JP2020019732A - γ-tubulin inhibitor - Google Patents

Abstract

To provide a novel compound with a microtubule polymerization inhibitory activity promising as an anticancer agent.SOLUTION: The invention provides a compound represented by formula (I) in the figure or a pharmaceutically acceptable salt thereof.

Description

  The present invention relates to a novel compound having a microtubule (tubulin) polymerization inhibitory activity and a pharmaceutical composition containing the compound.

  Microtubules are one of the organelles involved in various vital activities such as mass transport, cell movement, and cell division in eukaryotic cells.Because they are a major component of the spindle in cell division, Drugs that inhibit microtubule function are known to inhibit cell division.

  Since cancer cells repeat disordered cell division, microtubules, which are organelles essential for cell division, have been targets of anticancer drugs for some time, and microtubule inhibitors such as paclitaxel and vinblastine have been used as anticancer drugs. It is used clinically as a cancer drug. However, microtubule inhibitors also disrupt the microtubule structure (microtubule network) of undivided interphase cells, sometimes causing severe side effects, including peripheral neuralgia. Therefore, anticancer agents targeting mitotic phase-specific microtubules and microtubule-related factors have been demanded (Non-Patent Document 1).

For microtubule formation in higher eukaryotes, γ-tubulin, a substance around the centriole, and γ-tubulin ring complex (γ-TuRC) composed of GCPs 2 to 6 are essential. γ-TuRC is recruited to the central body by the action of plk1 during mitosis, and functions as a scaffold for forming microtubules (see FIG. 1) (Non-Patent Document 2).
Inhibition of γ-tubulin and γ-TuRC, which are important scaffolds for microtubule nucleation, is considered to inhibit mitotic phase-specific microtubule elongation and spindle formation, so γ-tubulin has side effects. It is thought that there is little possibility that it will be a target of new anticancer drugs.

Dumontet C, Jordan MA.Microtubule-binding agents: a dynamic field of cancer therapeutics.Nat Rev Drug Discov 2010; 9: 790-803.doi: 10.1038 / nrd3253 Kollman JM, Merdes A, Mourey L, Agard DA.Microtubule nucleation by γ-tubulin complexes. Nat Rev Mol Cell Biol 2011; 12: 709-21. Doi: 10.1038 / nrm3209

  An object of the present invention is to provide a novel compound having a microtubule polymerization inhibitory activity and promising as an anticancer agent.

  The present inventors searched for a compound having a higher microtubule polymerization inhibitory activity, and found that by introducing specific substituents at positions 6 and 7 of the benzene ring of the A ring of glaziovianin A, a very high The present inventors have found that they have tube polymerization inhibitory activity, and have completed the present invention.

（式中、
Ｒ_１は、Ｃ_２〜８のアルケニル基、Ｃ_２〜８のアルキニル基、置換又は無置換のアリール基又はヘテロシクリル基から選択され；
Ｒ_２は、存在する場合は、夫々独立して、ハロゲン原子、Ｃ_１〜８のアルキル基、Ｃ_１〜８のアルコキシ基、ニトロ基、シアノ基又はカルボキシル基から選択され；
ｍは、１〜８の整数であり：
ｎは、１〜８の整数である。）
［２］ Ｒ_１が、

で表される、［１］に記載の化合物又は医薬的に許容可能なその塩。
（式（１）〜（４）において、
Ｒ_３、Ｒ_４、Ｒ_５、及びＲ_６は、存在する場合は、夫々独立して、ハロゲン原子、ハロゲン原子、Ｃ_１〜８のアルキル基、Ｃ_１〜８のアルコキシ基、ニトロ基、シアノ基又はカルボキシル基から選択され、
Ｘは、窒素原子、酸素原子又は硫黄原子を表し、
Ｙは、炭素原子、窒素原子、酸素原子又は硫黄原子を表す。
［３］Ｒ_１が、Ｃ_２〜８のアルキニル基である、［１］に記載の化合物又は医薬的に許容可能なその塩。
［４］［１］〜［３］のいずれか１項に記載の化合物又は医薬的に許容可能なその塩を含む微小管重合阻害剤。
［５］［１］〜［３］のいずれか１項に記載の化合物又は医薬的に許容可能なその塩を含む、癌を治療又は予防するための医薬組成物。 That is, the present invention
[1] A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

(Where
R ₁ is selected from a C _2-8 alkenyl group, a C _2-8 alkynyl group, a substituted or unsubstituted aryl group or a heterocyclyl group;
R ₂ , when present, is each independently selected from a halogen atom, a C _1-8 alkyl group, a C _1-8 alkoxy group, a nitro group, a cyano group or a carboxyl group;
m is an integer from 1 to 8:
n is an integer of 1 to 8. )
[2] R ₁ is

Or the pharmaceutically acceptable salt thereof according to [1].
(In equations (1) to (4),
When present, R ₃ , R ₄ , R ₅ , and R ₆ each independently represent a halogen atom, a halogen atom, a C _1-8 alkyl group, a C _1-8 alkoxy group, a nitro group, a cyano group. Selected from a group or a carboxyl group,
X represents a nitrogen atom, an oxygen atom or a sulfur atom,
Y represents a carbon atom, a nitrogen atom, an oxygen atom or a sulfur atom.
[3] The compound according to [1], wherein R ₁ is C _2-8 alkynyl, or a pharmaceutically acceptable salt thereof.
[4] A microtubule polymerization inhibitor comprising the compound according to any one of [1] to [3] or a pharmaceutically acceptable salt thereof.
[5] A pharmaceutical composition for treating or preventing cancer, comprising the compound according to any one of [1] to [3] or a pharmaceutically acceptable salt thereof.

