
　上記工程１０で得られた化合物（９．４３ｇ，　４１．０ｍｍｏｌ）をエタノ－ル（３８０ｍｌ）に溶解し、１０％パラジウム炭素（Ｍ）（川研ファインケミカル株式会社）（１．８ｇ）を加え、水素雰囲気下、６０℃に加温して５時間撹拌した。触媒をセライトでろ去し、減圧下溶媒を留去して、標記化合物（７．７２ｇ，　３８．６ｍｍｏｌ，　９４％）を得た。
1H－NMR (CDCl3) δ : 7.40 (1H, d, J= 8.0 Hz), 6.81 (1H, s), 6.77－6.73 (1H, m), 6.14－5.43 (3H, br m), 1.88 (3H, t, J = 18.1 Hz). [Step 11] 2-Amino-4- (1,1-difluoroethyl) benzamide The compound (9.43 g, 41.0 mmol) obtained in the above step 10 was dissolved in etanol (380 ml) and 10% palladium. Carbon (M) (Kawaken Fine Chemical Co., Ltd.) (1.8 g) was added, and the mixture was heated to 60 ° C. and stirred for 5 hours under a hydrogen atmosphere. The catalyst was filtered off with Celite, and the solvent was evaporated under reduced pressure to give the title compound (7.72 g, 38.6 mmol, 94%).
1 H-NMR (CDCl 3 ) δ: 7.40 (1H, d, J = 8.0 Hz), 6.81 (1H, s), 6.77-6.73 (1H, m), 6.14-5.43 (3H, br m), 1.88 ( 3H, t, J = 18.1 Hz).
［工程１２］　７－（１，１－ジフルオロエチル）キナゾリン－４（３Ｈ）－オン
　上記工程１１で得られた化合物（２．６７ｇ，　１３．３ｍｍｏｌ）、ホルムアミジンアセテ－ト（４．１７ｇ，　４０．０ｍｍｏｌ）をエタノ－ル（８０ｍｌ）に溶解し、５時間加熱還流した。室温まで冷却した後、減圧下溶媒を留去した。得られた残留物に水を加え、超音波処理後、室温でしばらく撹拌した。固体をろ取して、標記化合物（２．５９ｇ，　１２．３ｍｍｏｌ，　９３％）を得た。
1H－NMR (DMSO－d6) δ : 12.42 (1H, br s), 8.22 (1H, d, J = 8.6 Hz), 8.18 (1H, s), 7.81 (1H, s), 7.71－7.67 (1H, m), 2.04 (3H, t, J = 19.0 Hz).　 [Step 12] 7- (1,1-difluoroethyl) quinazoline-4 (3H) -one The compound (2.67 g, 13.3 mmol) obtained in the above step 11 and form amidine acetate (4.17 g, 40.0 mmol) was dissolved in etanol (80 ml), and the mixture was heated under reflux for 5 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure. Water was added to the obtained residue, and after ultrasonic treatment, the mixture was stirred at room temperature for a while. The solid was collected by filtration to give the title compound (2.59 g, 12.3 mmol, 93%).
1 H-NMR (DMSO-d 6 ) δ: 12.42 (1H, br s), 8.22 (1H, d, J = 8.6 Hz), 8.18 (1H, s), 7.81 (1H, s), 7.71-7.67 ( 1H, m), 2.04 (3H, t, J = 19.0 Hz).
［工程１３］　４－クロロ－７－（１，１－ジフルオロエチル）キナゾリン
上記工程１２で得られた化合物（２．５９ｇ，　１２．３ｍｍｏｌ）をトルエン（５５ｍｌ）に懸濁し、氷冷下、Ｎ，Ｎ－ジイソプロピルエチルアミン（６．０２ｍｌ，　３４．６ｍｍｏｌ）、オキシ塩化リン（５．６９ｇ，　３７．０ｍｍｏｌ）を加えて、１００℃に加熱して４時間撹拌した。室温に戻した後、酢酸エチルで希釈した。氷冷下、飽和重曹水にゆっくりと注ぎ、酢酸エチルで抽出し、水、飽和食塩水で洗浄した。無水硫酸ナトリウムで乾燥後、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝０：１００－２０：８０）で精製して、標記化合物（２．６４ｇ，　１１．５ｍｍｏｌ，　９３％）を得た。
1H－NMR (CDCl3) δ : 9.11 (1H, s), 8.36 (1H, d, J = 8.6 Hz), 8.23 (1H, s), 7.88－7.84 (1H, m), 2.04 (3H, t, J = 18.4 Hz). [Step 13] 4-Chloro-7- (1,1-difluoroethyl) quinazoline The compound (2.59 g, 12.3 mmol) obtained in the above step 12 was suspended in toluene (55 ml), and N , N-diisopropylethylamine (6.02 ml, 34.6 mmol) and phosphorus oxychloride (5.69 g, 37.0 mmol) were added, and the mixture was heated to 100 ° C. and stirred for 4 hours. After returning to room temperature, it was diluted with ethyl acetate. Under ice-cooling, the mixture was slowly poured into saturated aqueous sodium hydrogen carbonate, extracted with ethyl acetate, and washed with water and saturated brine. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 0: 100-20: 80) to obtain the title compound (2.64 g, 11.5 mmol, 93%).
1 1 H-NMR (CDCl 3 ) δ: 9.11 (1H, s), 8.36 (1H, d, J = 8.6 Hz), 8.23 (1H, s), 7.88-7.84 (1H, m), 2.04 (3H, t) , J = 18.4 Hz).
［工程１４］　５－［（４－クロロ－３－フルオロフェニル）メチル］－２－［７－（１，１－ジフルオロエチル）キナゾリン－４－イル］－１０，１０－ジフルオロ－２，５，１３－トリアザジスピロ［３．２．５７．２４］テトラデカン－６，１４－ジオン（Compound B）
上記工程７で得られた化合物（５．７１ｇ，　１３．０ｍｍｏｌ）および上記工程１３で得られた化合物（３．１３ｇ，　１３．７ｍｍｏｌ）を２－プロパノ－ル（１３０ｍｌ）に懸濁し、Ｎ，Ｎ－ジイソプロピルエチルアミン（９．０８ｍｌ，　５２．１ｍｍｏｌ）を加えて、８０℃で５時間加熱還流した。室温に戻した後、減圧下溶媒を留去した。得られた残留物を酢酸エチルで希釈し、水、飽和食塩水で洗浄した。無水硫酸ナトリウムで乾燥し、減圧下溶媒を留去した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝０：１００－３５：６５）で精製して、標記化合物（６．７９ｇ，　１１．４ｍｍｏｌ，　８８％）を得た。
1H－NMR (CDCl3) δ : 8.71 (1H, s), 8.04 (1H, s), 7.66 (1H, d, J = 8.6 Hz), 7.56－7.53 (1H, m), 7.36－7.32 (1H, m), 7.01－6.97 (1H, m), 6.94－6.91 (1H, m), 6.69 (1H, br s), 5.11 (2H, d, J = 9.8 Hz), 5.02 (2H, s), 4.60 (2H, d, J = 9.8 Hz), 2.42－2.27 (4H, m), 2.06－1.92 (5H, m), 1.87－1.78 (2H, m). 
MS (m/z) : 594 (M+H)+. [Step 14] 5-[(4-Chloro-3-fluorophenyl) methyl] -2- [7- (1,1-difluoroethyl) quinazoline-4-yl] -10,10-difluoro-2,5 13-Triazadispiro [3.2.5 7 . 2 4] tetradecane -6,14- dione (Compound B)
The compound (5.71 g, 13.0 mmol) obtained in the above step 7 and the compound (3.13 g, 13.7 mmol) obtained in the above step 13 were suspended in 2-propanol (130 ml), and N, N-diisopropylethylamine (9.08 ml, 52.1 mmol) was added, and the mixture was heated under reflux at 80 ° C. for 5 hours. After returning to room temperature, the solvent was distilled off under reduced pressure. The obtained residue was diluted with ethyl acetate and washed with water and saturated brine. The mixture was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 0: 100-35: 65) to obtain the title compound (6.79 g, 11.4 mmol, 88%).
1 H-NMR (CDCl 3 ) δ: 8.71 (1H, s), 8.04 (1H, s), 7.66 (1H, d, J = 8.6 Hz), 7.56-7.53 (1H, m), 7.36-7.32 (1H) , m), 7.01-6.97 (1H, m), 6.94-6.91 (1H, m), 6.69 (1H, br s), 5.11 (2H, d, J = 9.8 Hz), 5.02 (2H, s), 4.60 (2H, d, J = 9.8 Hz), 2.42-2.27 (4H, m), 2.06-1.92 (5H, m), 1.87-1.78 (2H, m).
MS (m / z): 594 (M + H) + .
（実施例３）Compound Cの製造 (Example 3) Manufacture of Compound C
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
［工程１］　４，４－ジフルオロ－１－｛［（２，２，２－トリクロロエトキシ）カルボニル］アミノ｝シクロヘキサン－１－カルボン酸
　実施例２の工程３の方法で得られた化合物（３７．０ｇ，　１８１ｍｍｏｌ）を８規定水酸化カリウム水溶液（２００ｍｌ）に懸濁し、１２０℃に加熱して１８時間撹拌した。反応液を氷冷して、５規定塩酸水溶液（３２０ｍｌ）を加えて中和して懸濁液を得た。得られた懸濁液に氷冷下、４規定水酸化ナトリウム水溶液（４５ｍｌ，　１８１ｍｍｏｌ）と１，４－ジオキサン（２３０ｍｌ）を加えて撹拌した。－５℃に冷却しながらクロロぎ酸　２，２，２－トリクロロエチル（３６．４ｍｌ，　２７２ｍｍｏｌ）の１，４－ジオキサン（２７０ｍｌ）溶液と１規定水酸化ナトリウム水溶液（２７０ｍｌ）を同時に滴下し、室温で３６時間撹拌させた。氷冷下、１規定水酸化ナトリウム水溶液（２７２ｍｌ）を加えて液性をｐＨ１０付近に調整し、ジエチルエ－テルで洗浄した。氷冷下、２規定塩酸水溶液を加えて液性をｐＨ５付近に調整し、酢酸エチルで抽出した。飽和食塩水で洗浄後、無水硫酸ナトリウムで乾燥した。減圧下溶媒を留去して、標記化合物（５２．３ｇ，　１４８ｍｍｏｌ，　８１％）を得た。
1H－NMR (CDCl3) δ : 7.26 (1H, s), 5.31－5.26 (1H, br s), 4.75 (2H, s), 2.30－2.22 (4H, m), 2.20－2.06 (2H, m), 2.04－1.87 (2H, m). [Step 1] 4,4-Difluoro-1-{[(2,2,2-trichloroethoxy) carbonyl] amino} cyclohexane-1-carboxylic acid The compound obtained by the method of Step 3 of Example 2 (37. (0 g, 181 mmol) was suspended in an 8N aqueous potassium hydroxide solution (200 ml), heated to 120 ° C., and stirred for 18 hours. The reaction mixture was ice-cooled and neutralized by adding a 5N hydrochloric acid aqueous solution (320 ml) to obtain a suspension. To the obtained suspension, 4N aqueous sodium hydroxide solution (45 ml, 181 mmol) and 1,4-dioxane (230 ml) were added under ice-cooling, and the mixture was stirred. While cooling to -5 ° C, a solution of 2,2,2-trichloroethyl chloroformate (36.4 ml, 272 mmol) in 1,4-dioxane (270 ml) and a 1N aqueous solution of sodium hydroxide (270 ml) were added dropwise at the same time. The mixture was stirred at room temperature for 36 hours. Under ice-cooling, a 1N aqueous sodium hydroxide solution (272 ml) was added to adjust the liquid property to around pH 10, and the mixture was washed with diethyl ether. Under ice-cooling, a 2N hydrochloric acid aqueous solution was added to adjust the liquid property to around pH 5, and the mixture was extracted with ethyl acetate. After washing with saturated brine, it was dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain the title compound (52.3 g, 148 mmol, 81%).
1 H-NMR (CDCl 3 ) δ: 7.26 (1H, s), 5.31-5.26 (1H, br s), 4.75 (2H, s), 2.30-2.22 (4H, m), 2.20-2.06 (2H, m) ), 2.04-1.87 (2H, m).
［工程２］　ｔｅｒｔ－ブチル　３－｛［（４－クロロ－３－フルオロフェニル）メチル］（４，４－ジフルオロ－１－｛［（２，２，２－トリクロロエトキシ）カルボニル］アミノ｝シクロヘキサン－１－カルボニル）アミノ｝－３－｛［２－（モルホリン－４－イル）エチル］カルバモイル｝ピロリジン－１－カルボキシレ－ト
　ｔｅｒｔ－ブチル　３－オキソピロリジノン－１－カルボキシレ－ト（１５．６ｇ，　８１．６ｍｍｏｌ）をメタノ－ル（６００ｍｌ）に溶解し、１－（４－クロロ－３－フルオロフェニル）メタナミン（１３．７ｇ，　８１．６ｍｍｏｌ）を加えて５５℃に加温して１時間半撹拌した。室温に戻した後、上記工程２で得られた化合物（２８．９ｇ，　８１．６ｍｍｏｌ）、２－モルホリノエチルイソシアニド（１２．１ｍｌ，　８５．６ｍｍｏｌ）を加えて再び５５℃に加温して８時間撹拌した。減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（メタノ－ル－ジクロロメタン＝５：９５）で精製して、標記化合物（６７．０ｇ，　８１．６ｍｍｏｌ，　定量的）を得た。 [Step 2] tert-Butyl 3-{[(4-chloro-3-fluorophenyl) methyl] (4,4-difluoro-1-{[(2,2,2-trichloroethoxy) carbonyl] amino} cyclohexane- 1-carbonyl) amino} -3-{[2- (morpholin-4-yl) ethyl] carbamoyl} pyrrolidine-1-carboxylate tert-butyl 3-oxopyrrolidinone-1-carboxylate (15.6 g) , 81.6 mmol) is dissolved in methaneol (600 ml), 1- (4-chloro-3-fluorophenyl) methanamine (13.7 g, 81.6 mmol) is added, and the mixture is heated to 55 ° C. for 1 hour. Semi-stirred. After returning to room temperature, the compound (28.9 g, 81.6 mmol) obtained in the above step 2 and 2-morpholinoethyl isocyanide (12.1 ml, 85.6 mmol) were added, and the mixture was heated again to 55 ° C. 8 Stirred for hours. The residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography (methanol-dichloromethane = 5:95) to obtain the title compound (67.0 g, 81.6 mmol, quantitative). It was.
［工程３］　ｔｅｒｔ－ブチル　６－［（４－クロロ－３－フルオロフェニル）メチル］－１１，１１－ジフルオロ－７，１５－ジオキソ－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－２－カルボキシレ－ト
　亜鉛（粉末状，　２４．３ｇ，　３３４ｍｍｏｌ）をテトラヒドロフラン（３００ｍｌ）に懸濁し、酢酸（１５０ｍｌ）を加えた。氷冷下、上記工程３で得られた化合物（６７．０ｇ，　８１．６ｍｍｏｌ）のテトラヒドロフラン（３００ｍｌ）溶液を加え、室温に昇温して２４時間撹拌した。反応液をジクロロメタンで希釈し、セライトろ過して不溶物を除去した。減圧下溶媒を留去して得られた残留物をトルエン（６３０ｍｌ）に溶解し、室温で酢酸（３０ｍｌ）を加えた。８０℃に加温して３時間撹拌した後に、減圧下トルエンを留去した。得られた残留物をジクロロメタンに溶解し、氷冷下飽和重曹水を加えアルカリ性に調整した。ジクロロメタンで抽出し、無水硫酸ナトリウムで乾燥した。減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝０：１００－５０：５０）で精製して、標記化合物（５．７０ｇ，　１１．０ｍｍｏｌ，　１４％）を得た。
1H－NMR (CDCl3) δ : 7.38－7.33 (1H, m), 7.26－7.21 (1H, m), 6.98－6.86 (2H, m), 4.89－4.33 (2H, m), 3.98－3.86 (1H, m), 3.79－3.49 (3H, m), 2.55－1.95 (8H, m), 1.93－1.78 (2H, m), 1.45 (9H, br s). [Step 3] tert-Butyl 6-[(4-chloro-3-fluorophenyl) methyl] -11,11-difluoro-7,15-dioxo-2,6,14-triazadispiro [4.2.5 8 . 25 ] Pentadecane-2-carboxylate Zinc (powder, 24.3 g, 334 mmol) was suspended in tetrahydrofuran (300 ml) and acetic acid (150 ml) was added. Under ice-cooling, a solution of the compound (67.0 g, 81.6 mmol) obtained in the above step 3 in tetrahydrofuran (300 ml) was added, the temperature was raised to room temperature, and the mixture was stirred for 24 hours. The reaction mixture was diluted with dichloromethane and filtered through Celite to remove insoluble matter. The solvent was evaporated under reduced pressure and the obtained residue was dissolved in toluene (630 ml), and acetic acid (30 ml) was added at room temperature. After heating to 80 ° C. and stirring for 3 hours, toluene was distilled off under reduced pressure. The obtained residue was dissolved in dichloromethane, and saturated aqueous sodium hydrogen carbonate was added under ice-cooling to adjust the alkalinity. It was extracted with dichloromethane and dried over anhydrous sodium sulfate. The residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography (ethyl acetate-hexane = 0: 100-50: 50) to obtain the title compound (5.70 g, 11.0 mmol, 14%). ) Was obtained.
1 H-NMR (CDCl 3 ) δ: 7.38-7.33 (1H, m), 7.26-7.21 (1H, m), 6.98-6.86 (2H, m), 4.89-4.33 (2H, m), 3.98-3.86 ( 1H, m), 3.79-3.49 (3H, m), 2.55-1.95 (8H, m), 1.93-1.78 (2H, m), 1.45 (9H, br s).
［工程４］　６－［（４－クロロ－３－フルオロフェニル）メチル］－１１，１１－ジフルオロ－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－７，１５－ジオン　塩酸塩
　上記工程３で得られた化合物（５．７０ｇ，　１１．０ｍｍｏｌ）を１，４－ジオキサン（５０ｍｌ）に懸濁し、氷冷下、４規定塩酸／１，４－ジオキサン溶液（２５ｍｌ）を加えて室温で１４時間撹拌した。減圧下溶媒を留去し、ジエチルエ－テルを加えてしばらく撹拌した。生じた固体をろ取して、標記化合物（２．４８ｇ，　５．４９ｍｍｏｌ，　５０％）を得た。
1H－NMR (CD3OD) δ : 7.48－7.43 (1H, m), 7.14－7.10 (1H, m), 7.05－7.01 (1H, m), 4.96－4.91 (1H, m), 4.55－4.48 (1H, m), 3.78－3.73 (1H, m), 3.61－3.56 (2H, m), 3.52－3.45 (1H, m), 3.36－3.31 (1H, m), 2.63－2.55 (1H, m), 2.50－2.41 (1H, m), 2.41－1.92 (8H, m). [Step 4] 6-[(4-Chloro-3-fluorophenyl) methyl] -11,11-difluoro-2,6,14-triazadispiro [4.2.5 8 . 25 ] Pentadecane-7,15-dione hydrochloride The compound (5.70 g, 11.0 mmol) obtained in step 3 above was suspended in 1,4-dioxane (50 ml), and under ice-cooling, 4N hydrochloric acid / A 1,4-dioxane solution (25 ml) was added, and the mixture was stirred at room temperature for 14 hours. The solvent was evaporated under reduced pressure, diethyl ether was added, and the mixture was stirred for a while. The resulting solid was collected by filtration to give the title compound (2.48 g, 5.49 mmol, 50%).
1 H-NMR (CD 3 OD) δ: 7.48-7.43 (1H, m), 7.14-7.10 (1H, m), 7.05-7.01 (1H, m), 4.96-4.91 (1H, m), 4.55-4.48 (1H, m), 3.78-3.73 (1H, m), 3.61-3.56 (2H, m), 3.52-3.45 (1H, m), 3.36-3.31 (1H, m), 2.63-2.55 (1H, m) , 2.50-2.41 (1H, m), 2.41-1.92 (8H, m).
