WO2018199727A1 - Pharmaceutical composition, containing nm23 activator, for inhibiting cancer metastasis - Google Patents

Pharmaceutical composition, containing nm23 activator, for inhibiting cancer metastasis Download PDF

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Publication number
WO2018199727A1
WO2018199727A1 PCT/KR2018/005036 KR2018005036W WO2018199727A1 WO 2018199727 A1 WO2018199727 A1 WO 2018199727A1 KR 2018005036 W KR2018005036 W KR 2018005036W WO 2018199727 A1 WO2018199727 A1 WO 2018199727A1
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cancer
formula
pharmaceutical composition
metastasis
compound
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PCT/KR2018/005036
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French (fr)
Korean (ko)
Inventor
이공주
이희윤
이제진
서은경
이은선
김황석
이홍수
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이화여자대학교 산학협력단
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Priority claimed from KR1020180049329A external-priority patent/KR102034958B1/en
Application filed by 이화여자대학교 산학협력단 filed Critical 이화여자대학교 산학협력단
Priority to CN201880028168.XA priority Critical patent/CN110573147B/en
Priority to JP2019558495A priority patent/JP2020517710A/en
Priority to EP18789847.3A priority patent/EP3616692A4/en
Priority to US16/609,094 priority patent/US11389411B2/en
Publication of WO2018199727A1 publication Critical patent/WO2018199727A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon

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  • the present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising the novel Nm23 activator.
  • Cancer metastasis is one of the most important factors in determining the prognosis of cancer patients and is the main process of determining death from cancer. In the treatment of cancer, a lot of efforts have been made for the survival of patients such as surgery, radiation therapy, and chemotherapy, but efforts to improve the survival of cancer patients continue.
  • the field of studying cancer metastasis is one of the last strategies to overcome cancer, and the study of cancer metastasis suppressors is essential for developing metastasis suppressing drugs.
  • Nm23 is a gene encoding a protein involved in the development and differentiation of normal tissues, and a decrease in the expression of Nm23 has been reported in various metastatic cell lines.
  • the Nm23 protein which generally consists of 150 to 180 amino acids, comprises a leucine zipper motif and has a dinucleotide phosphate kinase (NDPK) activity.
  • NDPK dinucleotide phosphate kinase
  • Nm23-H1 has been found to play an important role in cancer metastasis and various other cellular mechanisms such as cell proliferation, embryonic development, differentiation, tumor formation, and the like.
  • 1 is a diagram showing the stages of cancer metastasis.
  • the metastasis of cancer begins at various stages, where cancer cells begin to infiltrate blood vessels in the primary tumor tissue, moving through the vessels and surviving to form new colonies at the secondary site. It happens through the process.
  • Nm23-H1 is involved in cancer metastasis inhibitory activity at various stages such as invasion / intravasation, extravasation, metastatic colonization, etc. in the process of cancer metastasis.
  • Horak CE et al., The role of metastasis suppressor genes in metastatic dormancy., APMIS. (2008) Jul-Aug; 116 (7-8): 586-601).
  • Nm23 affects cancer metastasis and development
  • NDPK Nucleotide diphosphate kinase
  • ATP is used to convert NDP (UDP, GDP, CDP) into NTP (UTP, GTP, CTP).
  • the protein that converts was found to be an enzyme that regulates the amount of NTP in the cell.
  • 2 is a schematic diagram showing NDPK activity of Nm23.
  • overexpression of Nm23-H1 has been shown to be closely associated with decreased cancer cell invasion (Lee E, et al., Multiple functions of Nm23-H1 are regulated by oxido-reduction system., PLoS One. (2009) ) Nov 23; 4 (11): e7949).
  • the present inventors completed the present invention by carrying out research and development on a small molecule substance that modulates the activity of Nm23 involved in the cancer metastasis process.
  • One object of the present invention to provide a pharmaceutical composition for inhibiting cancer metastasis comprising a novel activator of Nm23.
  • a pharmaceutical composition comprising a compound represented by Formula 1 of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient may act as an activator of Nm23-H1 and / or Nm23-H2 to metastasize cancer cells and Inhibits infiltration; Therefore, Nm23 activator according to the present invention can be very useful as a pharmaceutical composition for inhibiting metastasis of cancer.
  • 1 is a schematic diagram showing the stage of metastasis of cancer.
  • FIG. 2 is a schematic diagram showing NDPK activity of Nm23.
  • Figure 3 is a diagram confirming the NDPK activity for the compound of formula 2-1.
  • 5 is a diagram showing the results of plasma resonance analysis.
  • FIG. 6 is a diagram illustrating the change in cell morphology and treatment of Rac1 activity in each cell line by treating the compound of formula 2-1 to two breast cancer cell lines.
  • FIG. 7 is a diagram verifying the inhibition of invasion and migration of the compound of formula 2-1 on MDA-MB-231 cells.
  • FIG. 8 is a diagram showing the metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model.
  • FIG. 9 is a diagram showing the volume of cancer, and mouse weight change according to the treatment of the compound of formula 2-1.
  • FIG. 10 is a diagram showing metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model.
  • the present inventors completed the present invention by uncovering compounds that enhance the NDPK activity of Nm23.
  • One embodiment of the present invention is a pharmaceutical composition for inhibiting cancer metastasis comprising a compound represented by the following formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Ring A is cyclohexene, cyclohexane or benzene.
  • the compound represented by Chemical Formula 1 is a compound represented by any one of the following Chemical Formulas 2 to 4.
  • Stereoisomers in the present invention may exist as optical isomers, racemates, racemic mixtures, single enantiomers, diastereomeric mixtures and respective diastereomers. Such isomers can be separated by conventional techniques such as column chromatography or HPLC. Alternatively, the stereoisomers of each of the compounds represented by Formula 1 can be stereospecifically synthesized using optically pure starting materials and / or reagents in known arrangements.
  • the stereoisomer of the compound represented by Formula 1 is E form.
  • the compound represented by Chemical Formula 1 may be represented by any one of the following Chemical Formulas 2-1 to 4-1.
  • the compounds of Formulas 2-1 to 4-1 may be prepared by, for example, the process shown in Preparation Examples 1 to 3 of the present invention, but is not limited thereto.
  • pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, inorganic ions, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, prepared with calcium, potassium, sodium and magnesium, Inorganic acid salts prepared with iodic acid, perchloric acid and sulfuric acid; Acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid Organic acid salts prepared with acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid and the like; Sulfonic acid salts prepared with methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-tolu
  • the pharmaceutical composition of the present invention may act as an activator of Nm23-H1, Nm23-H2 or Nm23-H1 and Nm23-H2.
  • the compound of formula 1 of the present invention interacts with Nm23-H1 to form a hydrophobic pocket to expose the active site of Nm23-H1 and kpn-loop, which is known to be most important for regulating NDPK activity. And allostericly increase NDPK activity.
  • the compound of formula 1 of the present invention may exhibit the same activity for Nm23-H2, which is another subtype of Nm23 (FIG. 3 and Table 2).
  • the compound represented by Formula 1 of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof may further promote the activity of Nm23 in the presence of ATP (FIG. 4).
  • the cancer is breast cancer, lung cancer, melanoma, prostate cancer, colon cancer, bladder cancer, bone cancer, blood cancer, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, stomach cancer, It may be any one selected from the group consisting of tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, anal muscle cancer, central nervous system tumor, liver cancer and colon cancer.
  • the pharmaceutical composition of the present invention does not increase the efficacy, but may further include ingredients that are commonly used in the pharmaceutical composition to improve the smell, taste, time and the like.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive.
  • Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, Hydroxypropyl cellulose, opiodry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc, but are not limited thereto.
  • the pharmaceutical composition may further include one or more active ingredients exhibiting the same or similar medicaments in addition to the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and can be formulated for human or veterinary use for oral or parenteral use.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants can be used.
  • Solid form preparations for oral administration include tablets, pills, powders, granules and capsules and the like, and such solid form preparations may be used in pharmaceutical compositions comprising the compounds of the present invention at least one excipient such as starch, calcium carbonate. , Can be mixed with sucrose, lactose or gelatin.
  • Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.
  • injectable esters such as ethyl oleate
  • suppositories witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method, and may be external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection during parenteral administration. Injection may be selected, but is not limited thereto.
  • composition of the present invention may be used alone, but may be used in combination with various cancer treatment methods, such as radiation therapy and chemotherapy, in order to increase the treatment efficiency.
  • Another embodiment of the present invention is a method for inhibiting cancer metastasis comprising administering a therapeutically effective amount of a composition comprising a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  • Another embodiment of the present invention is a use for the manufacture of a drug for inhibiting cancer metastasis of the compound represented by the formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition of the present invention can be administered to a subject to inhibit cancer metastasis.
  • the term "individual” has a disease caused by cancer or a direct or indirect cause thereof, and includes a human including a disease whose symptoms may be improved by administering the pharmaceutical composition of the present invention. , But not limited to mammals such as sheep, pigs, goats, and dogs.
  • the term "administration" means introducing a pharmaceutical composition of the present invention to a subject in any suitable manner.
  • the route of administration can be administered orally or parenterally via any route as long as it can reach the desired tissue.
  • the pharmaceutical composition of the present invention may be administered by any device that allows migration to target cells.
  • the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is weight, sex, age, health condition, severity of the patient. , The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently, and other factors well known in the medical arts.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with the conventional therapeutic agent in combination administration. It may be single or multiple doses.
  • NBuLi (2.48 M in Hx) (1.95 equiv) was added to tetrahydrofuran in which diethyl 3,4-dimethoxybenzylphosphonate (2.0 equiv) was dissolved at 0 ° C.
  • the mixture was mixed at room temperature for 30 minutes and then lowered to 0 ° C. to (1 S , 2 S ) -2- (3,4-dimethoxyphenyl) cyclohex-3-enecarbaldehyde (1.0 equiv) DMPU (5.0 equiv) was dissolved in THF and added using cannula.
  • the mixture was mixed overnight at room temperature and then the reaction was terminated with ammonium chloride and water.
  • the mixture was extracted with ethyl acetate, dried over MgSO 4 , concentrated and separated on silica gel using cylindrical chromatography.
  • Formula 2-1 may also be prepared by the following Scheme 5.
  • o-bromobenzaldehyde (1.0 equiv) was dissolved in DMF, followed by 3,4-dimethoxyphenylboronic acid (1.0 equiv), 2 M Na 2 CO 3 (3.0 equiv) and Pd (PPh 3 ) 4 (0.01 equiv) ) was added at room temperature.
  • the mixture was mixed overnight at 80 ° C. and diluted with ethyl acetate. Then, the reaction was terminated by adding ammonium chloride and water. Then, the mixture was extracted with ethyl acetate and the organic layer was dried over MgSO 4 . The mixture was filtered to concentrate the combined organic layers, and then separated on silica gel using cylindrical chromatography.
  • NDPK assay buffer 20 mM HEPES, 3 mM MgCl 2
  • test substance compound 2-1, 3-1 or 4-1 compound
  • Cultured and cell based NDPK analysis was performed.
  • Cell lysates obtained from 5,000 K MDA-MB-231 cells lysed with protease inhibitor cocktail and NDPK assay buffer were centrifuged at 8,000 rpm for 10 minutes at 4 ° C. 40 ⁇ L of the lysate was incubated with the test material for 5 minutes and then reacted with NDPK by adding 50 ⁇ M UDP.
  • ATP consumption was assessed by the ATP Decision Kit (Molecular probe, USA).
  • ITC experiments were performed using the ITC200 instrument (Malvern Inc.) and data were analyzed using the ORIGIN 7.0 program.
  • the concentration of Nm23-H1 protein in the cells was 30 ⁇ M and the syringe contained 300 ⁇ M of activator or 1 mM ATP.
