WO2015102380A1 - Novel 8-oxoprotoberberine derivative or pharmaceutically acceptable salt thereof, preparation method therefor and pharmaceutical composition for preventing or treating diseases associated with activity of nfat5, containing same as active ingredient - Google Patents

Novel 8-oxoprotoberberine derivative or pharmaceutically acceptable salt thereof, preparation method therefor and pharmaceutical composition for preventing or treating diseases associated with activity of nfat5, containing same as active ingredient Download PDF

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Publication number
WO2015102380A1
WO2015102380A1 PCT/KR2014/013059 KR2014013059W WO2015102380A1 WO 2015102380 A1 WO2015102380 A1 WO 2015102380A1 KR 2014013059 W KR2014013059 W KR 2014013059W WO 2015102380 A1 WO2015102380 A1 WO 2015102380A1
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WIPO (PCT)
Prior art keywords
derivative
nfat5
oxoprotoberberine
dioxolo
isoquinoline
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PCT/KR2014/013059
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French (fr)
Korean (ko)
Inventor
조희영
임희종
이계형
박우규
김현영
정대영
김완욱
조철수
박수정
한은진
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한국화학연구원
가톨릭대학교 산학협력단
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Priority to US15/029,075 priority Critical patent/US9650389B2/en
Priority claimed from KR1020140193586A external-priority patent/KR101656394B1/en
Publication of WO2015102380A1 publication Critical patent/WO2015102380A1/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
    • A61K31/435—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
    • A61K31/4375—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 six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems

Definitions

  • the present invention relates to a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof, a method for preparing the same, and a pharmaceutical composition for preventing or treating NFAT5 and an activity-related disease containing the same as an active ingredient.
  • Autoimmunity refers to a condition in which the body recognizes its speculum or tissue as an externally derived antigen and causes an immune response.
  • the original immune response is a system made against external antigens such as pathogens.
  • autoimmunity attacks their own organs or tissues and causes various diseases.
  • Autoimmune diseases include rheumatoid arthritis, systemic scleroderma, thrush lupus, atopic dermatitis, Beche's disease, Sjogren's syndrome, multiple sclerosis, and Grave's hyperthyroidism.
  • Rheumatoid arthritis is an autoimmune disease that causes chronic remnant inflammation in many tissues and organs, but is mainly a symptomatic synovial attack, which causes pain and deformation and loss of function and mobility if not treated properly.
  • the joint swells with a large amount of synovial fluid, the fibrous tissue (pannus) develops in the synovial membrane, and the destruction of articular cartilage (ankylos is), the lungs, the pericardium, and the ribs. Inflammation spreads to the sclera of the eye, but the cause of autoimmunity that causes rheumatoid arthritis is not clear.
  • Non-Patent Document 1 Approximately 0.6% of American adults suffer from rheumatoid arthritis, with 2-3 times more women than men (Non-Patent Document 1).
  • the treatment of rheumatoid arthritis includes nonpharmacological physiotherapy, orthoses, occupational therapy, nutritional therapy, and pharmacological treatments such as analgesics and anti-inflammatory drugs.
  • the drugs used for pharmacological treatment are nonsteroidal anti-inflammatory drugs ( Non-steroidal anti-inflammatory drugs (NSAIDs) or anti-rheumatic drugs (disease-modified ant i -rheumatic drugs, DMARD) can be used.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • DMARD disease-modified ant i -rheumatic drugs
  • Anti-inflammatory drugs are mainly anti-inflammatory drugs that exhibit anti-inflammatory action by inhibiting the synthesis of prostaglandins by inhibiting cyclooxygenase (C0X) enzyme activity, an important mediator of inflammation, for example, diclofenac, piroxicam ), Indomethacin, meloloc 1 meloxicam, celecoxib, rofecoxib, and lumiracoxib, which can be used to stop the progression of joints. You can't.
  • Antirheumatic drugs that can slow or stop disease progression include methotrexate, lef hmomide,
  • NFAT nuclear factor of activated T cells
  • TonEBP tacrine binding protein
  • NFAT5 is composed of approximately 1,500 amino acids and is expressed in almost all tissues. In particular, NFAT5 is highly expressed in the kidneys, lungs, pituitary gland, placenta, testicles, thymus, etc., which are actively metabolized and proliferated (Non-Patent Document 4).
  • NFAT5 does not have a calcineurin domain, which is not directly affected by calcium concentration, and is activated by osmostress in hypertonic fluid with increased osmotic pressure to maintain homeostasis.
  • NFAT5 activated in T cells is known to bind to the promoter of CD24 to increase transcription of CD24, thereby enabling amplification of T cells.
  • Relevant factors at higher levels of NFAT5 include ROS reactive oxygen species and p38 MAPK, and it has been reported that R0S is related to NFAT5 activity induced by TLR (Toll-like receptor) (Non-Patent Document 5).
  • NO nitric oxide a type of R0S
  • iNOS lnducible nitric oxide synthase NO induced by iNOS is known to play an important role in various diseases, particularly inflammatory reactions, and the expression of iNOS has been reported to be related to various diseases.
  • Non-Patent Document 6 Recently, NFAT5 has been reported to be highly expressed in joint synovial fluid of rheumatoid patients with autoimmune diseases and to increase the secretion of inflammatory cytokines IL- ⁇ , TNF- ⁇ , and the like (Non-Patent Document 7).
  • Berberine is a quaternary ammonium salt of isoquinoline alkaloids contained in sulfur. It is well known as a herbal medicine and anti-cancer, anti-obesity and diabetes treatment effects have been reported. In addition, berberine is the main component of the commercial cold-form preparation Jeong, Ro-hwan, which has an inhibitory effect on intestinal bacteria, sedative nervous system, arteriosclerosis prevention, anti-inflammatory effect, biliary tract, and pancreatic fluid secretion.
  • a derivative having various substituents at the 13 position (protoberberine ring) of berberine, a protoberberine having a significantly improved NFAT5 inhibitory effect and a C0X enzyme inhibitory effect than berberine has been known to exhibit excellent inflammation inhibitory effect in arthritis animal models.
  • the protoberberine has a disadvantage in that it is difficult to develop as an oral administration agent because the solubility is not soaked upon oral administration. Accordingly, the present inventors recognize that arthritis or autoimmune disease is a disease that requires continuous treatment, and requires a new mechanism for the preparation of drug resistance and requires the development of an oral preparation rather than an injection, and shows the effect of an NFAT5 inhibitor.
  • the shangyu 8-oxoprotoberberine derivative according to the present invention has an excellent effect of inhibiting NFAT5, and has improved the physical properties and the oral absorption rate through pharmacokinetic studies, thereby treating oral administration of rheumatoid arthritis
  • the present invention has been completed by finding out that it can be useful.
  • Non-Patent Document 1 Handout on Health: Rheumatoid Arthr itis. Nat ional Inst i tute of Arthr itis and Musculoskeletal and Skin Di seases, Apr il 2013; [Non-Patent Document 2] Biochem. Pharm. 72 (2006) 1597-1604; [Non-Patent Document 3] Nuc lei c Acids Res. 33 (2005) 3845-3854; [Non-Patent Document 4] J. nmunol. 165 (2000) 4884-4894;
  • Non-Patent Document 6 Semin Cancer Biol. 15 (2005) 277-289.
  • Non-Patent Document 7 Arthr i t i s and Rheumat i sm. 63 (2011) 1843-1852.
  • An object of the present invention is to provide a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide a method for preparing the 8-oxoprotoberberine derivative.
  • Another object of the present invention is to provide a pharmaceutical composition containing the 8—oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Another object of the present invention to provide an activity-related inhibitor of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Another object of the present invention is to provide a dietary supplement for the prevention or improvement of NFAT5 activity-related diseases containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Another object of the present invention is to provide a method for preventing, ameliorating or treating NFTA5 related diseases, which comprises administering to a subject an effective amount of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof. It is another object of the present invention to provide the use of an 8-oxoprotobe.verberine derivative or a pharmaceutically acceptable salt thereof for use in the composition for preventing, ameliorating or treating NFTA5-related diseases.
  • the present invention provides an 8-oxoprotoberberine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
  • A is d- 6 alkylene, -C (O)-or -NHCO0)-;
  • R is hydroxy, amino, straight or branched d-6 alkoxy, C 6 - 12 aryl or a nitrogen (N), oxygen (O) and sulfur (S) be the same or different selected from the group consisting of an interrogating atoms 1 Heteroaryl of 5 to 8 membered single ring or 8 to 11 membered double ring containing at least one species, wherein the aryl or heteroaryl is unsubstituted or halogen and straight or branched Ci-6 alkyl or d- 6 alkoxy. It is substituted by 1 or more types from the group which consists of.
  • step 1 Reacting the compound represented by the formula (2) with the compound represented by the formula (3) to obtain a compound represented by the formula (4) (step 1);
  • step 2 Method of preparing the 8-oxoprotoberberine derivative of claim 1 represented by the formula (1) comprising the step (step 2) to obtain a compound represented by the formula (1) by reacting the compound represented by the formula (4) obtained in the step 1 under basic conditions
  • the present invention provides a pharmaceutical composition containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also provides a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5, which contains a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides an inhibitor related to the activity of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a nutraceutical composition for preventing or ameliorating the activity-related diseases of NFAT5 containing a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides an activity of NFAT5 comprising administering to a subject an effective amount of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof
  • an effective amount of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof Provides methods for preventing, ameliorating or treating related diseases.