［工程５］　２－（４－クロロ－２，６－ジフルオロベンゾイル）－６－［（４－クロロ－３－フルオロフェニル）メチル］－１１，１１－ジフルオロ－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－７，１５－ジオン
上記工程４で得られた化合物（２．０ｇ，　４．４２ｍｍｏｌ）をジメチルホルムアミド（６０ｍｌ）に溶解し、トリエチルアミン（１．２３ｍｌ，　８．８４ｍｍｏｌ）を加えて撹拌した。氷冷下、４－クロロ－２，６－ジフルオロ安息香酸（１．１１ｇ，　５．７５ｍｍｏｌ）、Ｎ，Ｎ－ジイソプロピルエチルアミン（１．５４ｍｌ，　８．８４ｍｍｏｌ）、Ｎ－［１－（シアノ－２－エトキシ－２－オキソエチリデンアミノオキシ）ジメチルアミノ（モルホリノ）］ウロニウム　ヘキサフルオロホスフェ－ト（２．０８ｇ，　４．８６ｍｍｏｌ）を加え、室温で２４時間撹拌した。反応液に水を加え、酢酸エチルで抽出し、飽和食塩水で洗浄した。無水硫酸ナトリウムで乾燥し、減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝１０：９０－１００：０）で精製して、標記化合物（ラセミ体）（２．１２ｇ，　３．６０ｍｍｏｌ，　８１％）を得た。 [Step 5] 2- (4-Chloro-2,6-difluorobenzoyl) -6-[(4-chloro-3-fluorophenyl) methyl] -11,11-difluoro-2,6,14-triazadispiro [4] .2.5 8 . 25 ] Pentadecane-7,15-dione The compound (2.0 g, 4.42 mmol) obtained in the above step 4 was dissolved in dimethylformamide (60 ml), and triethylamine (1.23 ml, 8.84 mmol) was added. Stirred. Under ice-cooling, 4-chloro-2,6-difluorobenzoic acid (1.11 g, 5.75 mmol), N, N-diisopropylethylamine (1.54 ml, 8.84 mmol), N- [1- (cyano-2) -Ethoxy-2-oxoethylideneaminooxy) dimethylamino (morpholino)] uronium hexafluorophosphate (2.08 g, 4.86 mmol) was added, and the mixture was stirred at room temperature for 24 hours. Water was added to the reaction mixture, the mixture was extracted with ethyl acetate, and washed with saturated brine. The residue obtained by drying over anhydrous sodium sulfate and distilling off the solvent under reduced pressure was purified by silica gel column chromatography (ethyl acetate-hexane = 10: 90-100: 0) to give the title compound (racemic). (2.12 g, 3.60 mmol, 81%) was obtained.
［工程６］　（－）－２－（４－クロロ－２，６－ジフルオロベンゾイル）－６－［（４－クロロ－３－フルオロフェニル）メチル］－１１，１１－ジフルオロ－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－７，１５－ジオン（Compound C）
　上記工程５で得られた化合物を以下の条件にて光学分割を行なった。
カラム：ダイセル　ＣＨＩＲＡＬＰＡＫ　ＩＢ　２０ｍｍＩＤｘ２５０ｍｍＬ
溶出溶媒：ヘキサン：２－プロパノ－ル＝５０：５０（Ｖ／Ｖ）
流速：１５．０ｍｌ／ｍｉｎ
温度：２５℃
ピ－ク（保持時間：９．７分）を分取して、標記化合物（７１７ｍｇ）を得た。
比旋光度［α］Ｄ ２０　＝　－４．７９８　（ｃ＝１．０，　クロロホルム）
1H－NMR (CDCl3) δ : 8.28－8.02 (1H, m), 7.40－7.29 (1H, m), 7.07－6.78 (4H, m), 4.92－4.70 (1H, m), 4.48－4.34 (1H, m), 4.22－4.11 (1H, m), 4.09－3.97 (1H, m), 3.81－3.66 (1H, m), 3.57－3.46 (1H, m), 2.61－1.99 (8H, m), 1.95－1.83 (2H, m).
MS (m/z) : 590 (M+H)+. [Step 6] (-)-2- (4-Chloro-2,6-difluorobenzoyl) -6-[(4-chloro-3-fluorophenyl) methyl]-11,11-difluoro-2,6,14 -Triazadispiro [4.2.5 8 . 25 ] Pentadecane-7,15-Zeon (Compound C)
The compound obtained in the above step 5 was optically resolved under the following conditions.
Column: Daicel CHIRALPAK IB 20mm IDx250mmL
Elution solvent: Hexane: 2-Propanol = 50: 50 (V / V)
Flow velocity: 15.0 ml / min
Temperature: 25 ° C
The peak (retention time: 9.7 minutes) was fractionated to obtain the title compound (717 mg).
Specific rotation [α] D 20 = -4.798 (c = 1.0, chloroform)
1 H-NMR (CDCl 3 ) δ: 8.28-8.02 (1H, m), 7.40-7.29 (1H, m), 7.07-6.78 (4H, m), 4.92-4.70 (1H, m), 4.48-4.34 ( 1H, m), 4.22-4.11 (1H, m), 4.09-3.97 (1H, m), 3.81-3.66 (1H, m), 3.57-3.46 (1H, m), 2.61-1.99 (8H, m), 1.95－1.83 (2H, m).
MS (m / z): 590 (M + H) + .
（実施例４）Compound Dの製造 (Example 4) Manufacture of Compound D
Figure JPOXMLDOC01-appb-C000004
［工程１］　ｔｅｒｔ－ブチル　３－｛（１－｛［（ベンジルオキシ）カルボニル］アミノ｝－４，４－ジフルオロシクロヘキサン－１－カルボニル）［（４－メトキシフェニル）メチル］アミノ｝－３－｛［２－（モルホリン－４－イル）エチル］カルバモイル｝ピロリジン－１－カルボキシレ－ト
　実施例３の工程２で得られた化合物（１．６５ｇ，　５．２７ｍｍｏｌ）、ｔｅｒｔ－ブチル　３－オキソピロリジノン－１－カルボキシレ－ト（９７５ｍｇ，　５．２７ｍｍｏｌ）、（４－メトキシフェニル）メチルアミン（６８２μｌ，　５．２７ｍｍｏｌ）をメタノ－ル（２５ｍｌ）に懸濁し、室温で１５分間撹拌した。反応液に２－モルホリノエチルイソシアニド（７２４μｌ，　５．２７ｍｍｏｌ）を加え、５５℃に加温して８時間撹拌した。減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－メタノ－ル－ジクロロメタン＝９５：０：５－８０：１５：５）で精製して、標記化合物（６０２ｍｇ，　０．７９４ｍｍｏｌ，　１５％）を得た。
Figure JPOXMLDOC01-appb-C000004
[Step 1] tert-Butyl 3-{(1-{[(benzyloxy) carbonyl] amino} -4,4-difluorocyclohexane-1-carbonyl) [(4-methoxyphenyl) methyl] amino} -3- { [2- (Morphorin-4-yl) ethyl] carbamoyl} pyrrolidine-1-carboxylate The compound (1.65 g, 5.27 mmol) obtained in step 2 of Example 3, tert-butyl 3-oxopyrrolidinone. -1-Carboxylate (975 mg, 5.27 mmol) and (4-methoxyphenyl) methylamine (682 μl, 5.27 mmol) were suspended in methaneol (25 ml) and stirred at room temperature for 15 minutes. 2-Molholinoethyl isocyanide (724 μl, 5.27 mmol) was added to the reaction mixture, and the mixture was heated to 55 ° C. and stirred for 8 hours. The residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography (ethyl acetate-methanol-dichloromethane = 95: 0: 5-80: 15: 5), and the title compound (602 mg, 0.794 mmol, 15%) was obtained.
［工程２］　ｔｅｒｔ－ブチル　１１，１１－ジフルオロ－６－［（４－メトキシフェニル）メチル］－７，１５－ジオキソ－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－２－カルボキシレ－ト
　上記工程１で得られた化合物（５９９ｍｇ，　０．７９０ｍｍｏｌ）を酢酸エチル（８ｍｌ）及び酢酸（２ｍｌ）に溶解し、２０％水酸化パラジウム炭素触媒（１５０ｍｇ）を加えて、水素雰囲気下、室温で２時間撹拌した。酢酸（４ｍｌ）を加え、さらに２時間反応させた。不溶物をセライトでろ過し、ろ液を減圧下濃縮した。得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝４０：６０－７０：３０）で精製して、標記化合物（１５６ｍｇ，　０．３１６ｍｍｏｌ，　４０％）を得た。
1H－NMR (DMSO－D6) δ : 8.84－8.78 (1H, m), 7.10－7.02 (2H, m), 6.87 (2H, d, J = 8.5 Hz), 4.76－4.31 (2H, m), 3.72 (3H, s), 3.68－3.59 (1H, m), 3.55－3.30 (2H, m), 2.33－2.03 (9H, m), 1.93－1.81 (2H, m), 1.42－1.29 (9H, m). [Step 2] tert-Butyl 11,11-difluoro-6-[(4-methoxyphenyl) methyl] -7,15-dioxo-2,6,14-triazadispiro [4.2.5 8 . 25 ] Pentadecane-2-carboxylate The compound (599 mg, 0.790 mmol) obtained in the above step 1 was dissolved in ethyl acetate (8 ml) and acetic acid (2 ml), and a 20% palladium hydroxide carbon catalyst (150 mg) was dissolved. ) Was added, and the mixture was stirred at room temperature for 2 hours under a hydrogen atmosphere. Acetic acid (4 ml) was added and the reaction was carried out for another 2 hours. The insoluble material was filtered through Celite, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 40: 60-70: 30) to give the title compound (156 mg, 0.316 mmol, 40%).
1 H-NMR (DMSO-D 6 ) δ: 8.84-8.78 (1H, m), 7.10-7.02 (2H, m), 6.87 (2H, d, J = 8.5 Hz), 4.76-4.31 (2H, m) , 3.72 (3H, s), 3.68-3.59 (1H, m), 3.55-3.30 (2H, m), 2.33-2.03 (9H, m), 1.93-1.81 (2H, m), 1.42-1.29 (9H, m).
［工程３］　１１，１１－ジフルオロ－６－［（４－メトキシフェニル）メチル］－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－７，１５－ジオン　塩酸塩
　上記工程２で得られた化合物（１５３ｍｇ，　０．３１０ｍｍｏｌ）を４規定塩酸／１，４－ジオキサン溶液（４ｍｌ）に溶解し、室温で６時間撹拌した。ジエチルエ－テルを加え、析出物をろ取し、ジエチルエ－テルで洗浄した。減圧下乾燥し、標記化合物（１３０ｍｇ，　０．３０２ｍｍｏｌ，　９８％）を得た。
1H－NMR (CD3OD) δ : 7.15－7.10 (2H, m), 6.93－6.88 (2H, m), 4.95 (1H, d, J = 15.9 Hz), 4.44 (1H, d, J = 15.9 Hz), 3.77 (3H, s), 3.68－3.63 (3H, m), 3.58－3.53 (2H, m), 3.44－3.38 (1H, m), 2.59－2.44 (2H, m), 2.43－1.91 (8H, m). [Step 3] 11,11-difluoro-6-[(4-methoxyphenyl) methyl] -2,6,14-triazadispiro [4.2.5 8 . 25 ] Pentadecane-7,15-dione hydrochloride The compound (153 mg, 0.310 mmol) obtained in the above step 2 was dissolved in 4N hydrochloric acid / 1,4-dioxane solution (4 ml) and stirred at room temperature for 6 hours. did. Diethyl ether was added, and the precipitate was collected by filtration and washed with diethyl ether. Drying under reduced pressure gave the title compound (130 mg, 0.302 mmol, 98%).
1 H-NMR (CD 3 OD) δ: 7.15-7.10 (2H, m), 6.93-6.88 (2H, m), 4.95 (1H, d, J = 15.9 Hz), 4.44 (1H, d, J = 15.9) Hz), 3.77 (3H, s), 3.68-3.63 (3H, m), 3.58-3.53 (2H, m), 3.44-3.38 (1H, m), 2.59-2.44 (2H, m), 2.43-1.91 ( 8H, m).
［工程４］　２－（４－クロロ－２，６－ジフルオロベンゾイル）－１１，１１－ジフルオロ－６－［（４－メトキシフェニル）メチル］－２，６，１４－トリアザジスピロ［４．２．５８．２５］ペンタデカン－７，１５－ジオン（Compound D）
　上記工程３で得られた化合物（１２７ｍｇ，　０．２９５ｍｍｏｌ）をＮ，Ｎ－ジメチルホルムアミド（４ｍｌ）に溶解し、４－クロロ－２，６－ジフルオロ安息香酸（５６．９ｍｇ，　０．２９５ｍｍｏｌ）、１－［ビス（ジメチルアミノ）メチレン］－１Ｈ－１，２，３－トリアゾロ［４，５－ｂ］ピリジニウム　３－オキシドヘキサフルオロホスファ－ト（１４６ｍｇ，　０．３８４ｍｍｏｌ）、ジイソプロピルエチルアミン（１８０μｌ，　１．０３ｍｍｏｌ）を加え、室温で７時間撹拌した。反応液に水を加え、酢酸エチルで抽出した。有機層を飽和重曹水、飽和食塩水で洗浄後、無水硫酸マグネシウムで乾燥した。減圧下溶媒を留去し、得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝６０：４０－１００：０，　アミノシリカゲル）で精製して、標記化合物（１４６ｍｇ，　０．２５７ｍｍｏｌ，　８７％）を得た。
1H－NMR (CDCl3) δ : 8.19 (1H, br s), 7.12－7.07 (2H, m), 7.05－7.01 (2H, m), 6.88－6.84 (2H, m), 4.92 (1H, d, J = 15.9 Hz), 4.36 (1H, d, J = 15.9 Hz), 4.17 (1H, d, J = 14.0 Hz), 4.04 (1H, d, J = 14.0 Hz), 3.79 (3H, s), 3.74－3.65 (1H, m), 3.55－3.47 (1H, m), 2.56－1.97 (8H, m), 1.94－1.82 (2H, m). 
MS (m/z) : 568 (M+H)+. [Step 4] 2- (4-Chloro-2,6-difluorobenzoyl) -11,11-difluoro-6-[(4-methoxyphenyl) methyl] -2,6,14-triazadispiro [4.2.5] 8 . 25 ] Pentadecane-7,15-Zeon (Compound D)
The compound (127 mg, 0.295 mmol) obtained in the above step 3 was dissolved in N, N-dimethylformamide (4 ml) to prepare 4-chloro-2,6-difluorobenzoic acid (56.9 mg, 0.295 mmol). 1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxide hexafluorophosphate (146 mg, 0.384 mmol), diisopropylethylamine (180 μl, 1.03 mmol) was added, and the mixture was stirred at room temperature for 7 hours. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium hydrogen carbonate and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (ethyl acetate-hexane = 60: 40-100: 0, amino silica gel) to obtain the title compound (146 mg, 0.257 mmol, 87). %) Was obtained.
1 H-NMR (CDCl 3 ) δ: 8.19 (1H, br s), 7.12-7.07 (2H, m), 7.05-7.01 (2H, m), 6.88-6.84 (2H, m), 4.92 (1H, d) , J = 15.9 Hz), 4.36 (1H, d, J = 15.9 Hz), 4.17 (1H, d, J = 14.0 Hz), 4.04 (1H, d, J = 14.0 Hz), 3.79 (3H, s), 3.74-3.65 (1H, m), 3.55-3.47 (1H, m), 2.56-1.97 (8H, m), 1.94-1.82 (2H, m).
MS (m / z): 568 (M + H) + .
（実施例５）Compound Eの製造 (Example 5) Manufacture of Compound E
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
［工程１］　（２Ｒ）－シクロヘキシル｛［（２，２，２－トリクロロエトキシ）カルボニル］アミノ｝酢酸
　Ｄ－α－シクロヘキシルグリシン（１０．０ｇ，　６２．０ｍｍｏｌ）を水（１２０ｍｌ）に懸濁し、１規定水酸化ナトリウム水溶液（７０ｍｌ），ジエチルエ－テル（６０ｍｌ）を加えて溶解した。－５℃に冷却しながらクロロぎ酸　２，２，２－トリクロロエチル（１２．０ｍｌ，　８７．０ｍｍｏｌ）の１，４－ジオキサン（９０ｍｌ）溶液と１規定水酸化ナトリウム水溶液（９０ｍｌ）を同時に滴下し、０℃で１時間、室温で１５時間撹拌させた。反応液に水を加え、ジエチルエ－テルで洗浄した。水層に氷冷下、１規定塩酸水溶液を加えて液性をｐＨ５付近に調整し、酢酸エチルで抽出し、無水硫酸ナトリウムで乾燥した。減圧下溶媒を留去して、標記化合物（２２．４ｇ，　６７．４ｍｍｏｌ，　定量的）を得た。
1H－NMR (CDCl3) δ : 5.53－5.45 (1H, m), 4.82－4.68 (2H, m), 4.38－4.32 (1H, m), 1.96－1.85 (1H, m), 1.84－1.73 (4H, m), 1.37－1.06 (6H, m). [Step 1] (2R) -cyclohexyl {[(2,2,2-trichloroethoxy) carbonyl] amino} acetate D-α-cyclohexylglycine (10.0 g, 62.0 mmol) was suspended in water (120 ml). 1 Aqueous sodium hydroxide solution (70 ml) and diethyl ether (60 ml) were added and dissolved. While cooling to -5 ° C, a solution of 2,2,2-trichloroethyl chloroformate (12.0 ml, 87.0 mmol) in 1,4-dioxane (90 ml) and a 1N aqueous solution of sodium hydroxide (90 ml) were added dropwise simultaneously. Then, the mixture was stirred at 0 ° C. for 1 hour and at room temperature for 15 hours. Water was added to the reaction mixture, and the mixture was washed with diethyl ether. Under ice-cooling, a 1N hydrochloric acid aqueous solution was added to the aqueous layer to adjust the liquid property to around pH 5, extracted with ethyl acetate, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain the title compound (22.4 g, 67.4 mmol, quantitative).
1 H-NMR (CDCl 3 ) δ: 5.53-5.45 (1H, m), 4.82-4.68 (2H, m), 4.38-4.32 (1H, m), 1.96-1.85 (1H, m), 1.84-1.73 ( 4H, m), 1.37-1.06 (6H, m).
［工程２］　ｔｅｒｔ－ブチル　３－｛［（２Ｒ）－２－シクロヘキシル－２－｛［（２，２，２－トリクロロエトキシ）カルボニル］アミノ｝アセチル］［（４－メトキシフェニル）メチル］アミノ｝－３－｛［２－（モルホリン－４－イル）エチル］カルバモイル｝ピロリジン－１－カルボキシレ－ト
　上記工程１で得られた化合物（２．４７ｇ，　７．４３ｍｍｏｌ）、ｔｅｒｔ－ブチル　３－オキソピロリジノン－１－カルボキシレ－ト（１．３８ｇ，　７．４３ｍｍｏｌ）、（４－メトキシフェニル）メチルアミン（１．０２ｇ，　７．４３ｍｍｏｌ）をメタノ－ル（１００ｍｌ）に懸濁し、室温で１０分間撹拌した。反応液に２－モルホリノエチルイソシアニド（１．０２ｍｌ，　７．４３ｍｍｏｌ）を加え、５５℃に加温して９時間撹拌した。減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（メタノ－ル－ジクロロメタン＝１：９９－１５：８５）で精製して、標記化合物（４．３３ｇ，　５．５７ｍｍｏｌ，　７５％）を得た。
MS (m/z) : 776, 777, 778, 779 (M+H)+. [Step 2] tert-butyl 3-{[(2R) -2-cyclohexyl-2-{[(2,2,2-trichloroethoxy) carbonyl] amino} acetyl] [(4-methoxyphenyl) methyl] amino} -3-{[2- (Morphorin-4-yl) ethyl] carbamoyl} pyrrolidine-1-carboxylate The compound (2.47 g, 7.43 mmol) obtained in the above step 1, tert-butyl 3-oxo. Pyrrolidinone-1-carboxylate (1.38 g, 7.43 mmol) and (4-methoxyphenyl) methylamine (1.02 g, 7.43 mmol) were suspended in methaneol (100 ml) and at room temperature for 10 minutes. Stirred. 2-Molholinoethyl isocyanide (1.02 ml, 7.43 mmol) was added to the reaction mixture, and the mixture was heated to 55 ° C. and stirred for 9 hours. The residue obtained by distilling off the solvent under reduced pressure was purified by silica gel column chromatography (methanol-dichloromethane = 1: 99-15: 85), and the title compound (4.33 g, 5.57 mmol, 75) was purified. %) Was obtained.