  Since the compound of the present invention has strong cytotoxicity, particularly γ-tubulin inhibitory activity, it can provide a promising anticancer agent.

Schematic diagram of microtubule formation in higher eukaryotes. 1 shows an NMR spectrum of Compound A. 1 shows an NMR spectrum of Compound B. Evaluation results of interphase microtubule morphology of compound A and compound B in animal cells. Evaluation results of in vitro tubulin polymerization inhibitory activity of compound A and compound B. Evaluation results of mitotic spindle morphology of animal cells for compound A and compound B. The evaluation result of the microtubule nucleus formation activity from the animal cell centrosome about the compound A and the compound B.

  One aspect of the present invention is a compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

In the formula (I), R ₁ is selected from a C _2-8 alkenyl group, a C _2-8 alkynyl group, a substituted or unsubstituted aryl group or a heterocyclyl group.

C _2-8 alkenyl groups include, for example, vinyl group, allyl group, 1-propenyl group, isopropenyl group, 1-butenyl group, 2-butenyl group, 3-butenyl group, 1,3-butanedienyl group, 1- Pentenyl group, 2-pentenyl group, 3-pentenyl group, 4-pentenyl group, 1,3-pentanedienyl group, 1-hexenyl group, 2-hexenyl group, 3-hexenyl group, 4-hexenyl group, 5-hexenyl And 1,4-hexanedienyl groups. The double bond may have either a cis configuration or a trans configuration.

The C _{2 to 8} alkynyl group, for example, Asechiniru groups include propargyl group, preferably a Asechiniru group.

The above C _2-8 alkenyl group and C _2-8 alkynyl group may have one or more optional substituents. Examples of the substituent include an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, an acyl group, an aromatic ring or a substituted aromatic ring (the substituent is an alkoxy group, a halogen atom, an amino group, Examples thereof include, but are not limited to, a mono- or di-substituted amino group, a substituted silyl group, an acyl group, etc. When the aromatic ring has two or more substituents, they may be the same. But they are not limited to these. When the alkenyl group and the alkynyl group have two or more substituents, they may be the same or different.

The aryl group for R ₁ is a monocyclic or condensed polycyclic aromatic hydrocarbon group, for example, a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a 1-indenyl group, a 2-indenyl group, a 2 , 3-dihydroinden-1-yl group, 2,3-dihydroinden-2-yl group, 2-anthryl group, indazolyl group and the like, and a phenyl group is preferable.

The heterocyclyl group for R ₁ is a saturated or unsaturated heterocyclic ring containing one or more hetero atoms as ring-constituting atoms, for example, an oxygen atom, a nitrogen atom, or a sulfur atom. Examples of the saturated heterocyclyl group include a pyrrolidinyl group, an oxolyl group, a thiolyl group, an azinyl group, an oxyl group, a thianyl group, and a morphonyl group.
The unsaturated heterocyclyl group is preferably an aromatic heterocycle (also referred to as a heteroaryl group) containing one or more heteroatoms as ring constituent atoms, for example, an oxygen atom, a nitrogen atom, a sulfur atom, or the like.
Examples of the heteroaryl group include a thienyl group (2- or 3-thienyl group), a pyridyl group, a furyl group, a thiazolyl group, an oxazolyl group, a pyrazolyl group, a 2-pyrazinyl group, a pyrimidinyl group, a pyrrolyl group, an imidazolyl group, a pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group or 1,2,4-oxadiazol-3-yl group, quinolyl group, isoquinolyl group, 1,2- Dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3 -Dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2,3- Tetrahydrobenzo-2-yl group, benzothiazolyl group, benzimidazolyl group, and a fluorenyl group or a thioxanthenyl group.
When the aryl group and the heterocyclyl group are either a single ring or a condensed ring, they can be bonded at all possible positions.

The aryl group and the heterocyclyl group may have one or more arbitrary substituents on the ring, and may not have the substituent. Examples of the substituent include a halogen atom, C _{1 to 8} alkyl group, an alkoxy group, a nitro group, a cyano group, a carboxyl group or the like. When it has two or more substituents, they may be the same or different.

  The halogen atom includes a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

The C _1-8 alkyl group may be any of a linear, branched, cyclic, or an aliphatic hydrocarbon group composed of a combination thereof, but is preferably a linear or branched chain. preferable. C _1-8 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo -Pentyl group, n-hexyl group, isohexyl group, n-heptyl group, n-octyl group and the like.

  Examples of the alkoxy group include a saturated alkoxy group which is linear, branched, cyclic, or a combination thereof. For example, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n- A pentyloxy group, a cyclopentyloxy group, a cyclopropylethyloxy group, a cyclobutylmethyloxy group, an n-hexyloxy group, a cyclohexyloxy group, a cyclopropylpropyloxy group, a cyclobutylethyloxy group or a cyclopentylmethyloxy group is preferable. As an example.

In the formula (I), R ₂ , when present, are each independently selected from a halogen atom, a C _1-8 alkyl group, a C _1-8 alkoxy group, a nitro group, a cyano group or a carboxyl group. You.

Halogen _atom, an alkyl group of _{C 1 to 8,} for the alkoxy group of _{C 1 to 8,} include those exemplified above with respect to _{R 1.}
The carboxyl group includes those having a carboxyl group at the terminal of the alkyl group, and those having a carboxyl group substituted on any carbon atom of the alkyl group.
In a preferred embodiment of the present invention, the substituent for R ₂ is absent and is an unsubstituted benzene ring.

  In the formula (I), m is an integer of 1 to 8, preferably an integer of 1 to 3, and more preferably 1.

  In the formula (I), n is an integer of 1 to 8, preferably an integer of 1 to 3, and more preferably 1.

In one embodiment of the present invention, R ₁ is represented by the following formulas (1) to (4).

In the formulas (1) to (4), R ₃ , R ₄ , R ₅ , and R ₆ , when present, each independently represent a halogen atom, a halogen atom, a C _1-8 alkyl group, C _{1 ~ 8} alkoxy, nitro, cyano or carboxyl groups.

Halogen _atom, an alkyl group of _{C 1 to 8,} for the alkoxy group of _{C 1 to 8,} include those exemplified above with respect to _{R 1.}
The carboxyl group includes those having a carboxyl group at the terminal of the alkyl group, and those having a carboxyl group substituted on any carbon atom of the alkyl group.

In the formulas (2) to (4), X represents a nitrogen atom, an oxygen atom or a sulfur atom.
In the formula (3), Y represents a carbon atom, a nitrogen atom, an oxygen atom or a sulfur atom. In the formula (3), X and Y may be the same or different.

  The groups represented by formulas (1) to (4) can be bonded at all possible positions.

In a preferred embodiment of the present invention, the group represented by the formula (1) includes the following.

In a preferred embodiment of the present invention, the group represented by the formula (2) includes the following.

In a preferred embodiment of the present invention, the group represented by the formula (3) includes the following.

In a preferred embodiment of the present invention, the group represented by the formula (4) includes the following.

In one preferred aspect of the invention, R ₁ in formula (I) is a C _2-8 alkynyl group.

In one preferred aspect of the invention, R ₁ in formula (I) is a substituted or unsubstituted phenyl group.

  Another aspect of the present invention is a microtubule polymerization inhibitor comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

  The microtubule polymerization inhibitor of the present invention may contain not only the compound represented by the formula (I) but also a salt thereof or a solvate or hydrate thereof. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and examples thereof include base addition salts, acid addition salts, and amino acid salts. Examples of the base addition salt include sodium salts, potassium salts, calcium salts, alkaline earth metal salts such as magnesium salts, ammonium salts, or triethylamine salts, piperidine salts, and organic amine salts such as morpholine salts. Examples of acid addition salts include mineral salts such as hydrochloride, hydrobromide, sulfate, nitrate and phosphate; methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid, acetic acid, propionate, Organic acid salts such as tartaric acid, fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid and salicylic acid Can be mentioned. Examples of the amino acid salt include a glycine salt, an aspartate salt, a glutamate and the like. Further, a metal salt such as an aluminum salt may be used.

  The type of the solvent that forms the solvate is not particularly limited, and examples thereof include solvents such as ethanol, acetone, and isopropanol.

  The compounds represented by the formula (I) also include stereoisomers such as tautomers, geometric isomers (for example, E-form and Z-form), and enantiomers, unless otherwise specified. That is, when one or two or more asymmetric carbons are contained in the compound represented by the formula (I), the stereochemistry of the asymmetric carbons is independently (R) or (S) It can take any of the isomers and may exist as stereoisomers, such as enantiomers or diastereoisomers of the derivative. Accordingly, as an active ingredient of the microtubule polymerization inhibitor of the present invention, any stereoisomer in a pure form, any mixture of stereoisomers, a racemate, and the like can be used. Included in the scope.

  Another aspect of the present invention is a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof.

  One preferred embodiment of the present invention includes a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, such as malignant lymphoma, non-small cell lung cancer, ovarian cancer, breast cancer, gastric cancer, head and neck cancer and the like. It also relates to a pharmaceutical composition for treating or preventing cancer (hereinafter, also referred to as “the anticancer agent of the present invention”).

  Another preferred embodiment of the present invention also relates to a pharmaceutical composition for the treatment or prevention of fungal infection, comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof (hereinafter "" Also referred to as "antifungal agent of the present invention").

  Pathogenic fungal infections that can be treated or prevented by the antifungal agents of the invention include, in particular, aspergillosis (including invasive pulmonary aspergillosis), blast mycosis (prominent or rapidly progressive infection and central nervous system infection). (Eg, renal stones, urinary tract obstruction, renal transplantation, or retrograde candidiasis in patients with poorly controlled diabetes mellitus), coccidioidomycosis (sufficient for other chemotherapy) Cryptococcus, histoplasmosis, mucormycosis (including, for example, craniofacial mucormycosis and mucorpneumonia), paracoccidioidomycosis and sporotrichum disease.

  The pharmaceutical composition, anticancer agent or antifungal agent of the present invention is obtained by administering a compound represented by the formula (I) as an active ingredient or a pharmaceutically acceptable salt, hydrate or solvate thereof. In general, it is desirable to administer the composition in the form of a pharmaceutical composition containing the above-mentioned substance as an active ingredient and one or more pharmaceutical additives. The term "composition" as in a pharmaceutical composition refers to any two or more products, as well as products containing the active ingredients and the inert ingredients (pharmaceutically acceptable excipients) that make up the carrier. Occurs directly or indirectly as a result of association, complexation or aggregation of components, or as a result of dissociation of one or more components, or as a result of another type of reaction or interaction of one or more components. Any products are also included.

  As the active ingredient of the pharmaceutical composition, anticancer agent or antifungal agent of the present invention, two or more of the above compounds can be used in combination, or other known active ingredients having microtubule polymerization inhibitory activity are blended. It is also possible.

  In addition, the anticancer agent of the present invention comprises a compound represented by the formula (I) as an active ingredient or a pharmaceutically acceptable salt, hydrate or solvate thereof, and a combination drug used in combination with an existing anticancer agent. It is also possible. As the existing anticancer agent, those known in the art can be used, and examples thereof include methotrexate, doxorubicin, and cisplatin.

  In addition, the antifungal agent of the present invention is used in combination with a compound represented by the formula (I) as an active ingredient or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and an existing antifungal agent. It is also possible to make a combined medicine. As the existing antifungal agents, those known in the art can be used. Examples thereof include amphotericin B, miconazole, fluconazole, and micafungin.

  The type of the pharmaceutical composition of the present invention, the anticancer agent or the antifungal agent is not particularly limited. Examples include gels, patches, inhalants, and injections. These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another suitable solvent at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the compound of the present invention is prepared by dissolving it in water, but may be dissolved in a physiological saline solution or a glucose solution as necessary, or may be added with a buffer or a preservative. Good. It is provided in any formulation for oral or parenteral administration. For example, pharmaceutical compositions for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions or solutions, for intravenous administration, for intramuscular administration, Alternatively, it can be prepared as a pharmaceutical composition for parenteral administration in the form of injections for subcutaneous administration, drops, transdermal absorbers, transmucosal absorbers, nasal drops, inhalants, suppositories and the like. Injections, drops, and the like can be prepared as a powdery dosage form such as a lyophilized form, and dissolved in an appropriate aqueous medium such as physiological saline before use. In addition, a sustained-release preparation coated with a polymer or the like can be directly administered into the brain.

  The pharmaceutical composition of the present invention, the type of pharmaceutical additive used for the production of an anticancer agent or an antifungal agent, the ratio of the pharmaceutical additive to the active ingredient, or the method for producing the pharmaceutical composition, depends on the form of the composition. Those skilled in the art can appropriately select them. As the pharmaceutical additives, inorganic or organic substances or solid or liquid substances can be used, and generally, they can be blended at 1% to 90% by weight based on the weight of the active ingredient. Specifically, examples of such substances include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethyl cellulose, hydroxypropyl starch, carboxymethyl cellulose calcium. , Ion exchange resins, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid esters, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysodium Bate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.

  To prepare a solid preparation for oral administration, the active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, silicic acid and the like are mixed together to form a powder, or if necessary, sucrose, A binder such as hydroxypropylcellulose and polyvinylpyrrolidone and a disintegrating agent such as carboxymethylcellulose and carboxymethylcellulose calcium are added, and the mixture is granulated by wet or dry granulation to obtain granules. In order to produce tablets, these powders and granules may be compressed as they are or by adding a lubricant such as magnesium stearate or talc. These granules or tablets may be coated with an enteric base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer and the like, or may be coated with an enteric coated preparation such as ethyl cellulose, carnauba wax, hydrogenated oil and the like to form a sustained release preparation. it can. To produce capsules, powders or granules are filled into hard capsules, or the active ingredient is dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, or the like, and then coated with a gelatin film to form soft capsules. Can be.

  In order to manufacture an injection, the active ingredient is used as necessary, such as a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, glucose, etc. It may be dissolved in distilled water for injection together with an isotonic agent and sterile-filtered and filled into an ampoule, or may further be subjected to vacuum freeze-drying with addition of mannitol, dextrin, cyclodextrin, gelatin, or the like to give a working-solution-type injection. . Also, reticin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like can be added to the active ingredient and emulsified in water to prepare an emulsion for injection.

  The dose and frequency of administration of the pharmaceutical composition of the present invention, the anticancer agent or the antifungal agent are not particularly limited, the purpose of preventing and / or treating the deterioration and progression of the disease to be treated, the type of the disease, the weight and age of the patient, It can be appropriately selected by a doctor according to conditions such as the severity of the disease. In general, the daily dose for an adult in oral administration is about 0.01 to 1000 mg (weight of the active ingredient), and it can be administered once or several times a day or every few days. it can. When used as an injection, it is desirable to continuously or intermittently administer a daily dose of 0.001 to 100 mg (weight of the active ingredient) to an adult.

  Although the method for producing the compound represented by the formula (I) is not particularly limited, a method for synthesizing a representative compound among the compounds included in the formula (I) is specifically shown in Examples of the present specification. Those skilled in the art can appropriately modify or modify starting materials, reaction reagents, reaction conditions, and the like, as necessary, with reference to the examples in the present specification and the following schemes, to thereby convert the compounds included in the formula (I). Can be manufactured.

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[Synthesis Example 1]
Compound A (7- (benzyloxy) -3- (4,7-dimethoxybenzo [d] [1,3] dioxol-5-yl) -6- (prop-2-yn-1-yloxy) -4H- Synthesis of chromen-4-one) Compound A of the present invention was synthesized by the following procedure.

(1) Synthesis of compound 2

In a nitrogen atmosphere, Reference 1 (“Practical synthesis of glaziovianin A, a cytotoxic isoflavone, and its O ⁷ -propargyl analogue” Ichiro Hayakawa, * Shuya Shioda, Akiyuki Ikedo, Hideo Kigoshi * Bull. Chem. Soc. Jpn. 2014, 87 (4), 544-549.), And 12.3 g (67.3 mmol) of Compound 1 synthesized in 300 mL of anhydrous acetonitrile were dissolved. To this solution, 18.6 g (135 mmol) of potassium carbonate, 7.80 mL (67.8 mmol) of benzyl chloride, and 30.0 g (81.2 mmol) of tetrabutylammonium iodide were added at room temperature. The solution was stirred at room temperature for 22 hours. After completion of the reaction, 200 mL of a saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution, and the mixture was extracted three times with 400 mL of ethyl acetate. The organic layers were combined and washed with 600 mL of saturated saline. The organic layer was dried over anhydrous sodium sulfate, and after removing the desiccant by filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 15: 1 → 6: 1 → 5: 1 → 3: 1) to obtain 16.4 g of compound 2.

mp: 124-126 ° C
IR (neat) 3020, 1631, 1506, 1265, 1215, 1063 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 12.59 (s, 1H), 7.44-7.33 (m, 5H), 7.09 (s, 1H), 6.49 (s, 1H), 5.18 (s, 2H), 3.87 ( s, 3H), 2.56 (s, 3H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 202.1, 159.8, 155.9, 142.1, 135.6, 128.7 (2C), 128.1, 127.2 (2C), 112.4, 111.9, 101.9, 70.7, 56.9, 26.3.
HRMS (ESI) m / z 273.1125, calcd for C ₁₆ H ₁₇ O ₄ [M + H] ⁺ 273.1127.

(2) Synthesis of compound 3

Under a nitrogen atmosphere, 204 mg (749 μmol) of Compound 2 was dissolved in 6.0 mL of acetonitrile. To this solution was added L-(+)-ascorbic acid (158 mg, 899 μmol). The solution was cooled to 0 ° C., and 1.50 mL (1.50 mmol) of a 1 M aqueous solution of cerium ammonium nitrate was added, followed by stirring for 1 hour. 199 mg (1.13 mmol) of L-(+)-ascorbic acid, 25 mL of chloroform, and 25 mL of water were added to the reaction solution, and the mixture was separated into an organic layer and an aqueous layer. The aqueous layer was further extracted twice with 30 mL of chloroform. The organic layers were combined, dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the solvent was distilled off under reduced pressure. The residue was dissolved in 5 mL of dichloromethane. To this solution, 5 mL of a saturated aqueous solution of Na ₂ S ₂ O ₄ was added at room temperature, and the mixture was stirred for 1 hour. The reaction solution was separated into an organic layer and an aqueous layer, and the aqueous layer was extracted twice with 6.0 mL of chloroform. The organic layers were combined, dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: acetone = 35: 1 → 10: 1 → 5: 1) to obtain 118 mg of compound 3.

mp: 159-161 ° C
IR (neat) 3566, 3020, 2974, 1699, 1635, 1506, 1458, 1219 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 12.46 (s, 1H), 7.43-7.40 (m, 5H), 7.24 (s, 1H), 6.53 (s, 1H), 5.14 (s, 2H), 2.54 ( s, 3H).
* No proton signal of one hydroxy group was observed.
¹³ C NMR (100 MHz, CDCl ₃ ) δ 202.7, 158.6, 152.7, 138.0, 134.9, 128.9 (2C), 128.8, 128.0 (2C), 114.1, 112.6, 100.8, 71.2, 26.5.
HRMS (ESI) m / z [M + H] ⁺ 259.0967, calcd for C ₁₅ H ₁₅ O ₄ [M + H] ⁺ 259.0970.

(3) Synthesis of compound 4

  Under a nitrogen atmosphere, 1.40 g (5.43 mmol) of Compound 3 was dissolved in 85.0 mL of dichloromethane. The solution was cooled to 0 ° C., 2.00 mL (22.1 mmol) of dihydropyran and 64.0 mg (273 μmol) of (+)-10-camphorsulfonic acid were added, and the mixture was stirred for 2 hours. After 150 μL (1.08 mmol) of triethylamine was added to the reaction solution and the mixture was stirred for 10 minutes, 100 mL of a saturated aqueous solution of sodium hydrogen carbonate was added to separate the organic layer and the aqueous layer. The aqueous layer was further extracted twice with 120 mL of ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 30: 1 → 20: 1 → 15: 1 → 8: 1) to obtain 1.74 g of compound 4.

mp: 102-105 ° C
IR (neat) 3566, 3020, 2976, 1635, 1508, 1373, 1329, 1254, 1217 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 12.60 (s, 1H), 7.43-7.32 (m, 6H), 6.50 (s, 1H), 5.26 (t, J = 3.2, 1H), 5.11 (s, 2H ), 4.05 (m, 1H), 3.61 (m, 1H), 2.52 (s, 3H), 2.00-1.81 (m, 3H), 1.69-1.60 (m, 3H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 202.3, 160.7, 157.3, 138.6, 135.8, 128.5 (2C), 128.0, 127.0 (2C), 120.8, 112.3, 101.8, 99.0, 70.3, 62.2, 30.2, 26.2, 25.2 , 18.6.
HRMS (ESI) m / z 343.1546, calcd for C ₂₀ H ₂₃ O ₅ [M + H] ⁺ 343.1546.

(4) Synthesis of compound 5

  Under a nitrogen atmosphere, 10.0 mL (75.3 mmol) of N, N-dimethylformamide dimethyl acetal was added to 1.00 g (2.93 mmol) of Compound 4 at room temperature. The solution was stirred at 90 ° C for 5 hours. After completion of the reaction, the solution was distilled off under reduced pressure to obtain 1.21 g of crude compound 5. The obtained crude compound 5 was used for the next reaction without purification.

(5) Synthesis of compound 6

  Under a nitrogen atmosphere, 205 mg of the crude compound 5 was dissolved in 12.0 mL of chloroform. The solution was cooled to 0 ° C., and 400 μL (4.97 mmol) of pyridine and 261 mg (2.07 mmol) of iodine were added. The solution was stirred at room temperature for 23 hours under light protection. After completion of the reaction, 15 mL of a saturated aqueous solution of sodium thiosulfate was added to the reaction solution, and the mixture was extracted three times with 20 mL of chloroform. The organic layers were combined and washed with 50 mL of a saturated saline solution. The organic layer was dried over anhydrous sodium sulfate, and after removing the desiccant by filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 12: 1 → 10: 1 → 8: 1 → 4: 1) to obtain 223 mg of compound 6.

mp: 166-170 ° C
IR (neat) 3020, 2976, 1647, 1616, 1496, 1456, 1367, 1215 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 8.18 (s, 1H), 7.84 (s, 1H), 7.46-7.34 (m, 5H), 6.90 (s, 1H), 5.54 (t, J = 3.0 Hz, 1H), 5.21 (s, 2H), 3.96 (m, 1H), 3.62 (m, 1H), 2.01-1.88 (m, 3H), 1.72-1.62 (m, 3H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 172.3, 156.8, 155.0, 152.8, 145.4, 135.6, 128.7 (2C), 128.2, 127.0 (2C), 115.5, 112.5, 101.4, 97.7, 86.6, 70.9, 62.2, 30.1 , 25.1, 18.6.
HRMS (ESI) m / z 479.0351, calcd for C ₂₁ H ₁₉ IO ₅ [M + H] ⁺ 479.0355.

(5) Synthesis of compound 8

Under an argon atmosphere, a mixture of 129 mg (270 μmol) of compound 6, 100 mg of compound 7 (325 μmol) synthesized with reference to Reference 1, and 11.5 mg (14.1 μmol) of PdCl ₂ (dppf) · CH ₂ Cl ₂ was frozen and degassed. 6.0 ml of the obtained 1,4-dioxane and 1.90 ml (1.90 mmol) of a 1 M aqueous sodium carbonate solution were added. This solution was stirred at room temperature for 15.5 hours under a stream of argon. After completion of the reaction, 10 mL of water was added to this solution, and the mixture was extracted three times with 12 mL of ethyl acetate. The organic layers were combined and washed with 30 mL of a saturated saline solution. The organic layer was dried over anhydrous sodium sulfate, and after removing the desiccant by filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 8: 1 → 6: 1 → 3: 1). 142 mg of the obtained compound 8 was dissolved in ethyl acetate. To this solution, 714 mg of Pd scavenger (SiliaMetsS (registered trademark) Thiourea) was added, and the mixture was stirred for 1 hour at room temperature. After removing the Pd scavenger by filtration, the solvent was distilled off under reduced pressure to obtain 141 mg of compound 8.

mp: 88-92 ° C
IR (neat) 3020, 2978, 1646, 1607, 1522, 1468, 1423, 1352, 1295 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.91 (s, 1H), 7.85 (s, 1H), 7.47-7.32 (m, 5H), 6.93 (s, 1H), 6.50 (s, 1H), 6.00 ( s, 2H), 5.57 (t, J = 3.0 Hz, 1H), 5.22 (s, 2H), 4.01 (s, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.62 (m, 1H ), 2.05-1.88 (m, 3H), 1.71-1.58 (m, 3H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 175.3, 154.6, 153.4, 152.8, 144.8, 139.0, 138.9, 136.9, 136.7, 135.8, 128.6 (2C), 128.1, 126.9 (2C), 121.5, 118.1, 118.0, 112.7 , 110.0, 101.7, 101.7, 97.7, 70.8, 62.1, 60.1, 56.8, 30.1, 25.1, 18.6.
HRMS (ESI) m / z 533.1806, calcd for C ₃₀ H ₂₈ O ₉ [M + H] ⁺ 533.1812.

(6) Synthesis of compound 9

  Under a nitrogen atmosphere, 17.1 mg (32.1 μmol) of compound 8 was dissolved in a mixed solvent (10.5 mL) of chloroform / methanol 2: 1. To this solution, 1.20 mg (6.31 μmol) of p-toluenesulfonic acid monohydrate was added at room temperature. The solution was stirred at room temperature for 1.5 hours. The reaction solution was extracted by adding 20.0 μL (143 μmol) of triethylamine and 3 mL of water. The aqueous layer was further extracted twice with 5 mL of chloroform. The organic layers were combined, dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 3: 1 → 1: 1) to obtain 10.8 mg of compound 9.

mp: 224-226 ° C
IR (neat) 3608, 3020, 2976, 1647, 1558, 1541, 1219 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88 (s, 1H), 7.74 (s, 1H), 7.46-7.45 (m, 5H), 6.96 (s, 1H), 6.52 (s, 1H), 6.02 ( s, 2H), 5.23 (s, 2H), 3.87 (s, 3H), 3.84 (s, 3H).
* No proton signal of one hydroxy group was observed.
¹³ C NMR (100 MHz, CDCl ₃ ) δ 175.5, 153.6, 151.5, 150.8, 144.1, 139.1, 139.0, 137.0, 136.7, 134.8, 128.9 (2C), 128.9, 127.9 (2C), 121.5, 118.9, 118.0, 110.0 , 109.0, 101.8, 100.3, 71.6, 60.2, 56.9.
HRMS (ESI) m / z 449.1233, calcd for C ₂₅ H ₂₀ O ₈ [M + H] ⁺ 449.1236.

(7) Synthesis of compound A

  Under a nitrogen atmosphere, 15.2 mg (33.9 μmol) of compound 9 was dissolved in acetone (6.0 mL). To this solution, 10.8 mg (78.1 μmol) of potassium carbonate and 3.90 μL (51.8 μmol) of propargyl bromide were added at room temperature. The solution was heated at reflux for 5 hours. To the reaction solution was added 10 mL of a saturated aqueous solution of sodium hydrogen carbonate, and the mixture was extracted three times with 12 mL of chloroform. The organic layers were combined and washed with 30 mL of a saturated saline solution. The organic layer was dried over anhydrous sodium sulfate, and after removing the desiccant by filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 6: 1 → 1: 1) to obtain 16.0 mg of compound A. FIG. 2 shows an NMR spectrum chart of the compound A.

mp: 174-176 ° C
IR (neat) 3307, 3020, 2976, 1635, 1608, 1506, 1456, 1419, 1295, 1261, 1215, 1097 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.87 (s, 1H), 7.78 (s, 1H) 7.48-7.33 (m, 5H), 6.92 (s, 1H), 6.51 (s, 1H), 6.01 (s , 2H), 5.26 (s, 2H), 4.87 (d, J = 2.2 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 2.54 (t, J = 2.2 Hz, 1H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 175.3, 153.7, 153.5, 152.6, 145.7, 139.1, 138.9, 137.0, 136.7, 135.5, 128.8 (2C), 128.3, 127.2 (2C), 121.7, 117.9, 117.9, 110.0 , 108.1, 101.8, 101.7, 77.8, 76.3, 71.1, 60.2, 57.0, 56.8.
HRMS (ESI) m / z 487.1385, calcd for C ₂₈ H ₂₂ O ₈ [M + H] ⁺ 487.1393.

[Synthesis Example 2]
Synthesis of Compound B (6,7-bis (benzyloxy) -3- (4,7-dimethoxybenzo [d] [1,3] dioxol-5-yl) -4H-chromen-4-one) Was used to synthesize the compound B of the present invention.

(1) Synthesis of compound B

  Under a nitrogen atmosphere, 15.2 mg (33.9 μmol) of compound 9 was dissolved in acetone (6.0 mL). To this solution, 10.5 mg (76.0 μmol) of potassium carbonate and 6.10 μL (51.4 μmol) of benzyl bromide were added at room temperature. The solution was heated at reflux for 6.5 hours. To the reaction solution was added 10 mL of a saturated aqueous solution of sodium hydrogen carbonate, and the mixture was extracted three times with 12 mL of chloroform. The organic layers were combined and washed with 30 mL of a saturated saline solution. The organic layer was dried over anhydrous sodium sulfate, and after removing the desiccant by filtration, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 6: 1 → 1: 1) to obtain 16.7 mg of compound B. FIG. 3 shows an NMR spectrum chart of the compound B.

mp: 154-158 ° C
IR (neat) 3020, 2976, 1608, 1456, 1296, 1245, 1153, 1037 cm- ¹ .
¹ H NMR (400 MHz, CDCl ₃ ) δ 7.87 (s, 1H), 7.73 (s, 1H), 7.51-7.32 (m, 10H), 6.92 (s, 1H), 6.51 (s, 1H), 6.01 ( s, 2H), 5.27 (s, 2H), 5.25 (s, 2H), 3.86 (s, 3H), 3.84 (s, 3H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ 175.4, 153.8, 153.4, 152.2, 147.1, 139.1, 138.9, 137.0, 136.7, 136.4, 135.8, 128.7 (2C), 128.5 (2C), 128.2, 128.0, 127.4 (2C ), 127.0 (2C), 121.6, 118.0, 118.0, 110.0, 107.5, 101.8, 101.7, 71.0, 71.0, 60.2, 56.8.
HRMS (ESI) m / z 539.1709, calcd for C ₃₂ H ₂₆ O ₈ [M + H] ⁺ 539.1706.

[Example 1]
Evaluation of cell killing activity The cell killing activity was evaluated using HeLa cells. After subcultured in a DMEM medium containing 10% fetal bovine serum and cultured at 37 ° C. in 5% CO ₂ , 100 μl of each 3 × 10 ⁴ cells / ml in each well was seeded on a 96-well plate, and then 18 After time, each drug was added. Forty-eight hours after the addition of the drug, the number of viable cells was quantified using the WST-8 reagent.
As shown in Table 1, Compound A and Compound B showed stronger cell killing activity than Gatastatin.

[Example 2]
Evaluation of Interphase Microtubule Morphology of Animal Cells and In Vitro Tubulin Polymerization Inhibitory Activity The effects on microtubule morphology during animal cell interphase were evaluated using HeLa cells. After subcultured in a DMEM medium containing 10% fetal bovine serum and cultured at 37 ° C. in 5% CO ₂ , 100 μl of each 3 × 10 ⁴ cells / ml in each well was seeded on a 96-well plate, and then 18 After time, each drug was added. Twelve hours later, the cells were fixed, and α-tubulin, the centrosome component factoricrin, and the chromosome were fluorescently stained and observed (FIG. 4).
Tubulin polymerization inhibitory activity in a test tube was measured by diluting tubulin purified from pig brain with RB buffer (100 mM MES, 1 mM EGTA, 0.5 mM MgCl ₂ , pH 6.8) to 1 mg / ml. After adding the drug and placing on ice for 5 minutes, 1 mM of GTP and 1 M of Glutamate were added, and the mixture was heated to 37 ° C. to start the polymerization reaction. After polymerizing the microtubules for 30 minutes, ultracentrifugation was performed at 75,000 rpm, and the sample was separated into a supernatant (α / β-tubulin) and a precipitate (microtube). After confirming each sample by SDS-PAGE, the band concentration was quantified using image analysis software Image J, and the ratio of the precipitate to the α / β-tubulin (Total) contained in the supernatant and the precipitate, that is, The rate at which the tubes polymerized was calculated (FIG. 5).
As shown in FIGS. 4 and 5, compound A (denoted OK12 in FIG. 4) and compound B (denoted OK13 in FIG. 4) did not affect the microtubule skeletal morphology of interphase cells and It was confirmed that the compound did not show a phosphorus polymerization inhibitory effect.

[Example 3]
Evaluation of mitotic spindle morphology of animal cells The effect of mitotic spindle morphology on animal cell division was evaluated using HeLa cells. Each cell cultured in a DMEM medium containing 10% fetal calf serum and cultured at 37 ° C. in 5% CO 2 was seeded in a 96-well plate at 3 × 10 ⁴ cells / ml, 100 μl per well, and then 18 hours. Later, each drug was added. After 12 hours, the cells were fixed and α-tubulin was fluorescently stained and observed (FIG. 6, Table 2).

  As shown in FIG. 6, compound A (denoted as OK12) and compound B (denoted as OK13) induced a multipolar spindle, similarly to gatastatin. When the concentration required for multipolar spindle formation was examined, catastatin required 30 μM, whereas compounds A and B induced a multipolar spindle at a concentration of 3 μM or more.

[Example 4]
Evaluation of microtubule nucleation activity from animal cell centrosome The influence on the nucleation activity from centrosome was examined by microtubule nucleation assay. STLC (S-Trityl-L-Cysteine) for inducing monopolar spindles was treated with 20 μM for 6 hours to form monopolar spindles, and then left on ice for 1 hour to depolymerize microtubules. . After treating each drug for 15 minutes, microtubule nucleation was performed by heating at 30 ° C. for 3 minutes, and then the cells were fixed. After fluorescent staining of the microtubules using the α-Tubulin antibody, the area where the microtubules were extended was quantified using the image analysis software Image J.
As shown in FIG. 7, Gatastatin strongly inhibited microtubule nucleation at 30 μM, whereas Compound A and Compound B inhibited microtubule nucleation at a lower concentration of 3 μM (FIG. 7).

  The results of Examples 1-4 show that compounds A and B inhibit microtubule elongation from the intracellular centrosome without directly inhibiting microtubule polymerization, resulting in abnormal spindle formation and tumor cell growth inhibition. It indicates that. Existing anticancer drugs that inhibit polymerization and depolymerization of microtubules have a problem of side effects due to inhibition of interphase microtubule function, but compounds A and B are recruited during cell division. Since it inhibits activating γ-Tubulin, it can be used as an anticancer agent with few side effects.

Claims (5)

A compound represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

(Where
R ₁ is selected from a C _2-8 alkenyl group, a C _2-8 alkynyl group, a substituted or unsubstituted aryl group or a heterocyclyl group;
R ₂ , when present, is each independently selected from a halogen atom, a C _1-8 alkyl group, a C _1-8 alkoxy group, a nitro group, a cyano group or a carboxyl group;
m is an integer from 1 to 8:
n is an integer of 1 to 8. ) R ₁ is

The compound according to claim 1, which is represented by the formula: or a pharmaceutically acceptable salt thereof.
(In equations (1) to (4),
When present, R ₃ , R ₄ , R ₅ , and R ₆ each independently represent a halogen atom, a halogen atom, a C _1-8 alkyl group, a C _1-8 alkoxy group, a nitro group, a cyano group. Selected from a group or a carboxyl group,
X represents a nitrogen atom, an oxygen atom or a sulfur atom,
Y represents a carbon atom, a nitrogen atom, an oxygen atom or a sulfur atom. The compound according to claim 1, wherein R ₁ is a C _2-8 alkynyl group, or a pharmaceutically acceptable salt thereof.   A microtubule polymerization inhibitor comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.   A pharmaceutical composition for treating or preventing cancer, comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

Images