Benzimidazolyl urea derivative and pharmaceutical application thereof

Abstract

The present disclosure relates to benzimidazolyl urea derivatives and pharmaceutical uses thereof. The benzimidazolyl urea derivative exhibits an excellent effect in the treatment and prevention of pathological conditions caused by excess glucocorticoids by inhibiting adrenal corticosteroid production and cholesterol biosynthesis, and can be used for the prevention and treatment of endogenous hypercortisolism.

Description

Technical Field

[0001] The present disclosure relates to benzimidazolyl urea derivatives and pharmaceutical uses thereof. Background Art

[0002] In humans, excessive secretion of adrenal glucocorticoids (commonly known as cortisol) can interfere with glucose / lipid metabolism, immune responses, cardiovascular function, mood, and cognitive function, leading to various pathological symptoms. For example, Cushing's syndrome is an endocrine disorder characterized by chronically elevated blood levels of glucocorticoids, which can be caused by a variety of causes (e.g., pituitary adenoma, adrenal hyperplasia, adrenal tumors, and ectopic adrenocorticotropic hormone secretion). This condition increases the risk of complications, including hypertension, hyperglycemia, osteoporosis, cardiovascular disease, sleep disorders, and susceptibility to infection, with a 5-year mortality rate of almost 50%.

[0003] For patients with an overdose of adrenocorticotropic hormone (ACTH) or glucocorticoids, surgical intervention is not always possible, so drug treatment is crucial not only before surgery but also when surgery is unsuccessful or impossible. Postoperative recurrence rates can reach 20%-30%. Treatments developed to date are either insufficiently effective or have limited use due to side effects. For example, ketoconazole, one of the most commonly used medications for Cushing's syndrome, was suspended in 2013 by the KFDA due to the risk of liver damage.

[0004] Therefore, there is an unmet medical need for treatment, and the development of new therapeutic approaches is urgently needed. Summary of the Invention Technical goals

[0005] The object of the present disclosure is to provide benzimidazolyl urea derivatives that inhibit corticosteroid production and cholesterol biosynthesis and their pharmaceutical uses. Technical Solution

[0006] In order to achieve the above object, the present disclosure provides a benzimidazolyl urea derivative represented by the following Chemical Formula 1:

[0007] [Chemical Formula 1]

[0008]

[0009] Wherein, in Chemical Formula 1,

[0010] R is hydrogen, halogen, (C1-C4) alkyl, tetrafluoromethyl (CF3), (C1-C4) alkoxy, amino (NH2), nitrogen dioxide (NO2), cyano (CN), cyanomethyl (CH2CN), phenyl, benzyl or naphthyl.

[0011] Furthermore, the present disclosure provides a pharmaceutical composition for preventing or treating hypercortisolism, the pharmaceutical composition comprising a benzimidazolyl urea derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Furthermore, the present disclosure provides a health functional food composition for preventing or improving hypercortisolism, the health functional food composition comprising a benzimidazolyl urea derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Beneficial effects

[0013] The benzimidazolyl urea derivatives according to the present disclosure exhibit excellent effects in the treatment and prevention of pathological symptoms caused by excessive secretion of glucocorticoids including Cortisol by inhibiting corticosteroid production and cholesterol biosynthesis, and are therefore useful for the prevention and treatment of endogenous hypercortisolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The structures of the benzimidazolyl urea derivatives disclosed herein and their effects on cortisol production are shown. Figure a shows the effects of halogenated benzimidazolyl urea derivatives as cortisol production inhibitors, b shows the structure-dependent effects of halogenated benzimidazolyl urea derivatives (CJ21-CJ32) on cortisol production, and c shows the dose-response curves for cortisol production of CJ22, CJ25, CJ28, and metyrapone.

[0015] Figure 2 The results show the effects of the benzimidazolyl urea derivatives according to the present disclosure on adrenal steroidogenesis. a shows the effects of the benzimidazolyl urea derivatives on the levels of major steroid intermediates and cortisol, and b shows the mRNA expression of steroidogenic genes after treatment with the benzimidazolyl urea derivatives.

[0016] Figure 3 Results show the effects of the benzimidazolyl urea derivatives of the present disclosure on forskolin-induced cortisol production and steroidogenic gene expression. Table a shows the dose-dependent effects of CJ28 on cortisol production in the presence or absence of 1 μM forskolin (hereinafter referred to as FSK), and table b shows the mRNA expression of steroidogenic genes when treated with CJ28 in the presence or absence of FSK.

[0017] Figure 4Figure 2 shows the characteristics of benzimidazolyl urea derivative (CJ28)-responsive gene transcripts in H295R cells. a shows a Venn diagram of differentially expressed genes (DEGs) after 24 h of treatment with CJ28 (30 μM) or vehicle (VEH, 0.1% DMSO), b shows the top five up-regulated or down-regulated gene transcripts after CJ28 treatment, and c shows CJ28-responsive DEGs involved in regulating corticosteroid production.

[0018] Figure 5 Figure 2 shows the analysis results of the pathway and upstream regulator enhancement of benzimidazolyl urea derivatives (CJ28) responsive DEGs according to the present disclosure. a shows the enhancement of pathways and biological processes of DEGs in CJ28-treated cells, b shows the top 10 upstream transcriptional regulators rich in DEGs suggested by IPA, c shows a Venn diagram for comparing the target DEGs contained in a given pathway / GO term and upstream regulator, d shows the changes in the mRNA expression levels of target genes NR5A1 (left) and SREBF1 / 2 (right) after CJ28 treatment.

[0019] Figure 6 Results are shown for the effects of the benzimidazolyl urea derivatives of the present disclosure on cholesterol biosynthesis and steroidogenesis. Figure a shows the effects of the benzimidazolyl urea derivatives on mRNA expression of genes regulating de novo cholesterol synthesis and transport, Figure b shows a comparison of the effects of CJ28, metyrapone, and cholesterol biosynthesis inhibition on cholesterol production, Figure c shows a comparison of the effects of CJ28, metyrapone, and cholesterol biosynthesis inhibition on cortisol production, and Figure d shows a comparison of the effects of CJ28, metyrapone, and cholesterol biosynthesis inhibition on steroidogenic gene expression. DETAILED DESCRIPTION

[0020] Hereinafter, the present disclosure will be described in detail.

[0021] As used herein, the term "prevention" refers to any action to inhibit or delay hypercortisolism (e.g., Cushing's syndrome) or at least one symptom thereof by administering a pharmaceutical composition, cosmetic composition, or health functional food composition according to the present disclosure. In addition, it also includes treating a subject who has shown efficacy of the disease to prevent or avoid recurrence.

[0022] As used herein, the term "treating" refers to any action that improves or beneficially alters the symptoms of hypercortisolism or at least one or more of its symptoms, for example, by alleviating, reducing or eliminating the symptoms by administering a pharmaceutical composition according to the present disclosure.