MS (m / z): 776, 777, 778, 779 (M + H) + .
［工程３］　ｔｅｒｔ－ブチル　（５Ｓ，８Ｒ）－８－シクロヘキシル－６－［（４－メトキシフェニル）メチル］－７，１０－ジオキソ－２，６，９－トリアザスピロ［４．５］デカン－２－カルボキシレ－ト　および　ｔｅｒｔ－ブチル　（５Ｒ，８Ｒ）－８－シクロヘキシル－６－［（４－メトキシフェニル）メチル］－７，１０－ジオキソ－２，６，９－トリアザスピロ［４．５］デカン－２－カルボキシレ－ト
　上記工程２で得られた化合物（４．３３ｇ，　５．５７ｍｍｏｌ）をテトラヒドロフラン（４０ｍｌ）に溶解し、酢酸（１０ｍｌ）を加えた。氷冷下、亜鉛（１．４６ｇ，　２２．３ｍｍｏｌ）を加えて、室温で３時間撹拌した。不溶物をセライトろ過して、テトラヒドロフランで洗浄した。減圧下ろ液を濃縮し、トルエンを加えて共沸させた。得られた残留物をトルエン（４０ｍｌ）に溶解し、酢酸（２ｍｌ）を加えて、８０℃で１時間撹拌した。減圧下溶媒を留去し、トルエンで共沸して得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝８０：２０－１００：０）で精製して、低極性フラクションから（５Ｓ，８Ｒ）体（７２０ｍｇ，　１．５３ｍｍｏｌ，　２７％）を、高極性フラクションから（５Ｒ，８Ｒ）体（５１７ｍｇ，　１．１０ｍｍｏｌ，　２０％）を得た。
MS (m/z) : 372 (M+H－Boc)+. [Step 3] tert-Butyl (5S, 8R) -8-cyclohexyl-6-[(4-methoxyphenyl) methyl] -7,10-dioxo-2,6,9-triazaspiro [4.5] decan-2 -Carboxylate and tert-butyl (5R, 8R) -8-cyclohexyl-6-[(4-methoxyphenyl) methyl] -7,10-dioxo-2,6,9-triazaspiro [4.5] decan -2-carboxylate The compound (4.33 g, 5.57 mmol) obtained in the above step 2 was dissolved in tetrahydrofuran (40 ml), and acetic acid (10 ml) was added. Zinc (1.46 g, 22.3 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 3 hours. The insoluble material was filtered through Celite and washed with tetrahydrofuran. The reduced pressure filtrate was concentrated, toluene was added, and the mixture was azeotropically heated. The obtained residue was dissolved in toluene (40 ml), acetic acid (2 ml) was added, and the mixture was stirred at 80 ° C. for 1 hour. The solvent was distilled off under reduced pressure, and the residue obtained by azeotroping with toluene was purified by silica gel column chromatography (ethyl acetate-hexane = 80: 20-100: 0) from a low-polarity fraction (5S, An 8R) form (720 mg, 1.53 mmol, 27%) was obtained from a highly polar fraction (5R, 8R) form (517 mg, 1.10 mmol, 20%).
MS (m / z): 372 (M + H－Boc) + .
［工程４］　（５Ｒ，８Ｒ）－８－シクロヘキシル－６－［（４－メトキシフェニル）メチル］－２，６，９－トリアザスピロ［４．５］デカン－７，１０－ジオン　塩酸塩
　上記工程３で得られた（５Ｒ，８Ｒ）体（５１７ｍｇ，　１．１０ｍｍｏｌ）を１，４－ジオキサン（１０ｍｌ）に溶解し、４規定塩酸／１，４－ジオキサン溶液（１０ｍｌ）を加えて室温で２時間撹拌した。ジエチルエ－テルで希釈し、析出した固体をろ取した。ジエチルエ－テルで洗浄し、減圧下乾燥して標記化合物（４２９ｍｇ，　１．０５ｍｍｏｌ，　９６％）を得た。
1H－NMR (DMSO－D6) δ : 9.92 (1H, br s), 9.16 (1H, br s), 8.66 (1H, s), 7.17－7.09 (2H, m), 6.92－6.84 (2H, m), 4.77－4.56 (2H, m), 4.04－3.99 (1H, m), 3.73 (3H, s), 3.60－3.19 (3H, m), 2.41－2.28 (1H, m), 2.25－2.11 (1H, m), 2.01－1.87 (1H, m), 1.81－1.00 (11H, m). [Step 4] (5R, 8R) -8-cyclohexyl-6-[(4-methoxyphenyl) methyl] -2,6,9-triazaspiro [4.5] decane-7,10-dione hydrochloride The (5R, 8R) compound (517 mg, 1.10 mmol) obtained in (1) was dissolved in 1,4-dioxane (10 ml), 4N hydrochloric acid / 1,4-dioxane solution (10 ml) was added, and the mixture was added at room temperature for 2 hours. Stirred. The solid was diluted with diethyl ether and the precipitated solid was collected by filtration. The mixture was washed with diethyl ether and dried under reduced pressure to give the title compound (429 mg, 1.05 mmol, 96%).
1 H-NMR (DMSO-D 6 ) δ: 9.92 (1H, br s), 9.16 (1H, br s), 8.66 (1H, s), 7.17-7.09 (2H, m), 6.92-6.84 (2H, m), 4.77-4.56 (2H, m), 4.04-3.99 (1H, m), 3.73 (3H, s), 3.60-3.19 (3H, m), 2.41-2.28 (1H, m), 2.25-2.11 ( 1H, m), 2.01-1.87 (1H, m), 1.81-1.000 (11H, m).
［工程５］　（５Ｒ，８Ｒ）－２－（２－アミノ－６－フルオロ－４－メチルベンゾイル）－８－シクロヘキシル－６－［（４－メトキシフェニル）メチル］－２，６，９－トリアザスピロ［４．５］デカン－７，１０－ジオン（Compound E）
　上記工程４で得られた化合物（５０．０ｍｇ，　０．１２３ｍｍｏｌ）をＮ，Ｎ－ジメチルホルムアミド（３ｍｌ）に溶解し、トリエチルアミン（３４．２μｌ，　０．２４５ｍｍｏｌ）を加えて撹拌した。氷冷下、２－アミノ－６－フルオロ－４－メチル安息香酸（２７．０ｍｇ，　０．１５９ｍｍｏｌ）、Ｎ，Ｎ－ジイソプロピルエチルアミン（４２．７μｌ，　０．２４５ｍｍｏｌ）、Ｎ－［１－（シアノ－２－エトキシ－２－オキソエチリデンアミノオキシ）ジメチルアミノ（モルホリノ）］ウロニウム　ヘキサフルオロホスフェ－ト（５７．７ｍｇ，　０．１３５ｍｍｏｌ）を加え、室温で７２時間撹拌した。反応液に水を加え、酢酸エチルで抽出し、飽和食塩水で洗浄した。無水硫酸ナトリウムで乾燥後、減圧下溶媒を留去して得られた残留物をシリカゲルカラムクロマトグラフィ－（酢酸エチル－ヘキサン＝２０：８０－１００：０；メタノ－ル－ジクロロメタン＝５：９５）で精製して、標記化合物（６０．４ｍｇ，　０．１１６ｍｍｏｌ，　９４％）を得た。
1H－NMR (CDCl3) δ : 7.17－7.02 (2H, m), 6.86－6.75 (2H, m), 6.32－6.27 (1H, m), 6.27－6.14 (1H, m), 6.02－5.90 (1H, m), 4.89－4.59 (1H, m), 4.49－4.36 (2H, m), 4.27－4.11 (1H, m), 4.04－3.96 (2H, m), 3.79－3.76 (3H, m), 3.76－3.57 (3H, m), 2.51－2.10 (5H, m), 1.89－1.76 (1H, m), 1.75－0.97 (10H, m). 
MS (m/z) : 523 (M+H)+. [Step 5] (5R, 8R) -2- (2-amino-6-fluoro-4-methylbenzoyl) -8-cyclohexyl-6-[(4-methoxyphenyl) methyl] -2,6,9-triazaspiro [4.5] Decane-7,10-Zeon (Compound E)
The compound (50.0 mg, 0.123 mmol) obtained in the above step 4 was dissolved in N, N-dimethylformamide (3 ml), triethylamine (34.2 μl, 0.245 mmol) was added, and the mixture was stirred. Under ice-cooling, 2-amino-6-fluoro-4-methylbenzoic acid (27.0 mg, 0.159 mmol), N, N-diisopropylethylamine (42.7 μl, 0.245 mmol), N- [1- (cyano) -2-ethoxy-2-oxoethylideneaminooxy) dimethylamino (morpholino)] uronium hexafluorophosphate (57.7 mg, 0.135 mmol) was added, and the mixture was stirred at room temperature for 72 hours. Water was added to the reaction mixture, the mixture was extracted with ethyl acetate, and washed with saturated brine. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography (ethyl acetate-hexane = 20: 80-100: 0; methanol-dichloromethane = 5: 95). Purification gave the title compound (60.4 mg, 0.116 mmol, 94%).
1 1 H-NMR (CDCl 3 ) δ: 7.17-7.02 (2H, m), 6.86-6.75 (2H, m), 6.32-6.27 (1H, m), 6.27-6.14 (1H, m), 6.02-5.90 ( 1H, m), 4.89-4.59 (1H, m), 4.49-4.36 (2H, m), 4.27-4.11 (1H, m), 4.04-3.96 (2H, m), 3.79-3.76 (3H, m), 3.76-3.57 (3H, m), 2.51-2.10 (5H, m), 1.89-1.76 (1H, m), 1.75-0.97 (10H, m).
MS (m / z): 523 (M + H) + .
（実施例６）Compound A、B、C、D、Eのがん細胞に対する増殖抑制活性評価
ヒト神経芽腫細胞株SH－SY5Y（European Collection of Authenticated Cell Cultures）は牛胎児血清（Hyclone ＃SH30910.03）（最終濃度15％）を含むMEMおよびF12混合培地（Thermo　Fisher　Scientific ＃11095ならびに＃11765）（以下、「MEM／F12培地」という。）で継代維持した。細胞株はTrypLE Express（Thermo Fisher Scientific ＃12605）で剥離回収した後、室温で1000 rpmで5分間遠心処理し、上清を除去した。MEM／F12培地で細胞を懸濁し、20000個／ 1 mLの細胞濃度に調製し、96穴プレ－ト（Corning ＃3904）の各ウェルへ100μLずつ分注し、37℃、5％CO2下で24時間培養した。続いてMEM／F12培地にてそれぞれ所定の濃度に希釈したCompound A、B、C、D、Eを、各ウェルへ50 μL添加し(day1)、37℃、5％ CO2下、3日間培養した。化合物添加当日(day 1)ならびに化合物添加3日後(day 4)にATP測定用試薬であるCellTiter－Glo 2.0 Assay(Promega ＃G9242)を30μL／wellずつ各ウェルに添加し、EnVision（PerkinElmer）で各ウェルの発光量を測定した。化合物添加当日の発光量(C1)、3日間培養後の化合物非添加群(C4)および化合物添加群(T4)の発光量より、次式に基づき細胞増殖率を算出した。 (Example 6) Evaluation of growth inhibitory activity of Compound A, B, C, D, and E against cancer cells Human neuroblastoma cell line SH-SY5Y (European Collection of Authenticated Cell Cultures) is fetal bovine serum (Hyclone # SH30910. 03) Subculture was maintained in MEM and F12 mixed medium (Thermo Fisher Scientific # 11095 and # 11765) containing (final concentration 15%) (hereinafter referred to as “MEM / F12 medium”). The cell line was exfoliated and collected by TrypLE Express (Thermo Fisher Scientific # 12605), and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant. Suspend cells in MEM / F12 medium, adjust to 20000 cells / 1 mL cell concentration, dispense 100 μL into each well of 96-well plate (Corning # 3904), 37 ° C., under 5% CO 2. Was cultured for 24 hours. Subsequently, 50 μL of Compound A, B, C, D, and E diluted to a predetermined concentration in MEM / F12 medium was added to each well (day 1), and cultured at 37 ° C. under 5% CO 2 for 3 days. did. On the day of compound addition (day 1) and 3 days after compound addition (day 4), CellTiter-Glo 2.0 Assay (Promega # G9242), which is a reagent for ATP measurement, was added to each well at 30 μL / well, and each well was added with EnVision (PerkinElmer). The amount of light emitted from the wells was measured. The cell proliferation rate was calculated based on the following formula from the luminescence amount on the day of compound addition (C1), the luminescence amount of the compound-free group (C4) and the compound-added group (T4) after culturing for 3 days.
Figure JPOXMLDOC01-appb-M000006
Figure JPOXMLDOC01-appb-M000006
各濃度における細胞増殖率と化合物濃度を片対数プロットした。結果を図１に示す。検討の結果、Compound A、B、C、Dは細胞増殖阻害活性を示したが、Compound Eは細胞増殖阻害活性を示さないことを見出した。 The cell proliferation rate and compound concentration at each concentration were semi-logarithmically plotted. The results are shown in FIG. As a result of the examination, it was found that Compound A, B, C and D showed cell growth inhibitory activity, but Compound E did not show cell growth inhibitory activity.
（実施例７）Compound A、B、Cが細胞周期に及ぼす影響
　実施例６において、細胞増殖阻害活性を有することが判明したCompound A、B、Cを用いて、これら化合物が細胞周期にどのような影響を及ぼしているのか検討を行った。
ヒト神経芽腫細胞株SH－SY5Yは、MEM／F12培地で細胞を懸濁し、20000個／ 1 mLの細胞濃度に調製し、60 mmディッシュ（Corning ＃430166）へ4 mLずつ分注し、37℃、5％CO2下で2日間培養した。続いて新鮮なMEM／F12培地4 mLに置換した後、Compound A、B、CをそれぞれDMSOで最終濃度の1000倍の濃度に希釈した溶液を各ディッシュへ4μL添加し、37℃、5％CO2下で24時間培養した。細胞をTrypLE Expressで剥離回収した後、室温で1000 rpmで5分間遠心処理し、上清を除去した。D－PBS(－)（富士フイルム和光純薬 ＃045－29795）を加え室温で1000 rpmで5分間遠心処理し、上清を除去した。その後BD Cyclestest Plus DNA Reagent kit（BD Biosciences、＃340242）を用いkitに添付のプロトコ－ルに従い細胞を染色した。続いてBD FACSCanto（BD Biosciences）を用い細胞周期を解析した。得られたデ－タはFlowJo 7.6.5を用いて解析した。結果を図２に示す。検討の結果、Compound A、B、Cは細胞分裂期停止作用を示すことを見出した。 (Example 7) Effect of Compounds A, B, and C on the cell cycle Using Compounds A, B, and C, which were found to have cell growth inhibitory activity in Example 6, how these compounds were added to the cell cycle. We examined whether it had a positive effect.
The human neuroblastoma cell line SH-SY5Y is prepared by suspending cells in MEM / F12 medium to a cell concentration of 20000 cells / 1 mL, and dispensing 4 mL each into a 60 mm dish (Corning # 430166), 37 The cells were cultured at 5% CO 2 for 2 days. Subsequently, after replacement with 4 mL of fresh MEM / F12 medium, 4 μL of a solution obtained by diluting Compound A, B, and C with DMSO to a concentration 1000 times the final concentration was added to each dish, and 37 ° C., 5% CO Incubated under 2 for 24 hours. The cells were exfoliated and collected by TrypLE Express, and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant. D-PBS (-) (Fujifilm Wako Pure Chemical Industries, Ltd. # 045-29795) was added, and the mixture was centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant. The cells were then stained using the BD Cyclestest Plus DNA Reagent kit (BD Biosciences, # 340242) according to the protocol attached to the kit. Subsequently, the cell cycle was analyzed using BD FACSCanto (BD Biosciences). The obtained data was analyzed using FlowJo 7.6.5. The results are shown in FIG. As a result of the examination, it was found that Compounds A, B, and C show a cell division arresting action.
（実施例８）Compound A、B、Cの紡錘体形成に及ぼす影響
ヒト神経芽腫細胞株SH－SY5YをMEM／F12培地で懸濁し、20000個／ 1 mLの細胞濃度に調製し、8ウェルカルチャ－スライド（Falcon、＃354118）の各ウェルへ250μLずつ分注し、37℃、5％CO2下で24時間培養した。続いてMEM／F12培地でそれぞれ最終濃度の2倍の濃度に希釈したCompound A、B、Cを、各ウェルへ250μL添加し、37℃、5％CO2下で24時間培養した。培地を除去し、4％ホルムアルデヒド固定液（和光純薬、＃163－20145）を200μL添加し室温で15分間処理した。固定液を除去し、D－PBS(－) 200μLを添加し5分間処理し洗浄した。この操作をさらに２回繰り返した。5％正常ヤギ血清（Life technologies、 ＃50062Z）／ 0.3％ Triton X－100（SIGMA、＃X100－500ML）／ D－PBS(－)をブロッキングバッファ－とし、各ウェルへ200μL添加し室温で60分間処理した。続いて抗体希釈バッファ－1％ BSA（SIGMA、＃A9205－50ML）／ 0.3％　Triton X－100／ D－PBS(－)を用いて１次抗体β－チューブリン rabbit polyclonal抗体（Cell Signaling Technologies、＃2146）およびPericentrin mouse monoclonal抗体（Abcam、＃ab28144）をそれぞれ80倍および1000倍希釈し、4℃で一晩処理した。翌日、上記と同様にD－PBS(－)で３度洗浄後、上記と同様の抗体希釈バッファ－を用いて２次抗体Goat anti－mouse IgG Alexa 488抗体（Life technologies、 ＃A11029）およびGoat anti－rabbit IgG Alexa 594抗体（Life technologies、 ＃A11012）をともに200倍希釈し、遮光下室温で60分間処理した。続いてD－PBS(－)で３度洗浄し、DAPI含有VECTASHIELD（Vector、＃H－1200）で封入し、共焦点顕微鏡（Leica TCS－SP8 STED）で観察しLASXソフトウェアで解析した。結果を図３に示す。検討の結果、Compound A、B、Cは、monopolar spindleを誘導することを見出した。 (Example 8) Effect of Compounds A, B, and C on spindle formation Human neuroblastoma cell line SH-SY5Y was suspended in MEM / F12 medium, prepared to a cell concentration of 20000 cells / 1 mL, and 8 wells. 250 μL of each well of the culture slide (Falcon, # 354118) was dispensed and cultured at 37 ° C. under 5% CO 2 for 24 hours. Subsequently, 250 μL of Compound A, B, and C diluted in MEM / F12 medium to twice the final concentration was added to each well, and the cells were cultured at 37 ° C. under 5% CO 2 for 24 hours. The medium was removed, 200 μL of 4% formaldehyde fixative (Wako Pure Chemical Industries, Ltd., # 163-20145) was added, and the mixture was treated at room temperature for 15 minutes. The fixative was removed, 200 μL of D-PBS (−) was added, treated for 5 minutes and washed. This operation was repeated two more times. Using 5% normal goat serum (Life technologies, # 50062Z) / 0.3% Triton X-100 (SIGMA, # X100-500ML) / D-PBS (-) as a blocking buffer, add 200 μL to each well for 60 minutes at room temperature. Processed. Subsequently, the antibody dilution buffer-1% BSA (SIGMA, # A9205-50ML) / 0.3% Triton X-100 / D-PBS (-) was used to use the primary antibody β-tubulin rabbit polyclonal antibody (Cell Signaling Technologies, #). 2146) and Pericentrin mouse monoclonal antibody (Abcam, # ab28144) were diluted 80-fold and 1000-fold, respectively, and treated overnight at 4 ° C. The next day, after washing 3 times with D-PBS (-) in the same manner as above, the secondary antibody Goat anti-mouse IgG Alexa 488 antibody (Life technologies, # A11029) and Goat anti using the same antibody dilution buffer as above. -Rabbit IgG Alexa 594 antibody (Life technologies, # A11012) was diluted 200-fold and treated at room temperature in the dark for 60 minutes. Subsequently, the cells were washed 3 times with D-PBS (-), sealed with VECTASHIELD (Vector, # H-1200) containing DAPI, observed with a confocal microscope (Leica TCS-SP8 STED), and analyzed with LASX software. The results are shown in FIG. As a result of the examination, it was found that Compounds A, B, and C induce monopolar spindles.
