KR102516896B1 - Benzimidazole derivatives or its pharmaceutically acceptable salts and use thereof - Google Patents

Abstract

The present invention relates to a benzimidazole derivative or a pharmaceutically acceptable salt thereof and a composition for preventing or treating cancer comprising the derivative as an active ingredient, wherein the benzimidazole derivative of the present invention is activated in cancer cells to cause tubulin polymerization (tubulin polymerization), when administered to a subject, blocks the cell cycle of cancer cells and induces apoptosis, thereby exhibiting cytotoxicity, so it can be used for the prevention or treatment of cancer, preferably for the prevention or treatment of triple-negative breast cancer. there is.

Description

Benzimidazole derivatives or its pharmaceutically acceptable salts and use thereof {Benzimidazole derivatives or its pharmaceutically acceptable salts and use thereof}

The present invention relates to benzimidazole derivatives or pharmaceutically acceptable salts thereof, uses thereof, and the like.

Triple-negative breast cancer (ER-, PR-, HER2-) patients account for 10-15% of all breast cancer patients, and hormone receptors (ER (estrogen receptor), PR (progesterone receptor)) and HER2 protein Because of this, they do not benefit from hormone therapy or HER2-targeted therapies. Currently, standard treatment for triple-negative breast cancer is completely dependent on general cytotoxic anticancer drugs (Taxene-based or Anthracycline-based). Because there are no established targeted therapies, treatment strategies for other subtypes are less diverse than for breast cancer. What is more serious is that after surgery or chemotherapy, recurrence occurs within 2 to 3 years in most patients, and metastasis to other organs such as the lungs, liver, brain, and bones is easily induced, which affects the survival rate of patients.

The 5-years overall survival of patients diagnosed with stage-III is less than 55%, and the 5-year survival rate of patients with advanced cancer metastasis is less than 30%. Very low. Most of these patients are very serious diseases that ultimately lead to death within a few years.

Microtubule is a major component of the cytoskeleton and is composed of a tubulin heteropolymer composed of α subunits and β subunits. Microtubules perform various cellular functions such as intracellular transport, polarity maintenance, intracellular signal transduction, and cell migration and proliferation. During mitosis, the chromosomes are arranged in the center of the cell by forming spindle threads, which separate into the poles. If the spindle does not function properly, cell division is inhibited and apoptosis occurs, so it is attracting attention as a target of anticancer drugs.

Drugs targeting microtubules are largely divided into two groups: drugs that stabilize microtubules and drugs that destabilize microtubules. First, microtubule stabilizers include taxane, paclitaxel (Taxol), and decetaxel, which prevent depolymerization of microtubules and enhance polymerization. Most microtubule stabilizing substances bind to the taxane-binding site or to the overlapping site of β tubulin. Second, microtubule destabilizers include colchicine and vinca alkaloids, which bind to colchicine binding sites or vinca binding sites. It is the same that drugs that target these microtubules themselves are more effective at lower concentrations than drugs that affect microtubule polymers, and consequently inhibit cell mitosis. Therefore, it is required to develop potential tubulin polymerization inhibitors as anticancer agents.

On the other hand, flubendazole and albendazole are tubulin polymerization inhibitors, which are commercially available drugs as repellents, but can confirm cell death by cell cycle arrest. It is attracting attention as a therapeutic agent. However, it is difficult to absorb in the body due to its low solubility in water, and side effects such as an increase in liver enzyme levels appear when ingested in large amounts, so the development of a derivative with a new structure is required.

Accordingly, the present inventors confirmed that a novel benzimidazole derivative or a pharmaceutically acceptable salt thereof is a tubulin polymerization inhibitor that induces apoptosis by stopping the cell cycle of cancer cells (cell cycle arrest) , which led to the completion of the present invention.

Indian Journal of Chemistry, Vol. 52B, April 2013, pp. 535-545

An object of the present invention is to provide a benzimidazole derivative or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the benzimidazole derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to solve the above problems, the present invention provides a benzimidazole derivative represented by the following [Formula 1] or a racemate, isomer, solvate or pharmaceutically acceptable salt thereof:

[Formula 1]

In Formula 1,

X is oxygen or nitrogen;

R ₁ is a substituted or unsubstituted amine group, a substituted or unsubstituted alkoxy group, halogen or hydrogen.

As an embodiment of the present invention, in [Formula 1], the substituted amine group or the substituted alkoxy group is a C ₁ to C ₁₂ chain or cyclic alkyl group or a substituted or unsubstituted C ₃ to C ₁₂ heterocyclic ring It may be substituted with a group.

As another embodiment of the present invention, the benzimidazole derivative represented by [Formula 1] may be any one selected from the group consisting of the following compounds:

[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

[Formula 1-4]

및

and

[Formula 1-5]

.

.

As another embodiment of the present invention, the benzimidazole derivative may inhibit tubulin polymerization.

In addition, the present invention provides a composition for preventing or treating cancer comprising the benzimidazole derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a method for preventing or treating cancer comprising administering the benzimidazole derivative or a pharmaceutically acceptable salt thereof to a subject.