  • the active agent was prepared in DMSO (Dimethyl sulfoxide) at a concentration of 50 mM and stored at -20 ° C.
  • ITC buffer contained 20 mM HEPES at pH 7.5, 150 mM NaCl, 3 mM MgCl 2 and up to 2% DMSO. Titration was injected 20 times, 2 ⁇ L each at 25 ° C., and 150 sec intervals.
  • the titration data is a nonlinear least-squares-fitting algorithm with three floating variables: stoichiometry (N), dissociation constant (KD), and change of enthalpy of interaction. curve-fitting algorithm).
  • Nm23-H1 (1 mg / mL) in 10 mM SA buffer at pH 4.5 was quenched at 20 ⁇ L / min on a carboxymethyl dextran hydrogel (CMDH) surface sensor chip (Reichert Technologies, Depew, NY). While standard amino coupling was used until saturation was achieved.
  • CMDH carboxymethyl dextran hydrogel
  • K D is a value obtained by calculating K d / K a .
  • NMac1 is a diagram confirming the activity of NDPK to the compound of formula 2-1.
  • the compound of formula 2-1 in each figure is represented by NMac1.
  • NDPK activity of Nm23-H1 is increased according to the concentration of the compound of Chemical Formula 2-1.
  • Km-type activity exhibited the same tendency as the existing enzyme activator.
  • the NDPK activator increases the activity of NDPK by increasing the affinity for the substrate NTP.
  • the cell-based NDPK activity is increased with the concentration of the compound of formula 2-1.
  • FIG. 4 is a diagram showing the results of performing isothermal titration calorimetry (isothermal calorimetry) on the compound of formula 2-1.
  • isothermal calorimetry isothermal calorimetry
  • FIG. 5 is a diagram showing the results of plasma resonance analysis. As a result of Figure 5, it was confirmed that Nm23-H1 and the compound of the present invention is directly bonded.
  • MDA-MB-231 cells were cultured in 100 mm dishes and treated with the indicated concentrations of Formula 2-1 compound or 0.05% DMSO for 16 hours.
  • the active Rac1 pulldown assay was performed according to the manufacturer's instructions (Thermo Fisher Scientific).
  • MDA-MB-231 cells were cultured to 50-70% levels on Secureslip TM (Sigma) cell culture glass cover slips and then treated for various hours in the presence or absence of the compound of Formula 2-1.
  • the cells were gently washed with cold HBSS and then fixed in RBS with HBSS containing 4% paraformaldehyde at room temperature for 10 minutes. After washing with HBSS, infiltration with 0.1% Triton X-100 was performed at room temperature for 10 minutes, and after washing twice with HBSS, HBSS containing 3% BSA, 0.2% Tween 20 and 0.2% gelatin for 1 hour at room temperature. Blocking was performed, and the primary antibody was incubated at 37 ° C. for 2 hours, followed by 1 hour of incubation with a fluorochrome-conjugated species-specific secondary antibody.
  • the cells were treated with the compound of formula 2-1 for 16 hours when the cells were 70% filled. 1 x 10 5 cells were suspended in media without FBS and added over a Boyden chamber membrane. Culture medium containing 10% FBS was added to the membrane bottom. The chamber was incubated at 37 ° C., 5% CO 2 for 4 hours. The migrated cells were fixed and stained with crystal violet / methanol. Photos were taken and the migrated cells were counted and normalized to control cells.
  • FIG. 6 is a diagram illustrating the change in cell morphology and treatment of Rac1 activity in each cell line by treating the compound of formula 2-1 to two breast cancer cell lines.
  • FIG. 6A illustrates a case where the compound of Formula 2-1 was treated using confocal microscopy, and when the F-actin change of MDA-MB-231 cells was observed, the ruffle was reduced. Increasing contact between cells can be seen. This means inhibiting the activity of Rac1 involved in cell migration of Nm23-H1 and reducing cell mobility.
  • Figure 6B is a diagram measuring the change in shape and the activity of Rac1 according to the compound of formula 2-1 in MDA-MB-231 cells, a triple negative breast cancer cell line.
  • the pull-down assay of the activated Rac1 it can be seen that the compound of formula 2-1 of the present invention lowers the activity of Rac1 in a concentration-dependent manner.
  • FIG. 6C and FIG. 6D it can be seen that disappearing by knockdown of Nm23-H1 inhibits Rac1 activity through Nm23-H1.
  • Figure 7 is a diagram verifying the inhibition of infiltration and migration of the compound of formula 2-1 to MDA-MB-231 cells.
  • Figure 7A is a diagram showing the results of infiltration analysis, transwell migration analysis in MDA-MB-231 cells, a triple negative breast cancer cell line.
  • FIG. 7B is a diagram showing Nm23-H1 activity of the compound of formula 2-1 in A549 cells, a lung cancer cell line. Referring to the results of FIG. 7B, it can be seen that the compound of Formula 2-1 of the present invention reduces the wound healing and Matrigel infiltration by concentration in A549 cells.
  • FIG. 7C is a diagram showing Nm23-H1 and Nm23-H2 activities of the compound of formula 2-1.
  • FIG. 7C when the effects of the compounds of Formula 2-1 are all reduced by knockdown of Nm23-H1 and Nm23-H2, the in vitro metastasis inhibitory activity of Formula 2-1 is determined via Nm23-H1 and Nm23-H2. You can see what happens.
  • the metastasis inhibitory activity of the compound of formula 2-1 of the present invention was applied to a breast cancer metastasis model using MDA-MB-231 luc cells in order to confirm in a real animal model. After injection of MDA-MB-231 cells into the mammary fat pad of nude mice, when the tumor size reached 100 mm, the compound of formula 2-1 was injected daily at 10 mg / kg. The metastasis to the lung was observed during the week.
  • FIG. 8 is a diagram showing metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model. As a result of Figure 8 it can be seen that the metastasis to the lung in the group treated with the compound of formula 2-1 of the present invention.
  • FIG. 9 is a view showing the changes in the volume and weight of cancer according to the treatment of the compound of formula 2-1. As a result of FIG. 9, it was confirmed that the volume of the cancer was reduced in the group treated with the compound of formula 2-1 of the present invention.
  • FIG. 10 is a diagram showing metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model. As a result of Figure 10, it was confirmed that the cells metastasized to the lung significantly reduced.
  • the compound represented by the formula (1) according to the present invention can be used as a pharmaceutical composition effective in inhibiting metastasis of cancer by increasing NDPK activity of Nm23.

Abstract

The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis, the pharmaceutical composition containing an activator compound of the cancer metastasis inhibitor Nm23, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient. The compound according to the present invention inhibits the metastasis of cancer cells by stimulating the activity of Nm23 associated with cancer metastasis, and thus can be favorably used as a pharmaceutical composition for inhibiting cancer metastasis.

Description

Nm23 활성제를 포함하는 암 전이 억제용 약학적 조성물Pharmaceutical composition for inhibiting cancer metastasis comprising Nm23 active agent
본 발명은 신규한 Nm23 활성제를 포함하는 암 전이 억제용 약학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for inhibiting cancer metastasis comprising the novel Nm23 activator.
암의 전이 (tumor metastasis)는 암 환자의 예후를 결정하는 가장 중요한 인자 중 하나로 암으로 인한 사망을 결정하는 주요 과정이다. 암의 치료에서 수술과 방사선 요법, 화학 요법 등 환자의 생존을 위해 많은 노력이 이루어지고 있지만 여전히 암 환자의 생존을 높이기 위한 노력은 계속되고 있다. 암 전이를 연구하는 분야는 암을 극복하는 마지막 전략 중 하나이고 암 전이 억제 단백질 (tumor metastasis suppressor)을 연구하는 것은 전이 억제 약물을 개발하는데 있어 필수적이다. Cancer metastasis is one of the most important factors in determining the prognosis of cancer patients and is the main process of determining death from cancer. In the treatment of cancer, a lot of efforts have been made for the survival of patients such as surgery, radiation therapy, and chemotherapy, but efforts to improve the survival of cancer patients continue. The field of studying cancer metastasis is one of the last strategies to overcome cancer, and the study of cancer metastasis suppressors is essential for developing metastasis suppressing drugs.
Nm23은 정상 조직의 발달 및 분화에 관여하는 단백질을 암호화하는 유전자로, 다양한 전이성 세포주에서 Nm23의 발현 감소가 보고되고 있다. 일반적으로 150 내지 180 개의 아미노산으로 구성되는 Nm23 단백질은 류우신 지퍼 모티프 (leucine zipper motif)를 포함하고 이인산 뉴클레오사이드 키나제 (nucleoside diphosphate kinase, NDPK) 활성을 갖는다. 특히, Nm23-H1은 암 전이 및 다른 다양한 세포 기작들, 예컨대 세포 증식, 배아 발달, 분화, 종양 형성 등에 중요한 역할을 담당하는 것으로 규명되었다. 도 1은 암의 전이가 이루어지는 단계를 나타낸 도이다. 암의 전이는 암 세포가 일차로 생성된 종양 조직 (primary tumor)에서 혈관을 침투하는 것을 시작으로 혈관을 통해 이동하고 생존하여 이차 지점 (secondary site)에서 새로운 콜로니 (colony)를 형성하는 여러 단계의 과정을 거쳐 일어난다. 상기 도 1을 보면, Nm23-H1가 암 전이의 과정에서 침윤 (Invasion)/ 혈관 침투 (Intravasation)와 혈관외유출 (Extravasation), 전이성 콜로니화 (Metastatic colonization) 등 여러 단계에서 암 전이 억제 활성에 관여하고 있는 것을 확인할 수 있다 (Horak CE, et al., The role of metastasis suppressor genes in metastatic dormancy., APMIS. (2008) Jul-Aug;116(7-8):586-601).Nm23 is a gene encoding a protein involved in the development and differentiation of normal tissues, and a decrease in the expression of Nm23 has been reported in various metastatic cell lines. The Nm23 protein, which generally consists of 150 to 180 amino acids, comprises a leucine zipper motif and has a dinucleotide phosphate kinase (NDPK) activity. In particular, Nm23-H1 has been found to play an important role in cancer metastasis and various other cellular mechanisms such as cell proliferation, embryonic development, differentiation, tumor formation, and the like. 1 is a diagram showing the stages of cancer metastasis. The metastasis of cancer begins at various stages, where cancer cells begin to infiltrate blood vessels in the primary tumor tissue, moving through the vessels and surviving to form new colonies at the secondary site. It happens through the process. Referring to FIG. 1, Nm23-H1 is involved in cancer metastasis inhibitory activity at various stages such as invasion / intravasation, extravasation, metastatic colonization, etc. in the process of cancer metastasis. (Horak CE, et al., The role of metastasis suppressor genes in metastatic dormancy., APMIS. (2008) Jul-Aug; 116 (7-8): 586-601).
Nm23이 암 전이 및 발달에 영향을 미치는 기작은 아직까지 명확하게 밝혀지지 않았으나, NDPK (Nucleotide diphosphate kinase)로서, ATP를 이용하여 NDP (UDP, GDP, CDP)를 NTP (UTP, GTP, CTP)로 전환하는 단백질로 세포 내 NTP의 양을 조절하고 있는 효소로 밝혀졌다. 도 2는 Nm23의 NDPK 활성을 나타낸 모식도이다. 또한, Nm23-H1의 과발현이 암 세포의 침윤 감소와 밀접한 연관을 갖는다고 밝혀진바 있다 (Lee E, et al., Multiple functions of Nm23-H1 are regulated by oxido-reduction system., PLoS One. (2009) Nov 23; 4(11):e7949). The mechanism by which Nm23 affects cancer metastasis and development is not yet clear, but as NDPK (Nucleotide diphosphate kinase), ATP is used to convert NDP (UDP, GDP, CDP) into NTP (UTP, GTP, CTP). The protein that converts was found to be an enzyme that regulates the amount of NTP in the cell. 2 is a schematic diagram showing NDPK activity of Nm23. In addition, overexpression of Nm23-H1 has been shown to be closely associated with decreased cancer cell invasion (Lee E, et al., Multiple functions of Nm23-H1 are regulated by oxido-reduction system., PLoS One. (2009) ) Nov 23; 4 (11): e7949).