（実施例９）Compound Aの結合タンパク質の同定
Compound Aは紫外線照射により近傍の蛋白質と共有結合する光反応性基、およびクリック反応で化学修飾するためのアルキンを導入した化合物である。Compound Aと結合する蛋白質の複合体を同定するため、光架橋と化学架橋を組み合わせたプルダウン試験を実施した。 (Example 9) Identification of compound A binding protein
Compound A is a compound in which a photoreactive group that covalently binds to a nearby protein by irradiation with ultraviolet rays and an alkyne for chemical modification by a click reaction are introduced. A pull-down test combining photocrosslinking and chemical crosslinking was performed to identify the protein complex that binds to Compound A.
［サンプル調製］
ヒト神経芽腫細胞株SK－N－SHをMEM培地（Thermo　Fisher　Scientific ＃11095）で懸濁し、2×107個の細胞を15 cmディッシュに播種し、37℃、5％ CO2下で一日培養した後、Eg5阻害薬Ispinesib（100 nM）で15時間処理することで細胞周期を分裂期に同調させた。続いてMEM培地で400μMに希釈したCompound D、Compound Eを、それぞれのディッシュの1／10量添加し30分間培養した。続いて全てのディッシュへMEM培地で10μMに希釈したCompound Aをディッシュの1／10量添加し、更に1時間培養した後、UVクロスリンカ－で紫外線を10分間照射した。培地を除去し細胞をPBSで洗浄した後、1％ホルマリン含有D－PBS（－）で10分間固定した。D－PBS（－）で3回洗浄した細胞をスクレ－パ－で回収し、遠心して上清を除去した。少量のD－PBS（－）で懸濁した細胞を超音波破砕し、最終濃度が1％となるように SDS （sodium dodecyl sulfate）を添加し溶解した。これをD－PBS（－）で10倍希釈して、銅触媒のクリック反応によりCompound AにPhoto cleavableビオチンアジド（PC Biotin Azide, Click chemistry Tools： 1119－25）を付加した。反応後にメタノ－ルとディッシュの1／4量のクロロホルムを添加し、遠心により蛋白質を沈殿分離するとともに未反応のビオチンアジドを除去した。沈殿した蛋白質を2％ SDS、70mM TCEP （tris （2－carboxyethyl）phosphine）含有D－PBS（－）で再溶解し、ストレプトアビジン標識ビ－ズを用いて、ビオチン標識されたCompound Aとそれに結合している蛋白質複合体をプルダウンした。ビ－ズを0.2％ SDS含有D－PBS（－）で3回洗浄し、紫外線照射によりCompound A複合体を溶出した。 [Sample preparation]
Human neuroblastoma cell line SK-N-SH was suspended in MEM medium (Thermo Fisher Scientific # 11095), 2 × 10 7 cells were seeded in a 15 cm dish, and 1 at 37 ° C. under 5% CO 2. After daily culture, the cell cycle was synchronized to the mitotic phase by treatment with the Eg5 inhibitor Ispinesib (100 nM) for 15 hours. Subsequently, Compound D and Compound E diluted to 400 μM in MEM medium were added in an amount of 1/10 of each dish and cultured for 30 minutes. Subsequently, 1/10 amount of Compound A diluted to 10 μM in MEM medium was added to all the dishes, and after further culturing for 1 hour, ultraviolet rays were irradiated for 10 minutes with a UV crosslinker. After removing the medium and washing the cells with PBS, the cells were fixed with D-PBS (-) containing 1% formalin for 10 minutes. Cells washed 3 times with D-PBS (-) were collected with a scraper and centrifuged to remove the supernatant. Cells suspended in a small amount of D-PBS (-) were crushed by ultrasonic waves, and SDS (sodium dodecyl sulfate) was added and lysed to a final concentration of 1%. This was diluted 10-fold with D-PBS (-), and Photo cleavable biotin azide (PC Biotin Azide, Click chemistry Tools: 1119-25) was added to Compound A by a copper-catalyzed click reaction. After the reaction, 1/4 of the amount of chloroform and dish was added, and the protein was precipitated and separated by centrifugation, and unreacted biotin azide was removed. Precipitated protein is redissolved in D-PBS (-) containing 2% SDS, 70 mM TCEP (tris (2-carboxyethyl) phosphine) and bound to biotin-labeled Compound A using streptavidin-labeled beads. The protein complex is pulled down. The beads were washed 3 times with D-PBS (-) containing 0.2% SDS, and the Compound A complex was eluted by irradiation with ultraviolet rays.
続いてメタノ－ル／クロロホルム抽出により、タンパク質と夾雑物を分離した。サンプルにメタノ－ル200 μL、クロロホルム 50μL、蒸留水 150μLを添加後、懸濁した。混合液を遠心分離（14,000 rpm、室温、2分）し、上層を取り除いた。下層にメタノ－ル 200μLを添加、遠心分離（14,000 rpm、室温、2分）し、上清を取り除き沈殿を乾固した。
乾固したサンプルを尿素／Tris溶液（50 mM Tris－HCl （pH8.0）、10 mM EDTA、8 M 尿素、0.005％ ドデシルマルトシド）12.5μLで溶解、DTT溶液（100 mM）1.25μL添加により還元（37℃、20分）し、ヨ－ドアセトアミド溶液（200 mM）1.25μL添加によりアルキル化（遮光下、25℃、20分）した。サンプルに蒸留水35μLを添加し、再びメタノ－ル／クロロホルム抽出した。沈殿物に尿素／HEPES溶液（8 M 尿素、200 mM HEPES（pH8.0）、0.005％ ドデシルマルトシド）25μLを添加し再溶解させ、HEPES緩衝液（200 mM HEPES（pH8.0）、0.005％ ドデシルマルトシド）75 μLを添加し尿素濃度を希釈、trypsin （Modified trypsin （Promega ＃V5111）、250 ng／μL trypsin dilution buffer）10μLを添加し、37℃で一晩反応させた。続いてTrypsin消化物をTMTシステム（Thermo Scientific ＃90111）により標識した。簡単には、それぞれのTMTタグを90μLのアセトニトリルで溶解し、trypsin消化物に溶解した41μL のTMTタグを添加、室温で1時間反応させた。反応後8μLの5％ hydroxylamineを添加、室温、15分間反応させTMTラベル化反応を停止させた。全サンプルを混合しスピ－ドバックによりアセトニトリルを取り除き、80μLの50％ギ酸を添加した。TMTラベル化ペプチドをStageTip（C18－SCX）により脱塩、ギ酸アンモニウムまたはアンモニアにより4分画し、さらにStageTip（C18）により脱塩した。 Subsequently, proteins and impurities were separated by metalnol / chloroform extraction. 200 μL of methanol, 50 μL of chloroform, and 150 μL of distilled water were added to the sample and then suspended. The mixture was centrifuged (14,000 rpm, room temperature, 2 minutes) and the upper layer was removed. 200 μL of methanol was added to the lower layer, and the mixture was centrifuged (14,000 rpm, room temperature, 2 minutes), the supernatant was removed, and the precipitate was dried.
Dissolve the dried sample in 12.5 μL of urea / Tris solution (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 8 M urea, 0.005% dodecyl maltoside), and add 1.25 μL of DTT solution (100 mM). It was reduced (37 ° C., 20 minutes) and alkylated by adding 1.25 μL of iodine acetamide solution (200 mM) (25 ° C., 20 minutes under shading). 35 μL of distilled water was added to the sample, and methanol / chloroform extraction was performed again. Add 25 μL of urea / HEPES solution (8 M urea, 200 mM HEPES (pH 8.0), 0.005% dodecyl maltoside) to the precipitate and redissolve it to make a HEPES buffer (200 mM HEPES (pH 8.0), 0.005%). Dodecyl maltoside) 75 μL was added to dilute the urea concentration, trypsin (Modified trypsin (Promega # V5111), 250 ng / μL trypsin dilution buffer) 10 μL was added, and the mixture was reacted overnight at 37 ° C. The Trypsin digest was subsequently labeled by the TMT system (Thermo Scientific # 90111). Briefly, each TMT tag was dissolved in 90 μL of acetonitrile, 41 μL of TMT tag dissolved in trypsin digest was added, and the mixture was reacted at room temperature for 1 hour. After the reaction, 8 μL of 5% hydroxylamine was added, and the reaction was carried out at room temperature for 15 minutes to stop the TMT labeling reaction. All samples were mixed, acetonitrile was removed by speedback and 80 μL of 50% formic acid was added. The TMT-labeled peptide was desalted with StageTip (C18-SCX), fractionated with ammonium formate or ammonia, and further desalted with StageTip (C18).
［質量分析］
LC－MS／MS装置はEASY－nLC1200（Thermo Fisher Scientific）を装備したOrbitrap Fusion lumos （Thermo Fisher Scientific）を使用した。分析カラムはチップカラム（100μm I.D.、長さ150 mm）にReproSil－Pur 120 C18－AQ （2.4μm、Dr. Maisch GmbH）を詰めたものを使用した。LCによる分離は、流速300 nL／min、159.5分間、8－34％アセトニトリル（0.125％ギ酸）の直線的な勾配により行った。測定条件としては、Orbitrap Fusion LumosのLC－MS3を用い、ポジティブモ－ド、data－dependent top speed acquisition mode, サイクルタイムは3秒を用いた。フルMSスキャンはOrbitrap、分解能120,000で取得、スキャンレンジはm／z 380－1500で行った。MS／MSはCIDにより取得、MS／MSスペクトル中最も強度の強い10個のプレカーサーイオンに対して、HCD （collision energy 65％、分解能 60,000、スキャンレンジm／z 120－500）を取得した。 [Mass spectrometry]
The LC-MS / MS device used was an Orbitrap Fusion lumos (Thermo Fisher Scientific) equipped with an EASY-nLC1200 (Thermo Fisher Scientific). The analytical column used was a chip column (100 μm ID, length 150 mm) packed with ReproSil-Pur 120 C18-AQ (2.4 μm, Dr. Maisch GmbH). Separation by LC was performed at a flow rate of 300 nL / min for 159.5 minutes with a linear gradient of 8-34% acetonitrile (0.125% formic acid). As the measurement conditions, LC-MS3 of Orbitrap Fusion Lumos was used, and positive mode, data-dependent top speed acquisition mode, and cycle time of 3 seconds were used. The full MS scan was acquired with Orbitrap and a resolution of 120,000, and the scan range was m / z 380-1500. MS / MS was acquired by CID, and HCD (collision energy 65%, resolution 60,000, scan range m / z 120-500) was acquired for the 10 strongest precursor ions in the MS / MS spectrum.
得られたRAWデ－タは改変したReAdW.exeによりmzXMLへ変換し、GFY Core 3.7を用いて解析した。デ－タベ－ス検索にはCometを使用し、デ－タベ－スにはSwiss－prot （human）を使用した。検索条件として、ペプチドの未切断2まで許容、fixed modificationにはシステイン残基のカルバモイドメチル化、ペプチドN末端のTMTタグ、リジンのTMTタグ、variable modificationにはメチオニンの酸化をそれぞれ選択した。ペプチドの計算質量との許容範囲を50 ppm、プロダクトイオンの計算値との許容範囲を0.8 Daに設定し、質量値はモノアイソトピックを選択した。ペプチド、タンパク質のFDRを1％以下に設定し、LDA（Core）を用いてフィルタリングを行った。すべてのタンパク質の定量値は、Coreにより得られた値を、必要な場合Rを用いてデ－タ正規化、p値の計算を行った。
その結果、紫外線照射依存的なCompound Aへの結合（UV－／UV＋ ＜ 0.5）を示し、Compound Eの処理に影響を受けない約2000タンパク質が同定された。さらに、Compound Dによる競合が認められる活性依存的なCompound Aへの結合タンパク質として、ALDH1B1、EXOC5、NUMA1、TUBB2B、TUBB4A、TUBB3、TUBB、TUBB2A、CKAP5が同定された。結果を図４に示す。 The obtained RAW data was converted to mzXML by the modified ReAdW.exe and analyzed using GFY Core 3.7. Comet was used for the database search, and Swiss-prot (human) was used for the database. As search conditions, up to 2 uncleaved peptides were allowed, carbamoid methylation of cysteine residue was selected for fixed modification, TMT tag at the N-terminal of peptide, TMT tag for lysine, and oxidation of methionine was selected for variable modification. The allowable range with the calculated mass of the peptide was set to 50 ppm, the allowable range with the calculated value of the product ion was set to 0.8 Da, and the monoisotopic was selected as the mass value. The FDR of peptides and proteins was set to 1% or less, and filtering was performed using LDA (Core). For the quantitative values of all proteins, the values obtained by Core were normalized by data using R if necessary, and the p-value was calculated.
As a result, about 2000 proteins showing UV irradiation-dependent binding to Compound A (UV− / UV + <0.5) and not affected by the treatment of Compound E were identified. Furthermore, ALDH1B1, EXOC5, NUMA1, TUBB2B, TUBB4A, TUBB3, TUBB, TUBB2A, and CKAP5 were identified as activity-dependent binding proteins to Compound A that are competing with Compound D. The results are shown in FIG.
（実施例１０）RNA干渉によるNuMA1ノックダウンがCompound BとIspinesib（ Eg5阻害剤）の細胞増殖抑制に及ぼす影響