In addition, the present invention provides a use of the benzimidazole derivative or a pharmaceutically acceptable salt thereof for the preparation of a drug for preventing or treating cancer.

In addition, the present invention provides a method for diagnosing cancer comprising administering the benzimidazole derivative or a pharmaceutically acceptable salt thereof to a subject.

In addition, the present invention provides a use of the benzimidazole derivative or a pharmaceutically acceptable salt thereof for the preparation of a drug for diagnosing cancer.

As one embodiment of the present invention, the pharmaceutical composition may induce apoptosis by stopping the cell cycle of cancer cells.

As another embodiment of the present invention, the cancer may be breast cancer.

The present invention relates to a benzimidazole derivative or a pharmaceutically acceptable salt thereof and a composition for preventing or treating cancer containing the derivative as an active ingredient, wherein the benzimidazole derivative of the present invention is activated in cancer cells to cause tubulin polymerization It inhibits tubulin polymerization and exhibits cytotoxicity by blocking the cancer cell cycle and inducing apoptosis when administered to a subject, so it can be used for the prevention or treatment of cancer, preferably for the prevention or treatment of triple-negative breast cancer.

Figure 1 shows cell viability after treatment with benzimidazole derivatives of Formulas 1-1, 1-2, 1-4 and 1-5 in MDA-MB-231, a triple-negative breast cancer (TNBC) cell line. It shows the result of checking .
Figure 2 confirms cell viability after treatment with benzimidazole derivatives of formulas 1-1, 1-2, 1-4 and 1-5 in JIMT-1, a HER2 positive breast cancer (HER2+ BC) cell line. that showed the result.

As a result of intensive research on benzimidazole derivatives or pharmaceutically acceptable salts thereof, the present inventors confirmed the anticancer activity of the derivatives and completed the present invention.

More specifically, it was confirmed that the benzimidazole derivative was activated in cancer cells, inhibited tubulin polymerization, blocked the cancer cell cycle, and induced apoptosis, thereby exhibiting cytotoxicity.

From the above results, the present invention can provide a benzimidazole derivative represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1,

X is oxygen or nitrogen;

R ₁ is a substituted or unsubstituted amine group, a substituted or unsubstituted alkoxy group, halogen or hydrogen.

In the present invention, the term "substitution" is a reaction in which an atom or group of atoms included in a molecule of a compound is replaced with another atom or group of atoms. In [Formula 1], the substituted amine group or the substituted alkoxy group is one or more hydrogen atoms, C ₁ to C ₁₂ Chain or cyclic alkyl group or substituted or unsubstituted C ₃ to C ₁₂ heterocyclic group substituted it could be

In the present invention, the term "chain-like" refers to a molecule having a chain-like structure, and the chain-like structure is a chemical structure in which carbon atoms are connected in a chain shape, and there are straight chain shapes and branched ones.

In the present invention, the term "cyclic" refers to a structure in which both ends linked in the backbone of an organic compound are connected to form a ring.

In the present invention, the term "chain or cyclic alkyl group" means a monovalent linear or branched or cyclic saturated hydrocarbon residue having 1 to 12 carbon atoms and consisting only of carbon and hydrogen atoms. Examples of such alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, 2-butyl, 3-butyl, pentyl, n-hexyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Not limited.

In the present invention, the term "alkoxy group" means an oxygen-bonded alkyl group (-O-R). Examples of such alkoxy groups include, but are not limited to, methoxy groups, ethoxy groups, propoxy groups, butoxy groups, and the like.

The halogen may be fluorine (F), chloride (Cl), bromine (Br), or iodine (I).

In the present invention, the term “heterocyclic group” refers to a compound having two or more ring elements among ring compounds having 3 to 12 carbon atoms. Examples of such a heterocyclic group may include piperazine, azetidine, and the like. The heterocyclic group may be in a substituted form, and may be, for example, methylpiperazine.

,

, -F 또는 -H일 수 있다.In the present invention, the R _One More specifically,

,

, -F or -H.

As a preferred embodiment of the present invention, the compound represented by Formula 1 is preferably at least one selected from the group consisting of the following compounds:

[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

[Formula 1-4]

및

and

[Formula 1-5]

.

.

The benzimidazole derivative of the present invention is specifically activated in cancer cells, inhibits tubulin polymerization, and induces cell death. It can be used as a pharmaceutical composition for prevention or treatment.

In addition, the present invention may provide a method for preventing, treating, and/or diagnosing cancer comprising administering the benzimidazole derivative or a pharmaceutically acceptable salt thereof to a subject.

As used herein, the term "prevention" refers to any activity that inhibits or delays the occurrence, spread, or recurrence of cancer by administration of the composition of the present invention, and "treatment" refers to symptoms of the disease by administration of the composition of the present invention. means any action that improves or changes favorably.

As used herein, the term "pharmaceutical composition" means prepared for the purpose of preventing or treating a disease, and may be formulated and used in various forms according to conventional methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated and used in the form of external preparations, suppositories and sterile injection solutions.