[0023] As used herein, the term "improvement" refers to any action that alleviates or beneficially changes the symptoms of hypercortisolism or at least one or more symptoms thereof, for example, by alleviating, reducing or eliminating the symptoms by administering the cosmetic composition or health functional food composition according to the present disclosure.

[0024] The present disclosure provides a benzimidazolyl urea derivative represented by the following Chemical Formula 1:

[0025] [Chemical Formula 1]

[0026]

[0027] Wherein, in Chemical Formula 1,

[0028] R is hydrogen, halogen, (C1-C4) alkyl, tetrafluoromethyl (CF3), (C1-C4) alkoxy, amino (NH2), nitrogen dioxide (NO2), cyano (CN), cyanomethyl (CH2CN), phenyl, benzyl or naphthyl. Specifically, R can be any halogen selected from the group consisting of fluorine, chlorine, bromine and iodine.

[0029] In Chemical Formula 1, R may be substituted at the ortho, meta, or para position, and specifically, R may be substituted at the meta position. More specifically, in Chemical Formula 1, R may be any halogen selected from the group consisting of fluorine, chlorine, bromine, and iodine substituted at the meta position.

[0030] Furthermore, the present disclosure provides a pharmaceutical composition for preventing or treating hypercortisolism, the pharmaceutical composition comprising a benzimidazolyl urea derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0031] In Chemical Formula 1, R is hydrogen, halogen, (C1-C4) alkyl, tetrafluoromethyl (CF3), (C1-C4) alkoxy, amino (NH2), nitrogen dioxide (NO2), cyano (CN), cyanomethyl (CH2CN), phenyl, benzyl, or naphthyl. Specifically, R can be any halogen selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[0032] In Chemical Formula 1, R may be substituted at the ortho, meta, or para position, and specifically, R may be substituted at the meta position. More specifically, in Chemical Formula 1, R may be any halogen selected from the group consisting of fluorine, chlorine, bromine, and iodine substituted at the meta position.

[0033] Pharmaceutically acceptable salts can be selected from the group consisting of hydrochloride, bromate, sulfate, phosphate, nitrate, citrate, acetate, lactate, tartrate, maleate, gluconate, succinate, formate, trifluoroacetate, oxalate, fumarate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, sodium salt, potassium salt, lithium salt, calcium salt and magnesium salt, but are not limited thereto.

[0034] The composition can contain 1 μM to 100 μM, 1 μM to 70 μM, or 10 μM to 50 μM of the benzimidazolyl urea derivative.

[0035] The composition can inhibit corticosteroid production and cholesterol biosynthesis.

[0036] The hypercortisolism may be selected from the group consisting of diseases caused by excessive secretion of glucocorticoids including Cushing's syndrome, Cushing's disease and ectopic ACTH syndrome.

[0037] As used herein, the term "pharmaceutical composition" refers to a composition that is administered for a specific purpose, specifically, for the purposes of the present disclosure, to prevent or treat the symptoms of hypercortisolism.

[0038] The pharmaceutical composition of the present disclosure can be prepared in the form of a unit dose by using a pharmaceutically acceptable carrier according to a method that can be easily performed by those skilled in the art to which the present disclosure pertains, or by encapsulating into a multi-volume container.

[0039] Pharmaceutically acceptable carriers are commonly used carriers in preparation, including but not limited to lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate / ester, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present disclosure may also contain lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents and preservatives.

[0040] In the present disclosure, the content of the additives included in the pharmaceutical composition is not particularly limited and can be appropriately adjusted within the content range used in conventional preparations.

[0041] The pharmaceutical composition can be formulated in the form of one or more external agents selected from the group consisting of injectable preparations (e.g., aqueous solutions, suspensions, and emulsions), pills, capsules, granules, tablets, creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes, and dressings.

[0042] Pharmaceutical composition of the present disclosure can include the pharmaceutically acceptable carrier and diluent for preparation that exist in addition.Pharmaceutically acceptable carrier and diluent include but are not limited to excipient (for example, starch, sugar and mannitol), filler and extender (extenders) (for example, calcium phosphate), cellulose derivative (for example, carboxymethyl cellulose and hydroxypropyl cellulose), adhesive (for example, gelatin, alginate / ester and polyvinyl pyrrolidone), lubricant (for example, talc, calcium stearate, hydrogenated castor oil and polyethylene glycol), disintegrant (for example, polyvidone and cross-linked polyvinylpyrrolidone) and surfactant (for example, polysorbate, cetyl alcohol and glycerol).Pharmaceutically acceptable carrier and diluent can have biology and physiology friendliness to experimenter.The example of diluent can include but is not limited to saline, aqueous buffer, solvent and / or dispersion medium.

[0043] Depending on the desired method, the pharmaceutical composition of the present disclosure can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically). For oral administration, the pharmaceutical composition can be formulated into tablets, lozenges, lozenges, aqueous suspensions, oily suspensions, powder formulations, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. For parenteral administration, the pharmaceutical composition can be formulated into injections, suppositories, powders for inhalation, aerosols for spraying, ointments, powders for application, oils, and emulsions.

[0044] The dosage range of the pharmaceutical composition of the present disclosure can be varied according to the patient's condition, weight, age, sex, health status, dietary structure specificity, the nature of the preparation, the extent of the disease, the duration of administration of the composition, the method of administration, the administration cycle or administration interval, the excretion rate and the form of the drug, and can be appropriately selected by those skilled in the art. For example, it can be in the range of about 0.1 mg / kg to 10 mg / kg, but is not limited thereto, and can be administered once to several times a day.

[0045] According to the desired method, the pharmaceutical composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). The pharmaceutically effective amount and effective dose of the pharmaceutical composition of the present disclosure can vary according to the preparation method of the pharmaceutical composition, the method of administration, the duration of administration and / or the route of administration, and those skilled in the art can easily determine and prescribe the dosage effective for the desired treatment. The administration of the pharmaceutical composition of the present disclosure can be performed once a day or in divided doses for several times.

[0046] Furthermore, the present disclosure provides a health functional food composition for preventing or improving hypercortisolism, the health functional food composition comprising a benzimidazolyl urea derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0047] The composition may have the effect of inhibiting corticosteroid production and cholesterol biosynthesis.

[0048] For the above parts, the corresponding features can be replaced.

[0049] The present disclosure can generally be used as a common food product.

[0050] As used herein, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients that have functions useful to the human body, has high medical or health effects, and is processed to effectively obtain bioregulatory functions other than nutritional supplements, and can be used interchangeably with terms known in the art (e.g., functional food). For example, it can be manufactured into health supplements, special nutritional supplements, and functional drinks, or the composition according to the present disclosure can be added to natural foods and processed foods.

[0051] The health functional food composition can be made into powder, granule, tablet, capsule, syrup or beverage, and the form that the health functional food composition can adopt is not restricted, and can comprise all foods in the traditional sense.For example, beverage and various drinks, fruit and processed food thereof (canned fruit, jam etc.), fish and meat and processed food thereof (ham, bacon etc.), bread and noodles, biscuits and snacks and dairy products (butter, cheese etc.) are all possible, and can comprise all functional foods in the traditional sense.It can also comprise the food as animal feed.

[0052] According to the health functional food composition of the present disclosure, can be manufactured by further comprising acceptable food additives on food science and other suitable auxiliary components commonly used in this area.For example, it can further comprise flavoring, natural carbohydrate, sweetener, vitamin, electrolyte, coloring agent, pectic acid, alginic acid, organic acid, protective colloid thickener, pH adjusting agent, stabilizing agent, preservative, glycerine, alcohol and carbonating agent.Especially, monosaccharide (for example, glucose and fructose), disaccharide (for example, maltose and sucrose), polysaccharide (for example, dextrin and cyclodextrin) and sugar alcohol (for example, xylitol, sorbitol and erythritol) can be used as natural carbohydrate, and natural sweetener (for example, thaumatin and stevia extract) and synthetic sweetener (for example, saccharin and aspartame) can be used as sweetener.Preferably add food additives and other auxiliary components for use with minimal amount, as long as reach the purpose of adding them. When expressed as a number, the trace amount may range from 0.0005 wt % to about 0.5 wt % based on the total weight of the health functional food composition. Example

[0053] Hereinafter, the present disclosure will be described in more detail by way of examples. These examples are only intended to explain the present disclosure in more detail, and those skilled in the art will understand that, based on the gist of the present disclosure, the scope of the present disclosure is not limited by these examples.

[0054] Experimental methods

[0055] 1. Cell Culture and Treatment

[0056] NCI-H295R (H295R) cells were maintained in Dulbecco's modified Eagle's medium-F12 (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% ITS+ premix (Corning, Corning, NY, USA), 2.5% Nu-Serum (Corning) and 1% antibiotic antimycotic solution (Thermo Fisher Scientific) at 37°C in a humidified incubator with 5% CO2. All experiments were performed in 6-well or 12-well cell culture plates (SPL Life Sciences, Pocheon, South Korea). Confluent cells were cultured in serum-free medium for 12 hours and then treated with vehicle (0.1% or 0.2% DMSO) or benzimidazolyl urea derivatives. Metyrapone (catalog number: M2696), mitotane (catalog number: SML1885) and forskolin (catalog number: F3917) are obtained from Merck (Merck) (Burlington, MA, USA). Ketoconazole (catalog number: 1103) and AY9944 (catalog number: 1639) are obtained from Tocris Bioscience (Bristol, UK). U-18666A (catalog number: 10009869) is obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). After 24 hours or 48 hours of cultivation, the culture medium is collected for steroid measurement. The culture medium is centrifuged (10,000 × g) for 5 minutes at 4°C, and the supernatant is quickly frozen. The cells are washed with cold Dulbecco's phosphate-buffered saline and stored at -80°C.

[0057] 2. Measurement of steroids

[0058] The concentrations of steroids secreted into the culture medium were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Hormone concentrations of cortisol (Arbor Assays, Ann Arbor, MI, USA), pregnenolone (ALPCO, Salem, NH, USA), 17α-hydroxyprogesterone (IBL-America, Minneapolis, MN, USA), and progesterone (ALPCO) were measured using ELISA kits according to the manufacturer's instructions.

[0059] 3. Measurement of total cholesterol content

[0060] Use a solution containing 1% Triton TM Cells were harvested in cold Tris-buffered saline (pH 7.4) of X-100, transferred to microcentrifuge tubes, and incubated on ice for 15 minutes. A portion of lysate was mixed with an equal volume of sodium dodecyl sulfate (SDS) sample buffer (20% glycerol, 100mM Tris-HCl [pH 6.8] and 8% SDS) to carry out protein quantification, and the remainder was centrifuged for 20 minutes (10,000 × g) at 4°C. Supernatant was diluted with cholesterol assay buffer using a cholesterol / cholesterol ester quantitative assay kit (catalog number: ab65359, Abcam, Cambridge, UK), and the total cholesterol content was measured according to the manufacturer's instructions.

[0061] 4. RNA Isolation and Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

[0062] Total RNA was extracted using the microRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For qRT-PCR, PrimeScript TM First-strand cDNA synthesis kit (PrimeScript TM 500 ng of each RNA sample was reverse transcribed using the 1st Strand cDNA Synthesis Kit (Takara Bio, Kusatsu, Japan). Green PCR Master Mix (Power Green PCR Master Mix (Thermo Fisher Scientific) was mixed with the specified primer sets. Relative mRNA expression levels were calculated using the comparative Ct method, with TBP as an internal control. The primer sequences used for qRT-PCR are shown in Table 1 below.

[0063] Table 1

[0064] 5. RNA Sequencing (RNA-Seq) and Bioinformatics Analysis

[0065] Total RNA was extracted from H295R cells treated with solvent (0.1% DMSO) or CJ28 (30 μM) for 24 h. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA-Seq for transcriptome analysis and subsequent bioinformatics analysis was performed as previously described with minor modifications. That is, using Ribo-Zero TM Total RNA was depleted of ribosomal RNA using the Illumina reagent (Illumina, San Diego, CA, USA) and the TruSeq TM Strand-specific total RNA preparation kit (TruSeq TM cDNA library was prepared using Stranded Total RNA Prep Kit (Illumina). TMInitial sequence data was obtained using paired-end sequencing on the 2500 sequencing platform (Illumina), and raw reads were preprocessed by removing adapters and low-quality sequences (<Q20) using Trimmomatic with default parameters. TopHat v2.0.13 and Cuffdiff v2.2.0 were used to align the processed reads to the human reference genome (Ensembl GRCh38.p13) and identify significantly detected transcripts for analysis of differentially expressed genes (DEGs). The initial dataset was constructed using DEGs with a >2-fold change (FC) and a P-value <0.05. To analyze the molecular pathways composed of DEGs, the selected DEGs were first compared with the previously reported adrenal steroidogenesis pathway. In addition, the overall molecular pathway regulation patterns were analyzed using ENRICHR and Ingenuity Pathway Analysis (IPA, Qiagen). The "Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway", "Wiki Pathway", and "Gene Ontology (GO) Biological Functions" databases for ENRICHR were used to estimate the biomolecular pathways composed of DEGs, and the "Top Regulatory Factor Search" function of IPA was used to estimate the transcription factors affected by compound CJ28 treatment.