ヒト肺がん細胞株LK－2（Health Science Research Resources Bank）は牛胎児血清（最終濃度10％）を含むRPMI1640培地（Thermo　Fisher　Scientific ＃11875）で、ヒト神経芽腫細胞株SH－SY5YはMEM／F12培地で継代維持した。まず、各細胞株を300000個／ 1 mLの細胞濃度に調製した。Negative control siRNA（Dharmacon、ON－TARGET plus Non－targeting Pool、＃D－001810－10－50）およびNuMA1 siRNA（Dharmacon、ON－TARGET plus NUMA1 siRNA、＃J－005272－05－0002）は、最終濃度10 nMとなるようにOpti－MEM培地（Thermo Fisher Scientific、＃11058－021）を用いてLipofectamine RNAiMAX（Thermo Fisher Scientific、＃13778－150）と混合した。 6ウェルプレ－ト（IWAKI、 ＃3810－006）の各ウェルへ細胞懸濁液を1 mL、各細胞株に対する上記培地を1 mL、siRNA－RNAiMAX reagent混合液を0.5 mLずつ分注し、37℃、5％CO2下で24時間培養した。続いて培養上清を取り除き、各細胞株に対する上記培地を2.5 mL添加し、先と同様に調製したsiRNA－RNAiMAX reagent混合液を0.5 mL添加した。４時間37℃、5％CO2下で培養した後、TrypLE Expressで剥離回収した後、室温で1000 rpmで5分間遠心処理し、上清を除去した。各細胞株に対する上記培地で細胞を懸濁し、20000個／ 1 mLの細胞濃度に調製し、96ウェルプレ－ト（Corning ＃3904）の各ウェルへ100μLずつ分注し、37℃、5％CO2下で24時間培養した。続いて各細胞株に対する上記培地で所定の濃度に希釈したCompound BもしくはIspinesibを各ウェルへ50μL添加し(day1)、37℃、5％ CO2下、3日間培養した。化合物添加当日(day 1)ならびに化合物添加3日後(day 4)にATP測定用試薬であるCellTiter－Glo 2.0 Assayを30μL／wellずつ各ウェルに添加し、EnVisionで各ウェルの発光量を測定した。化合物添加当日の発光量(C1)、3日間培養後の化合物非添加群(C4)および化合物添加群(T4)の発光量より、次式に基づき細胞増殖率を算出した。 (Example 10) Effect of NuMA1 knockdown due to RNA interference on suppression of cell proliferation of Compound B and Ispinesib (Eg5 inhibitor) Human lung cancer cell line LK-2 (Health Science Research Resources Bank) has fetal bovine serum (final concentration 10). %) In RPMI1640 medium (Thermo Fisher Scientific # 11875), the human neuroblastoma cell line SH-SY5Y was subcultured in MEM / F12 medium. First, each cell line was prepared to a cell concentration of 300,000 cells / 1 mL. Negative control siRNA (Dharmacon, ON-TARGET plus Non-targeting Pool, # D-001810-10-50) and NuMA1 siRNA (Dharmacon, ON-TARGET plus NUMA1 siRNA, # J-005272-05-0002) have final concentrations. It was mixed with Lipofectamine RNAiMAX (Thermo Fisher Scientific, # 13778-150) using Opti-MEM medium (Thermo Fisher Scientific, # 11058-021) to 10 nM. Dispense 1 mL of cell suspension into each well of 6-well plate (IWAKI, # 3810-006), 1 mL of the above medium for each cell line, and 0.5 mL of siRNA-RNAiMAX reagent mixture at 37 ° C. , Incubated for 24 hours under 5% CO 2 . Subsequently, the culture supernatant was removed, 2.5 mL of the above medium was added to each cell line, and 0.5 mL of the siRNA-RNAiMAX reagent mixture prepared in the same manner as above was added. After culturing under 5% CO 2 at 37 ° C. for 4 hours, the cells were peeled and recovered by TrypLE Express, and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant. Suspend the cells in the above medium for each cell line, prepare to a cell concentration of 20000 cells / 1 mL, dispense 100 μL into each well of 96-well plates (Corning # 3904), 37 ° C, 5% CO 2 Incubated underneath for 24 hours. Subsequently, 50 μL of Compound B or Ispine sib diluted to a predetermined concentration in the above medium for each cell line was added to each well (day 1), and the cells were cultured at 37 ° C. under 5% CO 2 for 3 days. On the day of compound addition (day 1) and 3 days after compound addition (day 4), CellTiter-Glo 2.0 Assay, a reagent for ATP measurement, was added to each well at a rate of 30 μL / well, and the amount of light emitted from each well was measured by EnVision. The cell proliferation rate was calculated based on the following formula from the luminescence amount on the day of compound addition (C1), the luminescence amount of the compound-free group (C4) and the compound-added group (T4) after culturing for 3 days.
Figure JPOXMLDOC01-appb-M000007
Figure JPOXMLDOC01-appb-M000007
各濃度における細胞増殖率と化合物濃度を片対数プロットした。結果を図５に示す。
検討の結果、NuMA1の発現抑制によりIspinesibの細胞増殖抑制効果には影響を及ぼさない一方で、Compound Bの細胞増殖抑制効果は顕著に減弱することを見出した。 The cell proliferation rate and compound concentration at each concentration were semi-logarithmically plotted. The results are shown in FIG.
As a result of the examination, it was found that the suppression of NuMA1 expression did not affect the cell growth inhibitory effect of Ispine sib, while the cell growth inhibitory effect of Compound B was significantly attenuated.
（実施例１１）Compound C依存的NuMA1結合タンパクの探索
［サンプル調製］
ヒト神経芽腫細胞株SH－SY5Yを10 cmディッシュ（Corning ＃430293）に播種し、37℃、5％CO2下で翌日までMEM／F12培地を用いて培養した。続いて、DMSOでそれぞれ最終濃度の1000倍の濃度に希釈したCompound C、Ispinesibを、各ディッシュへ添加し、37℃、5％CO2下で一晩培養した。翌日、D－PBS（－）で3回洗浄した後、0.5％ホルマリン（20％中性緩衝ホルマリン液（和光純薬工業株式会社＃060－01721）をD－PBS（－）で40倍希釈して調製）で室温15分間処理した。D－PBS（－）で3回洗浄した後、1mLの細胞溶解液（10mLを調製する際、HEPES（１M）（Thermo Fisher Scientific ＃15630－080）、1M MgCl2、Nonidet P－40（ナカライテスク株式会社 ＃25223－04）、1M KCl、1M ジチオトレイト－ル、蒸留水（日本薬局方注射用蒸留水、大塚製薬）をそれぞれ0.1mL、0.015mL、0.05mL、0.1mL、0.005mL、9.75mLの割合で混合したものに、cOmplete Tablets, Mini EASYpack（Roche ＃04 693 124 001）およびPhosSTOP EASYpack（Roche ＃04 906 837 001）をそれぞれ１錠加えた）を添加し細胞を溶解した。約20分間氷上でインキュベ－ションした後、マイジェクタ－注射針付シリンジ27G×1／2（テルモ株式会社 ＃SS－10M2713）で10回針を通すことで細胞を破砕した。15,000 × gで4℃で10分間遠心した。沈殿に0.1mLの1％ SDSを加え超音波破砕機 model UR－20P（Tomy Seiko Co. Ltd., Japan）で破砕した後、0.9mLの細胞溶解液を加え。15,000 × gで4℃で10分間遠心することにより上清を得て、これをSDS抽出画分として4℃で保存した。 (Example 11) Search for Compound C-dependent NuMA1-binding protein [Sample preparation]
The human neuroblastoma cell line SH-SY5Y was seeded on a 10 cm dish (Corning # 430293) and cultured at 37 ° C. under 5% CO 2 using MEM / F12 medium until the next day. Subsequently, Compound C and Ispinesib diluted with DMSO to a concentration 1000 times the final concentration were added to each dish and cultured overnight at 37 ° C. and 5% CO 2 . The next day, after washing 3 times with D-PBS (-), 0.5% formalin (20% neutral buffered formalin solution (Wako Pure Chemical Industries, Ltd. # 060-01721)) was diluted 40-fold with D-PBS (-). The treatment was carried out at room temperature for 15 minutes. After washing 3 times with D-PBS (-), 1 mL of cell lysate (when preparing 10 mL, HEPES (1M) (Thermo Fisher Scientific # 15630-080), 1M MgCl 2 , Nonidet P-40 (Nacalai Tesque) Co., Ltd. # 25223-04), 1M KCl, 1M dithiotrate, distilled water (distilled water for injection by Nippon Pharmacy, Otsuka Pharmaceutical) of 0.1 mL, 0.015 mL, 0.05 mL, 0.1 mL, 0.005 mL, 9.75 mL, respectively. The cells were lysed by adding cOmplete Tablets, Mini EASYpack (Roche # 04 693 124 001) and PhosSTOP EASYpack (Roche # 04 906 837 001) to the mixture in proportions. After incubating on ice for about 20 minutes, the cells were crushed by passing the needle 10 times with a syringe 27G × 1/2 (Terumo Corporation # SS-10M2713) with a Myjector injection needle. Centrifuge at 15,000 x g at 4 ° C for 10 minutes. Add 0.1 mL of 1% SDS to the precipitate, crush it with an ultrasonic crusher model UR-20P (Tomy Seiko Co. Ltd., Japan), and then add 0.9 mL of cell lysate. A supernatant was obtained by centrifuging at 15,000 × g at 4 ° C for 10 minutes, and this was stored as an SDS extract fraction at 4 ° C.
［免疫沈降実験］
Rabbit anti－NUMA Affinity Purified（Bethyl Laboratories, Inc. ＃A301－509A）（以後、抗NUMA1抗体と略す）を用いたSDS抽出画分の免疫沈降実験にはImmunoprecipitation Kit － Dynabeads Protein G（Thermo Fisher Scientific ＃10007D）（以後、IPキットと略す）を用いた。IPキット付属のDynabeads Protein GはIPキット付属のAb Binding & Washing Bufferで2回洗浄し元の容量にあわせて同Bufferで懸濁した（以後、洗浄済みDynabeadsと略す）。0.6 mLのSDS抽出画分に60μLの洗浄済みDynabeadsを加え4℃でゆるやかに回転させながら数時間攪拌した後、磁気スタンドでビ－ズを除くことにより非特異的にビ－ズに結合する成分を除去した（以後、プレクリア－済みSDS抽出画分と略す）。プレクリア－済みSDS抽出画分のそれぞれに抗NUMA1抗体を0.66μL（0.132μg）添加した。対照としてDMSO処理プレクリア－済みSDS抽出画分にNormal Rabbit IgG（Santa Cruz Biotechnology ＃sc－2027）を0.33μL（0.132μg）添加した。4℃でゆるやかに回転させながら終夜攪拌した後、20μLの洗浄済みDynabeadsを加え4℃でゆるやかに回転させながらさらに数時間攪拌した。続いて、0.1％ SDSを含む細胞溶解液を用いてビ－ズを3回洗浄した。最後にIPキット付属のWashing Bufferを用いてビ－ズを1回洗浄した。Washing Bufferを出来るだけ除いたビ－ズにサンプルバッファ－（NuPAGE Sample Reducing Agent （１０×）（Thermo Fisher Scientific、＃NP0009）、NuPAGE LDS Sample Buffer （４×）（Thermo Fisher Scientific、＃NP0008）、蒸留水を1：2.5：6.5で混合したもの）を120μL加え、70℃で60分間処理し攪拌した後、磁気スタンドでビ－ズを除き、サンプルとして4℃で保存した。 [Immunoprecipitation experiment]
Immunoprecipitation Kit-Dynabeads Protein G (Thermo Fisher Scientific #) for immunoprecipitation experiments of SDS extract fractions using Rabbit anti-NUMA Affinity Purified (Bethyl Laboratories, Inc. # A301-509A) (hereinafter abbreviated as anti-NUMA1 antibody) 10007D) (hereinafter abbreviated as IP kit) was used. Dynabeads Protein G included in the IP kit was washed twice with the Ab Binding & Washing Buffer included in the IP kit and suspended in the same Buffer according to the original capacity (hereinafter abbreviated as washed Dynabeads). Add 60 μL of washed Dynabeads to a 0.6 mL SDS extract fraction, stir for several hours while gently rotating at 4 ° C, and then remove the beads with a magnetic stand to nonspecifically bind to the beads. Was removed (hereinafter abbreviated as pre-cleared SDS extracted fraction). 0.66 μL (0.132 μg) of anti-NUMA1 antibody was added to each of the pre-cleared SDS extract fractions. As a control, 0.33 μL (0.132 μg) of Normal Rabbit IgG (Santa Cruz Biotechnology # sc-2027) was added to the DMSO-treated pre-cleared SDS extracted fraction. After stirring overnight while gently rotating at 4 ° C., 20 μL of washed Dynabeads was added, and the mixture was further stirred for several hours while gently rotating at 4 ° C. Subsequently, the beads were washed 3 times with a cell lysate containing 0.1% SDS. Finally, the beads were washed once using the Washing Buffer included in the IP kit. Sample buffer (NuPAGE Sample Reducing Agent (10 ×) (Thermo Fisher Scientific, # NP0009), NuPAGE LDS Sample Buffer (4 ×) (Thermo Fisher Scientific, # NP0008), distillation in beads with Washing Buffer removed as much as possible. 120 μL of water (mixed at 1: 2.5: 6.5) was added, treated at 70 ° C. for 60 minutes, stirred, and the beads were removed with a magnetic stand, and the sample was stored at 4 ° C.
［質量分析］
LC－MS／MS解析は実施例９に記載の方法と同様の方法で実施した。結果を図６に示す。
結果、Compound C処理特異的にNuMA1との結合が増加したタンパク質としてβ－チューブリン（TUBA1B、TUBB、TUBB3、TUBB4B）、CKAP5、TACC3を同定した。 [Mass spectrometry]
The LC-MS / MS analysis was performed by the same method as that described in Example 9. The results are shown in FIG.
As a result, β-tubulin (TUBA1B, TUBB, TUBB3, TUBB4B), CKAP5, and TACC3 were identified as proteins with increased binding to NuMA1 specifically for Compound C treatment.
（実施例１２）Compound B処理によるNuMA1とTACC3およびCKAP5の結合の増強
Compound B処理によりNuMA1とTACC3もしくはCKAP5との結合が増強することをNuMA1の免疫沈降後ウエスタンブロットにより確認した。サンプル調製法は、Compound CにかえてCompound Bを用いたこと、および実験に用いたディッシュを10 cmディッシュから15 cmディッシュ（IWAKI ＃3030－150）に変更した以外、実施例11に記載の方法に従った。免疫沈降実験においても、実施例11の方法に準じたが、各SDS抽出画分を0.85 mL、プレクリア－時及び免疫沈降時にそれぞれ85μLの洗浄済みDynabeadsを用い、免疫沈降時に抗NUMA1抗体を8.5μL（1.7μg）添加し、サンプルバッファ－を60μL添加した点のみ変更した。
［電気泳動］
サンプルをPerfect NT Gel 5－20％（D.R.C.、＃NTH－576HP10CD）を用いて10μL／レ－ンで電気泳動した。その後、ニトロセルロ－スメンブレンiBlot Gel Transfer Stacks Nitrocellulose, Regular（Thermo Fisher Scientific ＃IB301001）へiBlot Dry Blotting System（Thermo Fisher Scientific ＃IB1001）を用いて転写し、StartingBlock T20（TBS）Blocking Buffer（Thermo Fisher Scientific、＃37543）で室温1時間緩やかに攪拌しながらインキュベ－ションすることによりブロッキングした。続いて1次抗体として抗NUMA1抗体（5000倍希釈）、抗CKAP5抗体（Ch－TOG （D2Z8J）Rabbit mAb（Cell Signaling Technology ＃67774S）、500倍希釈）、抗TACC3抗体（TACC3（C－5）（Santa Cruz Biotechnology ＃sc－271165）、1000倍希釈）、抗β－チューブリン抗体（Monoclonal Anti－β－チューブリンantibody（Sigma－Aldrich ＃T8328）、1000倍希釈）及び抗β－Actin（Anti－β－Actin antibody, Mouse monoclonal（Sigma－Aldrich ＃A1978）40000倍希釈）を用いて、4℃で終夜緩やかに攪拌しながらインキュベ－ションした。続いて2次抗体として5000倍希釈したECL Anti－Rabbit IgG, Horseradish Peroxidase linked whole antibody from donky（GE Healthcare、＃NA934V）、もしくは5000倍希釈したECL Anti－Mouse IgG, Horseradish Peroxidase linked whole antibody from sheep（GE Healthcare、＃NA931V）を用いて室温で30分間緩やかに攪拌しながらインキュベ－ションした。なお、1次抗体および2次抗体の希釈はそれぞれCan Get Signal Solution1およびSolution2（TOYOBO Co., Ltd. ＃NKB－101）を用いた。また各ステップの間にTBS－Tweenで5回洗浄した。2次抗体処理及びそれに続く洗浄をすませたニトロセルロ－スメンブレンをImmobilon Forte Western HRP Substrate（Millipore、＃WBLUF0500）で約30秒処理した後、LAS4000（Fujifilm）で化学発光を撮影した。結果を図７に示す。
検討の結果、DMSO処理およびIspinesib処理に比較し、Compound B処理により顕著にNuMA1とTACC3およびCKAP5の結合量が増加することを見出した。 (Example 12) Enhancement of binding between NuMA1 and TACC3 and CKAP5 by Compound B treatment
It was confirmed by Western blotting after immunoprecipitation of NuMA1 that the binding between NuMA1 and TACC3 or CKAP5 was enhanced by Compound B treatment. As the sample preparation method, the method described in Example 11 except that Compound B was used instead of Compound C and the dish used in the experiment was changed from a 10 cm dish to a 15 cm dish (IWAKI # 3030-150). Followed. In the immunoprecipitation experiment, the method of Example 11 was followed, but each SDS extract fraction was 0.85 mL, 85 μL of washed Dynabeads was used during pre-clearing and immunoprecipitation, and 8.5 μL of anti-NUMA1 antibody was used during immunoprecipitation. (1.7 μg) was added, and only the point where 60 μL of the sample buffer was added was changed.
[Electrophoresis]