In the present invention, "included as an active ingredient" means that the component is included in an amount necessary or sufficient to realize a desired biological effect. The amount can be determined in consideration of other non-toxic factors, and can vary depending on various factors such as, for example, the disease or condition being treated, the type of composition to be administered, the size of the subject, or the severity of the disease or condition. Effective amounts of individual compositions can be determined empirically without undue experimentation by those skilled in the art to which the present invention pertains.

In addition, the pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients according to each formulation.

The pharmaceutically acceptable carrier may be saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and, if necessary, antioxidants, buffers, bacteriostatic agents Other common additives such as may be further included. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it may be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA).

In the present invention, the term "pharmaceutically acceptable salt" means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutically acceptable salt is a compound of the present invention hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, inorganic acids such as phosphoric acid, sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, tartaric acid, formic acid, citric acid, acetic acid, It can be obtained by reacting with organic carbonic acids such as trichloroacetic acid, trifluoroacetic acid, capric acid, isobutanoic acid, malonic acid, succinic acid, phthalic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, and salicylic acid. In addition, by reacting the compound of the present invention with a base, salts such as alkali metal salts such as ammonium salts, sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, dicyclohexylamine, N-methyl-D-glue It can also be obtained by forming salts of organic bases such as carmine and tris(hydroxymethyl)methylamine, and amino acid salts such as arginine and lysine.

In addition, the benzimidazole derivative or a pharmaceutically acceptable salt thereof may include not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates that can be prepared by conventional methods.

The composition of the present invention can be administered orally or parenterally in a pharmaceutically effective amount according to the desired method, and the term "pharmaceutically effective amount" of the present invention refers to a disease at a reasonable benefit/risk ratio applicable to medical treatment. The effective dose level is the patient's health condition, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, It may be determined according to factors including duration of treatment, combinations or drugs used concurrently, and other factors well known in the medical arts.

Therefore, the pharmaceutical composition of the present invention can be administered to a subject to prevent, treat, and/or diagnose cancer, and the type of cancer is not limited, but preferably has a higher acidity than normal cells and is a tubulin polymerization inhibitor It may be a cancer of which cytotoxicity is inhibited by, and non-limiting examples thereof include breast cancer, preferably triple negative breast cancer.

In the present invention, the term "individual" is not limited to any mammal such as a livestock or a human that requires cancer prevention, treatment, and/or diagnosis, but may preferably be a human.

The pharmaceutical composition of the present invention may be formulated in various forms for administration to a subject, and a representative formulation for parenteral administration is an injectable formulation, preferably an isotonic aqueous solution or suspension. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, it may be formulated for injection by dissolving each component in saline or a buffer solution. In addition, dosage forms for oral administration include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. , sucrose, mannitol, sorbitol, cellulose and/or glycine) and lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). The tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid or Disintegrants such as sodium salts, absorbents, colorants, flavors and/or sweeteners may further be included. The formulation may be prepared by conventional mixing, granulating or coating methods.

In addition, the pharmaceutical composition of the present invention may further include adjuvants such as preservatives, hydrating agents, emulsification accelerators, salts or buffers for osmotic pressure control, and other therapeutically useful substances, and may be formulated according to conventional methods. .

The pharmaceutical composition according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient depends on the route of administration, age, sex, weight and severity of the patient. It can be appropriately selected according to several factors. In addition, the composition of the present invention can be administered in parallel with a known compound capable of increasing the desired effect.

As the route of administration of the pharmaceutical composition according to the present invention, it can be administered to humans and animals orally or parenterally, such as intravenously, subcutaneously, intranasally or intraperitoneally. Oral administration also includes sublingual application. Parenteral administration includes injection methods such as subcutaneous injection, intramuscular injection and intravenous injection and drip methods.

In the pharmaceutical composition of the present invention, the total effective amount of the benzimidazole derivative or pharmaceutically acceptable salt thereof according to the present invention can be administered to the patient in a single dose, or in multiple doses This may be administered by a fractionated treatment protocol in which administration is administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease, but is typically administered repeatedly several times a day at an effective dose of 100 μg to 3,000 mg per administration based on adults. . However, the concentration of the benzimidazole derivative or its pharmaceutically acceptable salt takes into account various factors such as age, weight, health condition, sex, severity of disease, diet and excretion rate of the patient as well as the route of administration and number of treatments. Thus, an effective dosage for a patient can be determined.

In addition, the pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as it shows the effects of the present invention, and the pharmaceutical composition of the present invention is an active ingredient as the benzimidazole derivative or its pharmaceutical composition. A known anticancer agent may be further included in addition to the generally acceptable salt, and it may be used in combination with other known therapies for the treatment of these diseases.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it should be understood that this is not intended to limit the present invention to specific embodiments, and includes all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

[Example]

Example 1. Preparation of benzimidazole derivatives

All chemical reagents were commercially available. ¹ H NMR spectra were recorded on a Bruker Avance III 400 MHz and Bruker Fourier 300 MHz, with TMS used as an internal standard.

LCMS was taken on a quadrupole Mass Spectromete on an Agilent LC/MSD 1200 series (column: ODS 2000 (50 × 4.6 mm, 5 μm)) operating in ES (+) or (-) ionization mode; T = 30 °C, flow rate = 1.5 mL/min; Detected wavelength: 214 nm.

Synthesis of Formula 1-1

The synthesis process of the benzimidazole derivative of Chemical Formula 1-1 is shown in Reaction Scheme 1 below.

[Scheme 1]

Synthesis of 5-(4-fluorophenoxy)-2-nitrobenzeneamine

After dissolving 4-Fluorophenol (20 g, 0.178 mole) in 160 mL, slowly add 60%-NaH (7.86 g, 0.196 mole) in an ice-bath. After stirring for 1 hour at room temperature, 5-Chloro-2-nitroaniline (30.78 g, 0.178 mole) was dissolved in 30 mL and added slowly. After that, heat under reflux for 2 hours. After confirming the completion of the reaction by TLC, the reaction solution was slowly added dropwise to 500 mL of ice-water. After filtering the precipitate, the filter cake was again dissolved in 600 mL of ethylyl acetate, dried over anhydrous MgSO ₄ , filtered, and distilled under reduced pressure to obtain 5-(4-fluorophenoxy)-2-nitrobenzeneamine (42.49 g) as a yellow solid. .

yield 96%, R _f : 0.29 (EtOAc/n-Hexane, 1:4)

¹ H NMR (400 MHz. CDCl ₃ , δ, ppm): 6.15 (br s, 2H, -NH ₂ ), 6.33-8.13 (m, 7H, ArH)

Synthesis of 4-(4-fluorophenoxy)benzene-1,2-diamine

After dissolving 5-(4-Fluorophenoxy)-2-nitrobenzeneamine (40.17 g, 0.1618 mole) in 800 mL of acetic acid, Zn (74 g, 1.133 mole) was slowly added under an ice-bath. Then, after stirring at room temperature for 10 hours, completion of the reaction was confirmed by TLC, followed by filtration. Distill the filtrate under reduced pressure, correct the pH to 8 with 5M-NaOH solution, and extract with 600 mL of ethyl acetate. Then, after drying with anhydrous MgSO ₄ , filtration and distillation under reduced pressure to obtain a dark brown liquid, which was columned to obtain 4-(4-fluorophenoxy)benzene-1,2-diamine (17.34 g) as a brown liquid.

yield 49%, Melting Point: liquid, R _f : 0.33 (EtOAc/n-Hexane, 1:1)

¹ H NMR (400 MHz. CDCl ₃ , δ, ppm): 3.43 (brs, 4H, -NH ₂ ), 6.36-7.02 (m, 7H, ArH)

Synthesis of 1,3-Bis(methoxycarbonyl)-S-methylisothiourea

Put S-methylisothiourea sulfate (50 g, 0.1796 mole) in 300 mL of purified water and cool to 0 °C. Thereafter, methyl chloroformate (78 g, 0.8262 mole) was slowly added dropwise and stirred. Stir at the same temperature for 4 hours, check the pH from time to time and correct the pH to 8-9 with 25%-NaOH solution. When the reaction was terminated by TLC, white solids were formed, which were filtered and dried under vacuum at 50 °C to obtain 1,3-Bis(methoxycarbonyl)-S-methylisothiourea (31.12 g) as a white solid.

yield 84%, R _f : 0.24 (EtOAc/n-Hexane, 1:4)

¹ H NMR (400 MHz. CDCl ₃ , δ, ppm): 2.43 (s, 3H, S-CH ₃ ), 3.80 (s, 3H, -OCH ₃ ), 3.83 (s, 3H, -OCH ₃ ), 11.85 (brs, 1H, -NH)

Synthesis of methyl 5-(4-fluorophenoxy)-1H-benzo[d]imidazol-2-ylcarbamate

4-(4-fluorophenoxy)benzene-1,2-diamine (1.0 g, 4.58 mmole) and 1,3-Bis(methoxycarbonyl)-S-methylisothiourea (2.45 g, 11.91 mmole) were mixed with 5%-AcOH in Ethanol 10 mL. After dissolving it, it was heated under reflux for 5 hours. After the reaction was terminated with TLC, it was cooled to room temperature and the resulting precipitate was filtered. Upon filtration, the mixture was sufficiently washed with MeOH and dried under vacuum at 50 °C to obtain methyl 5-(4-fluorophenoxy)-1H-benzo[d]-imidazol-2-ylcarbamate (Formula 1-1, 1.02 g) as a white solid.

yield 77%, Melting Point: 266 ℃, R _f : 0.23 (EtOAc/n-Hexane, 1:1)

¹ H NMR (400 MHz. CDCl ₃ , δ, ppm): 3.75 (s, 3H, -OCH ₃ ), 6.78-7.40 (m, 7H, ArH), 11.61 (brs, 2H, (-NH) ₂ )

Synthesis of Formula 1-2

The synthesis process of the benzimidazole derivative of Chemical Formula 1-2 is shown in Reaction Scheme 2 below.