[0066] 6. Statistical Analysis

[0067] A simple comparison between the solvent and compound (benzimidazolyl urea derivative) treatment groups was performed using the Student's t-test. Post hoc comparisons were performed using analysis of variance (ANOVA) and Bonferroni tests for statistical evaluation of the dose-dependent effects or combined effects of multiple compounds. For the significance of the molecular pathways composed of DEGs, the Fisher's exact test was used to statistically evaluate the degree of overlap between the input DEG list and the gene sets of each molecular pathway in the presented databases. Inter-group differences with P < 0.05 were considered statistically significant.

[0068] <Example 1> Synthesis of Compounds

[0069] 1. 1-(1H-Benzimidazol-2-yl)-3-(3-bromophenyl)urea (CJ28)

[0070] The synthesis method of the benzimidazolyl urea derivative is as follows. 1,1′-Carbonyldiimidazole (96 mg, 0.58 mmol) was added in batches to a solution of 3-bromoaniline (100 mg, 0.58 mmol) dissolved in N,N-dimethylformamide (3 mL). Subsequently, the reaction mixture was stirred at room temperature for 2 hours. Then, 2-aminobenzimidazole (77 mg, 0.58 mmol) dissolved in N,N-dimethylformamide (1 mL) was added dropwise to the mixture and stirred overnight. The reaction was quenched with water and extracted with ethyl acetate. The organic layer was concentrated under reduced pressure and purified by medium pressure liquid chromatography (MPLC) to give 1-(1H-benzo[d]imidazol-2-yl)-3-(3-bromophenyl)urea (CJ28, 155 mg); yield 81%, white solid; 1 H NMR(500MHz,DMSO-d6)δ11.35(br,2H),9.56(br,1H),8.05(s,1H),7.46(d,J=8.30Hz,1H),7.36-7 .33(m,2H),7.23(t,J=8.05Hz,1H),7.14(d,J=7.95Hz,1H),7.07-7.06(m,2H); HRMS(FAB)m / z[M+H] + , C 14 H 12 BrN4O, calculated value: 331.0194, found: 331.0198.

[0071] Hereinafter, benzimidazolyl urea derivatives CJ01-CJ33 were prepared in the same manner as the production method of CJ28, except that the corresponding aniline substrates were used instead of 3-bromoaniline.

[0072] 2.1-(1H-Benzo[d]imidazol-2-yl)-3-(p-tolyl)urea (CJ01)

[0073] Yield 64%; white solid; 1 H NMR(500MHz,DMSO-d6)δ11.02(s,2H),9.53(s,1H),7.48-7.42(m,2H),7.38(dd,J=5.8,3 .2Hz,2H),7.12(d,J=8.2Hz,2H),7.06(dd,J=5.9,3.2Hz,2H),2.26(s,3H); LRMS(ESI)m / z 267[M+H] + .

[0074] 3. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-(trifluoromethyl)phenyl)urea (CJ02)

[0075] Yield 58%; white solid;1 H NMR(500MHz, DMSO-d6)δ8.09(d,J=10.25Hz,1H),7.74-7.67(m,2H),7.38-7.31(m,3H),7.09-7.07(m,2H); LRMS(ESI)m / z 321[M+H] + .

[0076] 4. 1-(1H-Benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (CJ03)

[0077] Yield 48%; white solid; 1 H NMR (500MHz, DMSO-d6) δ9.62 (s, 1H), 8.28 (s, 1H), 7.67 (d, J = 12.3Hz, 1H), 7.48 (t, J = 12.8Hz, 1H), 7.34-7.26 (m, 3H), 7.08-7.05 (m, 2H); LRMS (ESI) m / z 321[M+H] + .

[0078] 5. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea (CJ04)

[0079] Yield 66%; white solid; 1 H NMR (500MHz, DMSO-d6) δ9.73 (s, 1H), 7.82 (d, J = 14.1Hz, 2H), 7.62 (d, J = 14.7Hz, 2H), 7.36-7.31 (m, 2H), 7.09-7.06 (m, 2H); HRMS (FAB) m / z [M+H] + , C 15 H 12 F3N4O, calculated value: 321.0963, found: 321.0970.

[0080] 6. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-(tert-butyl)phenyl)urea (CJ05)

[0081] Yield 36%; white solid; 1 H NMR(500MHz, DMSO-d6)δ7.45(d,J=9.5Hz,1H),7.42-7.37(m,3H),7.25-7.15(m,2H),7.07-7.05(m,2H); HRMS(FAB)m / z[M+H] + , C 18 H 21N4O, calculated value: 309.1715, measured value: 309.1718.

[0082] 7. 1-(1H-Benzo[d]imidazol-2-yl)-3-(3-(tert-butyl)phenyl)urea (CJ06)

[0083] Yield 74%; white solid; 1 H NMR(500MHz,DMSO-d6)δ9.40(s,1H),7.66(s,1H),7.47-7.37(m,2H),7.31(d,J= 8.1Hz,1H),7.23(t,J=7.8Hz,1H),7.06-7.04(m,3H),1.29(s,9H); LRMS(ESI)m / z 309[M+H] + .

[0084] 8. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-(tert-butyl)phenyl)urea (CJ07)

[0085] Yield 65%; white solid; 1 H NMR (500MHz, DMSO-d6) δ9.47 (s, 1H), 7.48 (d, J = 14.35Hz, 2H), 7.38-7.30 (m, 4H), 7.04 (dd, J = 5.5Hz, 2H), 1.26 (s, 9H); HRMS (FAB) m / z [M+H] + , C 18 H 21 N4O, calculated value: 309.1715, found: 309.1722. LRMS (ESI) m / z 321 [M+H] + .

[0086] 9. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-methoxyphenyl)urea (CJ08)

[0087] Yield 84%; white solid; 1 H NMR (500MHz, DMSO-d6) δ8.20 (d, J = 1.0Hz, 1H), 7.40 (t, J = 4.3Hz, 2H), 7.07-7.00 (m, 4H), 6.95 (t, J = 1.1Hz, 1H), 3.92 (s, 3H); LRMS (ESI) m / z 283 [M+H] + .

[0088] 10. 1-(1H-Benzo[d]imidazol-2-yl)-3-(3-methoxyphenyl)urea (CJ09)

[0089] Yield 73%; white solid; 1 H NMR (500MHz, DMSO-d6) δ9.53 (s, 1H), 7.38-7.35 (m, 3H), 7.19 (t, J = 12.8Hz, 1H), 7.07-7.02 (m, 3H), 6.58 (d, J = 14.1Hz, 1H), 3.74 (s, 3H); LRMS (ESI) m / z 283[M+H] + .

[0090] 11. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-methoxyphenyl)urea (CJ10)

[0091] Yield 77%; white solid; 1 H NMR (500MHz, DMSO-d6) δ9.44 (s, 1H), 7.47-7.34 (m, 4H), 7.05-7.02 (t, J = 5.5Hz, 2H), 6.90 (d, J = 14.6Hz, 2H), 3.71 (s, 3H); HRMS (FAB) m / z [M+H] + , C 15 H 15 N4O2, calculated value: 283.1195, found value: 283.1197.

[0092] 12. 1-(2-Aminophenyl)-3-(1H-benzo[d]imidazol-2-yl)urea (CJ11)

[0093] CJ11 was obtained by reducing CJ14 using SnCl2 in ethanol.

[0094] Yield 84%; white solid; 1 H NMR(500MHz, DMSO-d6)δ7.41-7.33(m,3H),7.02-6.99(m,2H),6.87(t,J=14.1Hz,1H),6.75(d,J=13.1Hz,1H),6.58(t,J=12.2Hz,1H); HRMS(FAB)m / z[M+H] + , C 14 H 14 N5O, calculated value: 268.1198, found value: 268.1201.

[0095] 13. 1-(4-Aminophenyl)-3-(1H-benzo[d]imidazol-2-yl)urea (CJ12)

[0096] CJ12 was obtained by reducing CJ16 using SnCl2 in ethanol.

[0097] Yield 89%; white solid; 1 H NMR(500MHz,DMSO-d6)δ8.17(t,1H),7.37(dd,2H),7.27(dd,1H),7.18(t,1H),7.10-7.04(m,3H); HRMS(FAB)m / z[M+H] + , C 14 H 14 N5O, calculated value: 268.1198, found value: 268.1187.

[0098] 14.1-(1H-Benzo[d]imidazol-2-yl)-3-phenylurea (CJ13)

[0099] Yield 73%; white solid; 1 H NMR (500MHz, DMSO-d6) δ9.56 (s, 1H), 7.56 (d, J = 13.5Hz, 2H), 7.38-7.35 (m, 2H), 7.33-7.28 (m, 2H), 7.07-6.97 (m, 3H); LRMS (FAB) m / z 253 [M+H] + .

[0100] 15. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-nitrophenyl)urea (CJ14)

[0101] Yield 82%; yellow solid; 1 H NMR (500MHz, DMSO-d6) δ8.42(d,J=12.2Hz,1H),8.09(d,J=12.2Hz,1H),7.70(t,J=11.9H z,1H),7.36-7.34(m,2H),7.19(t,J=13.4Hz,1H),7.10-7.07(m,2H); HRMS(FAB)m / z[M+H] + , calculated value: C 14 H 12 N5O3 298.0940, measured value: 298.0934.

[0102] 16. 1-(1H-Benzo[d]imidazol-2-yl)-3-(3-nitrophenyl)urea (CJ15)

[0103] Yield 81%; yellow solid; 1H NMR (300MHz, DMSO-d6) δ9.73 (br, 1H), 8.86 (s, 1H), 7.79 (t, J = 15.9Hz, 2H), 7.53 (t, J = 13.5Hz, 1H), 7.34-7.31 (m, 2H), 7.10-7.07 (m, 2H); LRMS (FAB) m / z 298[M+H] + .

[0104] 17. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-nitrophenyl)urea (CJ16)

[0105] Yield 87%; yellow solid; 1 H NMR(300MHz, DMSO-d6)δ8.17(d,J=15.3Hz,2H),7.87(d,J=15.3Hz,2H),7.35-7.32(m,2H),7.12-7.09(m,2H); LRMS(ESI)m / z 298[M+H] + .

[0106] 18. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-cyanophenyl)urea (CJ17)

[0107] Yield 68%; white solid; 1 H NMR (300MHz, DMSO-d6) δ9.73 (br, 1H), 7.80 (d, J = 15.0Hz, 2H), 7.69 (d, J = 14.7Hz, 2H), 7.34-7.31 (m, 2H), 7.10-7.07 (m, 2H); LRMS (ESI) m / z 278 [M+H] + .

[0108] 19. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-(cyanomethyl)phenyl)urea (CJ18)

[0109] Yield 66%; white solid; 1 H NMR (500MHz, DMSO-d6) δ7.82 (d, J = 9.45Hz, 1H), 7.68-7.35 (m, 5H), 7.28-7.17 (m, 1H), 7.08-7.06 (m, 2H), 6.95-6.78 (m, 1H); LRMS (ESI) m / z 292 [M+H] + .

[0110] 20. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-benzoylphenyl)urea (CJ19)

[0111] Yield 69%; white solid; 1 H NMR(300MHz, DMSO-d6)δ7.78-7.55(m,9H),7.34(m,2H),7.09(m,2H); HRMS(FAB)m / z[M+H] + , calculated value: C 21 H 17 N4O2 357.1352, measured value: 357.1341.

[0112] 21. 1-([1,1'-Biphenyl]-4-yl)-3-(1H-benzo[d]imidazol-2-yl)urea (CJ20)

[0113] Yield 58%; white solid; 1 H NMR(300MHz, DMSO-d6)δ9.64(br,1H),7.69-7.60(m,6H),7.46-7.28(m,5H),7.07-7.04(m,2H); HRMS(FAB)m / z[M+H] + , calculated value: C 20 H 17 N4O 329.1402, measured value: 329.1395.

[0114] 22. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-fluorophenyl)urea (CJ21)

[0115] Yield 82%; white solid; 1 H NMR(300MHz, DMSO-d6)δ8.17(m,1H),7.91-7.88(m,2H),7.79(m,2H),7.34-7.19(m,5H); HRMS(FAB)m / z[M+H] + , C 14 H 12 FN4O, calculated value: 271.0995, found value: 271.1003.

[0116] 23. 1-(1H-Benzo[d]imidazol-2-yl)-3-(3-fluorophenyl)urea (CJ22)

[0117] Yield 68%; white solid; 1 H NMR (400MHz, DMSO-d6) δ9.63 (br, 1H), 7.64 (d, J = 11.8Hz, 1H), 7.35-7.28 (m, 4H), 7.07 (m, 2H), 6.78 (s, 1H); HRMS (FAB) m / z [M+H] + , calculated value: C14 H 12 FN4O271.0995, measured value: 271.0984.

[0118] 24. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-fluorophenyl)urea (CJ23)

[0119] Yield 64%; white solid; 1 H NMR (300MHz, DMSO-d6) δ9.57 (br, 1H), 7.60-7.56 (m, 2H), 7.37-7.34 (m, 2H), 7.13 (t, J = 14.7Hz, 2H), 7.06-7.03 (m, 2H); LRMS (ESI) m / z 271 [M+H] + .

[0120] 25. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-chlorophenyl)urea (CJ24)

[0121] Yield 54%; white solid; 1 H NMR (300MHz, DMSO-d6) δ8.26 (d, J = 7.1Hz, 1H), 7.50 (d, J = 8.1Hz, 1H), 7.41-7.32 (m, 3H), 7.32-7.05 (m, 3H); LRMS (ESI) m / z 287 [M+H] + .

[0122] 26. 1-(1H-Benzo[d]imidazol-2-yl)-3-(3-chlorophenyl)urea (CJ25)

[0123] Yield 68%; white solid; 1 H NMR(300MHz,DMSO-d6)δ9.56(br,1H),7.88(s,1H),7.43-7.26(m,4H),7.08-6.98(m,3H); HRMS(FAB)m / z[M+H] + , C 14 H 12 ClN4O, calculated value: 287.0700, found: 287.0701.

[0124] 27. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-chlorophenyl)urea (CJ26)

[0125] Yield 68%; white solid; 1H NMR(500MHz,DMSO-d6)δ11.26(s,2H),9.64(s,1H),7.68-7.61(m,2H),7.42-7.32(m,4H),7.13-7.05(m,2H); LRMS(ESI)m / z 287[M+H] + .

[0126] 28. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-bromophenyl)urea (CJ27)

[0127] Yield 73%; white solid; 1 H NMR(300MHz, DMSO-d6)δ8.19(d,J=12.2Hz,1H),7.65(d,J=13.4Hz,1H),7.41-7.38(m,3H),7.08-7.02(m,3H); HRMS(FAB)m / z[M+H] + , calculated value: C 14 H 12 BrN4O 331.0194, found: 331.0195.

[0128] 29. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-bromophenyl)urea (CJ29)

[0129] Yield 77%; white solid; 1 H NMR (300MHz, DMSO-d6) δ9.65 (br, 1H), 7.57 (d, J = 14.7Hz, 2H), 7.45 (d, J = 14.7Hz, 2H), 7.35-7.32 (m, 2H), 7.07-7.04 (m, 2H); LRMS (ESI) m / z 331,333[M+H] + .

[0130] 30. 1-(1H-Benzo[d]imidazol-2-yl)-3-(2-iodophenyl)urea (CJ30)

[0131] Yield 78%; white solid; 1 H NMR (300MHz, DMSO-d6) δ8.00 (d, J = 13.4Hz, 1H), 7.87 (d, J = 11.3Hz, 1H), 7.41-7.35 (m, 3H), 7.08-7.05 (m, 2H), 6.90-6.85 (m, 1H); LRMS (ESI) m / z 379[M+H] + .

[0132] 31. 1-(1H-Benzo[d]imidazol-2-yl)-3-(3-iodophenyl)urea (CJ31)

[0133] Yield 61%; white solid; 1 H NMR (300MHz, DMSO-d6) δ9.48 (br, 1H), 8.21 (s, 1H), 7.46 (d, J = 12.9Hz, 1H), 7.35-7.30 (m, 3H), 7.09-7.05 (m, 3H); HRMS (FAB) m / z [M+H] + , C 14 H 12 N4O, calculated value: 379.0056, found value: 379.0060.

[0134] 32. 1-(1H-Benzo[d]imidazol-2-yl)-3-(4-iodophenyl)urea (CJ32)

[0135] Yield 75%; white solid; 1 H NMR (500MHz, DMSO-d6) δ7.61 (d, J = 10.3Hz, 2H), 7.45 (d, J = 10.7Hz, 2H), 7.35 (d, J = 4.3Hz, 2H), 7.06 (d, J = 3.7Hz, 2H); LRMS (FAB) m / z 379 [M+H] + .

[0136] 33. 1-(1H-Benzo[d]imidazol-2-yl)-3-(naphthalen-2-yl)urea (CJ33)

[0137] Yield 64%; white solid; 1 H NMR(300MHz, DMSO-d6)δ8.21(s,1H),7.94-7.81(m,3H),7.60-7.57(m,5H),7.31(m,2H); HRMS(FAB)m / z[M+H] + , C 18 H 15 N4O, calculated value: 303.1246, measured value: 303.1252.

[0138] <Example 2> Identification of benzimidazolyl urea derivatives as cortisol production inhibitors

[0139] To identify small molecules that inhibit cortisol production in human adrenal cortex cells, a series of drug-like chemical scaffolds were screened. H295R cells were treated with compounds from an internal chemical library (20 μM each) for 24 hours, and then the level of cortisol secretion in the culture medium was measured. Based on the initial screening, benzimidazolyl urea compounds were identified that showed inhibitory activity on cortisol secretion in H295R cells.

[0140] Structurally similar benzimidazolyl urea derivatives exhibited substituent-dependent inhibitory effects on cortisol secretion (Table 2).

[0141] Table 2

[0142] Among the above compounds, halogenated benzimidazolyl urea derivatives (CJ21-CJ32) were selected as inhibitors of cortisol production ( Figure 1 a). Figure 1 As shown in b, most of these compounds were found to inhibit cortisol secretion in H295R cells.

[0143] Among the 12 selected benzimidazolyl urea derivatives, the inhibitory effects of CJ22, CJ25, and CJ28 on cortisol production in H259R cells were measured. Figure 1 As shown in c, all three compounds significantly reduced cortisol secretion in a dose-dependent manner (by one-way ANOVA, for CJ22, F (7,17) =14.78, p<0.01; for CJ25, F (7,17) =23.77, p<0.01 7,16; for CJ28, F (7,17) =29.46, p<0.01). IC values of CJ22, CJ25, and CJ28 50 The values were 24.13 μM, 9.95 μM and 4.96 μM, respectively. Compared with that observed with vehicle treatment, the minimum cortisol secretion levels after CJ22, CJ25 and CJ28 treatment were 19.40%, 39.98% and 33.76%, respectively. The inhibitory effects of all three compounds were similar to those of metyrapone (IC 50 =34.98 μM, lowest level =45.46%).

[0144] <Example 3> Effect of benzimidazolyl urea derivatives on adrenal steroidogenesis

[0145] It was investigated whether the benzimidazolyl urea derivatives according to the present disclosure affect the steroidogenic pathway for cortisol production in H295R cells. Various steroidogenic genes regulate the steroidogenic pathway of adrenal GC biosynthesis, which mediates the conversion of cholesterol to cortisol. Here, changes in the relative levels of major intermediate steroids in cortisol biosynthesis (including pregnenolone, progesterone, and 17α-hydroxyprogesterone) were investigated after 24 hours of treatment with solvent (0.1% DMSO), metyrapone, or the compound. The results are shown in FIG. Figure 2 a.

[0146] like Figure 2 As shown in Figure a, the synthesis of pregnenolone, the first metabolite of cholesterol, was significantly reduced when treated with all three benzimidazolyl urea derivatives, but not with metyrapone. Benzimidazolyl urea derivatives also reduced the levels of progesterone and 17α-hydroxyprogesterone. Metyrapone selectively inhibits the activity of CYP11B1 (11β-hydroxylase), which catalyzes the conversion of 11-deoxycortisol to cortisol. Therefore, treatment with metyrapone only reduced cortisol levels, while blocking the steroidogenesis pathway led to the accumulation of pregnenolone and progesterone. These results suggest that compounds CJ22, CJ25, and CJ28 affect higher stages of adrenal steroidogenesis compared to compounds affected by metyrapone.