Samples were electrophoresed at 10 μL / lane using Perfect NT Gel 5-20% (DRC, # NTH-576HP10CD). After that, it was transferred to the nitrocellulose membrane iBlot Gel Transfer Stacks Nitrocellulose, Regular (Thermo Fisher Scientific # IB301001) using the iBlot Dry Blotting System (Thermo Fisher Scientific # IB1001), and started Block T20 (TBS) Blocking Buffer (Thermo Fisher Scientific, Blocking was performed by incubating with gentle stirring at room temperature for 1 hour at # 37543). Subsequently, as the primary antibody, anti-NUMA1 antibody (5000-fold dilution), anti-CKAP5 antibody (Ch-TOG (D2Z8J) Rabbit mAb (Cell Signaling Technology # 67774S), 500-fold dilution), anti-TACC3 antibody (TACC3 (C-5)) (Santa Cruz Biotechnology # sc-271165), 1000-fold dilution), anti-β-tubulin antibody (Monoclonal Anti-β-tubulin antibody (Sigma-Aldrich # T8328), 1000-fold dilution) and anti-β-Actin (Anti-) Incubation was performed using β-Actin antibody and Mouse monoclonal (Sigma-Aldrich # A1978) 40,000-fold diluted) at 4 ° C. with gentle stirring overnight. Subsequently, as a secondary antibody, 5000-fold diluted ECL Anti-Rabbit IgG, Horseradish Peroxidase linked whole antibody from donky (GE Healthcare, # NA934V), or 5000-fold diluted ECL Anti-Mouse IgG, Horseradish Peroxidase linked whole antibody from sheep ( Incubation was performed using GE Healthcare (# NA931V) at room temperature for 30 minutes with gentle stirring. Can Get Signal Solution 1 and Solution 2 (TOYOBO Co., Ltd. # NKB-101) were used to dilute the primary antibody and the secondary antibody, respectively. It was also washed 5 times with TBS-Tween between each step. The nitrocellulose membrane that had undergone secondary antibody treatment and subsequent washing was treated with Immobilon Forte Western HRP Substrate (Millipore, # WBLUF0500) for about 30 seconds, and then chemiluminescence was photographed with LAS4000 (Fujifilm). The results are shown in FIG.
As a result of the examination, it was found that the binding amount of NuMA1, TACC3 and CKAP5 was significantly increased by the Compound B treatment as compared with the DMSO treatment and the Ispine sib treatment.
（実施例１３）Compound B処理によるNuMA1、CKAP5局在検討
Compound B処理により、NuMA1とCKAP5がどのような細胞内局在を示すのか、免疫蛍光染色により調べた。
抗NuMA1抗体（SantaCruz、＃sc－365532）、抗CKAP5抗体（ThermoFisherScientific、＃PA3－16835）、抗マウスIgG－Alexa488標識抗体（Abcam、＃ab150117）、抗ウサギIgG－Alexa－568標識抗体（Abcam、＃ab175695）を用いた。3×105個のヒト肺がん細胞株LK－2を3.5 cmガラスボトムディッシュに播種し、37℃、5％ CO2下で一日培養後、20 nMのCompound Bまたは1 nMのIspinesibで16時間処理した。細胞を100％メタノ－ルで5分間（CKAP5染色用）固定し、D－PBS（－）で3回洗浄した。0.1％ TritonX－100含有D－PBS（－）で5分間、膜透過処理をした後、D－PBS（－）で3回洗浄し、3％ ウシ血清アルブミン含有D－PBS（－）で30分間ブロッキングした。1次抗体はブロッキング溶液で希釈し、室温で30分間静置して反応させた。これをD－PBS（－）で3回洗浄し、2次抗体も同様に反応、洗浄し、ProLong Diamond Antifade Mountant（ThermoFisherScientific、＃P36965）で封入した。観察はLeica社製 TSC SP8 STED顕微鏡を用い、LASXソフトウェアで解析した。結果を図８に示す。
Compound BおよびIspinesib処理により同様のmonopolar spindleの形成が認められたが、Ispinesib処理によりNuMA1は中心体の周囲にリング状に局在し、CKAP5は明確な局在が認められなかったのに対し、Compound B処理によりNuMA1およびCKAP5は中心体近傍に強く共局在していた。 (Example 13) Localization study of NuMA1 and CKAP5 by Compound B treatment
The intracellular localization of NuMA1 and CKAP5 by Compound B treatment was investigated by immunofluorescent staining.
Anti-NuMA1 antibody (SantaCruz, # sc-365532), anti-CKAP5 antibody (ThermoFisherScientific, # PA3-16835), anti-mouse IgG-Alexa488-labeled antibody (Abcam, # ab150117), anti-rabbit IgG-Alexa-568-labeled antibody (Abcam, # Ab175695) was used. 3 × 10 5 human lung cancer cell lines LK-2 were seeded on a 3.5 cm glass bottom dish, cultured for 1 day at 37 ° C. under 5% CO 2 , and then in 20 nM Compound B or 1 nM Ispine sib for 16 hours. Processed. Cells were fixed with 100% metanol for 5 minutes (for CKAP5 staining) and washed 3 times with D-PBS (-). After membrane permeation treatment with D-PBS (-) containing 0.1% TritonX-100 for 5 minutes, washing 3 times with D-PBS (-) and 30 minutes with D-PBS (-) containing 3% bovine serum albumin. Blocked. The primary antibody was diluted with a blocking solution and allowed to stand at room temperature for 30 minutes for reaction. This was washed 3 times with D-PBS (-), and the secondary antibody was also reacted and washed in the same manner, and encapsulated with ProLong Diamond Antifade Mountant (ThermoFisherScientific, # P36965). Observations were analyzed with LASX software using a Leica TSC SP8 STED microscope. The results are shown in FIG.
Similar monopolar spindle formation was observed with Compound B and Ispine sib treatment, but NuMA1 was localized around the centrosome in a ring shape and CKAP5 was not clearly localized with Ispine sib treatment. NuMA1 and CKAP5 were strongly co-localized near the centrosome by Compound B treatment.
（実施例１４）RNA干渉によるNuMA1、TACC3もしくはCKAP5ノックダウンがCompound Aとβ－チューブリンとの結合に及ぼす影響の解析
ヒト肺がん細胞株LK－2を500000個／ 1 mLの細胞濃度に調製した。Negative control siRNA（Dharmacon、ON－TARGET plus Non－targeting Pool、＃D－001810－10－50）、NuMA1 siRNA（Dharmacon、ON－TARGET plus NUMA1 siRNA、＃J－005272－05－0002）、TACC3 siRNA（Dharmacon、ON－TARGET plus TACC3 siRNA、＃J－004155－07－0002、＃J－004155－08－0002）、CKAP5 siRNA（Dharmacon、ON－TARGET plus CKAP5 siRNA、＃J－006847－07－0002、＃J－006847－08－0002）は、最終濃度10 nMとなるようにOpti－MEM培地を用いてLipofectamine RNAiMAXと混合した。 10 cmディッシュへ細胞懸濁液を8 mL、siRNA－RNAiMAX reagent混合液を2 mLずつ分注し、37℃、5％CO2下で24時間培養した。続いて培養上清を取り除き、新鮮な牛胎児血清（最終濃度10％）を含むRPMI1640培地を9 mL添加し、100μM Ispinesibを9μL添加した（最終濃度100 nM）。一晩37℃、5％CO2下で培養し、顕微鏡下細胞が分裂期で停止していることを確認した後、100μM Compound Aを9μL添加し（最終濃度100 nM）、1時間37℃、5％CO2下で培養した。続いて細胞に紫外線 （365 nm）を4℃で10分間照射した。その後スクレ－パ－で細胞をかきとり、4℃、1500 rpmで10分間遠心処理し上清を除去し、1 mLのPBSで洗浄後、100μLのD－PBS（－）にペレットを懸濁した。氷上で超音波破砕機を用いて細胞を破砕し、4℃、15000 rpmで30分間遠心処理し上清を細胞ライセ－トとし使用時まで－80℃保存した。 (Example 14) Analysis of the effect of RNA interference-induced NuMA1, TACC3 or CKAP5 knockdown on the binding of Compound A to β-tubulin Human lung cancer cell line LK-2 was prepared at a cell concentration of 500,000 cells / mL. .. Negative control siRNA (Dharmacon, ON-TARGET plus Non-targeting Pool, # D-001810-10-50), NuMA1 siRNA (Dharmacon, ON-TARGET plus NUMA1 siRNA, # J-005272-05-0002), TACC3 siRNA ( Dharmacon, ON-TARGET plus TACC3 siRNA, # J-004155-07-0002, # J-004155-08-0002), CKAP5 siRNA (Dharmacon, ON-TARGET plus CKAP5 siRNA, # J-006847-07-0002, # J-006847-08-0002) was mixed with Lipofectamine RNAiMAX using Opti-MEM medium to a final concentration of 10 nM. 8 mL of the cell suspension and 2 mL of the siRNA-RNAiMAX reagent mixture were dispensed into a 10 cm dish and cultured at 37 ° C. under 5% CO 2 for 24 hours. Subsequently, the culture supernatant was removed, 9 mL of RPMI1640 medium containing fresh fetal bovine serum (final concentration 10%) was added, and 9 μL of 100 μM Ispinesib was added (final concentration 100 nM). After culturing overnight at 37 ° C. under 5% CO 2 and confirming that the cells were arrested during mitosis under a microscope, 9 μL of 100 μM Compound A was added (final concentration 100 nM), and 37 ° C. for 1 hour. Incubated under 5% CO 2 . The cells were then irradiated with ultraviolet light (365 nm) at 4 ° C for 10 minutes. The cells were then scraped with a scraper, centrifuged at 1500 rpm for 10 minutes at 4 ° C. to remove the supernatant, washed with 1 mL of PBS, and the pellet was suspended in 100 μL of D-PBS (-). The cells were crushed on ice using an ultrasonic crusher, centrifuged at 4 ° C. and 15000 rpm for 30 minutes, and the supernatant was used as a cell lysate and stored at -80 ° C until use.
各サンプルのタンパク濃度をDC Protein Assay（BIO－RAD）を用いてSpectraMax Plusプレ－トリ－ダ－（モレキュラ－デバイス）で測定し、2 mg／mLの濃度にD－PBS（－）を用いて調製した。45μLの2 mg／mL細胞ライセ－トと5μLのClick試薬ミックス（50 mM CuSO4（ナカライテスク、＃09604－85）、1.25 mM TAMRA－Azide（Click chemistry tools、＃AZ10905）、50 mM TCEP（Bond－Breaker TCEP Solution、Neutral pH（PCC）、Thermo Fisher Scientific、＃77720）、10 mM THPTA（Tris（3－hydroxypropyltriazolylmethyl）amine、SIGMA、＃762342－500MG）、10％ SDS（SIGMA、＃L6026－250G））を混合し、室温で2時間反応させた。その後、各サンプルに18.3μLのサンプルバッファ－（4× LDS sample buffer（Life technologies、＃NP0008）および2－Mercaptoethanol（SIGMA、＃6250）を10：1で混合）を添加し、70℃10分間処理した。サンプルをゲル（Perfect NT Gel、5－20％）（DRC、＃NTH－576HP10CD）を用いてtriplicateで10μL／レ－ンで電気泳動し、TAMRAの蛍光をTyphoon FLA9500（GEヘルスケア）で測定し、β－チューブリン （50 kDa）のバンドの濃さをImageQuant TL（GEヘルスケア）を用いて定量した。結果を図９に示す。
NuMA1およびCKAP5のノックダウンによりCompound Aのβ－チューブリンへの結合は顕著に減少した。また、TACC3のノックダウンによって中程度の結合の減弱が認められた。一方で、CLTCのノックダウンでは影響は認められなかった。 The protein concentration of each sample was measured with the SpectraMax Plus assayer (molecular device) using the DC Protein Assay (BIO-RAD) and with D-PBS (-) at a concentration of 2 mg / mL. Prepared. 45 μL of 2 mg / mL cell lysate and 5 μL of Click reagent mix (50 mM CuSO4 (Nacalai Tesque, # 09604-85), 1.25 mM TAMRA-Azide (Click chemistry tools, # AZ10905), 50 mM TCEP (Bond-) Breaker TCEP Solution, Neutral pH (PCC), Thermo Fisher Scientific, # 77720), 10 mM THPTA (Tris (3-hydroxypropyltriazolylmethyl) amine, SIGMA, # 762342-500MG), 10% SDS (SIGMA, # L6026-250G)) Was mixed and reacted at room temperature for 2 hours. Then, 18.3 μL of sample buffer (4 × LDS sample buffer (Life technologies, # NP0008) and 2-Mercaptoethanol (SIGMA, # 6250) mixed at 10: 1) was added to each sample, and the mixture was treated at 70 ° C. for 10 minutes. did. Samples were electrophoresed in triplicate at 10 μL / lane using a gel (Perfect NT Gel, 5-20%) (DRC, # NTH-576HP10CD) and TAMRA fluorescence was measured with Typhoon FLA9500 (GE Healthcare). , Β-Tubulin (50 kDa) band density was quantified using ImageQuant TL (GE Healthcare). The results are shown in FIG.
Knockdown of NuMA1 and CKAP5 markedly reduced the binding of Compound A to β-tubulin. In addition, knockdown of TACC3 showed moderate attenuation of binding. On the other hand, no effect was observed in the knockdown of CLTC.
（実施例１５）Compound B処理による微小管伸長阻害
CKAP5およびTACC3は複合体を形成し、微小管の安定に重要な役割を担っていることが知られている。実施例１３によりCKAP5の局在異常が引き起こされていることが判明したため、Compound B処理により中心体から伸びる微小管の伸長が抑制されるかどうかを、微小管の成長端マ－カ－であるEB1の免疫染色により検討した。
抗EB1抗体（Abcam、＃ab53358）、抗ラットIgG－Alexa 647標識抗体（Abcam、＃ab150155）、CellMask Green（Thermo Fisher Scientific、C37608）、DAPI Solution（4',6-Diamidino-2-phenylindole Dihydrochloride Solution）（同仁化学研究所、D523）を用いた。
6×104個のヒト肺がん細胞株LK－2をクリアボトム96ウェルプレートに播種し、37℃、5％ CO2下で一日培養後、100 nMのConpound Bまたは10 nMのIspinesibで16時間処理した。細胞を100％メタノ－ルで5分間固定し、D－PBS（－）で2回洗浄した。0.1％ TritonX－100含有D－PBS（－）で5分間、膜透過処理をした後、D－PBS（－）で3回洗浄し、3％ ウシ血清アルブミン含有D－PBS（－）で30分間ブロッキングした。1次抗体はブロッキング溶液で希釈し、室温で30分間静置して反応させた。これをD－PBS（－）で3回洗浄し、2次抗体とCellMask Green混合液でも同様に反応、洗浄した。D－PBS（－）に浸漬した状態で横河電機株式会社製、Cell Voyager 7000Sを用いて測定し（測定条件：60倍水浸レンズ、16視野/ウェル、Z軸沿い0.5μmごとに25断面を測定しMaximum projectionプロトコールで1画像に統合）、Cell path finderソフトウェアで分裂期の細胞あたりのEB1クラスター数を解析した。具体的には、DAPIによるDNA染色で輝度の高い細胞を分裂期細胞として選択し、CellMask Green染色で細胞質を分別、一細胞当たりのEB1ドット数を計測した。
その結果、Compound BおよびIspinesibの処理により同様のmonopolar spindleを誘導するが、Compound B処理群ではIspinesib処理群に比較しEB1のシグナルが顕著に減少していたことから、Compound Bの処理により微小管伸長が抑制されることを見出した。結果を図１０および図１１に示す。 (Example 15) Inhibition of microtubule elongation by Compound B treatment
It is known that CKAP5 and TACC3 form a complex and play an important role in the stability of microtubules. Since it was found that the abnormal localization of CKAP5 was caused by Example 13, it is a growth end marker of microtubules whether or not the elongation of microtubules extending from the centrosome is suppressed by Compound B treatment. It was examined by immunostaining of EB1.
Anti-EB1 antibody (Abcam, # ab53358), anti-rat IgG-Alexa 647-labeled antibody (Abcam, # ab150155), CellMask Green (Thermo Fisher Scientific, C37608), DAPI Solution (4', 6-Diamidino-2-phenylindole Dihydrochloride Solution) ) (Dojin Chemical Research Institute, D523) was used.
6 × 10 4 human lung cancer cell lines LK-2 were seeded on a clear bottom 96-well plate, cultured daily under 5% CO 2 at 37 ° C, and then in 100 nM Compound B or 10 nM Ispine sib for 16 hours. Processed. The cells were fixed in 100% metanol for 5 minutes and washed twice with D-PBS (-). After membrane permeation treatment with D-PBS (-) containing 0.1% TritonX-100 for 5 minutes, washing 3 times with D-PBS (-) and 30 minutes with D-PBS (-) containing 3% bovine serum albumin. Blocked. The primary antibody was diluted with a blocking solution and allowed to stand at room temperature for 30 minutes for reaction. This was washed 3 times with D-PBS (-), and was similarly reacted and washed with a mixed solution of the secondary antibody and CellMask Green. Measured using Cell Voyager 7000S manufactured by Yokogawa Electric Co., Ltd. while immersed in D-PBS (-) (Measurement conditions: 60x water immersion lens, 16 fields / wells, 25 cross sections every 0.5 μm along the Z axis) Was measured and integrated into one image using the Maximum projection protocol), and the number of EB1 clusters per mitotic cell was analyzed using Cell path finder software. Specifically, cells with high brightness were selected as mitotic cells by DNA staining with DAPI, the cytoplasm was separated by CellMask Green staining, and the number of EB1 dots per cell was measured.