[Scheme 2]

Synthesis of tert-butyl 3-((4-Hydroxyphenyl)amino)azetidine-1-carboxylate

To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (5.00 g, 45.9 mmol) in THF (200 mL) was added 4-aminophenol (6.54 g, 38.2 mmol) and AcOH (2.75 g, 45.9 mmol). The mixture was stirred at room temperature for 2.0 hours. Sodium triacetoxyborohydride (16.2 g, 91.8 mmol) was added portionwise to the mixture and stirred at room temperature for 16 hours. After the reaction was completed, the mixture was poured into aqueous NaHCO ₃ , extracted with EA (100 mL * 3), dried over Na ₂ SO ₄ , concentrated, and purified with a silica gel column (PE/EA = 5:1) to obtain 8.90 g of an off-white oil. tert-butyl 3-((4-Hydroxyphenyl)amino)azetidine-1-carboxylate was obtained (88% yield).

MS Calcd. :264; MS Found : 265 [M+H] +. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 6.75-6.71 (m, 2H), 6.44-6.40 (m, 2H), 4.58-4.55 (m, 1H), 4.27-4.23 (m, 2H), 3.82 -3.78 (m, 1H), 3.71-3.68 (m, 2H), 1.44 (s, 9H).

Synthesis of tert-butyl 3-((4-(3-Amino-4-nitrophenoxy)phenyl)amino)azetidine-1-carboxylate

5-chloro-2-nitroaniline (5.38 g, 31.2 mmol) and K in a solution of tert-butyl 3-((4-Hydroxyphenyl)amino)azetidine-1-carboxylate (8.25 g, 31.2 mmol) in DMSO (100 mL). ₂ CO ₃ (12.9 g, 93.6 mmol) was added. The mixture was stirred at 90 °C for 48 h. After the reaction was complete, the mixture was poured into water (300 mL), extracted with EA (200 mL * 3), dried over Na ₂ SO ₄ , concentrated, and purified by a silica gel column (PE/EA = 5:1) to give an orange solid of 4.00 g of tert-butyl 3-((4-(3-Amino-4-nitrophenoxy)phenyl)amino)azetidine-1-carboxylate was obtained (32%, yield). MS Calcd.:400; MS Found: 401 [M+H]+.

Synthesis of tert-butyl 3-((4-(3,4-diaminophenoxy)phenyl)amino)azetidine-1-carboxylate

Pd ( OH) ₂ /C (300 mg) was added. The mixture was stirred at 45 °C under H ₂ (1 atm) for 16 h. After the reaction was completed, the mixture was filtered and the filtrate was concentrated to obtain 925 mg of tert-butyl 3-((4-(3,4-diaminophenoxy)phenyl)amino)azetidine-1-carboxylate as a brown oil (90% yield). .

MS Calcd.:370; MS Found: 371 [M+H]+.

Synthesis of tert-butyl 3-((4-((2-((methoxycarbonyl)amino)-1H-benzo[d]imidazol-5-yl)oxy)phenyl)amino)azetidine-1-carboxylate

1,3-bis(methoxycarbonyl) was added to a solution of the compound tert-butyl 3-((4-(3,4-diaminophenoxy)phenyl)amino)azetidine-1-carboxylate (925 mg, 2.50 mmol) in AcOH (20 mL). -2-methyl-2-thiopseudoeura (566 mg, 2.75 mmol) was added. The mixture was stirred at 80 °C for 30 min. After the reaction was complete, AcOH was evaporated and 20 mL of H ₂ O was added and extracted with EA (50 mL * 3). The organic layer was dried over Na ₂ SO ₄ and concentrated. The solid was triturated with EA to yield 550 mg of tert-butyl 3-((4-((2-((methoxycarbonyl)amino)-1H-benzo[d]imidazol-5-yl)oxy)phenyl)amino as a light gray solid. )azetidine-1-carboxylate was obtained (yield: 49%).

MS Calcd.:453; MS Found: 454 [M+H]+.

Synthesis of methyl (5-(4-(azetidin-3-ylamino)phenoxy)-1H-benzo[d] imidazol-2-yl)carbamate

tert-butyl 3-((4-((2-((methoxycarbonyl)amino)-1H-benzo[d] imidazol-5-yl)oxy)phenyl)amino)azetidine-1-carboxylate ( 450 mg, 1.00 mmol) was added TFA (16 mL). The mixture was stirred at room temperature for 3 h. After the reaction was completed, TFA was removed and neutralized with 10% NaOH aqueous solution. The mixture was purified by prep-HPLC to obtain 120 mg of methyl (5-(4-(azetidin-3-ylamino)phenoxy)-1H-benzo[d]imidazol-2-yl)carbamate (Formula 1-2) as an off-white solid (34% yield).

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 7.31 (d, 1H, J = 8.4 Hz), 6.87 (s, 1H), 6.80 (d, 2H, J = 8.0 Hz), 6.73-6.70 ( m, 1H), 6.50 (d, 2H, J = 8.8 Hz), 5.98 (s, 1H), 4.13 (d, 1H, J = 8.0 Hz), 3.72–3.69 (m, 5H), 3.42–3.34 (m , 2H).