[0147] Then, the mRNA expression levels of steroidogenic genes involved in cortisol production were examined, and the results are shown in Figure 2 b.

[0148] like Figure 2 As shown in Figure 2b, all major steroidogenic genes, such as CYP11A1 (encoding cholesterol side-chain cleavage monooxygenase) and STAR (encoding steroidogenic acute regulatory protein), which mediate the initial phase, as well as HSD3B2 (encoding 3β-hydroxysteroid dehydrogenase) and CYP17A1 (encoding 17α-hydroxylase), showed reduced mRNA expression after treatment with benzimidazolyl urea derivatives (CJ22, CJ25, and CJ28). The fold change in mRNA expression of each gene induced by each derivative correlated with changes in cortisol and intermediate steroids, indicating that these benzimidazolyl urea derivatives affect the transcriptional network that comprehensively coordinates the expression of steroidogenic genes in adrenal cortical cells.

[0149] In addition, the effect of compound CJ28 on forskolin-induced cortisol production in H295R cells was investigated, and the results are shown in Figure 3a. Forskolin activates adenylate cyclase, increasing intracellular cyclic AMP (cAMP) levels in cells, mimicking the stimulatory effect of ACTH on adrenal steroidogenesis. After incubation with compound CJ28 for 24 hours in the presence of solvent or forskolin (1 μM), cortisol secretion was examined. Figure 3 As shown in a, compound CJ28 significantly inhibited the basal and stimulated cortisol secretion in H295R cells, and when treated with 30 μM compound CJ28, forskolin-induced cortisol production was completely inhibited (by two-way ANOVA, for CJ28, F (2,18) =1280, p<0.01; for FSK, F (1,18) = 2082, p < 0.01; for the interaction, F (2,18) =758.1, p<0.01). As described above, basal mRNA expression of key genes mediating adrenal steroidogenesis was decreased in response to compound CJ28 treatment.

[0150] Next, the steroidogenic genes that mediate troponin-induced adrenal steroidogenesis were investigated, and the results are shown in Figure 3 b.

[0151] like Figure 3 As shown in (b), compound CJ28 (30 μM) significantly attenuated the forskolin-induced increase in CYP11A1, STAR, and CYP17A1 mRNA expressions (CY11A1: by two-way ANOVA, for CJ28, F(1,8)=68.79, p<0.01, for FSK, F (1,8) =146.5, p < 0.01, by two-way ANOVA, for the interaction, F (1,8) =26.55, p<0.01; STAR: for CJ28, F (1,8) =90.31, p<0.01, for FSK, F (1,8) =737.1, p < 0.01, for the interaction, F (1,8) =58.48, p<0.01; CYP17A1: for CJ28, F (1,8) =113.2, p<0.01, for FSK, F (1,8) =294.0, p < 0.01, for the interaction, F (1,8) =39.04, p<0.01). It is noteworthy that under the same conditions, in the presence of compound CJ28, forskolin-induced HSD3B2 mRNA expression was completely blocked (by two-way ANOVA, for CJ28, F (1,8) =2121, p<0.01; for FSK, F (1,8)=1965, p < 0.01; for the interaction, F (1,8) =1776, p<0.01). In conclusion, compound CJ28 inhibits ACTH-stimulated cortisol production by suppressing transcriptional activation of steroidogenic gene expression.

[0152] <Example 4> Effect of CJ28 on RNA expression profile at the transcriptome level

[0153] To investigate the molecular biological mechanism mediating the effects of compound CJ28, RNA-Seq was used to compare the transcriptome-level RNA expression profiles of H295R cells treated with solvent (0.1% DMSO) or CJ28 (30 μM) for 24 h. The results are shown in Figure 4 middle.

[0154] Among the 14,167 genes expressed at significant levels, 1,278 differentially expressed transcripts (609 upregulated DEGs and 669 downregulated DEGs) were identified between the two groups using the criteria of >2-FC and P < 0.05 ( Figure 4 a). Figure 4 As shown in Figure b, the top 10 DEGs (5 up-regulated and 5 down-regulated) were identified based on the fold difference. First, the expression patterns of key genes constituting the steroid biosynthesis pathway in human adrenal cortical cells induced by compound CJ28 were subsequently studied. In particular, Figure 4 As shown in Figure c, treatment with compound CJ28 significantly affected the expression patterns of multiple steroidogenic genes. The expression of genes that regulate cholesterol uptake and intramitochondrial transport (e.g., low-density lipoprotein receptor (LDLR), steroidogenic acute regulatory protein (STAR), and steroid sulfatase (STS)) was statistically significantly inhibited. In addition, the expression of genes encoding key steroidogenic mediators (e.g., 3β-hydroxysteroid dehydrogenase (HSD3B2), cholesterol side chain cleavage enzyme (CYP11A1), steroid 17-α-hydroxylase (CYP17A1), and ACTH receptor (MC2R)) was also significantly reduced. The extensive inhibition of the expression of genes involved in corticosteroid biosynthesis indicates that compound CJ28 targets upstream transcriptional regulators required for basal steroidogenesis and hormone-induced steroidogenesis in adrenal cortical cells.

[0155] To obtain more systematic information on the molecular and cellular changes and steroid biosynthesis pathway induced by compound CJ28, the bioinformatic molecular pathways of the targeted DEGs were analyzed, and the results are summarized in Figure 5 In. Figure 5As shown in (a), comparison of DEGs with multiple biomolecular pathway databases using ENRICHR indicated that genes involved in steroid / cortisol biosynthesis and secretion (KEGG pathway) and cholesterol biosynthesis and metabolism (Wiki pathway and GO) were significantly affected. Figure 5 As shown in b, the "upstream transcription factor" analysis of IPA predicted a series of upstream transcription factors that are thought to be regulated by treatment with compound CJ28. In particular, it was predicted that the transcriptional activation of nuclear receptor subfamily 5 group A member 1 (NR5A1) and sterol response element binding factor 1 / 2 (SREBF1 / 2) would be significantly inhibited. NR5A1, known as steroidogenic factor-1 (SF-1), is a transcription factor belonging to the nuclear receptor superfamily that is known to comprehensively regulate multiple steroidogenic genes and is essential for the development and normal function of steroidogenic tissues (e.g., the adrenal cortex). SREBF1 and 2 (known as sterol response element binding proteins) are transcriptional regulators that coordinately control the expression of various genes that mediate lipid metabolism. Consistent with the prediction that the activity of these transcription factors would be inhibited, the mRNA levels of NR5A1 and SREBF1 were significantly reduced by treatment with compound CJ28 ( Figure 5 b). Figure 5 As shown in Figure c, more than two-thirds of the DEGs involved in the above major biomolecular pathways were identified as target genes of these transcription factors; 14 of the 21 DEGs related to steroid / cortisol biosynthesis and secretion, and 21 of the 27 DEGs related to cholesterol synthesis and metabolism were previously reported as transcriptional targets of NR5A1 or SREBF. Figure 5 As shown in Figure d, most target genes showed significantly reduced mRNA expression in response to treatment with compound CJ28, except for NR0B encoding DAX-1 (a transcription factor known to inhibit steroidogenesis). Overall, transcriptome analysis showed that treatment with compound CJ28 was able to simultaneously inhibit the biosynthesis of corticosteroids, including cortisol and cholesterol, by affecting the transcriptional regulatory network that regulates related molecular pathways in human adrenal cortex cells.

[0156] <Example 5> Co-inhibition of Cholesterol and Cortisol Biosynthesis by CJ28

[0157] Transcriptome analysis showed that compound CJ28 inhibited both the cortisol and cholesterol biosynthesis pathways by altering transcriptional networks. Therefore, we investigated whether benzimidazolyl urea derivatives could alter the expression profiles of key genes regulating intracellular cholesterol levels. The results are shown in Figure 6 middle.

[0158] Cholesterol biosynthesis begins with acetate / ester and proceeds through the sequential action of multiple enzymes in the endoplasmic reticulum of steroidogenic cells, including the adrenal cortex. Since cholesterol is a precursor of steroids, the intracellular cholesterol concentration is directly related to the efficiency of steroid synthesis. Figure 6 As shown in Figure a, when treated with 30 μM compound CJ28 for 24 hours, the mRNA levels of key cholesterol biosynthesis genes (e.g., HMGCS, MVK, FDPS, FDFT1, SQLE, LSS, and DHCR7) were significantly reduced. In addition, treatment with compound CJ25 significantly reduced the mRNA expression of HMGCS1, MKV, FDPS, and LSS, but compound CJ22 had little effect on the expression of these genes. In contrast, all benzimidazolyl urea derivatives strongly reduced the expression of ABCA1 mRNA, which encodes ATP-binding cassette subfamily A member 1, a key factor mediating cholesterol intracellular transport. These results suggest that the basic structure of benzimidazolyl urea derivatives can affect multiple stages of cholesterol homeostasis.

[0159] To test this hypothesis, we investigated whether compound CJ28 affects intracellular cholesterol levels in H295R cells, and the results are shown in Figure 6 b.

[0160] refer to Figure 6 b, Consistent with the gene expression profile, compound CJ28 significantly reduced total cholesterol content in adrenal cortical cells, reducing levels to approximately half of the control group when treated for up to 48 hours. Although the slow onset of action of compound CJ28 differs from that of the cholesterol biosynthesis inhibitors U1666A and AY9944, these inhibitors, which inhibit specific enzymes, reduced intracellular cholesterol levels more rapidly after 24 hours of treatment, and this reduction persisted for 48 hours thereafter. In contrast, the steroid synthase inhibitor metyrapone had little effect on intracellular cholesterol levels.

[0161] like Figure 6 As shown in (c), all of these inhibitors significantly attenuated cortisol production. In particular, CJ28 induced a sustained and robust suppression after 48 h of treatment.

[0162] Next, a test was conducted to determine whether inhibition of cholesterol biosynthesis could affect the mRNA levels of key steroidogenic genes to determine the effect of compound CJ28 on the transcriptional network of cholesterol synthesis and steroidogenesis, and the results are shown in Figure 6 d.

[0163] like Figure 6As shown in Figure d, consistent with previous results, compound CJ28 significantly reduced the mRNA expression of key steroidogenic genes (including CYP11A1, STAR, HSD3B2 and CYP17A1), but this effect was not observed when treated with U18666A and AY9944. In addition, metyrapone had no inhibitory effect on the expression of steroidogenic genes. In summary, these results show that compound CJ28 reduces cortisol levels by comprehensively regulating the expression of genes encoding key enzymes involved in cholesterol and cortisol biosynthesis at the upstream transcription factor level. Considering that cholesterol is a common precursor of steroid hormones, the unique mode of action of compound CJ28 suggests that it can be an effective and long-term cortisol production inhibition strategy for preventing, alleviating and treating various pathological conditions caused by excessive cortisol. It is also an important preventive strategy for hypercholesterolemia, which is a representative side effect caused by existing steroidogenic inhibitors.

[0164] Although the specific parts of the present disclosure have been described in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments and the scope of the present disclosure is not limited thereto. Therefore, the true scope of the present disclosure will be defined by the appended claims and their equivalents.

[0165] The scope of the present disclosure is indicated by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present disclosure.

Claims

1. A benzimidazolyl urea derivative represented by the following Chemical Formula 1: [Chemical Formula 1] in, In the chemical formula 1, R is hydrogen, halogen, (C1-C4) alkyl, tetrafluoromethyl (CF3), (C1-C4) alkoxy, amino (NH2), nitrogen dioxide (NO2), cyano (CN), cyanomethyl (CH2CN), phenyl, benzyl or naphthyl.

2. The benzimidazolyl urea derivative according to claim 1, wherein In the Chemical Formula 1, R is substituted at the meta position.

3. The benzimidazolyl urea derivative according to claim 1, wherein In the chemical formula 1, R is any halogen selected from the group consisting of fluorine, chlorine, bromine and iodine.

4. A pharmaceutical composition for preventing or treating hypercortisolism, comprising a benzimidazolyl urea derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] in, In the chemical formula 1, R is hydrogen, halogen, (C1-C4) alkyl, tetrafluoromethyl (CF3), (C1-C4) alkoxy, amino (NH2), nitrogen dioxide (NO2), cyano (CN), cyanomethyl (CH2CN), phenyl, benzyl or naphthyl.

5. The pharmaceutical composition according to claim 4, wherein In the Chemical Formula 1, R is substituted at the meta position.

6. The pharmaceutical composition according to claim 4, wherein In the chemical formula 1, R is any halogen selected from the group consisting of fluorine, chlorine, bromine and iodine.

7. The pharmaceutical composition according to claim 4, wherein The composition inhibits corticosteroid production and cholesterol biosynthesis.

8. The pharmaceutical composition according to claim 4, wherein The hypercortisolism is selected from the group consisting of Cushing's syndrome, Cushing's disease and ectopic ACTH syndrome.

9. A health functional food composition for preventing or improving hypercortisolism, comprising a benzimidazolyl urea derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] in, In the chemical formula 1, R is hydrogen, halogen, (C1-C4) alkyl, tetrafluoromethyl (CF3), (C1-C4) alkoxy, amino (NH2), nitrogen dioxide (NO2), cyano (CN), cyanomethyl (CH2CN), phenyl, benzyl or naphthyl.

10. The health functional food composition according to claim 9, wherein The composition inhibits corticosteroid production and cholesterol biosynthesis.

11. The health functional food composition according to claim 9, wherein The health functional food composition is in the form of powder, granules, tablets, capsules, syrups or beverages.

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