As a result, the same monopolar spindle was induced by the treatment of Compound B and Ispine sib, but the signal of EB1 was significantly reduced in the Compound B treatment group as compared with the Ispine sib treatment group. It was found that elongation was suppressed. The results are shown in FIGS. 10 and 11.
以上の結果から、Compound A、B、C、Dは細胞分裂時、中心体近傍においてNuMA1、CKAP5、TACC3、およびβ－チューブリンを含む複合体に結合して安定化させることで、CKAP5の局在異常を引き起こし、中心体から伸びる微小管の伸長を阻害することで、中心体分離を阻害していることが明らかとなった。
本結果は、NuMA1およびCKAP5を含む複合体を安定化させることで、がん細胞高選択的に細胞の増殖を妨げることが可能であることを示すものである。 From the above results, Compound A, B, C, and D bind to and stabilize a complex containing NuMA1, CKAP5, TACC3, and β-tubulin near the centrosome during cell division, thereby stabilizing the CKAP5 station. It was clarified that the centrosome separation was inhibited by causing an abnormality and inhibiting the elongation of microtubules extending from the centrosome.
This result indicates that by stabilizing the complex containing NuMA1 and CKAP5, it is possible to prevent the growth of cancer cells in a highly selective manner.
（実施例１６）多種類細胞に対する増殖阻害活性評価
　Compound B, Cを用いて種々のがん種に対する細胞増殖抑制効果の検討を行った。
ヒト前立腺がん細胞株LNCaP clone FGC （American Type Culture Collection）は牛胎児血清（最終濃度10％）を含むRPMI1640培地（富士フイルム和光純薬 ＃187－02705）、ヒト食道がん細胞株TE10（理化学研究所バイオリソ－スセンタ－）は牛胎児血清（最終濃度10％）を含むRPMI1640培地（Thermo　Fisher　Scientific ＃11875）、ヒト食道がん細胞株TE14（理化学研究所バイオリソ－スセンタ－）は牛胎児血清（最終濃度10％）を含むRPMI1640培地（Thermo　Fisher　Scientific ＃11875）、ヒト頭頸部がん細胞株Detroit 562 (American Type Culture Collection)は牛胎児血清（最終濃度10％）を含むEMEM培地（富士フイルム和光純薬 ＃051－07615）、ヒト頭頸部がん細胞株HSC3（理化学研究所バイオリソ－スセンタ－）は牛胎児血清（最終濃度10％）を含むEMEM培地（富士フイルム和光純薬 ＃051－07615）、ヒト白血病細胞株AML193 (American Type Culture Collection)は牛胎児血清（最終濃度5％）およびITS Liquid Media Supplement (SIGMA I3146)および最終濃度5 ng／mL human GM－CSF（Miltenyi Biotec ＃130－095－372）を含むIMDM培地（Thermo　Fisher　Scientific ＃12440）、ヒト肺がん細胞株NCI－H1395 (American Type Culture Collection)は牛胎児血清（最終濃度10％）を含むRPMI1640培地（富士フイルム和光純薬 ＃187－02705）、ヒト肺がん細胞株NCI－H23 (American Type Culture Collection)は牛胎児血清（最終濃度10％）を含むRPMI1640培地（富士フイルム和光純薬 ＃187－02705）、ヒト黒色腫細胞株A375（American Type Culture Collection）は牛胎児血清（最終濃度10％）を含むDMEM培地（富士フイルム和光純薬＃043－30085）、ヒト膵臓がん細胞株BxPC－3（American Type Culture Collection）は牛胎児血清（最終濃度10％）を含むRPMI1640培地（富士フイルム和光純薬 ＃187－02705）、ヒト肺がん細胞株LK－2は牛胎児血清（最終濃度10％）を含むRPMI1640培地（Thermo　Fisher　Scientific ＃11875）で継代維持した。各細胞株はTrypLE Express（Thermo Fisher Scientific ＃12605）で剥離回収した後、室温で1000 rpmで5分間遠心処理し、上清を除去した。同様の培地で細胞を懸濁し、各細胞を20000個／ 1 mLの細胞濃度に調製し、96穴プレ－ト（Corning ＃3904）の各ウェルへ100μLずつ分注し、37℃、5％CO2下で24時間培養した。続いて各培地で所定の濃度に希釈した実施例の化合物溶液を各ウェルへ50μLずつ添加し(day1)、37℃、5％ CO2下、3日間培養した。化合物添加当日(day 1)ならびに化合物添加3日後(day 4)にATP測定用試薬であるCellTiter－Glo 2.0 Assayを30μL／wellずつ各ウェルに添加し、EnVisionで各ウェルの発光量を測定した。化合物添加当日の発光量(C1)、3日間培養後の化合物非添加群(C4)および化合物添加群(T4)の発光量より、次式に基づき細胞増殖率を算出した。 (Example 16) Evaluation of growth inhibitory activity on multiple types of cells The cell growth inhibitory effect on various cancer types was examined using Compounds B and C.
The human prostate cancer cell line LNCaP clone FGC (American Type Culture Collection) is an RPMI1640 medium (Fujifilm Wako Junyaku # 187-02705) containing bovine fetal serum (final concentration 10%), and the human esophalized cell line TE10 (physical chemistry). The laboratory bioimmortalized cell line is the RPMI1640 cell line containing bovine fetal serum (final concentration 10%) (Thermo Fisher Scientific # 11875), and the human esophalized cell line TE14 (physical and chemical research institute biomortalized cell line) is the bovine immortalized cell line. RPMI 1640 medium (Thermo Fisher Scientific # 11875) containing 10% final concentration), Detroit 562 (American Type Culture Collection), a human head and neck cancer cell line, is an EMEM medium (Fuji Film Japanese) containing bovine fetal serum (10% final concentration). Kojunyaku # 051-07615), human head and neck cancer cell line HSC3 (Bio-mortalized cell line of the Institute of Physical and Chemical Research) is an EMEM medium containing bovine fetal serum (final concentration 10%) (Fujifilm Wako Junyaku # 051-07615) , Human immortalized cell line AML193 (American Type Culture Collection) is available in bovine fetal cell line (finalized 5%) and ITS Liquid Media Supplement (SIGMA I3146) and finalized 5 ng / mL human GM-CSF (Miltenyi Biotec # 130-095-). IMDM cell line containing 372) (Thermo Fisher Scientific # 12440), human lung cancer cell line NCI-H1395 (American Type Culture Collection) is an RPMI1640 cell line containing bovine fetal cell line (final concentration 10%) (Fujifilm Wako Pure Cell Line # 187-). 02705), human lung cancer cell line NCI-H23 (American Type Culture Collection) is an RPMI1640 medium (Fujifilm Wako Pure Drug # 187-02705) containing bovine fetal serum (final concentration 10%), human keratomortalized cell line A375 (American). Type Culture Collection) is a DMEM medium (Fujifilm Wako Junyaku # 043-30085) containing bovine fetal serum (final concentration 10%), human pancreatic cancer cell line. BxPC-3 (American Type Culture Collection) contains RPMI1640 medium (Fujifilm Wako Pure Chemical Industries, Ltd. # 187-02705) containing fetal bovine serum (final concentration 10%), and human lung cancer cell line LK-2 contains fetal bovine serum (final concentration 10%). %) Was subcultured in RPMI 1640 medium (Thermo Fisher Scientific # 11875). Each cell line was exfoliated and collected by TrypLE Express (Thermo Fisher Scientific # 12605), and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant. Suspend the cells in a similar medium, prepare each cell to a cell concentration of 20000 cells / 1 mL, dispense 100 μL into each well of a 96-well plate (Corning # 3904), 37 ° C., 5% CO Incubated under 2 for 24 hours. Subsequently, 50 μL of the compound solution of the example diluted in each medium to a predetermined concentration was added to each well (day 1), and the cells were cultured at 37 ° C. under 5% CO 2 for 3 days. On the day of compound addition (day 1) and 3 days after compound addition (day 4), CellTiter-Glo 2.0 Assay, a reagent for ATP measurement, was added to each well at a rate of 30 μL / well, and the amount of light emitted from each well was measured by EnVision. The cell proliferation rate was calculated based on the following formula from the luminescence amount on the day of compound addition (C1), the luminescence amount of the compound-free group (C4) and the compound-added group (T4) after culturing for 3 days.
Figure JPOXMLDOC01-appb-M000008
Figure JPOXMLDOC01-appb-M000008
化合物の各細胞の増殖を50％阻害する濃度（GI50値）は、各濃度における細胞増殖率と化合物濃度を片対数プロットして算出した。結果を表１に示す。Compound B, Cのいずれも各がん種について細胞増殖抑制効果を示した。 The concentration (GI50 value) that inhibits the growth of each cell of the compound by 50% was calculated by semi-logarithm plotting the cell growth rate and the compound concentration at each concentration. The results are shown in Table 1. Both Compound B and Compound C showed a cell growth inhibitory effect for each cancer type.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
（実施例１７）NuMA1発現量の違う細胞を用いた感受性差を指標とするNuMA1およびCKAP5を含む複合体を安定化する化合物のスクリーニング
ヒト肺がん細胞株LK－2（Health Science Research Resources Bank）は牛胎児血清（最終濃度10％）を含むRPMI1640培地（Thermo　Fisher　Scientific ＃11875）で継代維持した。まず、各細胞株を300000個／ 1 mLの細胞濃度に調製した。Negative control siRNA（Dharmacon、ON－TARGET plus Non－targeting Pool、＃D－001810－10－50）およびNuMA1 siRNA（Dharmacon、ON－TARGET plus NUMA1 siRNA、＃J－005272－05－0002）は、最終濃度10 nMとなるようにOpti－MEM培地（Thermo Fisher Scientific、＃11058－021）を用いてLipofectamine RNAiMAX（Thermo Fisher Scientific、＃13778－150）と混合した。 6ウェルプレ－ト（IWAKI、 ＃3810－006）の各ウェルへ細胞懸濁液を1 mL、各細胞株に対する上記培地を1 mL、siRNA－RNAiMAX reagent混合液を0.5 mLずつ分注し、37℃、5％CO2下で24時間培養した。続いて培養上清を取り除き、各細胞株に対する上記培地を2.5 mL添加し、先と同様に調製したsiRNA－RNAiMAX reagent混合液を0.5 mL添加した。４時間37℃、5％CO2下で培養した後、TrypLE Expressで剥離回収した後、室温で1000 rpmで5分間遠心処理し、上清を除去した。各細胞株に対する上記培地で細胞を懸濁し、20000個／ 1 mLの細胞濃度に調製し、96ウェルプレ－ト（Corning ＃3904）の各ウェルへ100μLずつ分注し、37℃、5％CO2下で24時間培養した。続いて各細胞株に対する上記培地で所定の濃度に希釈したCompound B、Ispinesib、Volasertib、Alisertib、その他細胞分裂阻害剤として微小管重合阻害剤であるVincristine（日本化薬、オンコビン注射用）、微小管脱重合阻害剤であるPaclitaxel（富士フイルム和光純薬、#167-28166）を各ウェルへ50μL添加し(day1)、37℃、5％ CO2下、3日間培養した。化合物添加3日後(day 4)にATP測定用試薬であるCellTiter－Glo 2.0 Assayを30μL／wellずつ各ウェルに添加し、EnVisionで各ウェルの発光量を測定した。3日間培養後の化合物非添加群(C4)および化合物添加群(T4)の発光量より、次式に基づき細胞抑制率を算出した。 (Example 17) Screening of a compound that stabilizes a complex containing NuMA1 and CKAP5 using cells having different NuMA1 expression levels as an index Human lung cancer cell line LK-2 (Health Science Research Resources Bank) is a cow. Subculture was maintained in RPMI 1640 medium (Thermo Fisher Scientific # 11875) containing fetal bovine serum (final concentration 10%). First, each cell line was prepared to a cell concentration of 300,000 cells / 1 mL. Negative control siRNA (Dharmacon, ON-TARGET plus Non-targeting Pool, # D-001810-10-50) and NuMA1 siRNA (Dharmacon, ON-TARGET plus NUMA1 siRNA, # J-005272-05-0002) have final concentrations. It was mixed with Lipofectamine RNAiMAX (Thermo Fisher Scientific, # 13778-150) using Opti-MEM medium (Thermo Fisher Scientific, # 11058-021) to 10 nM. Dispense 1 mL of cell suspension into each well of 6-well plate (IWAKI, # 3810-006), 1 mL of the above medium for each cell line, and 0.5 mL of siRNA-RNAiMAX reagent mixture at 37 ° C. , Incubated for 24 hours under 5% CO 2 . Subsequently, the culture supernatant was removed, 2.5 mL of the above medium was added to each cell line, and 0.5 mL of the siRNA-RNAiMAX reagent mixture prepared in the same manner as above was added. After culturing under 5% CO 2 at 37 ° C. for 4 hours, the cells were peeled and recovered by TrypLE Express, and then centrifuged at 1000 rpm for 5 minutes at room temperature to remove the supernatant. Suspend the cells in the above medium for each cell line, prepare to a cell concentration of 20000 cells / 1 mL, dispense 100 μL into each well of 96-well plates (Corning # 3904), 37 ° C, 5% CO 2 Incubated underneath for 24 hours. Subsequently, Compound B, Ispinesib, Volasertib, Alisertib, and other microtubule polymerization inhibitors Vincristine (for injection of Japanese chemicals and oncobin), which are microtubule polymerization inhibitors, and microtubules, which were diluted to a predetermined concentration in the above medium for each cell line, were used. Paclitaxel (Fujifilm Wako Pure Chemical Industries, Ltd., # 167-28166), a depolymerization inhibitor, was added to each well in an amount of 50 μL (day 1) and cultured at 37 ° C. under 5% CO 2 for 3 days. Three days after the addition of the compound (day 4), CellTiter-Glo 2.0 Assay, which is a reagent for ATP measurement, was added to each well at a rate of 30 μL / well, and the luminescence amount of each well was measured by EnVision. From the luminescence amount of the compound-free group (C4) and the compound-added group (T4) after culturing for 3 days, the cell suppression rate was calculated based on the following formula.
Figure JPOXMLDOC01-appb-M000010
Figure JPOXMLDOC01-appb-M000010
化合物の各細胞を50％抑制する濃度（IC50値）について、各濃度における細胞増殖率と化合物濃度を片対数プロットして算出し、siContorl処理群のIC50とsiNuMA1処理群のIC50の比率を表２に示す。Compound BはsiRNA処理によりNuMA1の発現を抑制することで、IC50値が１０倍以上大きくなる（薬理活性が１／１０以下に落ちる）のに対し、その他の細胞分裂阻害剤ではNuMA1の発現抑制によるIC50値の変化は認められなかった。つまり、NuMA1の発現量に差がある細胞を用い、細胞間で大きく細胞増殖抑制効果の異なる化合物を選抜することで、NuMA1およびCKAP5を含む複合体を安定化する化合物をスクリーニングすることができることを見出した。 For the concentration that suppresses each cell of the compound by 50% (IC50 value), calculate by semi-log plotting the cell proliferation rate and compound concentration at each concentration, and the ratio of IC50 in the siContorl treatment group to IC50 in the siNuMA1 treatment group is shown in Table 2. Shown in. Compound B suppresses the expression of NuMA1 by siRNA treatment, which increases the IC50 value by 10 times or more (the pharmacological activity drops to 1/10 or less), whereas other cell division inhibitors suppress the expression of NuMA1. No change in IC50 value was observed. In other words, by using cells with different expression levels of NuMA1 and selecting compounds with significantly different cell growth inhibitory effects among the cells, it is possible to screen for compounds that stabilize the complex containing NuMA1 and CKAP5. I found it.
Figure JPOXMLDOC01-appb-T000011
  