이러한 발견을 토대로 Nm23의 발현을 증가시키거나, 세포 침투성 (cell permeable) Nm23-H1을 처리하는 방향으로 연구가 진행되어 왔다. 구체적으로, MPA (Medroxyprogesterone acetate)를 처리하면 Nm23-H1의 발현 양이 증가하는 것을 확인하였고 이러한 현상은 MPA 처리에 의해 암 전이가 억제되는 메커니즘으로 이해되고 있다 (Palmieri D et al., Medroxyprogesterone acetate elevation of Nm23-H1 metastasis suppressor expression in hormone receptor-negative breast cancer., J Natl Cancer Inst. (2005) May 4;97(9):632-42). 그러나, MPA의 처리는 Nm23-H1의 수준을 높이는 것 이외에도 예상치 못한 세포 내 반응을 일으키기 때문에, MPA는 약물로서 사용되지 못하고 있다.Based on these findings, studies have been conducted to increase the expression of Nm23 or to treat cell permeable Nm23-H1. Specifically, it was confirmed that the treatment of MPA (Medroxyprogesterone acetate) increases the expression level of Nm23-H1 and this phenomenon is understood as a mechanism of inhibiting cancer metastasis by MPA treatment (Palmieri D et al., Medroxyprogesterone acetate elevation). of Nm23-H1 metastasis suppressor expression in hormone receptor-negative breast cancer., J Natl Cancer Inst. (2005) May 4; 97 (9): 632-42). However, MPA has not been used as a drug because treatment of MPA causes unexpected intracellular responses in addition to raising the level of Nm23-H1.
또한, 최근에는 세포 침투성 Nm23-H1을 이용하여 암의 전이를 억제하는 방법이 제안되었다 (Lim J, et al., Cell-permeable NM23 blocks the maintenance and progression of established pulmonary metastasis. Cancer Res. (2011) Dec 1; 71 (23): 7216-25). 세포 침투성 Nm23-H1의 경우에는 원형질막을 통과할 수 있는 운반자 펩타이드 (transporter peptide)를 융합하는 방식으로 세포에 투입시켰다. 그 결과, 세포 침투성 Nm23-H1 처리에 의해 암 전이 억제 활성이 나타나는 것을 확인하였다. 다만, 이러한 세포 침투성 Nm23-H1이 단백질 의약품으로 적용되기까지는 생체 내 안정성을 극복해야 하는 과제가 남아있고, 암 전이 억제 약물은 단기간 처리로 큰 효과를 볼 수 없기 때문에 단백질 의약품과 같은 고비용의 약제를 선택할 수 없다는 현실적인 문제를 가지고 있다. Recently, a method of inhibiting cancer metastasis using cell permeable Nm23-H1 has been proposed (Lim J, et al., Cell-permeable NM23 blocks the maintenance and progression of established pulmonary metastasis. Cancer Res. (2011) Dec 1; 71 (23): 7216-25). In the case of cell permeability Nm23-H1 was introduced into the cell by fusion of a transporter peptide (transporter peptide) that can pass through the plasma membrane. As a result, it was confirmed that cancer metastasis inhibiting activity was exhibited by cell permeability Nm23-H1 treatment. However, until such cell-permeable Nm23-H1 is applied as a protein drug, it remains a challenge to overcome the stability in vivo, and since cancer metastasis suppressing drug does not show a great effect with a short-term treatment, it is necessary to use a high-cost drug such as protein drug. It has a realistic problem of not being able to choose.
본 발명자들은 암 전이 과정에 관여하는 Nm23의 활성을 조절하는 저분자 물질에 관한 연구 개발을 수행하여 본 발명을 완성하였다. The present inventors completed the present invention by carrying out research and development on a small molecule substance that modulates the activity of Nm23 involved in the cancer metastasis process.
본 발명의 하나의 목적은 신규한 Nm23의 활성제를 포함하는 암 전이 억제용 약학적 조성물을 제공하는 것이다. One object of the present invention to provide a pharmaceutical composition for inhibiting cancer metastasis comprising a novel activator of Nm23.
본 발명의 화학식 1로 표시되는 화합물, 이의 입체이성질체 또는 이의 약제학적으로 허용 가능한 염을 유효성분으로 포함하는 약학적 조성물은 Nm23-H1 및/또는 Nm23-H2의 활성제로 작용하여 암 세포의 전이 및 침윤을 억제한다. 따라서 본 발명에 따른 Nm23 활성제는 암의 전이 억제용 약학적 조성물로 매우 유용하게 활용될 수 있다. A pharmaceutical composition comprising a compound represented by Formula 1 of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient may act as an activator of Nm23-H1 and / or Nm23-H2 to metastasize cancer cells and Inhibits infiltration; Therefore, Nm23 activator according to the present invention can be very useful as a pharmaceutical composition for inhibiting metastasis of cancer.
도 1은 암의 전이가 이루어지는 단계를 나타낸 모식도이다. 1 is a schematic diagram showing the stage of metastasis of cancer.
도 2는 Nm23의 NDPK 활성을 나타낸 모식도이다. 2 is a schematic diagram showing NDPK activity of Nm23.
도 3은 화학식 2-1 화합물에 대한 NDPK 활성을 확인한 도이다.Figure 3 is a diagram confirming the NDPK activity for the compound of formula 2-1.
도 4는 화학식 2-1 화합물에 대한 등온선 열량 측정법 (isothermal calorimetry)을 수행한 결과를 나타낸 도이다. 4 is a view showing the results of performing isothermal calorimetry (isothermal calorimetry) for the compound of formula 2-1.
도 5는 플라즈마 공명 분석의 결과를 나타낸 도이다. 5 is a diagram showing the results of plasma resonance analysis.
도 6은 화학식 2-1 화합물을 두 가지의 유방암 세포주 (breast cancer cell line)에 처리하여 세포 모양 변화를 관찰하고 각각의 세포주에서 Rac1 활성을 관찰한 도이다. 6 is a diagram illustrating the change in cell morphology and treatment of Rac1 activity in each cell line by treating the compound of formula 2-1 to two breast cancer cell lines.
도 7은 MDA-MB-231 세포에 대한 화학식 2-1 화합물의 침윤 (invasion) 및 이동 (migration) 억제를 검증한 도이다.7 is a diagram verifying the inhibition of invasion and migration of the compound of formula 2-1 on MDA-MB-231 cells.
도 8은 유방암 전이 모델 (breast cancer metastasis model)에서의 화학식 2-1 화합물의 전이 억제 활성을 나타낸 도이다. 8 is a diagram showing the metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model.
도 9는 화학식 2-1 화합물의 처리에 따른 암의 부피, 및 마우스 체중 변화를 나타낸 도이다. 9 is a diagram showing the volume of cancer, and mouse weight change according to the treatment of the compound of formula 2-1.
도 10은 유방암 전이 모델에서의 화학식 2-1 화합물의 전이 억제 활성을 나타낸 도이다.10 is a diagram showing metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model.
본 발명자들은 Nm23의 NDPK 활성을 높이는 화합물을 밝힘으로써 본 발명을 완성하였다. The present inventors completed the present invention by uncovering compounds that enhance the NDPK activity of Nm23.
이하, 본 명세서에 대하여 더욱 상세하게 설명한다.Hereinafter, this specification is demonstrated in detail.
본 명세서에서 어떤 부분이 어떤 구성요소를 "포함" 한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다. In the present specification, when a part "contains" a certain component, this means that the component may further include other components, except for the case where there is no contrary description.
본 발명의 하나의 양태는, 하기 화학식 1로 표시되는 화합물, 이의 입체이성질체 또는 이의 약제학적으로 허용 가능한 염을 유효성분으로 포함하는 암 전이 억제용 약학적 조성물이다. One embodiment of the present invention is a pharmaceutical composition for inhibiting cancer metastasis comprising a compound represented by the following formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient.
[화학식 1][Formula 1]
Figure PCTKR2018005036-appb-I000001
Figure PCTKR2018005036-appb-I000001
화학식 1에 있어서, In Chemical Formula 1,
고리 A는 사이클로헥센 (cyclohexene), 사이클로헥산 (cyclohexane) 또는 벤젠 (benzene) 이다. Ring A is cyclohexene, cyclohexane or benzene.
본 발명의 일 구현예에 있어서, 상기 화학식 1로 표시되는 화합물은 하기 화학식 2 내지 4 중 어느 하나로 표시되는 화합물이다. In one embodiment of the present invention, the compound represented by Chemical Formula 1 is a compound represented by any one of the following Chemical Formulas 2 to 4.
[화학식 2] [Formula 2]
Figure PCTKR2018005036-appb-I000002
Figure PCTKR2018005036-appb-I000002
[화학식 3][Formula 3]
Figure PCTKR2018005036-appb-I000003
Figure PCTKR2018005036-appb-I000003
[화학식 4][Formula 4]
Figure PCTKR2018005036-appb-I000004
Figure PCTKR2018005036-appb-I000004
본 발명에서 입체이성질체 (stereoisomer)는 광학이성질체, 라세미체, 라세믹 혼합물, 단일의 이넨티오머, 부분입체이성체 혼합물 및 각각의 부분입체이성체로서 존재할 수 있다. 이러한 이성질체는 종래기술, 예를 들어 관 크로마토그래피 또는 HPLC 등의 분할에 의해 분리가 가능하다. 또는, 화학식 1로 표시되는 화합물 각각의 입체이성질체는 공지된 배열의 광학적으로 순수한 출발 물질 및/또는 시약을 사용하여 입체 특이적으로 합성할 수 있다. Stereoisomers in the present invention may exist as optical isomers, racemates, racemic mixtures, single enantiomers, diastereomeric mixtures and respective diastereomers. Such isomers can be separated by conventional techniques such as column chromatography or HPLC. Alternatively, the stereoisomers of each of the compounds represented by Formula 1 can be stereospecifically synthesized using optically pure starting materials and / or reagents in known arrangements.
본 발명의 또 하나의 구현예에 있어서, 상기 화학식 1로 표시되는 화합물의 입체이성질체는 E form이다. In another embodiment of the present invention, the stereoisomer of the compound represented by Formula 1 is E form.
본 발명의 일 구현예에 있어서, 상기 화학식 1로 표시되는 화합물은 하기 화학식 2-1 내지 4-1 중 어느 하나로 표시될 수 있다. In one embodiment of the present invention, the compound represented by Chemical Formula 1 may be represented by any one of the following Chemical Formulas 2-1 to 4-1.
[화학식 2-1][Formula 2-1]
Figure PCTKR2018005036-appb-I000005
Figure PCTKR2018005036-appb-I000005
[화학식 3-1][Formula 3-1]
Figure PCTKR2018005036-appb-I000006
Figure PCTKR2018005036-appb-I000006
[화학식 4-1][Formula 4-1]
Figure PCTKR2018005036-appb-I000007
Figure PCTKR2018005036-appb-I000007
상기 화학식 2-1 내지 4-1의 화합물은, 예컨대 본 발명의 제조예 1 내지 3에 제시된 공정을 통해 제조될 수 있으나, 이에 제한되는 것은 아니다.The compounds of Formulas 2-1 to 4-1 may be prepared by, for example, the process shown in Preparation Examples 1 to 3 of the present invention, but is not limited thereto.
본 발명에서, 약제학적으로 허용 가능한 염은 의약업계에서 통상적으로 사용되는 염을 의미하며, 예를 들어 칼슘, 포타슘, 소듐 및 마그네슘 등으로 제조된 무기이온염, 염산, 질산, 인산, 브롬산, 요오드산, 과염소산 및 황산 등으로 제조된 무기산염; 아세트산, 트라이플루오로아세트산, 시트르산, 말레인산, 숙신산, 옥살산, 벤조산, 타르타르산, 푸마르산, 만데르산, 프로피온산, 젖산, 글리콜산, 글루콘산, 갈락투론산, 글루탐산, 글루타르산, 글루쿠론산, 아스파르트산, 아스코르브산, 카본산, 바닐릭산, 하이드로 아이오딕산 등으로 제조된 유기산염; 메탄설폰산, 에탄설폰산, 벤젠설폰산, p-톨루엔설폰산, 나프탈렌설폰산 등으로 제조된 설폰산염; 글리신, 아르기닌, 라이신 등으로 제조된 아미노산염; 및 트리메틸아민, 트라이에틸아민, 암모니아, 피리딘, 피콜린 등으로 제조된 아민염 등이 있으나, 열거된 이들 염에 의해 본 발명에서 의미하는 염의 종류가 한정되는 것은 아니다.In the present invention, pharmaceutically acceptable salts mean salts commonly used in the pharmaceutical industry, for example, inorganic ions, hydrochloric acid, nitric acid, phosphoric acid, bromic acid, prepared with calcium, potassium, sodium and magnesium, Inorganic acid salts prepared with iodic acid, perchloric acid and sulfuric acid; Acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid Organic acid salts prepared with acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid and the like; Sulfonic acid salts prepared with methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid and the like; Amino acid salts prepared with glycine, arginine, lysine and the like; And amine salts made of trimethylamine, triethylamine, ammonia, pyridine, picoline, and the like, but the salts used in the present invention are not limited by these salts.
또한, 본 발명의 약학적 조성물은 Nm23-H1, Nm23-H2 또는 Nm23-H1과 Nm23-H2의 활성제로 작용할 수 있다. 구체적으로, 본 발명의 화학식 1의 화합물은 Nm23-H1과 상호 작용을 통하여, 소수성 포켓 (hydrophobic pocket)을 형성하여 Nm23-H1의 활성 부위와 NDPK 활성 조절에 가장 중요하다고 알려져 있는 kpn-loop의 노출을 조절하고, 알로스테릭 (allosteric)하게 NDPK 활성을 증가시킬 수 있다. 또한, 본 발명의 화학식 1의 화합물은 Nm23의 다른 서브 타입인 Nm23-H2에 대해서도 같은 활성을 나타낼 수 있다 (도 3 및 표 2).In addition, the pharmaceutical composition of the present invention may act as an activator of Nm23-H1, Nm23-H2 or Nm23-H1 and Nm23-H2. Specifically, the compound of formula 1 of the present invention interacts with Nm23-H1 to form a hydrophobic pocket to expose the active site of Nm23-H1 and kpn-loop, which is known to be most important for regulating NDPK activity. And allostericly increase NDPK activity. In addition, the compound of formula 1 of the present invention may exhibit the same activity for Nm23-H2, which is another subtype of Nm23 (FIG. 3 and Table 2).
본 발명의 상기 화학식 1로 표시되는 화합물, 이의 입체이성질체 또는 이의 약제학적으로 허용 가능한 염은 ATP 존재 하에서 Nm23의 활성을 더욱 촉진시킬 수 있다 (도 4). The compound represented by Formula 1 of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof may further promote the activity of Nm23 in the presence of ATP (FIG. 4).
본 발명의 일 구현예에서, 상기 암은 유방암, 폐암, 흑색종, 전립선암, 대장암, 방광암, 뼈암, 혈액암, 갑상선암, 부갑상선암, 골수암, 직장암, 인후암, 후두암, 식도암, 췌장암, 위암, 설암, 피부암, 뇌종양, 자궁암, 두부 또는 경부암, 담낭암, 구강암, 결장암, 항문부근암, 중추신경계 종양, 간암 및 대장암으로 이루어진 군으로부터 선택된 어느 하나일 수 있다. In one embodiment of the invention, the cancer is breast cancer, lung cancer, melanoma, prostate cancer, colon cancer, bladder cancer, bone cancer, blood cancer, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, stomach cancer, It may be any one selected from the group consisting of tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, anal muscle cancer, central nervous system tumor, liver cancer and colon cancer.
본 발명의 일 구현예에 있어서, 본 발명의 약학적 조성물은 약효를 증가시키지는 않으나 약학적 조성물에 통상 사용되어 냄새, 맛, 시각 등을 향상시킬 수 있는 성분을 추가로 포함할 수 있다. 또한, 본 발명의 약학적 조성물은 약학적으로 허용 가능한 첨가제를 추가적으로 포함할 수 있다. 약학적으로 허용 가능한 첨가제는 예컨대, 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 있으나, 이를 한정하지 않는다. In one embodiment of the present invention, the pharmaceutical composition of the present invention does not increase the efficacy, but may further include ingredients that are commonly used in the pharmaceutical composition to improve the smell, taste, time and the like. In addition, the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, for example, starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, Hydroxypropyl cellulose, opiodry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, white sugar, dextrose, sorbitol and talc, but are not limited thereto.
추가로, 상기 약학적 조성물은 상기 화학식 1로 표시되는 화합물, 이의 입체이성질체 또는 이의 약제학적으로 허용 가능한 염 외에 동일 또는 유사한 약효를 나타내는 유효성분을 1 종 이상 더 포함할 수 있다. In addition, the pharmaceutical composition may further include one or more active ingredients exhibiting the same or similar medicaments in addition to the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
본 발명의 상기 약학적 조성물은 약학적으로 허용 가능한 담체를 포함하고 경구 또는 비경구용의 인체 또는 수의용으로 제형화될 수 있다. 본 발명의 약학적 조성물을 제제화하는 경우 충진제, 증량제, 결합제, 습윤제, 붕해제 및 계면활성제 등의 희석제 또는 부형제를 사용할 수 있다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제 및 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 화합물을 포함하는 약학적 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 칼슘카보네이트, 수크로스, 락토오스 또는 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘, 스티레이트, 탈크 같은 윤활제를 사용할 수 있다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제 및 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물 및 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 및 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제가 포함된다. 비수성용제 및 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름 및 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔 (witepsol), 마크로골, 트윈 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and can be formulated for human or veterinary use for oral or parenteral use. When formulating the pharmaceutical composition of the present invention, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants can be used. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules and the like, and such solid form preparations may be used in pharmaceutical compositions comprising the compounds of the present invention at least one excipient such as starch, calcium carbonate. , Can be mixed with sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium, styrate, and talc may be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents, water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As a base of suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여할 수 있으며, 비경구 투여 시 피부 외용 또는 복강 내 주사, 직장 내 주사, 피하 주사, 정맥 주사, 근육 내 주사 또는 흉부 내 주사 주입방식을 선택할 수 있으나, 이에 제한되는 것은 아니다.The pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method, and may be external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection during parenteral administration. Injection may be selected, but is not limited thereto.
또한, 본 발명의 약학적 조성물은 단독으로 사용할 수 있으나, 치료 효율을 증가시키기 위하여 추가적으로 방사선 요법, 화학 요법 등의 다양한 암 치료 방법들과 병용하여 사용할 수 있다.In addition, the pharmaceutical composition of the present invention may be used alone, but may be used in combination with various cancer treatment methods, such as radiation therapy and chemotherapy, in order to increase the treatment efficiency.
본 발명의 다른 하나의 양태는, 상기 화학식 1로 표시되는 화합물, 이의 입체이성질체 또는 이의 약제학적으로 허용 가능한 염을 포함하는 조성물의 치료학적으로 유효한 양의 투여를 포함하는 암 전이 억제 방법이다. Another embodiment of the present invention is a method for inhibiting cancer metastasis comprising administering a therapeutically effective amount of a composition comprising a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
본 발명의 또 다른 하나의 양태는, 상기 화학식 1로 표시되는 화합물, 이의 입체이성질체 또는 이의 약제학적으로 허용 가능한 염의 암 전이 억제용 약물의 제조를 위한 용도이다.Another embodiment of the present invention is a use for the manufacture of a drug for inhibiting cancer metastasis of the compound represented by the formula (1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof.
본 발명의 상기 약학적 조성물은 개체에 투여되어 암 전이를 억제할 수 있다. 본 발명에서 사용된 용어, "개체"는 암 또는 이의 직, 간접적 원인에 의해 유발된 질환을 가지고 있으며, 본 발명의 상기 약학적 조성물을 투여하여 증상이 호전될 수 있는 질환을 가진 인간을 포함한 말, 양, 돼지, 염소, 개 등의 포유동물을 의미하나, 이에 제한되는 것은 아니다. The pharmaceutical composition of the present invention can be administered to a subject to inhibit cancer metastasis. As used herein, the term "individual" has a disease caused by cancer or a direct or indirect cause thereof, and includes a human including a disease whose symptoms may be improved by administering the pharmaceutical composition of the present invention. , But not limited to mammals such as sheep, pigs, goats, and dogs.
본 발명에서 사용된 용어, "투여"는 어떠한 적절한 방법으로 개체에 본 발명의 약학적 조성물을 도입하는 것을 의미한다. 투여 경로는 목적 조직에 도달할 수 있는 한 임의의 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또한, 본 발명의 약학적 조성물이 표적 세포로 이동할 수 있도록 하는 임의의 장치에 의해 투여될 수 있다.As used herein, the term "administration" means introducing a pharmaceutical composition of the present invention to a subject in any suitable manner. The route of administration can be administered orally or parenterally via any route as long as it can reach the desired tissue. In addition, the pharmaceutical composition of the present invention may be administered by any device that allows migration to target cells.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여될 수 있다. 본 발명에서 사용된 용어, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 체중, 성별, 연령, 건강 상태, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 병용 투여 시 종래의 치료제와 순차적 또는 동시에 투여될 수 있다. 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다. 1 일 1 회 투여할 수도 있고, 수 회 나누어 투여할 수도 있다. 다만, 상기 투여량, 투여 용법이 본 발명의 범위를 한정하는 것은 아니다. The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. As used herein, the term pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is weight, sex, age, health condition, severity of the patient. , The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with the conventional therapeutic agent in combination administration. It may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art. It may be administered once a day or may be divided several times. However, the dosage and administration regimen do not limit the scope of the present invention.
이하, 하기 실시예 및 실험예에 의해 본 발명을 보다 상세하게 설명한다. 그러나, 하기 실시예는 본 발명의 범위를 제한하는 것은 아니며, 이는 본 발명의 이해를 돕기 위한 것으로 해석되어야 할 것이다.Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following examples are not intended to limit the scope of the present invention, which will be construed as to aid the understanding of the present invention.