LCMS [mobile phase: 98% water (0.1% TFA) and 5% CH ₃ CN - 40% water (0.1% TFA) and 60% CH ₃ CN 6.0 min, finally 0.5 min under these conditions], purity is 95%, Rt = 2.873 min; MS Calcd.:353; MS Found: 354 [M+H]+.

Synthesis of Formulas 1-3

The synthetic process of the benzimidazole derivative of Chemical Formula 1-3 is shown in Reaction Scheme 3 below.

[Scheme 3]

tert Synthesis of -butyl 3-((4-hydroxyphenyl)(methyl)amino) azetidine-1-carboxylate

To a solution of tert-butyl 3-oxoazetidine-1-carboxylate (10.0 g, 48.0 mmol) in THF (100 mL) was added 4-(methylamino)phenol (6.84 g, 40.0 mmol) and AcOH (4.20 g, 70.0 mmol) did The mixture was stirred at room temperature for 2 hours. Sodium triacetoxyborohydride (17.0 g, 80.0 mmol) was added portionwise to the mixture and stirred at room temperature for 16 hours. After the reaction was complete, the mixture was poured into aqueous NaHCO ₃ , extracted with EA (100 mL * 3), dried over Na ₂ SO ₄ , concentrated and purified by reverse column (CH ₃ CN/H ₂ O) to yield 13.0 g of tert-butyl 3-((4-hydroxyphenyl)(methyl)amino)azetidine-1-carboxylate was obtained as a yellow solid

MS Calcd.: 278; MS Found: 279 [M+H] ⁺ .

tert Synthesis of -butyl 3-((4-(3-amino-4-nitrophenoxy)phenyl)(methyl)amino)azetidine-1-carboxylate

To a solution of tert-butyl 3-((4-hydroxyphenyl)(methyl)amino) azetidine-1-carboxylate (13.0 g, 47.0 mmol) in DMSO (200 mL) was added 5-chloro-2-nitroaniline (8.10 g, 47.0 mmol). ) and Cs ₂ CO ₃ (22.8 g, 70.0 mmol) were added. The mixture was stirred at 80 °C for 16 hours. After the reaction was complete, the mixture was poured into water (800 mL), extracted with EA (400 mL * 3), dried over Na ₂ SO ₄ , concentrated, and purified by reverse column (CH ₃ CN/H ₂ O) to give a red oil form to obtain 11.0 g of tert -butyl 3-((4-(3-amino-4-nitrophenoxy)phenyl)(methyl)amino)azetidine-1-carboxylate.

MS Calcd.:414; MS Found: 415 [M+H] ⁺ .

tert Synthesis of -butyl 3-((4-(3,4-diaminophenoxy)phenyl)(methyl)amino)azetidine-1-carboxylate

To a solution of tert -butyl 3-((4-(3-amino-4-nitrophenoxy)phenyl)(methyl)amino)azetidine-1-carboxylate (2.50 g, 6.00 mmol) in MeOH (50 mL), Pd(OH) ₂ /C (500 mg) was added. The mixture was stirred at 30 °C under H ₂ (1 atm) for 16 h. After the reaction was completed, the mixture was filtered and the filtrate was concentrated to obtain 2.20 g of tert-butyl 3-((4-(3,4-diaminophenoxy)phenyl)(methyl)amino)azetidine-1-carboxylate in the form of a yellow oil.

MS Calcd.: 384; MS Found: 385 [M+H] ⁺ .

Synthesis of tert-butyl 3-((4-((2-((methoxycarbonyl)amino)-1H-benzo[d]imidazol-5-yl)oxy)phenyl)(methyl)amino)azetidine-1-carboxylate

To a solution of tert-butyl 3-((4-(3,4-diaminophenoxy)phenyl)(methyl)amino)azetidine-1-carboxylate (2.20 g, 5.70 mmol) in AcOH (40 mL) was added 1,3-bis( methoxycarbonyl)-2-methyl-2-thiopseudoeura (1.30 g, 5.70 mmol) was added. The mixture was stirred at 85 °C for 16 h. After the reaction was complete, AcOH was removed and extracted with EA (50 mL * 3). The organic layer was dried over Na ₂ SO ₄ and concentrated. The solid was purified by prep-HPLC to yield 1.10 g of tert-butyl 3-((4-((2-((methoxycarbonyl)amino)-1H-benzo[d]imidazol-5-yl)oxy)phenyl) as an off-white solid. (methyl)amino)azetidine-1-carboxylate was obtained (41% yield).

MS Calcd.: 467; MS Found: 468 [M+H] ⁺ .

Synthesis of methyl (5-(4-(azetidin-3-yl(methyl)amino)phenoxy)-1H-benzo[d]imidazol-2-yl)carbamate

tert-butyl 3-((4-((2-((methoxycarbonyl)amino)-1H-benzo[d]imidazol-5-yl)oxy)phenyl)(methyl)amino)azetidine-1 in DCM (10 mL) To a solution of -carboxylate (350 mg, 0.750 mmol) was added TFA (5 mL). The mixture was stirred at room temperature for 35 min. After the reaction was complete, TFA was removed and TEA was added. The mixture was purified by prep-HPLC to obtain 90.0 mg of methyl (5-(4-(azetidin-3-yl(methyl)amino)phenoxy)-1H-benzo[d]imidazol-2-yl)carbamate (Formula 1-3 ) was obtained as a white solid (yield 32%).

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 7.35-7.33 (m, 1H), 6.94 (s, 1H), 6.87-6.85 (m, 2H), 6.76-6.72 (m, 3H), 4.20 -4.16 (m, 1H), 3.73 (s, 3H), 3.60-3.57 (m, 3H), 2.76 (s, 3H). LCMS [Mobile phase: 90% water (0.1% TFA) and 10% CH ₃ CN - 50% water (0.1% TFA) and 50% CH ₃ CN 6.0 min, finally 0.5 min under these conditions.], purity is 98% , Rt = 2.876 min; MS Calcd.:367; MS Found: 368 [M+H] ⁺ .

Synthesis of Formulas 1-4

The synthetic process of the benzimidazole derivative of Chemical Formula 1-4 is shown in Reaction Scheme 4 below.

[Scheme 4]

Synthesis of N1-(4-fluorophenyl)-4-nitrobenzene-1,3-diamine

5-chloro-2-nitroaniline (3.00 g, 17.3 mmol), 4-fluoroaniline (2.30 g, 20.7 mmol), Pd(OAc) ₂ in a solution of Cs ₂ CO ₃ (10.1 g, 31 mmol) in toluene (100 mL). (100 mg) and Xphos (600 mg) were added and the mixture was stirred at 115 °C for 20 h. After completion of the reaction, it was concentrated and purified with a silica gel column (PE/EA=20:1~5:1~2:1) to obtain 2.00 g of N1-(4-fluorophenyl)-4-nitrobenzene-1,3-diamine was obtained as a yellow solid (46% yield).

MS Calcd.: 247; MS Found: 248 [M+H] ⁺ .

Synthesis of N4-(4-fluorophenyl)benzene-1,2,4-triamine

Pd(OH) 2 /C (0.40 g) was added to a solution of N1-(4-fluorophenyl)-4-nitrobenzene-1,3-diamine (2.00 g, 8.1 mmol) in THF/MeOH ( ₂₀ mL/20 mL). added. The mixture was stirred at 50 °C for 20 h under H ₂ (1 atm). After the reaction was completed, the mixture was filtered and the filtrate was concentrated to obtain 1.80 g of N4-(4-fluorophenyl)benzene-1,2,4-triamine as a black oil (90% yield).

MS Calcd.: 217; MS Found: 218 [M+H] ⁺ .

Synthesis of methyl (6-((4-fluorophenyl)amino)-1H-benzo[d]imidazol-2-yl)carbamate

1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudoeura (3.30 g, 16.2 mmol) was added. The mixture was stirred at 80 °C for 1 h. After the reaction was complete, the mixture was filtered and the cake was triturated with DMSO/H ₂ O/Na ₂ CO ₃ to obtain 300 mg of methyl (6-((4-fluorophenyl)amino)-1H-benzo[d]imidazol-2-yl )carbamate (formula 1-4) was obtained as a light gray solid (90% yield).

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 7.31 (d, 1H, J = 8.4 Hz), 6.87 (s, 1H), 6.80 (d, 2H, J = 8.0 Hz), 6.73-6.70 ( m, 1H), 6.50 (d, 2H, J = 8.8 Hz), 5.98 (s, 1H), 4.13 (d, 1H, J = 8.0 Hz), 3.72–3.69 (m, 5H), 3.42–3.34 (m , 2H). LCMS [mobile phase: 70% water (0.02% NH ₄ HCO ₃ ) and 30% CH ₃ CN - 40% water (0.02% NH ₄ HCO ₃ ) and 60% CH ₃ CN at 6.0 min, finally 0.5 min under these conditions ], purity 94%, Rt = 2.393 min; MS Calcd.:300; MS Found: 301 [M+H] ⁺ .

Synthesis of Formulas 1-5

The synthetic process of the benzimidazole derivative of Chemical Formula 1-5 is shown in Reaction Scheme 5 below.

[Scheme 5]

Synthesis of 4-nitro-N1-phenylbenzene-1,3-diamine

Aniline (1.90 g, 20.8 mmol), 5-chloro- ₂ -nitroaniline ( _3.00 g, 17.3 mmol), Pd(OAc) ₂ (100 mg) and Xphos (600 mg) were added and the mixture was stirred at 115 °C for 20 h. After completion of the reaction, it was concentrated and purified with a silica gel column (PE/EA = 20:1~5:1~2:1) to obtain 2.00 g of 4-nitro-N1-phenylbenzene-1,3-diamine in the form of a yellow solid ( yield 50%).

MS Calcd.: 229; MS Found: 230 [M+H] ⁺ .