Figure JPOXMLDOC01-appb-T000011
  

Claims (52)

  1. 　NuMA1およびCKAP5を含む複合体を安定化する化合物。 A compound that stabilizes the complex containing NuMA1 and CKAP5.
  2. 　請求項１に記載の化合物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化する化合物。 The compound according to claim 1, which stabilizes a complex containing NuMA1, CKAP5 and β-tubulin.
  3. 　請求項１に記載の化合物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化する化合物。 The compound according to claim 1, which stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  4. 　がんの治療に用いられる請求項１～３のいずれか１項に記載の化合物。 The compound according to any one of claims 1 to 3, which is used for the treatment of cancer.
  5. 　NuMA1およびCKAP5を含む複合体を安定化する化合物を含有する医薬組成物。 A pharmaceutical composition containing a compound that stabilizes a complex containing NuMA1 and CKAP5.
  6. 　請求項５に記載の医薬組成物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化する化合物を含有する医薬組成物。 The pharmaceutical composition according to claim 5, which contains a compound that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin.
  7. 　請求項５に記載の医薬組成物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化する化合物を含有する医薬組成物。 The pharmaceutical composition according to claim 5, which contains a compound that stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  8. 　がんの治療に用いられる請求項５～７のいずれか１項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 5 to 7, which is used for treating cancer.
  9. 　NuMA1およびCKAP5を含む複合体を安定化させる方法。 A method of stabilizing a complex containing NuMA1 and CKAP5.
  10. 　請求項９に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化させる方法。 The method according to claim 9, wherein the complex containing NuMA1, CKAP5 and β-tubulin is stabilized.
  11. 　請求項９に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化させる方法。 The method according to claim 9, wherein the complex containing NuMA1, CKAP5, β-tubulin and TACC3 is stabilized.
  12. 　がんの治療に用いられる請求項９～１１のいずれか１項に記載の方法。 The method according to any one of claims 9 to 11, which is used for treating cancer.
  13. 　NuMA1およびCKAP5を含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。 A method for suppressing the growth of cancer cells, which is characterized by stabilizing a complex containing NuMA1 and CKAP5.
  14. 　請求項１３に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。 The method according to claim 13, wherein the method for suppressing the growth of cancer cells is characterized by stabilizing a complex containing NuMA1, CKAP5 and β-tubulin.
  15. 　請求項１３に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体を安定化させることを特徴とするがん細胞の増殖を抑制する方法。 The method according to claim 13, wherein the method for suppressing the growth of cancer cells is characterized by stabilizing a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  16. 　以下の工程：
    　（a）化合物をNuMA1およびCKAP5を含む複合体と接触させる工程
    　（b）化合物によりNuMA1およびCKAP5を含む複合体が安定化されているか否かを確認する工程
    を含むことを特徴とするNuMA1およびCKAP5を含む複合体を安定化する化合物のスクリ－ニング方法。     The following steps:
    (A) A step of contacting the compound with the complex containing NuMA1 and CKAP5 (b) A step of confirming whether or not the complex containing NuMA1 and CKAP5 is stabilized by the compound is included. A method for screening a compound that stabilizes a complex containing.
  17. 　以下の工程：
    　（a）NuMA1の発現量が高い細胞およびNuMA1の発現量が低い細胞のそれぞれに化合物を接触させる工程
    　（b）それぞれの細胞における化合物の細胞増殖抑制作用および／または細胞分裂阻害作用を測定する工程
    　（c）NuMA1の発現量の高い細胞における細胞増殖抑制作用の方がNuMA1の発現量の低い細胞における細胞増殖抑制作用よりも高い場合に、当該化合物をNuMA1およびCKAP5を含む複合体を安定化する化合物であると同定する工程
    を含むことを特徴とするNuMA1およびCKAP5を含む複合体を安定化する化合物のスクリ－ニング方法。     The following steps:
    (A) Step of contacting the compound with cells having a high expression level of NuMA1 and cells having a low expression level of NuMA1 (b) Step of measuring the cell growth inhibitory action and / or cell division inhibitory action of the compound in each cell (C) Stabilize the complex containing NuMA1 and CKAP5 when the cell growth inhibitory effect in cells with high NuMA1 expression is higher than the cell growth inhibitory effect in cells with low NuMA1 expression. A method for screening a compound that stabilizes a complex containing NuMA1 and CKAP5, which comprises the step of identifying it as a compound.
  18. 　NuMA1およびCKAP5を含む複合体を安定化させることを特徴とするがんの治療方法。 A cancer treatment method characterized by stabilizing a complex containing NuMA1 and CKAP5.
  19. 　請求項１８に記載の方法であって、NuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化させることを特徴とするがんの治療方法。 The method according to claim 18, which is a method for treating cancer, which comprises stabilizing a complex containing NuMA1, CKAP5 and β-tubulin.
  20. 　請求項１８に記載の方法であって、NuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化させることを特徴とするがんの治療方法。 The method according to claim 18, which is a method for treating cancer, which comprises stabilizing a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  21. 　がんの治療に使用するためのNuMA1およびCKAP5を含む複合体を安定化する化合物。 A compound that stabilizes a complex containing NuMA1 and CKAP5 for use in the treatment of cancer.
  22. 　請求項２１に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化する化合物。 The compound according to claim 21, which stabilizes a complex containing NuMA1, CKAP5 and β-tubulin for use in the treatment of cancer.
  23. 　請求項２１に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化する化合物。 The compound according to claim 21, which stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3 for use in the treatment of cancer.
  24. 　がんの治療のための医薬の製造におけるNuMA1およびCKAP5を含む複合体を安定化する化合物の使用。 Use of compounds that stabilize the complex containing NuMA1 and CKAP5 in the manufacture of pharmaceuticals for the treatment of cancer.
  25. 　請求項２４に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体を安定化する化合物の使用。 The use of a compound according to claim 24 that stabilizes a complex containing NuMA1, CKAP5 and β-tubulin in the manufacture of a medicament for the treatment of cancer.
  26. 　請求項２４に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体を安定化する化合物の使用。 The use of the compound according to claim 24, which stabilizes a complex containing NuMA1, CKAP5, β-tubulin and TACC3 in the manufacture of a medicament for the treatment of cancer.
  27. 　NuMA1およびCKAP5を含む複合体の機能を調整する化合物。 A compound that regulates the function of the complex containing NuMA1 and CKAP5.
  28. 　請求項２７に記載の化合物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整する化合物。 The compound according to claim 27, which regulates the function of a complex containing NuMA1, CKAP5 and β-tubulin.
  29. 　請求項２７に記載の化合物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整する化合物。 The compound according to claim 27, which regulates the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  30. 　がんの治療に用いられる請求項２７～２９のいずれか１項に記載の化合物。 The compound according to any one of claims 27 to 29 used for the treatment of cancer.
  31. 　NuMA1およびCKAP5を含む複合体の機能を調整する化合物を含有する医薬組成物。 A pharmaceutical composition containing a compound that regulates the function of a complex containing NuMA1 and CKAP5.
  32. 　請求項３１に記載の医薬組成物であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整する化合物を含有する医薬組成物。 The pharmaceutical composition according to claim 31, which contains a compound that regulates the function of a complex containing NuMA1, CKAP5 and β-tubulin.
  33. 　請求項３１に記載の医薬組成物であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整する化合物を含有する医薬組成物。 The pharmaceutical composition according to claim 31, which contains a compound that regulates the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  34. 　がんの治療に用いられる請求項３１～３３のいずれか１項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 31 to 33, which is used for treating cancer.
  35. 　NuMA1およびCKAP5を含む複合体の機能を調整する方法。 A method of adjusting the function of the complex containing NuMA1 and CKAP5.
  36. 　請求項３５に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整する方法。 The method according to claim 35, wherein the function of the complex containing NuMA1, CKAP5 and β-tubulin is adjusted.
  37. 　請求項３５に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整する方法。 The method according to claim 35, wherein the function of the complex containing NuMA1, CKAP5, β-tubulin and TACC3 is adjusted.
  38. 　がんの治療に用いられる請求項３５～３７のいずれか１項に記載の方法。 The method according to any one of claims 35 to 37, which is used for treating cancer.
  39. 　NuMA1およびCKAP5を含む複合体の機能を調整することを特徴とするがん細胞の増殖を抑制する方法。 A method for suppressing the growth of cancer cells, which is characterized by regulating the function of a complex containing NuMA1 and CKAP5.
  40. 　請求項３９に記載の方法であって、NuMA1、CKAP5およびβ－チューブリンを含む複合体の機能を調整することを特徴とするがん細胞の増殖を抑制する方法。 The method according to claim 39, which suppresses the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1, CKAP5 and β-tubulin.
  41. 　請求項３９に記載の方法であって、NuMA1、CKAP5、β－チューブリンおよびTACC3を含む複合体の機能を調整することを特徴とするがん細胞の増殖を抑制する方法。 The method according to claim 39, which suppresses the growth of cancer cells, which comprises regulating the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  42. 　以下の工程：
    　（a）化合物をNuMA1およびCKAP5を含む複合体と接触させる工程
    　（b）化合物によりNuMA1およびCKAP5を含む複合体の機能が調整されているか否かを確認する工程
    を含むことを特徴とするNuMA1およびCKAP5を含む複合体の機能を調整する化合物のスクリ－ニング方法。     The following steps:
    (A) A step of contacting the compound with the complex containing NuMA1 and CKAP5 (b) A step of confirming whether or not the function of the complex containing NuMA1 and CKAP5 is adjusted by the compound. A method for screening a compound that regulates the function of a complex containing CKAP5.
  43. 　以下の工程：
    　（a）NuMA1の発現量が高い細胞およびNuMA1の発現量が低い細胞のそれぞれに化合物を接触させる工程
    　（b）それぞれの細胞における化合物の細胞増殖抑制作用および／または細胞分裂阻害作用を測定する工程
    　（c）NuMA1の発現量の高い細胞における細胞増殖抑制作用の方がNuMA1の発現量の低い細胞における細胞増殖抑制作用よりも高い場合に、当該化合物をNuMA1およびCKAP5を含む複合体の機能を調整する化合物であると同定する工程
    を含むことを特徴とするNuMA1およびCKAP5を含む複合体の機能を調整する化合物のスクリ－ニング方法。     The following steps:
    (A) Step of contacting the compound with cells having a high expression level of NuMA1 and cells having a low expression level of NuMA1 (b) Step of measuring the cell growth inhibitory action and / or cell division inhibitory action of the compound in each cell (C) When the cell growth inhibitory effect in cells with high NuMA1 expression is higher than the cell growth inhibitory effect in cells with low NuMA1 expression, the compound adjusts the function of the complex containing NuMA1 and CKAP5. A method for screening a compound that regulates the function of a complex containing NuMA1 and CKAP5, which comprises the step of identifying the compound to be used.
  44. 　NuMA1およびCKAP5を含む複合体の機能を調整することを特徴とするがんの治療方法。 A cancer treatment method characterized by adjusting the function of a complex containing NuMA1 and CKAP5.
  45. 　請求項４４に記載の方法であって、NuMA1、CKAP5およびβ－チュ－ブリンを含む複合体の機能を調整することを特徴とするがんの治療方法。 The method according to claim 44, which is a method for treating cancer, which comprises adjusting the function of a complex containing NuMA1, CKAP5 and β-tubulin.
  46. 　請求項４４に記載の方法であって、NuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体の機能を調整することを特徴とするがんの治療方法。 The method according to claim 44, which is a method for treating cancer, which comprises adjusting the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3.
  47. 　がんの治療に使用するためのNuMA1およびCKAP5を含む複合体の機能を調整する化合物。 A compound that regulates the function of a complex containing NuMA1 and CKAP5 for use in the treatment of cancer.
  48. 　請求項４７に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体の機能を調整する化合物。 The compound according to claim 47, which regulates the function of a complex containing NuMA1, CKAP5 and β-tubulin for use in the treatment of cancer.
  49. 　請求項４７に記載の化合物であって、がんの治療に使用するためのNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体の機能を調整する化合物。 The compound according to claim 47, which regulates the function of a complex containing NuMA1, CKAP5, β-tubulin and TACC3 for use in the treatment of cancer.
  50. 　がんの治療のための医薬の製造におけるNuMA1およびCKAP5を含む複合体の機能を調整する化合物の使用。 Use of compounds that regulate the function of complexes containing NuMA1 and CKAP5 in the manufacture of drugs for the treatment of cancer.
  51. 　請求項５０に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5およびβ－チュ－ブリンを含む複合体の機能を調整する化合物の使用。 The use of a compound according to claim 50 that regulates the function of a complex comprising NuMA1, CKAP5 and β-tubulin in the manufacture of a medicament for the treatment of cancer.
  52. 　請求項５０に記載の使用であって、がんの治療のための医薬の製造におけるNuMA1、CKAP5、β－チュ－ブリンおよびTACC3を含む複合体の機能を調整する化合物の使用。
     
          The use of a compound according to claim 50 that regulates the function of a complex comprising NuMA1, CKAP5, β-tubulin and TACC3 in the manufacture of a medicament for the treatment of cancer.
PCT/JP2020/021850 2019-06-04 2020-06-03 COMPOUND STABILIZING COMPLEX CONTAINING NuMA1 AND CKAP5 WO2020246488A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2019-104414 2019-06-04
JP2019104414 2019-06-04

Publications (1)

Publication Number Publication Date
WO2020246488A1 true WO2020246488A1 (en) 2020-12-10

Family

ID=73653185

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/021850 WO2020246488A1 (en) 2019-06-04 2020-06-03 COMPOUND STABILIZING COMPLEX CONTAINING NuMA1 AND CKAP5

Country Status (2)

Country Link
TW (1) TW202110851A (en)
WO (1) WO2020246488A1 (en)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CASSIMERIS, L. ET AL.: "TOGp, the human homolog of XMAP215/Disl, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly", MOLECULAR BIOLOGY OF THE CELL, vol. 15, no. 4, 2004, pages 1580 - 1590, XP002401401, ISSN: 1059-1524 *
FORAKER AMY B., CAMUS STÉPHANE M., EVANS TIMOTHY M., MAJEED SOPHIA R., CHEN CHIH-YING, TANER SABRINA B., CORRÊA IVAN R., DOXSEY ST: "Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG", JOURNAL OF CELL BIOLOGY, vol. 198, no. 4, 2012, pages 591 - 605, XP055770334, ISSN: 0021-9525 *
HOOD FIONA E., WILLIAMS SAMANTHA J., BURGESS SELENA G., RICHARDS MARK W., ROTH DANIEL, STRAUBE ANNE, PFUHL MARK, BAYLISS RICHARD, : "Coordination of adjacent domains mediates TACC3-ch-TOG- clathrin assembly and mitotic spindle binding", JOURNAL OF CELL BIOLOGY, vol. 202, no. 3, 2013, pages 463 - 478, XP055770337, ISSN: 0021-9525 *
ROSAS-SALVANS MIQUEL, CAVAZZA TOMMASO, ESPADAS GUADALUPE, SABIDO EDUARD, VERNOS ISABELLE: "Proteomic profiling of microtubule self- organization in M-phase", MOLECULAR & CELLULAR PROTEOMICS, vol. 17, no. 10, 2018, pages 1991 - 2004, XP055770331, ISSN: 1535-9484 *
THAKUR HARISH C., SINGH MADHURENDRA, NAGEL-STEGER LUITGARD, PRUMBAUM DANIEL, FANSA EYAD KALAWY, GREMER LOTHAR, EZZAHOINI HAKIMA, A: "Role of centrosomal adaptor proteins of the TACC family in the regulation of microtubule dynamics during mitotic cell division", BIOLOGICAL CHEMISTRY, vol. 394, no. 11, 2013, pages 1411 - 1423, XP055770341, ISSN: 1431-6730 *

Also Published As

Publication number Publication date
TW202110851A (en) 2021-03-16

Similar Documents

Publication Publication Date Title
Sayan et al. Fra-1 controls motility of bladder cancer cells via transcriptional upregulation of the receptor tyrosine kinase AXL
JP7474269B2 (en) Compositions and methods for treating cancer
CN111789956B (en) Drug therapy, screening technology and kit for inhibiting side effects caused by chemotherapy
JP2022050418A (en) Theramutein modulators
Yoon et al. Benproperine, an ARPC2 inhibitor, suppresses cancer cell migration and tumor metastasis
KR20160043534A (en) Selective grp94 inhibitors and uses thereof
JP2021152022A (en) Cell penetrating antibodies
JP7104462B2 (en) Cell-permeable protein-antibody conjugate and usage
WO2007101710A1 (en) Use of inhibitors of scavenger receptor class proteins for the treatment of infectious diseases
JP2019508496A (en) TAF1 inhibitors for the treatment of cancer
Shatz et al. Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells
JP2018515563A (en) Compound
Bonezzi et al. Inhibition of SIRT2 potentiates the anti-motility activity of taxanes: implications for antineoplastic combination therapies
US20170334863A1 (en) Cdc42 INHIBITOR AND USES THEREOF
Greig et al. Effects of activating mutations on EGFR cellular protein turnover and amino acid recycling determined using SILAC mass spectrometry
US20200278355A1 (en) Conjugated proteins and uses thereof
Li et al. SENP1-mediated deSUMOylation of JAK2 regulates its kinase activity and platinum drug resistance
EP2857392B1 (en) Small compound targeting at tacc3
WO2019206203A1 (en) Method for adjusting ras ubiquitination
WO2020246488A1 (en) COMPOUND STABILIZING COMPLEX CONTAINING NuMA1 AND CKAP5
CN113543852A (en) Pyrrolopyrazole derivatives
US11427543B2 (en) Compounds for targeting cancer stem cells
WO2021202137A1 (en) Therapeutic uses of inhibitors of the rna-binding protein hur
Roblek et al. The Solute Carrier MFSD1 Decreases the Activation Status of β1 Integrin and Thus Tumor Metastasis
JP2019085381A (en) Apoptosis inducers and therapeutic agents for cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20818852

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20818852

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP