WO2020040591A1 - Novel use of pyrimidine derivative comprising lrrk kinase inhibitor as effective ingredient - Google Patents

Novel use of pyrimidine derivative comprising lrrk kinase inhibitor as effective ingredient Download PDF

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WO2020040591A1
WO2020040591A1 PCT/KR2019/010759 KR2019010759W WO2020040591A1 WO 2020040591 A1 WO2020040591 A1 WO 2020040591A1 KR 2019010759 W KR2019010759 W KR 2019010759W WO 2020040591 A1 WO2020040591 A1 WO 2020040591A1
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cancer
group
lrrk2
pancreatic cancer
pyrimidin
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PCT/KR2019/010759
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French (fr)
Korean (ko)
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최환근
박종배
고은화
손정범
김남두
이승훈
남도현
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재단법인 대구경북첨단의료산업진흥재단
국립암센터
사회복지법인 삼성생명공익재단
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Publication of WO2020040591A1 publication Critical patent/WO2020040591A1/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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
    • C07D213/08—Preparation by ring-closure
    • C07D213/09—Preparation by ring-closure involving the use of ammonia, amines, amine salts, or nitriles
    • C07D213/12—Preparation by ring-closure involving the use of ammonia, amines, amine salts, or nitriles from unsaturated compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/30—Halogen atoms or nitro radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • Protein kinases are enzymes that catalyze the transfer of the terminal phosphate group of adenosine triphosphate (ATP) to specific residues of the protein, tyrosine, serine, or threonine, and regulate the activity, growth and differentiation of cells against changes in cell mediators and the environment. Involved in the signal. Inappropriately high protein kinase activity is directly or indirectly associated with a number of diseases resulting from abnormal cell action. For example, the disease may be caused by failure of appropriate regulatory mechanisms of kinases related to mutations, over-expression, or inappropriate enzyme activity, or overproduction or lack of factors involved in signal transduction upstream or downstream of cytokines, kinases. Can be.
  • ATP adenosine triphosphate
  • the leucin-rich repeat kinase-2 (LRRK2) protein belongs to the leucin-rich repeat kinase family and consists of 2527 amino acid sequences with high similarity between species. LRRK2 protein is characterized by having both GTPase and Serine-threonine kinase activity in one protein. The expressed LRRK2 protein has been observed in various organs and tissues, including the pancreas. At the cellular level, it is present in the cytoplasm or cell membrane and mitochondrial outer membrane.
  • the LRRK2 protein has five functionally important domains, and is expected to regulate cell function through self-activation regulation by protein autophosphorylation, protein interaction and enzymatic action.
  • chaperone machinery, cytoskelecton arrangement, protein translational machinery, synaptic vesicle endocytosis, mitogen-activated protein kinase signal transduction Activated protein kinases signaling cascades and ubiquitin / autophage protein degradation pathways are known to be regulated by LRRK2 proteins.
  • LRRK2 protein has been reported to be associated with the imputation of mild cognitive impairment associated with Alzheimer's disease, L-Dopa induced dyskinesia, and CNS disorders associated with neuronal precursor differentiation.
  • the G2019S mutation of the LRRK2 protein has been reported to increase the incidence of acute myeloid leukemia (AML) as well as non-skin cancers such as kidney cancer, breast cancer, lung cancer, prostate cancer and the like. Specifically, the G2019S mutation of the LRRK2 protein increases the catalytic activity of the LRRK2 kinase domain. Furthermore, LRRK2 protein has also been reported to be associated with amyotrophic lateral sclerosis, rheumatoid arthritis and ankylosing spondylitis (WO 2011/038572).
  • An object in one aspect of the present invention is to provide a pharmaceutical composition for the prophylaxis or treatment of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer containing an LRRK2 protein inhibitor as an active ingredient.
  • An object in another aspect of the present invention is to provide a use of an LRRK2 protein inhibitor for use in the manufacture of a medicament for the treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer. It is.
  • One aspect of the present invention provides a pharmaceutical composition for the prophylaxis or treatment of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer containing an LRRK2 protein inhibitor as an active ingredient.
  • another aspect of the present invention provides a method for treating one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, comprising administering an LRRK2 protein inhibitor to a subject in need thereof.
  • another aspect of the present invention provides an LRRK2 protein inhibitor for use in the prevention or treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer.
  • Another aspect of the invention also provides the use of an LRRK2 protein inhibitor for use in the manufacture of a medicament for the treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer. .
  • the pharmaceutical composition for preventing or treating at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer provided in one aspect of the present invention exhibits excellent anticancer effect in vitro, in vivo, and therefore, pancreatic cancer, breast cancer and lung cancer. It can be usefully used as a therapeutic agent.
  • FIG. 1 is a result of confirming the P-LRRK2 inhibitory activity of Example 1, compound 2, which is an LRRK2 protein inhibitor.
  • Figure 2 is a graph showing the enzyme-substrate kinetics results of the compound of Example 1 calculated using Michaelis-Menten formula.
  • 3 is a graph showing the results of evaluating the LRRK2 inhibitory activity of the compound of Example 1.
  • Figure 4 is a result confirming the anti-cancer treatment effect of the pancreatic cancer animal model of Example 1, 2 compound which is an LRRK2 protein inhibitor.
  • 5 is a graph showing the results of evaluating the organoid target gemcitabine reactivity derived from the pancreatic cancer xenograft model (PDX).
  • Figure 6 is a graph showing the results of evaluating the anticancer activity of the test drugs against GUN # 13 organoids.
  • Figure 7 is a graph showing the results of evaluating the anticancer activity of test drugs for HPT # 19 organoids.
  • 8 is a graph showing the results of evaluating the anticancer activity of test drugs against HPT # 22 organoid.
  • FIG. 9 shows the organoid morphology identified when GUN # 13 organoids were treated with a test drug at 100 nM concentration.
  • FIG. 10 shows the organoid morphology confirmed when treated with 1000 nM concentration of test drug for GUN # 13 organoid.
  • FIG. 11 shows the organoid morphology confirmed when the test drug was treated at a concentration of 100 nM for HPT # 19 organoids.
  • FIG. 9 shows the organoid morphology identified when GUN # 13 organoids were treated with a test drug at 100 nM concentration.
  • FIG. 10 shows the organoid morphology confirmed when treated with
  • FIG. 12 shows the organoid morphology confirmed when the test drug was treated at a concentration of 1000 nM for HPT # 19 organoids.
  • FIG. 13 is a graph showing the results of evaluation of anticancer activity of each compound of Example 1 on breast cancer cell line MDA-MB231.
  • 14 is a graph showing the results of evaluation of anticancer activity of each compound of Example 1 on lung cancer cell line A549.
  • One aspect of the present invention provides a method for the prevention of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, containing as an active ingredient at least one Leucine Rich Repeat Kinase 2 (LRRK2) protein inhibitor selected from the following compound group:
  • LRRK2 Leucine Rich Repeat Kinase 2
  • therapeutic pharmaceutical compositions (1) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino ) Methanone; And (2) 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile.
  • the LRRK2 protein inhibitor according to the present invention can be used in the form of a pharmaceutically acceptable salt, in which acid addition salts formed by pharmaceutically acceptable free acid are useful.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanediodes.
  • Non-toxic organic acids such as acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like.
  • These types of pharmaceutically harmless salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide.
  • the acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving a derivative of the LRRK2 protein inhibitor in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and adding an organic acid or an inorganic acid.
  • the precipitate may be prepared by filtration and drying, or the solvent and excess acid may be distilled under reduced pressure, dried, and crystallized in an organic solvent.
  • the LRRK2 protein inhibitors of the present invention may be made as pharmaceutically acceptable metal salts using bases.
  • alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • Corresponding salts may also be obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate).
  • a suitable negative salt eg, silver nitrate
  • the LRRK2 protein inhibitor, the optical isomer thereof, or the pharmaceutically acceptable salt thereof may be administered in various dosage forms, orally or parenterally, in the case of clinical administration.
  • Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, and the like. , Dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), glidants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols.
  • Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine and the like, and optionally disintegrants such as starch, agar, alginic acid or its sodium salt or the like, or Boiling mixtures, absorbents, colorants, flavoring agents, and sweetening agents.
  • Pharmaceutical compositions comprising an LRRK2 protein inhibitor, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention may be administered parenterally, and parenteral administration may be administered by subcutaneous injection, intravenous injection, intramuscular injection or It can be administered via intrathoracic injection.
  • the LRRK2 protein inhibitor, its optical isomer, or pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or a buffer to prepare a formulation for parenteral administration into a solution or suspension, which is an ampoule or vial unit. It may be prepared in a dosage form.
  • the composition is sterile and may further contain preservatives, stabilizers, emulsifiers or emulsifiers, auxiliaries such as salts and buffers for the control of osmotic pressure, and other therapeutically useful substances, and mixing, granulating or It may be formulated according to the coating method.
  • the dosage to the human body of a pharmaceutical composition comprising the LRRK2 protein inhibitor of the present invention, its optical isomer, or a pharmaceutically acceptable salt thereof as an active ingredient is determined by the age, weight, sex, dosage form, health condition and Depending on the extent of the disease, based on an adult patient weighing 70 Kg, generally 0.1-1000 mg / day, preferably 1-500 mg / day, and also at the discretion of the physician or pharmacist It may be administered once a day to divided doses at regular time intervals.
  • another aspect of the present invention provides a method for treating one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, comprising administering the LRRK2 protein inhibitor to a subject in need thereof. do.
  • another aspect of the present invention provides the LRRK2 protein inhibitor for use in the prevention or treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer.
  • another aspect of the present invention provides a use of the LRRK2 protein inhibitor for use in the manufacture of a medicament for the treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer. do.
  • Step 1 After dissolving 2,4-dichloro-5-iodopyrimidine (1.0 equiv) in THF, methylamine (3.5 wt% in EtOH, 1.1 equiv) was added at 0 ° C. The mixture was stirred at 0 ° C. for 2 hours, then the solvent was removed and used in the next step without further purification (yield: 100%).
  • Step 2 After filling the two-necked round-bottom flask with nitrogen, CuI (5.0 equiv) and KF (5.0 equiv) were added. The mixture was heated to 150 ° C. and then stirred for 2 hours under reduced pressure. After the reaction, the temperature was lowered to room temperature and trimethyl (trifluoromethyl) silane (5.0 equiv) dissolved in DMF / NMP (1: 1) under nitrogen was added using a syringe. After reacting for 30 minutes, 2-chloro-5-iodo-N-methylpyrimidin-4-amine (1.0 equiv) dissolved in DMF / NMP (1: 1) was added using a syringe, and 50 ° C. Reaction was carried out for 18 hours.
  • Table 1 shows the structures of the compounds prepared in Examples 1 and 2.
  • LRRK2 protein inhibitor which is an active ingredient of a pharmaceutical composition for the prevention or treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, can effectively inhibit phosphorylated LRRK2 protein expression.
  • pancreatic cancer patient-derived cell GUN # 13 obtained from a 44-year-old pancreatic ductal adenocarcinoma patient was prepared. After 1 day of seeding, 7.5 ⁇ 10 5 to 1.0 ⁇ 10 6 cells were treated with Examples 1 and 2 at a concentration of 1 and 2 ⁇ M, respectively, and after 1 day, samples were collected and collected through Western Blot.
  • Example A group not treated with a compound was treated as a vehicle.
  • the results are shown in FIG.
  • the compounds of Examples 1 and 2 which are LRRK2 protein inhibitors, were excellent in phosphorylated LRRK2 protein expression inhibitory activity.
  • Enzyme-substrate reaction rates using the compounds according to the invention as enzymes and LRRK2 protein as substrates were calculated using the Michaelis-Menten equation.
  • the initial rate of the enzyme reaction was proportional to the concentration of the enzyme when the substrate (reactant) was small, and the enzyme approached a constant limit rate when the substrate concentration was excessive.
  • the Michaelis-Menten curve for the substrate reaction was satisfied (FIG. 2). Therefore, it was confirmed that the compound according to the present invention binds to LRRK2.
  • FIG. 3 it was confirmed whether the activity of LRRK2 was inhibited by the binding of these compounds and LRRK2.
  • the activity of LRRK2 is inhibited in proportion to the concentration of the compound.
  • the prepared oral preparations were dispensed 1 ml each and stored at -20 ° C. to be used every time they were tested. From day 25 after pancreatic cancer cells were injected, Examples 1 and 2 compounds were orally administered to a mouse model injected with pancreatic cancer cells at 60 mg / kg. After administration of the compound, MRI was used to measure the size of tumors produced in the mice. The group which was not treated with the compound of Example was set as a vehicle. The results are shown in FIG. 4 and Table 2 below. As shown in FIG. 4 and Table 2, the compounds of Examples 1 and 2, which are LRRK2 protein inhibitors, were excellent in anticancer activity against pancreatic cancer animal models.
  • Patient-derived in situ transplant mouse model (patient-derived orthotopic xenograft, PDX) obtained from patients who agreed to the study and approved directly by the pancreas of immunodeficient mice after approval from the Medical Life Research Review Board (IRB).
  • HPT surgically resected primary tumor specimen
  • GUN biopsy tissue
  • athymus Nude-Foxn1 nude mice were excised and sutured into the pancreas of the pancreas (PDX Generation 1, F1).
  • the size of the tumor was measured periodically through abdominal palpation and MRI imaging equipment, and when the size of the tumor reached 3000 mm 3 , the tumor was sacrificed to obtain the tumor tissue, and then a certain size (3 mm ⁇ 3 mm ⁇ 3 mm) was obtained.
  • a human cell dissociation kit (Miltenyi Biotech Inc.) containing collagenase and reacted for 1 hour in a tissue dissociation apparatus (Gentle Macs, Miltenyi Biotech Inc)
  • RPMI medium containing (FBS) Inhibiting enzymatic activity with RPMI medium containing (FBS) followed by centrifugation can yield precipitates of cells dissociated from tissues.
  • PDX-derived cancer cell line for each pancreatic cancer patient.
  • Established PDX-derived cancer cell lines and patient clinical information are shown in Table 3 below.
  • Example 1 Compounds and control drugs were dissolved in 5% DMSO, 0.2% TWEEN 80, and 1% AVICELL. Control drugs GNE7915, PF-06447475, MLi-2 and Example 1 compound were treated with 100 nM, 1000 nM. For vehicles, only excipients containing no test substance were treated.
  • Organisms formed by incubating in 96-well plates for 7 days were treated with gemcitabine by concentration (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 uM).
  • the medium containing the drug was exchanged every 3 days, and after 7 days, the survival rate of the organoids was evaluated by ATP assay. This method can be used to analyze the resistance of organoids to gemcitabine.
  • test drug was also treated with 0, 100, 1000 nM concentrations after 7 days of incubation by seeding at 2 ⁇ 10 3 cells / well in 96-well plates. After 7 days, the survival rate of organoids was evaluated by ATP assay, and the shape of organoids was observed using a microscope. In this way, the evaluation of morphological changes and evaluation of the inhibitory effect of pancreatic cancer cells on test drugs of organoids can be compared.
  • Example 1 showed a concentration-dependent anticancer activity against the pancreatic cancer-derived organoids, like the control drug.
  • the compound of Example 1 also showed anticancer activity in a concentration-dependent manner to the organoids of GUN # 13 and HPT # 19 patients resistant to gemcitabine. It is shown in Figures 9 to 12 that the form of GUN # 13 and HPT # 19 organoids form small according to the test drug treatment.
  • the breast cancer cell line MDA-MB-231 was cultured one day before the cell lines were allowed to repeat three times with 1000 cells in one well in a 96 well plate. The following day, the compound of Example 1 was dissolved in DMSO for 24 hours, and then grown for 24 hours. Growth was measured using ProTitle-GloLuminescent Cell Viability Assay. This method uses energy ATP, which is an indicator that cells are growing, so a higher value means that the cell is growing, and a lower value means that the cell is dying. Therefore, with the ATP measurement, the effect on the breast cancer cell line following the treatment of the compound of Example 1 can be measured graphically. The results are shown in FIG. As shown in FIG. 13, the compound of Example 1 was shown to exhibit concentration-dependent anticancer activity against breast cancer cell lines.
  • Example 1 of the Invention When applying a compound as a drug to a living body, an indicator of pharmacokinetics was used to evaluate the movement and effect of the drug in a living body. These pharmacokinetic indicators influence the determination of the route of administration and dosage of the drug. Specifically, when the compound of Example 1 of the present invention was quantitatively administered to mouse, rat, dog or monkey by intravenous (i.v.) or oral administration (PO), the pharmacokinetic index of the compound was evaluated. The indicators of the pharmacokinetics and the results are shown in Table 6 below.
  • T 1/2 the time it takes for the drug to decrease to half its original concentration
  • AUC integral of the concentration-time curve (area under the curve), blood concentration over time
  • MRT the average time a drug stays in the body
  • the pharmaceutical composition for preventing or treating at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer provided in one aspect of the present invention exhibits excellent anticancer effect in vitro, in vivo, and therefore, pancreatic cancer, breast cancer and lung cancer. Useful as a therapeutic.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to a novel use of a pyrimidine derivative comprising an LRRK kinase inhibitor as an effective ingredient. A pharmaceutical composition, provided according to one aspect of the present invention, for prevention or treatment of at least one selected from pancreatic cancer, breast cancer, and lung cancer exhibits an excellent therapeutic effect on cancer in vitro and in vivo and, as such, can be advantageously used as a therapeutic agent for pancreatic cancer, breast cancer, and lung cancer.

Description

LRRK 키나아제 저해제를 유효성분으로 함유하는 피리미딘 유도체의 신규용도Novel use of pyrimidine derivatives containing LRRK kinase inhibitors as active ingredients
LRRK 키나아제 저해제를 유효성분으로 함유하는 피리미딘 유도체의 신규 용도에 관한 것이다.A novel use of pyrimidine derivatives containing LRRK kinase inhibitors as active ingredients.
단백질 키나아제는 아데노신삼인산(ATP)의 말단 인산기를 단백질의 특정 잔기인 타이로신, 세린 또는 트레오닌에 전이시키는 반응을 촉매하는 효소로서, 세포 매개체 및 환경의 변화에 대한 세포의 활성이나 성장 및 분화를 조절하는 신호에 관여한다. 부적절하게 높은 단백질 키나아제 활성은 비정상적인 세포의 작용으로부터 기인하는 다수의 질병과 직접적 또는 간접적으로 연관이 있다. 예를 들면, 돌연변이, 과잉-발현 또는 부적절한 효소 활성에 관련된 키나아제의 적절한 조절 메카니즘의 실패나, 사이토카인, 키나아제의 업스트림 또는 다운스트림의 신호 전달에 참여하는 인자들의 과잉생성 또는 결핍에 의해 질병이 야기될 수 있다. 따라서, 키나아제 활성의 선택적인 억제는 질병 치료를 위한 신약 개발의 유익한 표적이 될 수 있다. LRRK2(leucin-rich repeat kinase-2) 단백질은 류신 풍부 반복 키나아제 집단(leucin-rich repeat kinase family)에 속하는 단백질이며, 종간 유사성이 높은 2527개의 아미노산 배열로 구성되어 있다. LRRK2 단백질은 하나의 단백질 안에 GTP 가수분해효소(GTPase)와 세린-트레오닌 키나아제(Serine-threonine kinase) 활성을 모두 갖는 특징이 있다. 발현된 LRRK2 단백질은 췌장을 포함한 다양한 기관과 조직에서 관찰되고 있으며, 세포수준에서는 세포질 또는 세포막 및 미토콘드리아 외막에서 존재한다. 또한, LRRK2 단백질은 기능상 중요한 5개의 도메인(domain)을 가지고 있어, 자가 인산화 작용(Autophosphorylation)에 의한 자가활성조절작용과 단백질 상호작용 및 효소작용을 통해 세포의 기능을 조절할 것으로 예상된다. 특히, 샤페론 기전(chaperone machinery), 세포골격 배열(cytoskelecton arrangement), 단백질번역기전(protein translational machinery), 시냅스 소포의 세포 내 유입(synaptic vesicle endocytosis), 미토젠-활성화 단백질 키나아제 신호 전달과정(mitogen-activated protein kinases signaling cascades) 및 유비퀴틴/오토파지단백질 분해과정(ubiquitin/autophage protein degradation pathways) 등이 LRRK2 단백질에 의해 조절되는 것으로 알려졌다. LRRK2 단백질은 알츠하이머병과 관련된 경도인지 손상의 전가, L-도파(Dopa) 유도된 운동 이상증, 및 뉴런 전구 분화와 관련된 CNS 장애와 관련됨이 보고되었다. 또한, LRRK2 단백질의 G2019S 돌연변이는 급성 골수성 백혈병(AML)뿐만 아니라, 신장암, 유방암, 폐암, 전립선암 등과 같은 비-피부암의 발생을 증가시키는 것이 보고되었다. 구체적으로, LRRK2 단백질의 G2019S 돌연변이는 LRRK2 키나아제 도메인의 촉매 활성을 증가시킨다. 나아가, LRRK2 단백질은 근위축성 측삭 경화증, 류마티스 관절염 및 강직 척추염과도 관련된 것이 보고되어 있다(국제공개특허 WO 2011/038572).Protein kinases are enzymes that catalyze the transfer of the terminal phosphate group of adenosine triphosphate (ATP) to specific residues of the protein, tyrosine, serine, or threonine, and regulate the activity, growth and differentiation of cells against changes in cell mediators and the environment. Involved in the signal. Inappropriately high protein kinase activity is directly or indirectly associated with a number of diseases resulting from abnormal cell action. For example, the disease may be caused by failure of appropriate regulatory mechanisms of kinases related to mutations, over-expression, or inappropriate enzyme activity, or overproduction or lack of factors involved in signal transduction upstream or downstream of cytokines, kinases. Can be. Thus, selective inhibition of kinase activity may be a beneficial target of drug development for disease treatment. The leucin-rich repeat kinase-2 (LRRK2) protein belongs to the leucin-rich repeat kinase family and consists of 2527 amino acid sequences with high similarity between species. LRRK2 protein is characterized by having both GTPase and Serine-threonine kinase activity in one protein. The expressed LRRK2 protein has been observed in various organs and tissues, including the pancreas. At the cellular level, it is present in the cytoplasm or cell membrane and mitochondrial outer membrane. In addition, the LRRK2 protein has five functionally important domains, and is expected to regulate cell function through self-activation regulation by protein autophosphorylation, protein interaction and enzymatic action. In particular, chaperone machinery, cytoskelecton arrangement, protein translational machinery, synaptic vesicle endocytosis, mitogen-activated protein kinase signal transduction Activated protein kinases signaling cascades and ubiquitin / autophage protein degradation pathways are known to be regulated by LRRK2 proteins. LRRK2 protein has been reported to be associated with the imputation of mild cognitive impairment associated with Alzheimer's disease, L-Dopa induced dyskinesia, and CNS disorders associated with neuronal precursor differentiation. In addition, the G2019S mutation of the LRRK2 protein has been reported to increase the incidence of acute myeloid leukemia (AML) as well as non-skin cancers such as kidney cancer, breast cancer, lung cancer, prostate cancer and the like. Specifically, the G2019S mutation of the LRRK2 protein increases the catalytic activity of the LRRK2 kinase domain. Furthermore, LRRK2 protein has also been reported to be associated with amyotrophic lateral sclerosis, rheumatoid arthritis and ankylosing spondylitis (WO 2011/038572).
한편, 본 출원인은 LRRK2 단백질의 저해제로 사용되는 화합물이 췌장암, 유방암, 폐암의 예방 또는 치료에 효과가 우수함을 in vitro, in vivo 실험을 통해 직접적으로 입증하고, 본 발명을 완성하였다.On the other hand, the applicant directly demonstrated in vitro, in vivo experiment that the compound used as an inhibitor of LRRK2 protein is excellent in the prevention or treatment of pancreatic cancer, breast cancer, lung cancer, and completed the present invention.
본 발명의 일 측면에서의 목적은 LRRK2 단백질 저해제를 유효성분으로 함유하는 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.An object in one aspect of the present invention is to provide a pharmaceutical composition for the prophylaxis or treatment of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer containing an LRRK2 protein inhibitor as an active ingredient.
본 발명의 다른 일 측면에서의 목적은 LRRK2 단백질 저해제를 이를 필요로 하는 대상(subject)에게 투여하는 단계를 포함하는, 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료방법을 제공하는 것이다.It is another object of the present invention to provide a method for treating one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, comprising administering an LRRK2 protein inhibitor to a subject in need thereof. It is.
본 발명의 또 다른 일 측면에서의 목적은 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료에 사용하기 위한, LRRK2 단백질 저해제를 제공하는 것이다.It is an object in another aspect of the present invention to provide an LRRK2 protein inhibitor for use in the prevention or treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer.
본 발명의 다른 일 측면에서의 목적은 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료를 위한 약제(medicament)의 제조에 사용하기 위한, LRRK2 단백질 저해제의 용도(use)를 제공하는 것이다.An object in another aspect of the present invention is to provide a use of an LRRK2 protein inhibitor for use in the manufacture of a medicament for the treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer. It is.
상기 목적을 달성하기 위하여,In order to achieve the above object,
본 발명의 일 측면은 LRRK2 단백질 저해제를 유효성분으로 함유하는 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention provides a pharmaceutical composition for the prophylaxis or treatment of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer containing an LRRK2 protein inhibitor as an active ingredient.
또한, 본 발명의 다른 일 측면은 LRRK2 단백질 저해제를 이를 필요로 하는 대상(subject)에게 투여하는 단계를 포함하는, 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료방법을 제공한다.In addition, another aspect of the present invention provides a method for treating one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, comprising administering an LRRK2 protein inhibitor to a subject in need thereof. .
나아가, 본 발명의 또 다른 일 측면은 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료에 사용하기 위한, LRRK2 단백질 저해제를 제공한다.Furthermore, another aspect of the present invention provides an LRRK2 protein inhibitor for use in the prevention or treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer.
또한, 본 발명의 다른 일 측면은 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료를 위한 약제(medicament)의 제조에 사용하기 위한, LRRK2 단백질 저해제의 용도(use)를 제공한다.Another aspect of the invention also provides the use of an LRRK2 protein inhibitor for use in the manufacture of a medicament for the treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer. .
본 발명의 일 측면에서 제공되는 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료용 약학적 조성물은 in vitro, in vivo에서 우수한 항암효과를 나타내므로, 췌장암, 유방암 및 폐암 치료제로 유용하게 사용될 수 있다.The pharmaceutical composition for preventing or treating at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer provided in one aspect of the present invention exhibits excellent anticancer effect in vitro, in vivo, and therefore, pancreatic cancer, breast cancer and lung cancer. It can be usefully used as a therapeutic agent.
도 1은 LRRK2 단백질 저해제인 실시예 1, 2번 화합물의 P-LRRK2 저해 활성을 확인한 결과이다. 도 2는 미하엘리스-멘텐식을 사용하여 계산한 실시예 1 화합물의 효소-기질 반응속도 결과를 나타내는 그래프이다. 도 3은 실시예 1 화합물의 LRRK2 저해활성을 평가한 결과를 나타내는 그래프이다. 도 4는 LRRK2 단백질 저해제인 실시예 1, 2번 화합물의 췌장암 동물모델에 대한 항암 치료효과를 확인한 결과이다. 도 5는 췌장암 이종이식 모델(PDX)에서 유래된 오가노이드 대상 젬시타빈 반응성을 평가한 결과를 나타내는 그래프이다. 도 6은 GUN #13 오가노이드에 대한 시험약물들의 항암활성을 평가한 결과를 나타내는 그래프이다. 도 7은 HPT #19 오가노이드에 대한 시험약물들의 항암활성을 평가한 결과를 나타내는 그래프이다. 도 8은 HPT #22 오가노이드에 대한 시험약물들의 항암활성을 평가한 결과를 나타내는 그래프이다. 도 9는 GUN #13 오가노이드에 대하여 시험 약물을 100 nM 농도로 처리하였을때 확인되는 오가노이드 형태를 나타낸다. 도 10은 GUN #13 오가노이드에 대하여 시험 약물을 1000 nM 농도로 처리하였을때 확인되는 오가노이드 형태를 나타낸다. 도 11은 HPT #19 오가노이드에 대하여 시험 약물을 100 nM 농도로 처리하였을때 확인되는 오가노이드 형태를 나타낸다. 도 12는 HPT #19 오가노이드에 대하여 시험 약물을 1000 nM 농도로 처리하였을때 확인되는 오가노이드 형태를 나타낸다. 도 13은 유방암 세포주 MDA-MB231에 대한 실시예 1 화합물의 농도별 항암 활성을 평가한 결과를 나타내는 그래프이다. 도 14는 폐암 세포주 A549에 대한 실시예 1 화합물의 농도별 항암 활성을 평가한 결과를 나타내는 그래프이다.1 is a result of confirming the P-LRRK2 inhibitory activity of Example 1, compound 2, which is an LRRK2 protein inhibitor. Figure 2 is a graph showing the enzyme-substrate kinetics results of the compound of Example 1 calculated using Michaelis-Menten formula. 3 is a graph showing the results of evaluating the LRRK2 inhibitory activity of the compound of Example 1. Figure 4 is a result confirming the anti-cancer treatment effect of the pancreatic cancer animal model of Example 1, 2 compound which is an LRRK2 protein inhibitor. 5 is a graph showing the results of evaluating the organoid target gemcitabine reactivity derived from the pancreatic cancer xenograft model (PDX). Figure 6 is a graph showing the results of evaluating the anticancer activity of the test drugs against GUN # 13 organoids. Figure 7 is a graph showing the results of evaluating the anticancer activity of test drugs for HPT # 19 organoids. 8 is a graph showing the results of evaluating the anticancer activity of test drugs against HPT # 22 organoid. FIG. 9 shows the organoid morphology identified when GUN # 13 organoids were treated with a test drug at 100 nM concentration. FIG. 10 shows the organoid morphology confirmed when treated with 1000 nM concentration of test drug for GUN # 13 organoid. FIG. 11 shows the organoid morphology confirmed when the test drug was treated at a concentration of 100 nM for HPT # 19 organoids. FIG. 12 shows the organoid morphology confirmed when the test drug was treated at a concentration of 1000 nM for HPT # 19 organoids. FIG. 13 is a graph showing the results of evaluation of anticancer activity of each compound of Example 1 on breast cancer cell line MDA-MB231. 14 is a graph showing the results of evaluation of anticancer activity of each compound of Example 1 on lung cancer cell line A549.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일 측면은 하기 화합물 군으로부터 선택되는 1종 이상의 LRRK2 (Leucine Rich Repeat Kinase 2) 단백질 저해제를 유효성분으로 함유하는, 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료용 약학적 조성물을 제공한다: (1) (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논; 및 (2) 3-(4-모폴리노-7H-피롤로[2,3-d]피리미딘-5-일)벤조니트릴.One aspect of the present invention provides a method for the prevention of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, containing as an active ingredient at least one Leucine Rich Repeat Kinase 2 (LRRK2) protein inhibitor selected from the following compound group: Provided are therapeutic pharmaceutical compositions: (1) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino ) Methanone; And (2) 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile.
본 발명에 따른 LRRK2 단백질 저해제는 약학적으로 허용 가능한 염의 형태로 사용될 수 있으며, 이때 염으로는 약학적으로 허용가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요오드화수소산, 아질산, 아인산 등과 같은 무기산류, 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸디오에이트, 방향족 산류, 지방족 및 방향족 설폰산류 등과 같은 무독성 유기산, 아세트산, 안식향산, 구연산, 젖산, 말레인산, 글루콘산, 메탄설폰산, 4-톨루엔설폰산, 주석산, 푸마르산 등과 같은 유기산으로부터 얻을 수 있다. 이러한 약학적으로 무독한 염의 종류에는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 다이하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, β-하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트, 만델레이트 등이 포함될 수 있다. 본 발명에 따른 산 부가염은 통상의 방법으로 제조할 수 있으며, 예를 들면 LRRK2 단백질 저해제의 유도체를 메탄올, 에탄올, 아세톤, 메틸렌클로라이드, 아세토니트릴 등과 같은 유기용매에 녹이고 유기산 또는 무기산을 가하여 생성된 침전물을 여과, 건조시켜 제조하거나, 용매와 과량의 산을 감압 증류한 후 건조시켜 유기용매 하에서 결정화시켜셔 제조할 수 있다. 또한, 본 발명의 LRRK2 단백질 저해제는 염기를 사용하여 약학적으로 허용가능한 금속염으로서 만들어질 수 있다. 구체적으로, 알칼리 금속 또는 알칼리 토금속 염은 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻을 수 있다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻을 수 있다. 본 발명에 따른 약학적 조성물에서, 상기 LRRK2 단백질 저해제, 이의 광학 이성질체, 또는 이의 약학적으로 허용가능한 염은 임상 투여시에 경구 또는 비경구의 여러 가지 제형으로 투여될 수 있는데, 제제화할 경우에는 보통 사용하는 충전제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조될 수 있다. 경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제, 트로키제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 함유할 수 있다. 정제는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및 폴리비닐피롤리딘 등과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염 등과 같은 붕해제 또는 비등 혼합물, 흡수제, 착색제, 향미제, 및 감미제를 함유할 수 있다. 본 발명에 따른 LRRK2 단백질 저해제, 이의 광학 이성질체, 또는 이의 약학적으로 허용가능한 염을 유효 성분으로 하는 약학적 조성물은 비경구 투여될 수 있으며, 비경구 투여는 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사를 통해 투여될 수 있다. 이때, 비경구 투여용 제형으로 제제화하기 위하여 상기 LRRK2 단백질 저해제, 이의 광학 이성질체, 또는 이의 약학적으로 허용가능한 염을 안정제 또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알 단위 투여형으로 제조할 수 있다. 상기 조성물은 멸균되고 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질이 추가로 함유될 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅 방법에 따라 제제화될 수 있다. 또한, 본 발명의 LRRK2 단백질 저해제, 이의 광학 이성질체, 또는 이의 약학적으로 허용가능한 염을 유효 성분으로 하는 약학적 조성물의 인체에 대한 투여량은 환자의 나이, 몸무게, 성별, 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있으며, 몸무게가 70 Kg인 성인 환자를 기준으로 할 때, 일반적으로 0.1-1000 mg/일이며, 바람직하게는 1-500 mg/일이며, 또한 의사 또는 약사의 판단에 따라 일정시간 간격으로 1일 1회 내지 수회로 분할 투여될 수도 있다.The LRRK2 protein inhibitor according to the present invention can be used in the form of a pharmaceutically acceptable salt, in which acid addition salts formed by pharmaceutically acceptable free acid are useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanediodes. Non-toxic organic acids such as acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like. These types of pharmaceutically harmless salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chloro Zensulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1-sulfonate, naphthalene-2-sulfonate, mandelate and the like can be included. The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving a derivative of the LRRK2 protein inhibitor in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and adding an organic acid or an inorganic acid. The precipitate may be prepared by filtration and drying, or the solvent and excess acid may be distilled under reduced pressure, dried, and crystallized in an organic solvent. In addition, the LRRK2 protein inhibitors of the present invention may be made as pharmaceutically acceptable metal salts using bases. Specifically, alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding salts may also be obtained by reacting alkali or alkaline earth metal salts with a suitable negative salt (eg, silver nitrate). In the pharmaceutical composition according to the present invention, the LRRK2 protein inhibitor, the optical isomer thereof, or the pharmaceutically acceptable salt thereof may be administered in various dosage forms, orally or parenterally, in the case of clinical administration. Can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like. Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, troches, and the like. , Dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine), glidants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine and the like, and optionally disintegrants such as starch, agar, alginic acid or its sodium salt or the like, or Boiling mixtures, absorbents, colorants, flavoring agents, and sweetening agents. Pharmaceutical compositions comprising an LRRK2 protein inhibitor, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention may be administered parenterally, and parenteral administration may be administered by subcutaneous injection, intravenous injection, intramuscular injection or It can be administered via intrathoracic injection. In this case, the LRRK2 protein inhibitor, its optical isomer, or pharmaceutically acceptable salt thereof is mixed with water together with a stabilizer or a buffer to prepare a formulation for parenteral administration into a solution or suspension, which is an ampoule or vial unit. It may be prepared in a dosage form. The composition is sterile and may further contain preservatives, stabilizers, emulsifiers or emulsifiers, auxiliaries such as salts and buffers for the control of osmotic pressure, and other therapeutically useful substances, and mixing, granulating or It may be formulated according to the coating method. In addition, the dosage to the human body of a pharmaceutical composition comprising the LRRK2 protein inhibitor of the present invention, its optical isomer, or a pharmaceutically acceptable salt thereof as an active ingredient is determined by the age, weight, sex, dosage form, health condition and Depending on the extent of the disease, based on an adult patient weighing 70 Kg, generally 0.1-1000 mg / day, preferably 1-500 mg / day, and also at the discretion of the physician or pharmacist It may be administered once a day to divided doses at regular time intervals.
또한, 본 발명의 다른 일 측면은 상기 LRRK2 단백질 저해제를 이를 필요로 하는 대상(subject)에게 투여하는 단계를 포함하는, 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료방법을 제공한다.In addition, another aspect of the present invention provides a method for treating one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, comprising administering the LRRK2 protein inhibitor to a subject in need thereof. do.
나아가, 본 발명의 또 다른 일 측면은 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료에 사용하기 위한, 상기 LRRK2 단백질 저해제를 제공한다.Furthermore, another aspect of the present invention provides the LRRK2 protein inhibitor for use in the prevention or treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer.
또한, 본 발명의 다른 일 측면은 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료를 위한 약제(medicament)의 제조에 사용하기 위한, 상기 LRRK2 단백질 저해제의 용도(use)를 제공한다.In addition, another aspect of the present invention provides a use of the LRRK2 protein inhibitor for use in the manufacture of a medicament for the treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer. do.
이하, 본 발명을 후술하는 실시예와 실험예를 통해 상세히 설명한다. 단, 후술하는 실시예와 실험예는 본 발명을 일부 예시하는 것일 뿐, 본 발명이 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples and experimental examples. However, Examples and Experimental Examples to be described later are merely illustrative of some of the present invention, the present invention is not limited thereto.
<실시예 1> (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논의 제조Example 1 Preparation of (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone
Figure PCTKR2019010759-appb-I000001
Figure PCTKR2019010759-appb-I000001
1-1. (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논의 제조-11-1. Preparation of (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone-1
표제 화합물을 WO 2011151360 특허문헌을 참조하여 준비하였다.The title compound was prepared with reference to WO 2011151360 patent.
1-2. (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논의 제조-21-2. Preparation of (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone-2
Figure PCTKR2019010759-appb-I000002
Figure PCTKR2019010759-appb-I000002
단계 1. 2,4-디클로로-5-요오도피리미딘(1.0 당량)을 THF에 녹인 후, 0℃에서 메틸아민(3.5 wt% in EtOH, 1.1 당량)을 첨가하였다. 상기 혼합물을 0℃에서 2시간 동안 교반한 뒤, 용매를 제거하고 더 이상의 정제과정 없이 다음 단계에서 사용하였다(수율: 100%). Step 1. After dissolving 2,4-dichloro-5-iodopyrimidine (1.0 equiv) in THF, methylamine (3.5 wt% in EtOH, 1.1 equiv) was added at 0 ° C. The mixture was stirred at 0 ° C. for 2 hours, then the solvent was removed and used in the next step without further purification (yield: 100%).
단계 2. Two-necked round-bottom flask를 질소 기채로 채운 후, CuI(5.0 당량) 및 KF(5.0 당량)를 넣었다. 상기 혼합물을 150℃로 온도를 가열한 뒤, 감압 상태에서 2시간 동안 교반하였다. 반응 후, 온도를 상온으로 낮추고 질소하에서 DMF/NMP(1:1)에 용해된 트리메틸(트리플로로메틸)실란(5.0 당량)을 실린지(syringe)를 이용하여 첨가하였다. 30분 동안 반응시킨 뒤, DMF/NMP(1:1)에 용해된 2-클로로-5-요오도-N-메틸피리미딘-4-아민(1.0 당량)을 실린지를 이용하여 첨가하고, 50℃에서 18시간 동안 반응시켰다. 반응 후, 상기 반응물에 물을 첨가하여 침전물을 형성시키고, 형성된 침전물을 여과하여 제거하였다. 수득된 여과액으로부터 EtOAc를 이용하여 유기물을 추출하였다(x3). 모아진 유기층은 브라인(brine)으로 세척하고, Na2SO4로 남은 물을 제거하였다. 물이 제거된 혼합물을 MPCL(EtOAc:Hex)를 이용하여 정제함으로써, 노란빛 고체의 목적 화합물을 수득하였다(수율: 70%). Step 2. After filling the two-necked round-bottom flask with nitrogen, CuI (5.0 equiv) and KF (5.0 equiv) were added. The mixture was heated to 150 ° C. and then stirred for 2 hours under reduced pressure. After the reaction, the temperature was lowered to room temperature and trimethyl (trifluoromethyl) silane (5.0 equiv) dissolved in DMF / NMP (1: 1) under nitrogen was added using a syringe. After reacting for 30 minutes, 2-chloro-5-iodo-N-methylpyrimidin-4-amine (1.0 equiv) dissolved in DMF / NMP (1: 1) was added using a syringe, and 50 ° C. Reaction was carried out for 18 hours. After the reaction, water was added to the reaction to form a precipitate, and the formed precipitate was removed by filtration. The organics were extracted from the filtrate obtained with EtOAc (x3). The combined organic layers were washed with brine and water remaining with Na 2 SO 4 was removed. The water-free mixture was purified using MPCL (EtOAc: Hex) to give the desired compound as a yellowish solid (yield: 70%).
단계 3. 2-클로로-N-메틸-5-(트리프루오로메틸)피리미딘-4-아민(1.0 당량)과 (4-아미노-3-메톡시페닐)(모폴리노)메타논(1.0 당량)을 다이옥세인(dioxane)에 녹이고, 상온에서 p-톨루엔술폰산(p-toluensulfonic acid)(1.0 당량)을 첨가하였다. 이를 100℃에서 16시간 교반한 뒤, 상온으로 식힌다음 용매를 제거하고 얼음물을 첨가하였다. 유기물은 EtOAc를 이용하여 추출하였다(x3). 모아진 유기층은 브라인으로 세척하고, Na2SO4로 남은 물을 제거하였다. 물이 제거된 혼합물을 MPCL(EtOAc:Hex)를 이용하여 정제함으로써, 흰색 고체의 목적 화합물을 수득하였다(수율: 70%).Step 3. 2-Chloro-N-methyl-5- (trifluoromethyl) pyrimidin-4-amine (1.0 equiv) and (4-amino-3-methoxyphenyl) (morpholino) methanone ( 1.0 equivalent) was dissolved in dioxane, and p-toluensulfonic acid (1.0 equivalent) was added at room temperature. After stirring for 16 hours at 100 ℃, cooled to room temperature, the solvent was removed and ice water was added. The organics were extracted with EtOAc (x3). The combined organic layers were washed with brine and the remaining water was removed with Na 2 SO 4 . The water-free mixture was purified using MPCL (EtOAc: Hex) to give the title compound as a white solid (yield: 70%).
<실시예 2> 3-(4-모폴리노-7H-피롤로[2,3-d]피리미딘-5-일)벤조니트릴의 제조Example 2 Preparation of 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile
Figure PCTKR2019010759-appb-I000003
Figure PCTKR2019010759-appb-I000003
표제 화합물을 WO 2014/001973 문헌을 참조하여 준비하였다.The title compound was prepared with reference to WO 2014/001973 document.
하기 표 1에 상기 실시예 1, 2에서 준비한 화합물의 구조를 나타내었다.Table 1 shows the structures of the compounds prepared in Examples 1 and 2.
실시예Example 화학구조Chemical structure 실시예Example 화학구조Chemical structure
1One
Figure PCTKR2019010759-appb-I000004
Figure PCTKR2019010759-appb-I000004
 		22
Figure PCTKR2019010759-appb-I000005
Figure PCTKR2019010759-appb-I000005
<실험예 1> 인산화된 LRRK2 단백질 발현 억제활성 평가Experimental Example 1 Evaluation of Inhibitory Activity of Phosphorylated LRRK2 Protein
본 발명의 일 측면에서 제공되는 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료용 약학적 조성물의 유효성분인 LRRK2 단백질 저해제가, 인산화된 LRRK2 단백질 발현을 효과적으로 억제할 수 있는지 평가하기 위하여 하기와 같은 실험을 수행하였다. 먼저, 44세의 췌장 관 세포암(pancreatic ductal adenocarcinoma) 환자로부터 수득한 췌장암 환자 유래 세포 GUN#13을 준비하였다. seeding 1일 후, 7.5×105 내지 1.0×106개의 상기 세포에 실시예 1, 2 화합물을 1, 2μM의 농도로 각각 처리하고, 1일이 경과한 후, 샘플을 수집하여 Western Blot을 통해 LRRK2 단백질 인산화 억제 활성을 평가하였다. 실시예 화합물을 처리하지 않은 군을 무처리군(Vehicle)으로 하였다. 그 결과를 도 1에 나타내었다. 도 1에 나타난 바와 같이, LRRK2 단백질 저해제인 실시예 1, 2번 화합물은 인산화된 LRRK2 단백질 발현 억제활성이 우수한 것으로 나타났다.In one aspect of the present invention, LRRK2 protein inhibitor, which is an active ingredient of a pharmaceutical composition for the prevention or treatment of one or more cancers selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, can effectively inhibit phosphorylated LRRK2 protein expression. In order to evaluate the presence of the following experiment was carried out. First, pancreatic cancer patient-derived cell GUN # 13 obtained from a 44-year-old pancreatic ductal adenocarcinoma patient was prepared. After 1 day of seeding, 7.5 × 10 5 to 1.0 × 10 6 cells were treated with Examples 1 and 2 at a concentration of 1 and 2 μM, respectively, and after 1 day, samples were collected and collected through Western Blot. LRRK2 protein phosphorylation inhibitory activity was evaluated. Example A group not treated with a compound was treated as a vehicle. The results are shown in FIG. As shown in FIG. 1, the compounds of Examples 1 and 2, which are LRRK2 protein inhibitors, were excellent in phosphorylated LRRK2 protein expression inhibitory activity.
<실험예 2> LRRK2 단백질에 대한 결합도 및 억제활성 평가Experimental Example 2 Evaluation of Binding and Inhibitory Activity to LRRK2 Protein
본 발명에 따른 화합물을 효소로, LRRK2 단백질을 기질로 하는 효소-기질 반응속도를 미하엘리스-멘텐식을 사용하여 계산하였다. 본 발명의 실시예 1 화합물과 LRRK2 단백질 간의 반응결과에 있어서, 효소 반응의 초기 속도는 기질(반응물)이 소량 일때 효소의 농도에 비례하며, 기질의 농도가 과량일 때 일정한 한계 속도에 접근한다는 효소-기질 반응에 대한 미하엘리스-멘텐 곡선을 만족하였다(도 2). 따라서, 본 발명에 따른 화합물이 LRRK2와 결합함을 확인할 수 있었다. 그 다음으로, 이러한 화합물 및 LRRK2 결합에 의해 LRRK2의 활성이 억제되는지 여부를 확인하였다. 그 결과, 도 3에 나타난 바와 같이, 화합물의 농도에 비례하여 LRRK2의 활성이 억제됨을 확인할 수 있었다.Enzyme-substrate reaction rates using the compounds according to the invention as enzymes and LRRK2 protein as substrates were calculated using the Michaelis-Menten equation. In the result of the reaction between the compound of Example 1 of the present invention and the LRRK2 protein, the initial rate of the enzyme reaction was proportional to the concentration of the enzyme when the substrate (reactant) was small, and the enzyme approached a constant limit rate when the substrate concentration was excessive. The Michaelis-Menten curve for the substrate reaction was satisfied (FIG. 2). Therefore, it was confirmed that the compound according to the present invention binds to LRRK2. Next, it was confirmed whether the activity of LRRK2 was inhibited by the binding of these compounds and LRRK2. As a result, as shown in Figure 3, it was confirmed that the activity of LRRK2 is inhibited in proportion to the concentration of the compound.
<실험예 3> 췌장암 동물모델에 대한 항암활성 평가Experimental Example 3 Evaluation of Anticancer Activity in Animal Models of Pancreatic Cancer
본 발명에 따른 화합물이 동물 모델에서 췌장암 세포에 대한 성장 억제 활성을 나타내는지 확인하기 위하여 실험을 수행하였다. 보다 구체적으로, 췌장암 환자 유래 세포주인 GUN13을 4.5×105 세포/μl 농도로 100 μl를 Balb/c-nude 마우스의 옆구리(flank)에 주입하였다. 그리고, 실시예 1 및 2 화합물을 각각 5% DMSO에 투명한 용액이 될 때까지 먼저 녹인 후, 5% Tween-80을 잘 섞은 다음 투명한 용액에 89%의 DW를 넣고 마지막으로 1%의 Avicel을 넣어 잘 혼합하여 동물 모델에 경구 투여할 제제를 제조하였다. 제조된 경구 투여용 제제는 1ml씩 분주하여 -20℃에 보관하여 시험할 때마다 녹여서 사용하였다. 췌장암 세포를 주입하고 25일 후부터 매일 실시예 1 및 2 화합물을 60 mg/kg으로 췌장암 세포가 주입된 마우스 모델에 경구 투여하였다. 화합물을 투여한 이후 MRI를 이용하여 상기 마우스에 생성된 종양의 크기를 측정하였다. 실시예의 화합물을 처리하지 않은 군을 무처리군(Vehicle)으로 하였다. 그 결과를 도 4와 하기 표 2에 나타내었다. 도 4와 하기 표 2에 나타난 바와 같이, LRRK2 단백질 저해제인 실시예 1, 2번 화합물은 췌장암 동물모델에 대한 항암활성이 우수한 것으로 나타났다.Experiments were conducted to determine whether the compounds according to the invention exhibit growth inhibitory activity against pancreatic cancer cells in animal models. More specifically, 100 μl of GUN13, a cell line derived from a pancreatic cancer patient, was injected into the flank of Balb / c-nude mice at a concentration of 4.5 × 10 5 cells / μl. Then, the compounds of Examples 1 and 2 were first dissolved in 5% DMSO until a clear solution was added. Then, 5% Tween-80 was mixed well, 89% DW was added to the clear solution, and finally 1% Avicel was added. Mixtures were prepared to prepare formulations for oral administration in animal models. The prepared oral preparations were dispensed 1 ml each and stored at -20 ° C. to be used every time they were tested. From day 25 after pancreatic cancer cells were injected, Examples 1 and 2 compounds were orally administered to a mouse model injected with pancreatic cancer cells at 60 mg / kg. After administration of the compound, MRI was used to measure the size of tumors produced in the mice. The group which was not treated with the compound of Example was set as a vehicle. The results are shown in FIG. 4 and Table 2 below. As shown in FIG. 4 and Table 2, the compounds of Examples 1 and 2, which are LRRK2 protein inhibitors, were excellent in anticancer activity against pancreatic cancer animal models.
End point (42day)평균 암 조직 크기 (mm3)End point (42day) Average cancer tissue size (mm 3 )
무처리군No treatment 569569
실시예 1Example 1 374374
실시예 2Example 2 277277
<실험예 4> 췌장암 환자유래 오가노이드를 이용한 항암활성 평가본 발명에 따른 화합물이 췌장암 환자유래 오가노이드에 대한 성장 억제 활성을 나타내는지 확인하기 위하여 실험을 수행하였다. Experimental Example 4 Evaluation of Anticancer Activity Using Organoids Derived from Pancreatic Cancer Patients Experiments were conducted to determine whether the compounds according to the present invention exhibited growth inhibitory activity against pancreatic cancer patient derived organoids.
4-1. 췌장암 환자유래 세포확보4-1. Acquiring cells derived from pancreatic cancer
의생명연구심의위원회(IRB)의 승인을 받은 후, 연구에 동의한 환자를 대상으로 검체를 확보하여 면역결핍마우스의 췌장에 직접 이식한 환자 유래 제자리 이식 마우스 모델(patient-derived orthotopic xenograft, PDX)을 제작하였다. 수술적으로 절제된 원발성 종양검체 (이후 HPT로 명명)와 수술이 불가능한 환자에서 시행한 생검조직(이후 GUN으로 명명)을 채취한 후, 배지에 담긴 튜브에 담아 이동하여 최대한 신속하게 암컷 Hsd: 무흉선 누드-Foxn1 누드마우스의 췌장의 췌미에 절제 및 봉합하여 이식하였다 (PDX 1세대, F1). 이후 주기적으로 복부 촉진 및 MRI 영상 장비를 통하여 종양의 크기를 측정하였고, 종양의 크기가 3000 mm3에 도달하면, 마우스를 희생하여 종양 조직을 수득 후, 일정크기 (3 mm × 3 mm × 3mm)로 조각내었다. 세포를 결합조직으로부터 분리해내기 위하여 콜라겐분해효소가 포함된 Human cell dissociation kit (Miltenyi Biotech Inc.)과 혼합하여 조직해리장치(Gentle Macs, Miltenyi Biotech Inc)에서 1시간 동안 반응한 후, 소 태아 혈청 (FBS)이 포함된 RPMI 배지로 효소활성을 억제하고 이어 원심분리하면 조직으로부터 해리된 세포의 침전물을 얻을 수 있다. 혈청이 포함된 RPMI 배지로 이를 현탁하여 10 cm 배양접시에 2×106 세포 수준으로 세포를 고르게 깔고 이틀마다 배지를 교환하면서 섬유아세포와 죽은 세포들을 제거하여 췌장암 환자별 PDX 유래 암세포주를 확립하였다. 확립한 PDX 유래 암세포주와 환자 임상정보를 하기 표 3에 나타내었다.Patient-derived in situ transplant mouse model (patient-derived orthotopic xenograft, PDX) obtained from patients who agreed to the study and approved directly by the pancreas of immunodeficient mice after approval from the Medical Life Research Review Board (IRB). Was produced. A surgically resected primary tumor specimen (hereinafter referred to as HPT) and a biopsy tissue (hereinafter referred to as GUN) from an inoperable patient are taken and transported in a tube of medium to be as fast as possible for female Hsd: athymus Nude-Foxn1 nude mice were excised and sutured into the pancreas of the pancreas (PDX Generation 1, F1). After that, the size of the tumor was measured periodically through abdominal palpation and MRI imaging equipment, and when the size of the tumor reached 3000 mm 3 , the tumor was sacrificed to obtain the tumor tissue, and then a certain size (3 mm × 3 mm × 3 mm) was obtained. To pieces. In order to separate the cells from connective tissue, mixed with a human cell dissociation kit (Miltenyi Biotech Inc.) containing collagenase and reacted for 1 hour in a tissue dissociation apparatus (Gentle Macs, Miltenyi Biotech Inc) Inhibiting enzymatic activity with RPMI medium containing (FBS) followed by centrifugation can yield precipitates of cells dissociated from tissues. Suspended it with serum-containing RPMI medium to evenly spread the cells at the level of 2 × 10 6 cells in a 10 cm dish and exchange the medium every two days to remove fibroblasts and dead cells to establish a PDX-derived cancer cell line for each pancreatic cancer patient. . Established PDX-derived cancer cell lines and patient clinical information are shown in Table 3 below.
No.No. Patient number(=cell line)Patient number (= cell line) Clinical informationClinical information
1One GUN #13 GUN # 13 수술만 진행. 약물사용하지 않음Surgery only. No drug use
22 HPT #19 HPT # 19 Gemcitabine-약효없음Gemcitabine-no effect
33 HPT #22 HPT # 22 수술만 진행. 약물사용하지 않음Surgery only. No drug use
4-2. 췌장암 환자유래 오가노이드 형성 및 배양상기 확립한 췌장암 환자별 PDX 유래 암세포주를 이용하여 오가노이드를 형성 및 배양하였다. 췌장암 PDX 유래 오가노이드의 초기 배양은 안정적인 형성을 위해, 24-웰 플레이트 내 마트리겔/웰의 3×104 세포/50 ul로 하기 표 4 조성의 배지 내 37℃, 5% CO2 인큐베이터에서 배양하였다. 4-2. Organoid Formation and Cultivation of Pancreatic Cancer Patients Organoids were formed and cultured using the PDX-derived cancer cell lines for the pancreatic cancer patients. Initial culture of pancreatic cancer PDX-derived organoids was cultured in 37 ° C., 5% CO 2 incubator in medium of Table 4 composition below with 3 × 10 4 cells / 50 ul of Matrigel / well in 24-well plates for stable formation. It was.
No.No. 성분ingredient 농도density
1One Growth factor reduced MatrigelGrowth factor reduced Matrigel 8mg/ml8mg / ml
22 Advanced DMEM/F12Advanced DMEM / F12
33 B27 B27 2%2%
44 N-acetylcysteineN-acetylcysteine 1.25mM1.25mM
55 Gastrin-1Gastrin-1 50nM 50nM
66 Recombinant Human EGFRecombinant Human EGF 50ng/ml50ng / ml
77 Recombinant Human R-Spondin-1Recombinant Human R-Spondin-1 50ng/ml50ng / ml
88 Conditioned R-Spondin 1 mediaConditioned R-Spondin 1 media 10%10%
99 Recombinant Human NogginRecombinant Human Noggin 100ng/ml100ng / ml
1010 Recombinant Human FGF10Recombinant Human FGF10 100ng/ml100ng / ml
1111 Recombinant Wnt3aRecombinant Wnt3a 2ng/ul2ng / ul
1212 Zellshield Zelshield 1×1 ×
1313 Y-27632Y-27632 10uM10 uM
4-3. 시험물질 조제 및 관리실시예 1 화합물 및 대조 약물들을 DMSO 5%, TWEEN 80 0.2%, AVICELL 1%에 녹여 사용하였다. 대조 약물인 GNE7915, PF-06447475, MLi-2와, 실시예 1 화합물을 100 nM, 1000 nM 처리하였다. 무처리군(vehicle)의 경우, 시험 물질을 포함하지 않은 부형제만 처리하였다. 4-3. Test Material Preparation and Management Example 1 Compounds and control drugs were dissolved in 5% DMSO, 0.2 % TWEEN 80, and 1% AVICELL. Control drugs GNE7915, PF-06447475, MLi-2 and Example 1 compound were treated with 100 nM, 1000 nM. For vehicles, only excipients containing no test substance were treated.
4-4. 시험관찰 항목4-4. Test observation items
96-웰 플레이트에서 7일간 배양되어 형성된 오가노이드를 대상으로 젬시타빈(Gemcitabine)을 농도별(0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 uM)로 처리하였다. 3일 간격으로 약물이 포함되어있는 배지를 교환하고, 7일 후, ATP assay를 통해 오가노이드의 생존율을 평가하였다. 해당 방법으로 오가노이드의 젬시타빈에 대한 내성을 분석할 수 있다.Organisms formed by incubating in 96-well plates for 7 days were treated with gemcitabine by concentration (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 uM). The medium containing the drug was exchanged every 3 days, and after 7 days, the survival rate of the organoids was evaluated by ATP assay. This method can be used to analyze the resistance of organoids to gemcitabine.
또한, 96-웰 플레이트에 2×103 세포/웰로 시딩(seeding)하여 7일 배양 후, 0, 100, 1000 nM 농도로 시험 약물을 처리하였다. 7일 후 ATP assay를 통해 오가노이드의 생존율의 평가와, 현미경을 이용하여 오가노이드의 형태를 관찰하였다. 해당 방법으로 오가노이드의 시험 약물에 대한 췌장암세포 억제효능 평가 및 형태 변화를 비교할 수 있다.The test drug was also treated with 0, 100, 1000 nM concentrations after 7 days of incubation by seeding at 2 × 10 3 cells / well in 96-well plates. After 7 days, the survival rate of organoids was evaluated by ATP assay, and the shape of organoids was observed using a microscope. In this way, the evaluation of morphological changes and evaluation of the inhibitory effect of pancreatic cancer cells on test drugs of organoids can be compared.
4-5. 결과4-5. result
먼저, 췌장암 이종이식 모델(PDX)에서 유래된 오가노이드 대상 젬시타빈 반응성을 평가한 결과를 도 5와 하기 표 5에 나타내었다.First, the results of evaluating the organoid target gemcitabine reactivity derived from the pancreatic cancer xenograft model (PDX) are shown in FIG. 5 and Table 5 below.
No.No. Patient number(=cell line)Patient number (= cell line) ClinicalinformationClinicalinformation Gem-responsein organoidGem-responsein organoid IC50(uM)IC 50 (uM)
1One GUN #13 GUN # 13 수술만 진행,약물사용하지 않음Surgery only, no medication Gem-resistantGem-resistant 2424
22 HPT #19 HPT # 19 Gem-약효없음Gem-no effect Gem-resistantGem-resistant 1010
33 HPT #22 HPT # 22 수술만 진행,약물사용하지 않음Surgery only, no medication Gem-sensitiveGem-sensitive 0.30.3
도 5와 상기 표 5에 나타난 바와 같이, GUN #13 및 HPT #19 환자는 젬시타빈에 내성을 보임을 알 수 있다. 다음으로, 췌장암 이종이식 모델(PDX)에서 유래된 오가노이드를 대상으로 시험 약물의 효능을 평가한 결과를 도 6 내지 8에 나타내었다. 도 6 내지 8에 나타난 바와 같이, 실시예 1 화합물은 대조 약물과 같이 췌장암 유래 오가노이드에 대하여 농도 의존적인 항암활성을 나타냈다. 특히, 젬시타빈에 내성을 보인 GUN #13 및 HPT #19 환자의 오가노이드에 대하여도 실시예 1 화합물은 농도 의존적으로 항암활성을 나타냈다. 시험 약물 처리에 따른 GUN #13 및 HPT #19 오가노이드의 형태가 작게 형성됨을 도 9 내지 12에 나타내었다.<실험예 5> 유방암 세포주에 대한 항암활성 평가 As shown in FIG. 5 and Table 5, it can be seen that GUN # 13 and HPT # 19 patients are resistant to gemcitabine. Next, the results of evaluating the efficacy of test drugs in organoids derived from pancreatic cancer xenograft model (PDX) are shown in FIGS. 6 to 8. As shown in Figures 6 to 8, Example 1 compound showed a concentration-dependent anticancer activity against the pancreatic cancer-derived organoids, like the control drug. In particular, the compound of Example 1 also showed anticancer activity in a concentration-dependent manner to the organoids of GUN # 13 and HPT # 19 patients resistant to gemcitabine. It is shown in Figures 9 to 12 that the form of GUN # 13 and HPT # 19 organoids form small according to the test drug treatment. Experimental Example 5 Evaluation of Anticancer Activity against Breast Cancer Cell Line
본 발명에 따른 화합물이 유방암 세포주에 대한 성장 억제 활성을 나타내는지 확인하기 위하여 실험을 수행하였다. 유방암 세포주 MDA-MB-231을 96 웰 플레이트에 1000개의 세포가 1개의 웰에 들어가서 3번 반복실험을 할 수 있도록 세포 주들을 하루 전에 배양하여 놓았다. 다음 날 농도별로 실시예 1 화합물을 DMSO에 녹여 24시간 동안 처리한 후, 생장측정을 Promega사의 CellTiter-GloLuminescent Cell Viability Assay를 사용하여 실시하였다. 이 방법은 세포가 생장하고 있다는 지표인 에너지 ATP를 사용하는 것이므로 그래프의 값이 높다는 것은 세포가 생장하고 있다는 의미이고, 그래프의 값이 낮다는 것은 세포가 사멸하고 있다는 것을 뜻한다. 따라서 ATP 측정값을 가지고 실시예 1 화합물의 처리에 따른 유방암 세포주에 대한 영향을 그래프로 측정할 수 있다. 결과를 도 13에 나타내었다. 도 13에 나타난 바와 같이, 실시예 1 화합물은 유방암 세포주에 대하여 농도 의존적인 항암활성을 나타내는 것으로 나타났다.Experiments were conducted to determine if the compounds according to the invention exhibit growth inhibitory activity against breast cancer cell lines. The breast cancer cell line MDA-MB-231 was cultured one day before the cell lines were allowed to repeat three times with 1000 cells in one well in a 96 well plate. The following day, the compound of Example 1 was dissolved in DMSO for 24 hours, and then grown for 24 hours. Growth was measured using ProTitle-GloLuminescent Cell Viability Assay. This method uses energy ATP, which is an indicator that cells are growing, so a higher value means that the cell is growing, and a lower value means that the cell is dying. Therefore, with the ATP measurement, the effect on the breast cancer cell line following the treatment of the compound of Example 1 can be measured graphically. The results are shown in FIG. As shown in FIG. 13, the compound of Example 1 was shown to exhibit concentration-dependent anticancer activity against breast cancer cell lines.
<실험예 6> 폐암 세포주에 대한 항암활성 평가Experimental Example 6 Evaluation of Anticancer Activity against Lung Cancer Cell Lines
본 발명에 따른 화합물이 폐암 세포주에 대한 성장 억제 활성을 나타내는지 확인하기 위하여, 실험예 5에서 유방암 세포주 MDA-MB-231 대신 폐암 세포주 A549를 사용하여 동일한 실험을 수행하였다. 결과를 도 14에 나타내었다. 도 14에 나타난 바와 같이, 실시예 1 화합물은 폐암 세포주에 대하여 농도 의존적인 항암활성을 나타내는 것으로 나타났다.In order to confirm that the compound according to the present invention exhibits growth inhibitory activity against lung cancer cell lines, the same experiment was performed using Lung cancer cell line A549 instead of breast cancer cell line MDA-MB-231 in Experimental Example 5. The results are shown in FIG. As shown in FIG. 14, the compound of Example 1 was shown to exhibit concentration-dependent anticancer activity against lung cancer cell lines.
<실험예 7> 약물동태학 평가Experimental Example 7 Pharmacokinetic Evaluation
본 발명의 실시예 1 화합물을 생체에 약물로 적용할 때, 생체 내에서 약물의 움직임 및 영향을 평가하기 위하여 약물동태학의 지표를 사용하였다. 이러한 약물동태학 지표는 약물의 투여 경로 및 투여량을 결정하는데 영향을 미친다. 구체적으로, 본 발명의 실시예 1 화합물을 정맥주사(i.v.) 또는 경구투여(PO)로 마우스, 랫트, 개 또는 원숭이에 정량 투여하였을 때, 상기 화합물의 약물동태학 지표를 평가하였다. 상기 약물 동태학의 지표 및 그 결과를 하기 표 6에 나타내었다.Example 1 of the Invention When applying a compound as a drug to a living body, an indicator of pharmacokinetics was used to evaluate the movement and effect of the drug in a living body. These pharmacokinetic indicators influence the determination of the route of administration and dosage of the drug. Specifically, when the compound of Example 1 of the present invention was quantitatively administered to mouse, rat, dog or monkey by intravenous (i.v.) or oral administration (PO), the pharmacokinetic index of the compound was evaluated. The indicators of the pharmacokinetics and the results are shown in Table 6 below.
실시예Example ParametersParameters T1/2 T 1/2 Tmax T max Cmax C max AUC(0-t) AUC (0-t) Vss_obsVss_obs Cl_obsCl_obs MRT(0-t) MRT (0-t) FF
hrhr hrhr ng/mLng / mL hr*ng/mLhr * ng / mL L/KgL / Kg mL/min/kgmL / min / kg hrhr %%
1One MouseMouse IV(5mg/kg)IV (5 mg / kg) 1.21.2 -- -- 870870 7.97.9 86.686.6 1.51.5 159159
PO(10mg/kg)PO (10 mg / kg) 1.41.4 0.30.3 1.41.4 2,7652,765 -- -- 2.22.2
RatRat IV(5mg/kg)IV (5 mg / kg) 1.41.4 -- -- 4,1884,188 1.81.8 17.717.7 1.71.7 79.979.9
PO(10mg/kg)PO (10 mg / kg) 1One 0.80.8 1624.31624.3 6,6966,696 -- -- 2.52.5
DogDog IV(1mg/kg)IV (1 mg / kg) 0.9070.907 -- -- 248248 -- 70.570.5 0.6530.653 127127
PO(10mg/kg)PO (10 mg / kg) 1.721.72 0.50.5 15491549 3,1393,139 -- -- 2.132.13
MonkeyMonkey IV(1mg/kg)IV (1 mg / kg) 2.972.97 -- 250250 996996 3.563.56 14.414.4 4.124.12 140140
PO(10mg/kg)PO (10 mg / kg) 2.992.99 1.251.25 600600 13,97813,978 -- -- 6.686.68
Vehicle (i) i.v.용: 5%NMP, 10%PEG400, 50%(10% Solutol HS in saline), 35% Saline; (ii) PO용:10%DMSO, 5%Tween-80, 85% DWFor Vehicle (i) i.v .: 5% NMP, 10% PEG400, 50% (10% Solutol HS in saline), 35% Saline; (ii) for PO: 10% DMSO, 5% Tween-80, 85% DW
*Ct/Cp ratio in mouse (n=3) * C t / C p ratio in mouse (n = 3)
CMAX: 약물이 투여된 이후 약물의 최고 농도C MAX : the highest concentration of drug since the drug was administered
T1/2: 약물의 농도가 원래 농도의 절반으로 줄어드는데 걸리는 시간T 1/2 : the time it takes for the drug to decrease to half its original concentration
TMAX: CMAX에 도달하는데 걸리는 시간T MAX : time to reach C MAX
AUC: 농도-시간 곡선의 적분값(곡선 아래 면적), 시간에 따른 혈중농도AUC: integral of the concentration-time curve (area under the curve), blood concentration over time
Cl: 단위 시간당 약물이 제거되는 혈장의 부피Cl: volume of plasma from which drug is removed per unit time
MRT: 약물이 체내에 머무르는 평균 시간MRT: the average time a drug stays in the body
F(%): 생체이용률, 복용한 약물 중 체순환으로 이행하는 약물의 비율F (%): bioavailability, the proportion of drugs taken to the body circulation
상기 표 6에 나타난 바와 같이, 본 발명의 화합물은 정맥주사보다 경구로 투여하는 경우 약물의 혈중 농도가 높게 유지되면서 체내에 머무르는 평균 시간이 길고, 생체 이용률이 높음을 확인하였다.As shown in Table 6, when the compound of the present invention is administered orally than intravenous injection, it was confirmed that the average time to stay in the body while maintaining the blood concentration of the drug is high, the bioavailability is high.
본 발명의 일 측면에서 제공되는 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료용 약학적 조성물은 in vitro, in vivo에서 우수한 항암효과를 나타내므로, 췌장암, 유방암 및 폐암 치료제로 유용하다.The pharmaceutical composition for preventing or treating at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer provided in one aspect of the present invention exhibits excellent anticancer effect in vitro, in vivo, and therefore, pancreatic cancer, breast cancer and lung cancer. Useful as a therapeutic.

Claims (4)

  1. 하기 화합물 군으로부터 선택되는 1종 이상의 LRRK2(Leucine Rich Repeat Kinase 2) 단백질 저해제를 유효성분으로 함유하는,It contains at least one LRRK2 (Leucine Rich Repeat Kinase 2) protein inhibitor selected from the following compound group as an active ingredient,
    췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료용 약학적 조성물:A pharmaceutical composition for the prophylaxis or treatment of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer:
    (1) (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논; 및(1) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone; And
    (2) 3-(4-모폴리노-7H-피롤로[2,3-d]피리미딘-5-일)벤조니트릴.(2) 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile.
  2. 하기 화합물 군으로부터 선택되는 1종 이상의 LRRK2 단백질 저해제를, 이를 필요로 하는 대상에게 약학적으로 유효한 양으로 투여하는 단계를 포함하는, 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료방법:Treating at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer, comprising administering at least one LRRK2 protein inhibitor selected from the group of compounds to a subject in need thereof: Way:
    (1) (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논; 및(1) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone; And
    (2) 3-(4-모폴리노-7H-피롤로[2,3-d]피리미딘-5-일)벤조니트릴.(2) 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile.
  3. 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 예방 또는 치료에 사용하기 위한, 하기 화합물 군으로부터 선택되는 1종 이상의 LRRK2 단백질 저해제:At least one LRRK2 protein inhibitor selected from the group of compounds for use in the prevention or treatment of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer:
    (1) (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논; 및(1) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone; And
    (2) 3-(4-모폴리노-7H-피롤로[2,3-d]피리미딘-5-일)벤조니트릴.(2) 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile.
  4. 췌장암, 유방암 및 폐암으로 이루어진 군으로부터 선택되는 1종 이상 암의 치료를 위한 약제(medicament)의 제조에 사용하기 위한, 하기 화합물 군으로부터 선택되는 1종 이상의 LRRK2 단백질 저해제의 용도(use):Use of at least one LRRK2 protein inhibitor selected from the group of compounds for use in the manufacture of a medicament for the treatment of at least one cancer selected from the group consisting of pancreatic cancer, breast cancer and lung cancer:
    (1) (3-메톡시-4-(4-(메틸아미노)-5-(트리플루오로메틸)피리미딘-2-일아미노)페닐)(모폴리노)메타논; 및(1) (3-methoxy-4- (4- (methylamino) -5- (trifluoromethyl) pyrimidin-2-ylamino) phenyl) (morpholino) methanone; And
    (2) 3-(4-모폴리노-7H-피롤로[2,3-d]피리미딘-5-일)벤조니트릴.(2) 3- (4-morpholino-7H-pyrrolo [2,3-d] pyrimidin-5-yl) benzonitrile.
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