Synthesis of N4-phenylbenzene-1,2,4-triamine

To a solution of 4-nitro-N1-phenylbenzene-1,3-diamine (2.00 g, 8.7 mmol) in THF/MeOH (20 mL/20 mL) was added Pd(OH) ₂ /C (0.40 g). The mixture was stirred at 50 °C for 20 h under H ₂ (1 atm). After the reaction was complete, the mixture was filtered and the filtrate was concentrated to obtain 1.80 g of N4-phenylbenzene-1,2,4-triamine as a black oil (90% yield).

MS Calcd.: 199; MS Found: 200 [M+H] ⁺ .

Synthesis of methyl (6-(phenylamino)-1H-benzo[d]imidazol-2-yl)carbamate

To a solution of N4-phenylbenzene-1,2,4-triamine (1.80 g, 8.7 mmol) in AcOH (30 mL) was added 1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudoeura (2.15 g, 10.4 mmol). was added. The mixture was stirred at 80 °C for 1 hour. After the reaction was complete, the mixture was filtered and the cake was pulverized with DMF/H ₂ O/Na ₂ CO ₃ to obtain methyl (6-(phenylamino)-1H-benzo[d]imidazol-2-yl)carbamate (Formula 1-5 ) 700 mg as a light gray solid (29% yield).

¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 11.74, (bs, 2H), 7.86 (s, 1H), 7.25 (d, 1H, J = 8.4 Hz), 7.17-7.14 (m, 3H) , 6.96 (d, 2H, J = 7.6 Hz), 6.83–6.81 (m, 1H), 6.70–6.67 (m, 1H), 3.69 (s, 3H). LCMS [mobile phase: 80% water (0.02% NH ₄ HCO ₃ ) and 20% CH ₃ CN - 6.0 min in 30% water (0.02% NH ₄ HCO ₃ ) and 70% CH ₃ CN, finally 0.5 min under these conditions ], purity 98%, Rt = 3.179 min; MS Calcd.:282; MS Found: 283 [M+H] ⁺ .

Example 2. Measurement of cell viability of benzimidazole derivatives in breast cancer cell lines

Human triple-negative breast cancer (TNBC) cell line MDA-MB-231 (Cell seeding numbers: 1 (M231) x 10 ⁴ cells / wells (confluency ≥ 25%) and HER2 positive breast cancer , HER2+ BC) cell line JIMT-1 (Cell seeding numbers: 1.2 (JIMT) x 10 ⁴ cells / wells (confluency ≥ 25%)) was used in the experiment.

The cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), streptomycin-penicillin (100 U/ml) and Fungizone (0.625 μg/ml) at 5% CO ₂ and 37 °C. cultured.

In MDA-MB-231 and JIMT-1, the benzimidazole derivatives of Chemical Formulas 1-1, 1-2, 1-4 and 1-5 were treated for 72 hours at various concentrations of 0, 0.5, 1, and 5 μM, respectively. After that, cell viability was measured by MTS assay technique. MTS assay is performed by attaching cells to a 96 well plate for 24 hours, then treating the benzimidazole derivative for 72 hours, and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl )-2-(4-sulfophenyl)-2H-tetrazolium) for 4 hours, and then the absorbance was measured at 490 nm using a Spectramax Plus384 microplate analyzer.

The measured results are shown in Figures 1 and 2. Figure 1 confirms the cell viability for MDA-MB-231, Figure 2 confirms the cell viability for JIMT-1.

As a result, it was confirmed that the benzimidazole derivatives inhibited cell viability in a concentration-dependent manner in MDA-MB-231 and JIMT-1. From the above results, it was confirmed that the benzimidazole derivatives of the present invention have the effect of inhibiting the survival of trastuzumab-resistant cancer cells.

As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

A pharmaceutical composition for preventing or treating breast cancer, comprising a benzimidazole derivative represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

In Formula 1,
X is oxygen or nitrogen;
R ₁ is a substituted amine group, halogen or hydrogen;
The substituted amine group is characterized in that it is substituted with a C ₁ to C ₆ chain or cyclic alkyl group, or a nitrogen-substituted C ₃ to C ₆ heterocycloalkyl.

delete According to claim 1,
A pharmaceutical composition for preventing or treating breast cancer, wherein the benzimidazole derivative represented by Formula 1 is any one selected from the group consisting of the following compounds:
[Formula 1-1]

[Formula 1-2]

[Formula 1-3]

[Formula 1-4]

and
[Formula 1-5]

.
According to claim 1,
A pharmaceutical composition for preventing or treating breast cancer, wherein the benzimidazole derivative represented by Formula 1 is any one selected from the group consisting of the following compounds:
[Formula 1-1]

[Formula 1-2]

[Formula 1-4]

and
[Formula 1-5]

.
According to claim 1,
The benzimidazole derivative is a pharmaceutical composition for preventing or treating breast cancer, characterized in that inhibits tubulin polymerization.
delete According to any one of claims 1 to 5,
The pharmaceutical composition is characterized by inducing apoptosis by stopping the cancer cell cycle (cell cycle arrest), a pharmaceutical composition for preventing or treating breast cancer.
delete According to claim 1,
The breast cancer is a pharmaceutical composition for preventing or treating breast cancer, characterized in that triple-negative breast cancer.

Images