제조예Production Example 1. 화학식 2-1 화합물 1.Compound 2-1
(1) 공정 A(1) step A
[반응식 1] Scheme 1
Figure PCTKR2018005036-appb-I000008
Figure PCTKR2018005036-appb-I000008
(E)-4-(부타-1,3-디에닐)-1,2-디메톡시벤젠 (1.0 당량)과 메틸 아크릴레이트 (1.5 당량)를 디클로로메탄에 녹인 후, Me2AlCl (1.0 M in Hx)(1.1 당량)을 -78 ℃에서 천천히 넣어주었다. 혼합물을 실온으로 올려준 후 밤새 섞어주었다. 다시 0 ℃로 온도를 낮추고 혼합물에 염화 암모늄과 증류수를 넣어 반응을 종료시킨 후 디클로로메탄으로 추출하였다. 추출한 유기층을 MgSO4로 건조시킨 후 필터하여 농축시켰다. 이 혼합물을 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다.( E ) -4- (buta-1,3-dienyl) -1,2-dimethoxybenzene (1.0 equiv) and methyl acrylate (1.5 equiv) were dissolved in dichloromethane and then Me 2 AlCl (1.0 M in Hx) (1.1 equiv) was added slowly at -78 ° C. The mixture was raised to room temperature and mixed overnight. The temperature was lowered to 0 ° C., and ammonium chloride and distilled water were added to the mixture to terminate the reaction. The extracted organic layer was dried over MgSO 4 , filtered and concentrated. This mixture was separated on silica gel using cylindrical chromatography.
(2) 공정 B(2) process B
[반응식 2] Scheme 2
Figure PCTKR2018005036-appb-I000009
Figure PCTKR2018005036-appb-I000009
-78 ℃에서 (1R,2S)메틸 2-(3,4-디메톡시페닐)사이클로헥스-3 -엔카복실레이트 (1.0 당량)를 녹인 디클로로메탄에 DIBAL-H (1.0 M in DCM 1.05 당량)를 천천히 첨가하였다. -78 ℃에서 두 시간 동안 섞어준 후 증류수 (10 당량)와 NaF (5.0 당량)를 첨가한 후, 30 분간 실온에서 섞어주었다. 혼합물을 실리카와 셀라이트로 필터해준 후 농축시켜 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다.DIBAL-H (1.0 M in DCM 1.05 equivalents) in dichloromethane dissolved in (1 R , 2 S ) methyl 2- (3,4-dimethoxyphenyl) cyclohex-3-encarboxylate (1.0 equiv) at -78 ° C. ) Was added slowly. After mixing for 2 hours at -78 ° C, distilled water (10 equivalents) and NaF (5.0 equivalents) were added, followed by mixing at room temperature for 30 minutes. The mixture was filtered through silica and celite, concentrated and separated on silica gel using cylindrical chromatography.
(3) 공정 C(3) process C
[반응식 3] Scheme 3
Figure PCTKR2018005036-appb-I000010
Figure PCTKR2018005036-appb-I000010
메탄올에 녹아있는 (1R,2S)-2-(3,4-디메톡시페닐)사이클로헥스-3- 엔카르발데히드 (1.0 당량)에 K2CO3를 실온에서 첨가하였다. 36 시간에서 72 시간 정도 에피머화 (epimerization) 반응을 진행한 후, 아세트산 에틸로 희석시키고, 염화 암모늄과 물로 반응을 종료시켰다. 아세트산 에틸로 추출한 후 농축시켜 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다.Dissolved in methanol (1 R, 2 S) -2- (3,4- dimethoxyphenyl) cyclopropyl the K 2 CO 3 was added at room temperature, the hex-3-Enka Levallois formaldehyde (1.0 eq.). After the epimerization reaction was carried out for 36 to 72 hours, the mixture was diluted with ethyl acetate, and the reaction was terminated with ammonium chloride and water. Extraction with ethyl acetate was followed by concentration and separation on silica gel using cylindrical chromatography.
(4) 공정 D(4) process D
[반응식 4] Scheme 4
Figure PCTKR2018005036-appb-I000011
Figure PCTKR2018005036-appb-I000011
디에틸 3,4-디메톡시벤질포스포네이트 (2.0 당량)가 녹아있는 테트라하이드로퓨란에 nBuLi (2.48 M in Hx)(1.95 당량)을 0 ℃에서 첨가하였다. 30 분 동안 이 혼합물을 실온에서 섞어준 후 다시 0 ℃로 온도를 낮추어 (1S,2S)-2-(3,4-디메톡시페닐)사이클로헥스-3-엔카르발데히드 (1.0 당량)와 DMPU (5.0 당량)를 THF에 녹여 캐뉼라를 사용해 첨가해주었다. 이 혼합물을 밤새 실온에서 섞어준 후 염화 암모늄과 물을 사용해 반응을 종료시켰다. 아세트산 에틸로 추출한 후 MgSO4로 건조한 후, 농축시켜 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다. NBuLi (2.48 M in Hx) (1.95 equiv) was added to tetrahydrofuran in which diethyl 3,4-dimethoxybenzylphosphonate (2.0 equiv) was dissolved at 0 ° C. The mixture was mixed at room temperature for 30 minutes and then lowered to 0 ° C. to (1 S , 2 S ) -2- (3,4-dimethoxyphenyl) cyclohex-3-enecarbaldehyde (1.0 equiv) DMPU (5.0 equiv) was dissolved in THF and added using cannula. The mixture was mixed overnight at room temperature and then the reaction was terminated with ammonium chloride and water. The mixture was extracted with ethyl acetate, dried over MgSO 4 , concentrated and separated on silica gel using cylindrical chromatography.
제조된 화학식 2-1 화합물의 1H NMR 결과는 다음과 같다. 1 H NMR results of the prepared compound 2-1 are as follows.
[화학식 2-1][Formula 2-1]
Figure PCTKR2018005036-appb-I000012
Figure PCTKR2018005036-appb-I000012
1H NMR (599 MHz, CDCl3) δ 6.81 - 6.75 (m, 2H), 6.78 - 6.72 (m, 2H), 6.71 (dd, J = 8.2, 1.8 Hz, 1H), 6.68 (d,J = 1.8 Hz, 1H), 6.07 (d, J = 15.9 Hz, 1H), 6.00 (dd, J = 15.9, 7.4 Hz, 1H), 5.88 (ddt, J = 9.6, 4.9, 2.8 Hz, 1H),5.66 (dd, J = 10.0, 2.1 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.16 (dt, J = 8.2, 2.6 Hz,1H), 2.33 (dtd, J = 10.7, 7.7, 2.8 Hz, 1H), 2.20 (ddp, J = 6.5, 4.5, 2.4 Hz, 2H), 1.90 (dq, J = 12.3, 4.5 Hz, 1H), 1.65 (dtd, J = 12.9, 10.1, 6.2 Hz, 1H). 1 H NMR (599 MHz, CDCl 3 ) δ 6.81-6.75 (m, 2H), 6.78-6.72 (m, 2H), 6.71 (dd, J = 8.2, 1.8 Hz, 1H), 6.68 (d, J = 1.8 Hz, 1H), 6.07 (d, J = 15.9 Hz, 1H), 6.00 (dd, J = 15.9, 7.4 Hz, 1H), 5.88 (ddt, J = 9.6, 4.9, 2.8 Hz, 1H), 5.06 (dd, J = 10.0, 2.1 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.16 (dt, J = 8.2, 2.6 Hz, 1H), 2.33 (dtd, J = 10.7, 7.7, 2.8 Hz, 1H), 2.20 (ddp, J = 6.5, 4.5, 2.4 Hz, 2H), 1.90 (dq, J = 12.3, 4.5 Hz, 1H) , 1.65 (dtd, J = 12.9, 10.1, 6.2 Hz, 1H).
(5) 대량 합성 공정(5) bulk synthesis process
화학식 2-1 화합물은 하기 반응식 5에 의해서도 제조될 수 있다.Formula 2-1 may also be prepared by the following Scheme 5.
[반응식 5] Scheme 5
Figure PCTKR2018005036-appb-I000013
Figure PCTKR2018005036-appb-I000013
4-((1S,6S)-6-에티닐사이클로헥스-2-에닐)-1,2-디메톡시벤젠 (1.0 당량) (다음 논문의 방법을 사용하여 합성할 수 있음; J. Chu et al., J. Nat. Prod. 2011, 74, 1817)을 녹인 후 루테니움 촉매 (0.1 당량)를 실온에서 첨가하였다. 15 분 동안 혼합물을 탈기 (degassing)해준 후 트리부틸틴 하이드라이드 (2.0 당량)를 첨가하였다. 혼합물을 2 시간 동안 LED 빛 아래에서 섞어준 후 용매를 진공상태에서 증발시켰다. 얇은 실리카 패드에서 필터하여 준 후 진공으로 농축시킨 뒤, 바로 다음 반응에 사용하였다.4-((1S, 6S) -6-ethynylcyclohex-2-enyl) -1,2-dimethoxybenzene (1.0 equiv) (can be synthesized using the method of the following paper; J. Chu et al. , J. Nat. Prod. 2011, 74, 1817) and then ruthenium catalyst (0.1 equiv) was added at room temperature. The mixture was degassed for 15 minutes and then tributyltin hydride (2.0 equiv) was added. The mixture was mixed under LED light for 2 hours and then the solvent was evaporated in vacuo. Filter on a thin silica pad, concentrated in vacuo, and immediately used for the next reaction.
비닐 스탄난 (1.0 당량)을 NMP에 녹인 후, 4-브로모-1,2-디메톡시벤젠 (1.2 당량), LiCl (1.2 당량)과 Pd(PPh3)4를 첨가하고 실온에서 섞어주었다. 5 시간 후 아세트산 에틸로 희석시킨 후 브라인 (brine)으로 씻어주었다. 추출된 유기층은 MgSO4로 건조시킨 후 필터하여 농축시킨 뒤, 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다. After dissolving vinyl stannane (1.0 equiv) in NMP, 4-bromo-1,2-dimethoxybenzene (1.2 equiv), LiCl (1.2 equiv) and Pd (PPh 3 ) 4 were added and mixed at room temperature. After 5 hours, the mixture was diluted with ethyl acetate and washed with brine. The extracted organic layer was dried over MgSO 4 , filtered and concentrated, and then separated from silica gel using cylindrical chromatography.
제조예Production Example 2. 화학식 3-1 화합물 2. Compound of Formula 3-1
[반응식 6] Scheme 6
Figure PCTKR2018005036-appb-I000014
Figure PCTKR2018005036-appb-I000014
o-브로모벤즈알데히드 (1.0 당량)를 DMF에 녹인 후 3,4-디메톡시페닐보로닉 산 (1.0 당량)과 2 M Na2CO3 (3.0 당량)과 Pd(PPh3)4 (0.01 당량)를 실온에서 첨가해주었다. 혼합물을 80 ℃에서 밤새 섞어준 후 아세트산 에틸로 희석시킨 다음, 염화 암모늄과 물을 넣어 반응을 종료하였다. 그 다음, 아세트산 에틸로 추출하고 유기층을 MgSO4로 건조시켰다. 이 혼합물을 필터해서 모아진 유기층을 농축시킨 후, 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다. o-bromobenzaldehyde (1.0 equiv) was dissolved in DMF, followed by 3,4-dimethoxyphenylboronic acid (1.0 equiv), 2 M Na 2 CO 3 (3.0 equiv) and Pd (PPh 3 ) 4 (0.01 equiv) ) Was added at room temperature. The mixture was mixed overnight at 80 ° C. and diluted with ethyl acetate. Then, the reaction was terminated by adding ammonium chloride and water. Then, the mixture was extracted with ethyl acetate and the organic layer was dried over MgSO 4 . The mixture was filtered to concentrate the combined organic layers, and then separated on silica gel using cylindrical chromatography.
[반응식 7] Scheme 7
Figure PCTKR2018005036-appb-I000015
Figure PCTKR2018005036-appb-I000015
상기 제조예 1의 공정 D와 동일한 방법으로 화학식 3-1의 화합물을 합성하였다. 제조된 화학식 3-1 화합물의 1H NMR 결과는 다음과 같다.In the same manner as in Process D of Preparation Example 1, a compound of Formula 3-1 was synthesized. 1 H NMR results of the prepared compound 3-1 are as follows.
[화학식 3-1][Formula 3-1]
Figure PCTKR2018005036-appb-I000016
Figure PCTKR2018005036-appb-I000016
1H NMR (599 MHz, CDCl3) δ 7.70 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.1 Hz, 2H), 7.30 (d, J = 6.2 Hz, 1H), 7.03- 6.91 (m, 6H), 6.89 (d, J = 1.6 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.83 (s,3H). 1 H NMR (599 MHz, CDCl 3) δ 7.70 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.1 Hz, 2H), 7.30 (d, J = 6.2 Hz, 1H), 7.03- 6.91 ( m, 6H), 6.89 (d, J = 1.6 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3 H).
제조예Production Example 3. 화학식 4-1 화합물 3. Compound of Formula 4-1
[반응식 8] Scheme 8
Figure PCTKR2018005036-appb-I000017
Figure PCTKR2018005036-appb-I000017
상기 제조예 1의 공정 A-C를 따라 제조한 (1S,2S)-2-(3,4-디메톡시페닐)사이클로헥스-3-엔카르발데히드 (1.0 당량)를 아세트산 에틸에 녹인 후, Pd/C 10 중량% (0.1 당량)를 실온에서 첨가하였다. 6 시간 후 실리카와 셀라이트에 걸러준 뒤, 아세트산 에틸로 씻어주었다. 그 다음, 모아진 유기 혼합물을 농축시킨 후, 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다.After a one (1 S, 2 S) -2- (3,4- dimethoxyphenyl) hex-3-cyclopenten Enka Levallois formaldehyde (1.0 eq.) Prepared in accordance with the AC process of Preparative Example 1 was dissolved in ethyl acetate, Pd 10 wt% / C (0.1 equiv) was added at room temperature. After 6 hours, the mixture was filtered through silica and celite and washed with ethyl acetate. The combined organic mixtures were then concentrated and separated on silica gel using cylindrical chromatography.
[반응식 9] Scheme 9
Figure PCTKR2018005036-appb-I000018
Figure PCTKR2018005036-appb-I000018
디메틸 (3,4-디메톡시벤질)포스포네이트 (2.0 당량)를 DMF에 녹인 후, NaH (60 % in mineral oil)(1.95 당량)를 0 ℃에서 첨가하였다. 이후, 혼합물을 30 분 동안 실온에서 섞어준 다음, (1S,2S)-2-(3,4-디메톡시페닐) 사이클로헥산카르발데히드 (1.0 당량)를 DMF에 녹여 캐뉼라를 사용해 첨가하였다. 혼합물을 밤새 실온에서 섞어준 후, 염화 암모늄과 물을 넣어 반응을 종료시켰다. 그 다음, 아세트산 에틸로 추출한 후 MgSO4로 건조시켰다. 필터로 걸러준 후 유기층을 농축시키고, 원통 크로마토그래피를 사용하여 실리카겔에서 분리하였다. 제조된 화학식 4-1 화합물의 1H NMR 결과는 다음과 같다.Dimethyl (3,4-dimethoxybenzyl) phosphonate (2.0 equiv) was dissolved in DMF and then NaH (60% in mineral oil) (1.95 equiv) was added at 0 ° C. Then, the mixture was dissolved in the following standard mix at room temperature for 30 minutes, (1 S, 2 S) -2- (3,4-dimethoxyphenyl) cyclohexane carbaldehyde (1.0 eq.) DMF was added using a cannula . After the mixture was mixed overnight at room temperature, ammonium chloride and water were added to terminate the reaction. Then, the mixture was extracted with ethyl acetate and dried over MgSO 4 . After filtration, the organic layer was concentrated and separated on silica gel using cylindrical chromatography. 1 H NMR results of the compound of Formula 4-1 are as follows.
[화학식 4-1][Formula 4-1]
Figure PCTKR2018005036-appb-I000019
Figure PCTKR2018005036-appb-I000019
1H NMR (400 MHz, Chloroform-d) δ 7.07 - 6.98 (m, 4H), 6.91 (s, 2H), 6.84 (d, J = 8.2 Hz, 2H), 6.74 (d, J =8.0 Hz, 1H), 6.72 - 6.65 (m, 5H), 6.04 (d, J = 15.9 Hz, 1H), 5.77 (dd, J = 15.9, 6.9 Hz, 1H), 3.93 (s, 6H), 3.88 (s,6H), 3.82 (s, 3H), 3.81 (s, 9H), 2.35 - 2.21 (m, 2H), 1.87 (td, J = 19.9, 16.5, 9.8 Hz, 5H), 1.38 (t, J = 10.8 Hz,9H). 1 H NMR (400 MHz, Chloroform- d ) δ 7.07-6.98 (m, 4H), 6.91 (s, 2H), 6.84 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 8.0 Hz, 1H ), 6.72-6.65 (m, 5H), 6.04 (d, J = 15.9 Hz, 1H), 5.77 (dd, J = 15.9, 6.9 Hz, 1H), 3.93 (s, 6H), 3.88 (s, 6H) , 3.82 (s, 3H), 3.81 (s, 9H), 2.35-2.21 (m, 2H), 1.87 (td, J = 19.9, 16.5, 9.8 Hz, 5H), 1.38 (t, J = 10.8 Hz, 9H ).
실시예Example 1.  One. NDPK의Of NDPK 활성에 대한 확인Confirmation of activity
(1) (One) NDPKNDPK 분석 analysis
5 ng의 재조합 Nm23-H1을 상온에서 1 분 동안 5 μM ADP 및 시험물질 (화학식 2-1, 3-1 또는 4-1 화합물)과 함께 NDPK 분석 완충액 (20 mM HEPES, 3 mM MgCl2)에서 배양하였고, 세포 기반 NDPK 분석을 수행 하였다. 프로테아제 억제제 칵테일 (protease inhibitor cocktail)과 NDPK 분석 완충액 (NDPK assay buffer)으로 용해시킨 5,000K MDA-MB-231 세포에서 얻은 세포 용해물을 4 ℃에서 10 분 동안 8,000 rpm으로 원심분리하였다. 40 μL의 용해물을 시험물질과 함께 5 분간 배양한 다음, 50 μM UDP를 첨가하여 NDPK와 반응시켰다. ATP 소비는 ATP 결정 키트 (Molecular probe, USA)에 의해 평가되었다. 5 ng of recombinant Nm23-H1 in NDPK assay buffer (20 mM HEPES, 3 mM MgCl 2 ) with 5 μM ADP and test substance (compound 2-1, 3-1 or 4-1 compound) at room temperature for 1 minute. Cultured and cell based NDPK analysis was performed. Cell lysates obtained from 5,000 K MDA-MB-231 cells lysed with protease inhibitor cocktail and NDPK assay buffer were centrifuged at 8,000 rpm for 10 minutes at 4 ° C. 40 μL of the lysate was incubated with the test material for 5 minutes and then reacted with NDPK by adding 50 μM UDP. ATP consumption was assessed by the ATP Decision Kit (Molecular probe, USA).
(2) 등온 적정형 열량계 분석 (Isothermal Titration Calorimetry)(2) Isothermal Titration Calorimetry
ITC 실험은 ITC200 장비 (Malvern Inc.)를 사용하여 수행되었고, 데이터는 ORIGIN 7.0 프로그램을 사용하여 분석하였다. 세포에서 Nm23-H1 단백질의 농도는 30 μM이었고 주사기에는 300 μM의 활성제 또는 1 mM ATP가 포함되었다. 활성제는 DMSO (Dimethyl sulfoxide)에 50 mM 농도로 준비하고, -20 ℃에서 보관하였다. ITC 완충액은 pH 7.5, 150 mM NaCl, 3 mM MgCl2 및 최대 2 % DMSO에서 20 mM HEPES를 포함하였다. 적정은 25 ℃에서 각각 2 μL를 20 회 주입하고, 150 초 간격으로 주입하였다. 뉴클레오타이드 결합 부위에서 DMSO의 결합에 대한 어떠한 증거도 관찰되지 않았다. 실험 로우데이터는 완충액에 대한 값을 빼서 희석하여 보정한 후 단일 부위 결합 모델로 적합시켰다. 적정 데이터는 화학양론 (stoichiometry; N), 해리 상수 (dissociation constant; KD) 및 상호 작용 엔탈피 변화 (change of enthalpy of interaction)의 3 가지 부동 변수를 갖는 비선형 최소 자승 곡선-피팅 알고리즘 (nonlinear least-squares curve-fitting algorithm)을 사용하여 적합화하였다.ITC experiments were performed using the ITC200 instrument (Malvern Inc.) and data were analyzed using the ORIGIN 7.0 program. The concentration of Nm23-H1 protein in the cells was 30 μM and the syringe contained 300 μM of activator or 1 mM ATP. The active agent was prepared in DMSO (Dimethyl sulfoxide) at a concentration of 50 mM and stored at -20 ° C. ITC buffer contained 20 mM HEPES at pH 7.5, 150 mM NaCl, 3 mM MgCl 2 and up to 2% DMSO. Titration was injected 20 times, 2 μL each at 25 ° C., and 150 sec intervals. No evidence of DMSO binding at the nucleotide binding site was observed. Experimental raw data were corrected by subtracting the values for the buffer and then fitted into a single site binding model. The titration data is a nonlinear least-squares-fitting algorithm with three floating variables: stoichiometry (N), dissociation constant (KD), and change of enthalpy of interaction. curve-fitting algorithm).
(3) 표면 (3) surface 플라즈마plasma 공명 분석(Surface  Resonance Analysis PlasmonPlasmon Resonance analysis) Resonance analysis
화학식 2-1 화합물과 Nm23-H1의 상호 작용을 25 ℃에서 표면 플라스몬 공명장치 (SR7500 DC, Reichert Inc., NY)를 사용하여 분석하였다. The interaction of the compound of formula 2-1 with Nm23-H1 was analyzed using a surface plasmon resonance apparatus (SR7500 DC, Reichert Inc., NY) at 25 ℃.
pH 4.5의 10 mM S.A 완충액 중의 Nm23-H1 (1 mg/mL)을 카복시메틸 덱스트란 하이드로겔 (carboxymethyl dextran hydrogel; CMDH) 표면 센서 칩 (Reichert Technologies, Depew, NY)에서 20 μL/분으로 10 분 동안 표준 아미노 커플링 (standard amino coupling)을 사용하여 포화도가 달성될 때까지 고정시켰다. Nm23-H1 (1 mg / mL) in 10 mM SA buffer at pH 4.5 was quenched at 20 μL / min on a carboxymethyl dextran hydrogel (CMDH) surface sensor chip (Reichert Technologies, Depew, NY). While standard amino coupling was used until saturation was achieved.
고정 버퍼 (10 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM MgCl2, 2 mM ATP 포함 또는 포함하지 않음)에서 화학식 2-1 화합물의 농도를 달리하여 (10-160 μM) 고정화된 Nm23-H1 (약 8200 RU)을 30 μL/분의 속도로 공급하였다. Nm23-H1 immobilized at different concentrations (10-160 μM) of the compound of formula 2-1 in fixed buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM MgCl 2 , 2 mM ATP) (About 8200 RU) was fed at a rate of 30 μL / min.
2 M NaCl을 1 분간 주입하여 각각의 결합 (association) 및 해리 (dissociation) 사이클 후에 센서 표면을 재생시켰다. 센서 그램은 데이터 분석 프로그램 Scrubber 2.0 (BioLogic Software, Australia, and KaleidaGraph Software, Australia)을 사용하였고, 간단한 1 : 1 Langmuir 상호 작용 모델(Langmuir interaction model) (A + B ↔ AB)에 적합하였다. 2 M NaCl was injected for 1 min to regenerate the sensor surface after each association and dissociation cycle. The sensorgrams used the data analysis program Scrubber 2.0 (BioLogic Software, Australia, and KaleidaGraph Software, Australia) and fit a simple 1: 1 Langmuir interaction model (A + B ↔ AB).
SPR 분석을 독립적으로 적어도 3 회 수행하여 평균화한 값을 하기 표 1에 나타내었다. KD는 Kd/Ka을 계산한 값이다. The values averaged by performing SPR analysis independently at least three times are shown in Table 1 below. K D is a value obtained by calculating K d / K a .
Figure PCTKR2018005036-appb-T000001
Figure PCTKR2018005036-appb-T000001
상기 표 1의 결과를 보아, ATP가 존재할 때, 해리가 느려져 KD 값이 감소하는 것을 확인할 수 있다. 이와 같은 결과로 ATP 존재 하에서 Nm23-H1과 본원 발명의 화합물의 상호작용이 더욱 강화되는 것을 확인할 수 있다. Looking at the results of Table 1, when ATP is present, it can be seen that the dissociation is slowed to decrease the K D value. As a result, it can be seen that the interaction of Nm23-H1 with the compound of the present invention is further enhanced in the presence of ATP.
도 3은 화학식 2-1 화합물에 대한 NDPK의 활성을 확인한 도이다. 이하, 각 도면에서의 화학식 2-1 화합물은 NMac1으로 표시하였다. 3 is a diagram confirming the activity of NDPK to the compound of formula 2-1. Hereinafter, the compound of formula 2-1 in each figure is represented by NMac1.
구체적으로 도 3A의 결과로 화학식 2-1 화합물의 농도에 따라 Nm23-H1의 NDPK 활성이 증가되는 것을 확인할 수 있으며, 도 3B로 UDP 농도에 따른 NDPK 활성을 측정하여 enzyme의 V0, Km value를 측정해본 결과 기존의 엔자임 활성제 (enzyme activator)와 같은 경향인 Km-type 활성을 나타내는 것을 확인할 수 있었다. 이와 같은 결과로 NDPK 활성제는 기질인 NTP에 대한 친화도를 높이는 방법으로 NDPK의 활성을 높이는 것을 알 수 있다. 또한, 도 3C의 결과로 세포 기반 NDPK 활성이 화학식 2-1 화합물의 농도에 따라 증가되는 것을 확인하였다. 또한 도 3D로 본 발명의 화학식 2-1 화합물은 Nm23-H1뿐만 아니라, Nm23의 다른 서브 타입인 Nm23-H2에 대해서도 같은 활성을 나타내는 것으로 확인할 수 있었다. 더욱 구체적으로 본 발명의 화학식 2-1 화합물은 Nm23-H1의 활성형인 헥사머릭 Nm23-H1만을 특이적으로 활성화하는 것을 도 3E를 통하여 확인할 수 있다. Specifically, as a result of FIG. 3A, it can be seen that NDPK activity of Nm23-H1 is increased according to the concentration of the compound of Chemical Formula 2-1. As shown in FIG. As a result, it was confirmed that Km-type activity exhibited the same tendency as the existing enzyme activator. As a result, it can be seen that the NDPK activator increases the activity of NDPK by increasing the affinity for the substrate NTP. In addition, as a result of Figure 3C it was confirmed that the cell-based NDPK activity is increased with the concentration of the compound of formula 2-1. In addition, it can be seen from FIG. 3D that the compound of Chemical Formula 2-1 of the present invention exhibits the same activity against Nm23-H1 as well as Nm23-H2, which is another subtype of Nm23. More specifically, the compound of formula 2-1 of the present invention can be confirmed through FIG. 3E that specifically activates only hexameric Nm23-H1, which is an active form of Nm23-H1.
또한, 상기 화학식 2-1 화합물의 유도체로서, 화학식 3-1 및 4-1의 화합물을 제조하고, 상기 유도체들의 NDPK 활성을 확인하였다. 각 시험물질의 DMSO 처리군 대비 상대적 NDPK 활성을 하기 표 2에 나타내었다. In addition, as a derivative of the compound of Formula 2-1, compounds of Formulas 3-1 and 4-1 were prepared, and the NDPK activity of the derivatives was confirmed. The relative NDPK activity compared to DMSO treatment group of each test substance is shown in Table 2 below.
화학식Chemical formula 화합물compound 상대적 NDPK 활성(± S.D)Relative NDPK Activity (± S.D)
-- DMSODMSO 1One
2-12-1
Figure PCTKR2018005036-appb-I000020
Figure PCTKR2018005036-appb-I000020
 		4.04 (± 0.13)4.04 (± 0.13)
3-13-1
Figure PCTKR2018005036-appb-I000021
Figure PCTKR2018005036-appb-I000021
 		4.11 (± 0.16)4.11 (± 0.16)
4-14-1
Figure PCTKR2018005036-appb-I000022
Figure PCTKR2018005036-appb-I000022
 		2.41 (± 0.29)2.41 (± 0.29)
위 결과로부터, 화학식 1에 해당하는 화합물들 (즉, 화학식 2-1 화합물, 및 이의 변이체 화학식 3-1 및 4-1의 화합물)은 모두 우수한 NDPK 활성 (Nm23에 대한 활성 증가)을 가짐을 확인하였다. 따라서 이하에서는, 대표적으로 화학식 2-1 화합물의 Nm23 활성과 관련된 추가 실험을 수행하였다.From the above results, it was confirmed that the compounds corresponding to Formula 1 (ie, the compounds of Formula 2-1, and variants thereof, the compounds of Formulas 3-1 and 4-1) all have excellent NDPK activity (increase in activity against Nm23). It was. Therefore, in the following, further experiments related to the Nm23 activity of the compound of formula 2-1 were performed.
도 4는 화학식 2-1 화합물에 대한 등온 적정형 열량계 분석 (isothermal calorimetry)을 수행한 결과를 나타낸 도이다. 도 4의 결과로 보아, Nm23-H1과 본원 발명의 화학식 2-1 화합물에 대한 직접적인 상호작용 (interaction)을 확인할 수 있었고, ATP 존재 하에서 더욱 수행되는 것을 확인할 수 있었다. 4 is a diagram showing the results of performing isothermal titration calorimetry (isothermal calorimetry) on the compound of formula 2-1. As a result of Figure 4, it was confirmed that the direct interaction (interaction) with Nm23-H1 and the compound of formula 2-1 of the present invention, it was confirmed that the further performed in the presence of ATP.
도 5는 플라즈마 공명 분석의 결과를 나타낸 도이다. 도 5의 결과로 보아, Nm23-H1과 본 발명의 화합물은 직접적으로 결합하는 것을 확인할 수 있었다. 5 is a diagram showing the results of plasma resonance analysis. As a result of Figure 5, it was confirmed that Nm23-H1 and the compound of the present invention is directly bonded.
실시예Example 2. 화학식 2-1 화합물의 전이 억제 활성 검증  2. Verification of Transition Inhibitory Activity of Compound of Chemical Formula 2-1
(1) (One) Rac1Rac1 활성 분석  Activity analysis
MDA-MB-231 세포를 100 mm 디쉬에서 배양하였고, 16 시간 동안 표시된 농도의 화학식 2-1 화합물 또는 0.05 % DMSO로 처리하였다. 활성 Rac1 풀다운 검정은 제조자의 지시 (Thermo Fisher Scientific)에 따라 수행 하였다.MDA-MB-231 cells were cultured in 100 mm dishes and treated with the indicated concentrations of Formula 2-1 compound or 0.05% DMSO for 16 hours. The active Rac1 pulldown assay was performed according to the manufacturer's instructions (Thermo Fisher Scientific).
(2) (2) 면역형광Immunofluorescence 분석 analysis
MDA-MB-231 세포를 SecureslipTM (Sigma) 세포 배양 유리 커버 슬립 상에서 50 내지 70 % 수준까지 배양한 뒤, 화학식 2-1 화합물의 존재 또는 부재 하에 다양한 시간 동안 처리하였다. 세포를 차가운 HBSS로 부드럽게 세척한 후, RBS에서 10 분 동안 상온에서 4 % 파라포름알데히드 함유 HBSS로 고정시켰다. HBSS로 세척 한 후, 10 분간 상온에서 0.1 % Triton X-100에 의한 침투를 수행하였고, HBSS로 2 회 세척한 후, 상온에서 1 시간 동안 3 % BSA, 0.2 % 트윈 20 및 0.2 % 젤라틴 함유 HBSS로 블로킹을 하고, 37 ℃에서 2 시간 동안 1 차 항체 배양을 한 후, 형광 색소 결합 종 특이적인 2 차 항체 (fluorochrome-conjugated species-specific secondary antibodies)와 1 시간 배양하였다. MDA-MB-231 cells were cultured to 50-70% levels on Secureslip ™ (Sigma) cell culture glass cover slips and then treated for various hours in the presence or absence of the compound of Formula 2-1. The cells were gently washed with cold HBSS and then fixed in RBS with HBSS containing 4% paraformaldehyde at room temperature for 10 minutes. After washing with HBSS, infiltration with 0.1% Triton X-100 was performed at room temperature for 10 minutes, and after washing twice with HBSS, HBSS containing 3% BSA, 0.2% Tween 20 and 0.2% gelatin for 1 hour at room temperature. Blocking was performed, and the primary antibody was incubated at 37 ° C. for 2 hours, followed by 1 hour of incubation with a fluorochrome-conjugated species-specific secondary antibody.
모든 항체를 1 % BSA 함유 HBSS로 희석하였다. 커버 슬립 (Cover slips)은 안티 페이딩 용액 (anti-fading solution)으로 고정시키고, LSM510 META (Zeiss) 레이저 스캐닝 공초점 현미경 (confocal microscope)의 x63 대물렌즈를 사용하여 관찰하였다. All antibodies were diluted with HBSS containing 1% BSA. Cover slips were fixed with anti-fading solution and observed using the x63 objective of the LSM510 META (Zeiss) laser scanning confocal microscope.
(3) 침윤 분석(3) infiltration analysis
세포가 70 % 채워졌을 때 16 시간 동안 화학식 2-1 화합물로 처리하였다. 1 x 105 개의 세포를 FBS 없이 배지에 부유시키고 보이든 챔버 멤브레인 (Boyden chamber membrane) 위에 첨가하였다. 10 % FBS를 함유한 배양 배지를 멤브레인 바닥에 첨가하였다. 챔버를 37 ℃, 5 % CO2에서 4 시간 동안 배양하였다. 이동한 세포를 고정하고 크리스탈 바이올렛/메탄올로 염색하였다. 사진을 찍고 이동한 세포를 세고 대조군 세포로 표준화하였다.The cells were treated with the compound of formula 2-1 for 16 hours when the cells were 70% filled. 1 x 10 5 cells were suspended in media without FBS and added over a Boyden chamber membrane. Culture medium containing 10% FBS was added to the membrane bottom. The chamber was incubated at 37 ° C., 5% CO 2 for 4 hours. The migrated cells were fixed and stained with crystal violet / methanol. Photos were taken and the migrated cells were counted and normalized to control cells.
도 6은 화학식 2-1 화합물을 두 가지의 유방암 세포주 (breast cancer cell line)에 처리하여 세포 모양 변화를 관찰하고 각각의 세포주에서 Rac1 활성을 관찰한 도이다. 6 is a diagram illustrating the change in cell morphology and treatment of Rac1 activity in each cell line by treating the compound of formula 2-1 to two breast cancer cell lines.
도 6A은 공초점 레이저 현미경 (confocal microscopy)을 이용하여 화학식 2-1 화합물을 처리한 경우를 관찰한 것으로, MDA-MB-231 세포의 F-actin의 변화를 보면, 러플 (Ruffle)이 줄고, 세포 간 접촉이 증가하는 것을 확인할 수 있다. 이는 Nm23-H1의 세포 이동에 관여 하는 Rac1의 활성을 억제하고, 세포의 이동성을 감소시키는 것을 의미한다. FIG. 6A illustrates a case where the compound of Formula 2-1 was treated using confocal microscopy, and when the F-actin change of MDA-MB-231 cells was observed, the ruffle was reduced. Increasing contact between cells can be seen. This means inhibiting the activity of Rac1 involved in cell migration of Nm23-H1 and reducing cell mobility.
또한, 도 6B는 삼중 음성 유방암 세포주 (Triple negative breast cancer cell line)인 MDA-MB-231 세포에서의 화학식 2-1 화합물에 따른 모양 변화와 Rac1의 활성 변화를 측정한 도이다. 도 6B를 보면, 활성화된 Rac1의 풀-다운 어세이를 하였을 때, 본원 발명의 화학식 2-1 화합물은 농도 의존적으로 Rac1의 활성을 낮추는 것을 확인할 수 있다. 도 6C 및 도 6D에서 Nm23-H1의 넉다운에 의해서 사라지는 것으로 보아 Rac1의 활성을 저해하는 것은 Nm23-H1을 경유하여 일어나는 것임을 확인할 수 있다. In addition, Figure 6B is a diagram measuring the change in shape and the activity of Rac1 according to the compound of formula 2-1 in MDA-MB-231 cells, a triple negative breast cancer cell line. Referring to Figure 6B, when the pull-down assay of the activated Rac1, it can be seen that the compound of formula 2-1 of the present invention lowers the activity of Rac1 in a concentration-dependent manner. In FIG. 6C and FIG. 6D, it can be seen that disappearing by knockdown of Nm23-H1 inhibits Rac1 activity through Nm23-H1.
도 7은 MDA-MB-231 세포에 대한 화학식 2-1 화합물의 침윤 및 이동 억제를 검증한 도이다.Figure 7 is a diagram verifying the inhibition of infiltration and migration of the compound of formula 2-1 to MDA-MB-231 cells.
도 7A는 삼중 음성 유방암 세포주 (Triple negative breast cancer cell line)인 MDA-MB-231 세포에서의 침윤 분석, 트랜스웰 이동 분석을 수행한 결과를 나타낸 도이다. Figure 7A is a diagram showing the results of infiltration analysis, transwell migration analysis in MDA-MB-231 cells, a triple negative breast cancer cell line.
도 7A를 보면, MDA-MB-231 세포에서 화학식 2-1 화합물의 농도를 달리하여 처리한 결과, 농도에 따라 트렌스웰 이동과 매트리겔 침윤이 감소하는 것을 확인할 수 있다. Referring to FIG. 7A, as a result of treatment with different concentrations of the compound of Formula 2-1 in MDA-MB-231 cells, it can be seen that the transwell migration and the matrigel infiltration are reduced according to the concentration.
도 7B는 폐암 세포주 (lung cancer cell line)인 A549 세포에서의 화학식 2-1 화합물의 Nm23-H1 활성을 나타낸 도이다. 도 7B의 결과를 보면, A549 세포에서 본 발명의 화학식 2-1 화합물은 상처 치유와 매트리겔 침윤을 모두 농도 별로 감소시키는 것을 확인할 수 있다. 7B is a diagram showing Nm23-H1 activity of the compound of formula 2-1 in A549 cells, a lung cancer cell line. Referring to the results of FIG. 7B, it can be seen that the compound of Formula 2-1 of the present invention reduces the wound healing and Matrigel infiltration by concentration in A549 cells.
도 7C는 화학식 2-1 화합물의 Nm23-H1 및 Nm23-H2 활성을 나타낸 도이다. 도 7C에서 화학식 2-1 화합물의 효과는 Nm23-H1 및 Nm23-H2의 넉다운에 의해 모두 감소되는 것으로 보았을 때, 화학식 2-1의 in vitro 전이 억제 활성은 Nm23-H1 및 Nm23-H2를 경유하여 일어나는 것을 확인할 수 있다. 7C is a diagram showing Nm23-H1 and Nm23-H2 activities of the compound of formula 2-1. In FIG. 7C, when the effects of the compounds of Formula 2-1 are all reduced by knockdown of Nm23-H1 and Nm23-H2, the in vitro metastasis inhibitory activity of Formula 2-1 is determined via Nm23-H1 and Nm23-H2. You can see what happens.
실시예Example 3. 유방암 전이 모델을 이용한 화학식 2-1 화합물의 전이 억제 활성 검증 3. Verification of metastasis inhibitory activity of the compound of formula 2-1 using breast cancer metastasis model
본 발명의 화학식 2-1 화합물의 전이 억제 활성을 실제 동물 모델에서 확인하기 위해 MDA-MB-231 luc 세포를 이용한 유방암 전이 모델에 적용하였다. MDA-MB-231 세포를 누드 마우스의 유방 지방체 (mammary fat pad)에 주사한 후 종양 사이즈 (tumor size)가 100 mm에 이르렀을 때 화학식 2-1 화합물을 10 mg/kg으로 매일 주사하여 4 주 동안 폐로 전이되는 양상을 관찰 하였다. The metastasis inhibitory activity of the compound of formula 2-1 of the present invention was applied to a breast cancer metastasis model using MDA-MB-231 luc cells in order to confirm in a real animal model. After injection of MDA-MB-231 cells into the mammary fat pad of nude mice, when the tumor size reached 100 mm, the compound of formula 2-1 was injected daily at 10 mg / kg. The metastasis to the lung was observed during the week.
도 8은 유방암 전이 모델에서의 화학식 2-1 화합물의 전이 억제 활성을 나타낸 도이다. 도 8의 결과로 본 발명의 화학식 2-1 화합물을 처리한 군에서 폐로의 전이가 감소함을 확인할 수 있다. 8 is a diagram showing metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model. As a result of Figure 8 it can be seen that the metastasis to the lung in the group treated with the compound of formula 2-1 of the present invention.
도 9는 화학식 2-1 화합물의 처리에 따른 암의 부피, 체중 변화를 나타낸 도이다. 상기 도 9의 결과로, 본 발명의 화학식 2-1 화합물을 처리한 군에서 암의 부피가 감소하는 것을 확인할 수 있었다. 9 is a view showing the changes in the volume and weight of cancer according to the treatment of the compound of formula 2-1. As a result of FIG. 9, it was confirmed that the volume of the cancer was reduced in the group treated with the compound of formula 2-1 of the present invention.
도 10은 유방암 전이 모델에서의 화학식 2-1 화합물의 전이 억제 활성을 나타낸 도이다. 도 10의 결과로 보았을 때, 폐로 전이된 세포는 통계적으로 유의하게 감소한 것을 확인할 수 있었다. 10 is a diagram showing metastasis inhibitory activity of the compound of formula 2-1 in a breast cancer metastasis model. As a result of Figure 10, it was confirmed that the cells metastasized to the lung significantly reduced.
상기 실시예의 결과로, 본 발명에 따른 화학식 1로 표시되는 화합물은 Nm23의 NDPK 활성을 높여, 암의 전이 억제에 효과적인 약학적 조성물로 사용될 수 있음을 확인할 수 있다. As a result of the above embodiment, it can be seen that the compound represented by the formula (1) according to the present invention can be used as a pharmaceutical composition effective in inhibiting metastasis of cancer by increasing NDPK activity of Nm23.
본 명세서는 본 발명의 기술 분야에서 통상의 지식을 가진 자이면 충분히 인식하고 유추할 수 있는 내용은 그 상세한 기재를 생략하였으며, 본 명세서에 기재된 구체적인 예시들 이외에 본 발명의 기술적 사상이나 필수적 구성을 변경하지 않는 범위 내에서 보다 다양한 변형이 가능하다. 따라서 본 발명은 본 명세서에서 구체적으로 설명하고 예시한 것과 다른 방식으로도 실시될 수 있으며, 이는 본 발명의 기술 분야에 통상의 지식을 가진 자이면 이해할 수 있는 사항이다.In the present specification, those skilled in the art of the present invention can fully recognize and infer the details that have been omitted, and the technical spirit or essential configuration of the present invention in addition to the specific examples described in this specification are changed. Many more variations are possible without departing. Therefore, the present invention may be implemented in a manner different from that specifically described and illustrated herein, which can be understood by those skilled in the art.

Claims (5)

  1. 하기 화학식 1로 표시되는 화합물, 이의 입체이성질체 또는 이의 약제학적으로 허용 가능한 염을 유효성분으로 포함하는 암 전이 억제용 약학적 조성물:A pharmaceutical composition for inhibiting cancer metastasis comprising a compound represented by the following Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof as an active ingredient:
    [화학식 1][Formula 1]
    Figure PCTKR2018005036-appb-I000023
    Figure PCTKR2018005036-appb-I000023
    화학식 1에 있어서, In Chemical Formula 1,
    고리 A는 사이클로헥센 (cyclohexene), 사이클로헥산 (cyclohexane) 또는 벤젠 (benzene)이다. Ring A is cyclohexene, cyclohexane or benzene.
  2. 제1항에 있어서, The method of claim 1,
    상기 화학식 1로 표시되는 화합물은 하기 화학식 2 내지 4 중 어느 하나로 표시되는 것인, 암 전이 억제용 약학적 조성물: Compound represented by Formula 1 is represented by any one of the following formulas 2 to 4, pharmaceutical composition for inhibiting cancer metastasis:
    [화학식 2] [Formula 2]
    Figure PCTKR2018005036-appb-I000024
    Figure PCTKR2018005036-appb-I000024
    [화학식 3][Formula 3]
    Figure PCTKR2018005036-appb-I000025
    Figure PCTKR2018005036-appb-I000025
    [화학식 4][Formula 4]
    Figure PCTKR2018005036-appb-I000026
    .
    Figure PCTKR2018005036-appb-I000026
    .
  3. 제1항에 있어서, The method of claim 1,
    상기 화학식 1로 표시되는 화합물의 입체이성질체는 E form인 것인, 암 전이 억제용 약학적 조성물. Stereoisomer of the compound represented by Formula 1 is E form, pharmaceutical composition for inhibiting cancer metastasis.
  4. 제1항에 있어서, The method of claim 1,
    상기 약학적 조성물은 Nm23-H1, Nm23-H2 또는 Nm23-H1과 Nm23-H2의 활성제인 것인, 암 전이 억제용 약학적 조성물.The pharmaceutical composition is Nm23-H1, Nm23-H2 or Nm23-H1 and Nm23-H2 will be an active agent of the pharmaceutical composition for inhibiting cancer metastasis.
  5. 제1항에 있어서, The method of claim 1,
    상기 암은 유방암, 폐암, 흑색종, 전립선암, 대장암, 방광암, 뼈암, 혈액암, 갑상선암, 부갑상선암, 골수암, 직장암, 인후암, 후두암, 식도암, 췌장암, 위암, 설암, 피부암, 뇌종양, 자궁암, 두부 또는 경부암, 담낭암, 구강암, 결장암, 항문부근암, 중추신경계 종양, 간암 및 대장암으로 이루어진 군으로부터 선택된 어느 하나인 것인, 암 전이 억제용 약학적 조성물.The cancer may be breast cancer, lung cancer, melanoma, prostate cancer, colon cancer, bladder cancer, bone cancer, blood cancer, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, esophageal cancer, pancreatic cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, Head or neck cancer, gallbladder cancer, oral cancer, colon cancer, anal muscle cancer, central nervous system tumor, liver cancer and any one selected from the group consisting of colon cancer, pharmaceutical composition for inhibiting cancer metastasis.
PCT/KR2018/005036 2017-04-28 2018-04-30 Pharmaceutical composition, containing nm23 activator, for inhibiting cancer metastasis WO2018199727A1 (en)

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