  • the present invention provides the use of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof for use in the composition for preventing, ameliorating or treating NFTA5-related diseases.
  • novel 8-oxoprotoberberine derivatives or pharmaceutically acceptable salts thereof according to the present invention have significantly improved oral absorption rate compared to conventional protoberberine due to the improvement of physical properties, and also inhibit the secretion of NFAT5 activity and inflammatory cytokines and rheumatoid arthritis.
  • NFAT5 activity-related diseases particularly rheumatoid arthritis or inflammatory diseases.
  • Figure 2 is a graph showing the effect of the derivative according to the invention on the transcriptional activity other than NFAT 3 ⁇ 4 ⁇ factor, specifically, A is NF- ⁇ , B is NFATc, C is C EB (CHO-Kl), D is CREBCHEK293 ), E is a graph showing the inhibition of transcriptional activity against ELK;
  • FIG. 3 is an image of a Western blot expressing p38 protein and active state p38 protein (phosphorylation) of the derivatives according to the invention
  • A is BGTl
  • B is A graph of the transcript of AR
  • A represents GM-CSF2
  • B represents MCP-1
  • C represents IL-6
  • Figure 7 is a graph showing the effect on the immune response of the mouse according to the derivative of the present invention, a control of the joint region of the mouse leg and a photograph after the injection of the derivative according to the present invention.
  • FIG. 8 is a diagram showing the inhibition of the expression of inflammatory cytokines (TNF- ⁇ , IL-6) induced by LPS in collagen-induced rheumatoid arthritis mice by the compound of Example 1.
  • FIG. 8 is a diagram showing the inhibition of the expression of inflammatory cytokines (TNF- ⁇ , IL-6) induced by LPS in collagen-induced rheumatoid arthritis mice by the compound of Example 1.
  • FIG. 9 is a diagram showing the inhibition of LPS-induced NFAT expression in collagen-induced rheumatoid arthritis mice by the compound of Example 1.
  • FIG. 9 is a diagram showing the inhibition of LPS-induced NFAT expression in collagen-induced rheumatoid arthritis mice by the compound of Example 1.
  • Figure 10a Figure 10b is a diagram showing metabolic stability and pharmacokinetics in hepatic microsomes by the compounds of Examples 1 and 3.
  • FIG. 11 is a diagram showing the inhibitory effect of inflammatory cytokine secretion by the compound of Example 1 in human mononuclear blood cells.
  • FIG. 12 is a diagram showing inhibition of differentiation into Thl7 cells by the compound of Example 1.
  • the present invention provides an 8-oxoprotoberberine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
  • R is one or more of the same or different hetero atoms selected from the group consisting of hydroxy, amino, straight or branched d- 6 alkoxy, C 6 — 12 aryl or nitrogen (N), oxygen (0) and sulfur (S)
  • R is preferably a hydroxy, amino, linear or Ci-4 alkoxy side chains, C 6 - 10 aryl or a nitrogen (N), oxygen (0) and the same sulfur is selected from the group consisting of (S) or different hetero A heteroaryl of 5 to 8 membered single ring or 8 to 11 membered dicyclic ring containing one subphase, wherein the aryl or heteroaryl consists of unsubstituted or halogen and linear or branched d- 6 alkyl or alkoxy.
  • One or more selected from the group more preferably one selected from the group consisting of hydroxy, amino, straight or branched d- 2 alkoxy, phenyl, pyridinyl, thiazolyl and benzoimidazolyl, wherein , with said phenyl, pyridinyl, thiazolyl or benzo-imidazolyl are unsubstituted or halogen, and straight chain or the group consisting of Cw alkyl or alkoxyalkyl side chain d- 4 Site is substituted with at least one member.
  • R is unsubstituted or halogen, methyl and Selected from the group consisting of phenyl, pyridinyl, thiazolyl and benzoimidazolyl substituted with one or more substituents selected from the group consisting of methoxy
  • the 8-oxoprotoberberine derivative represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.
  • Acid addition salts include inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono and dicarboxylates, phenylsubstituted alkanoates, hydroxy alkanoates and alkanedioates.
  • non-toxic organic acids such as aromatic acids, aliphatic and aromatic sulfonic acids, acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-luluenesulfonic acid, tartaric acid, fumaric acid and the like.
  • Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, eye Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, ' heptanoate, propiolate, oxalate, malonate, succinate, Suberates, Sebacates, Fumarates, Maleates, Butyne-1, 4-Diates, Nucleic Acids-1, 6 ⁇ dioates, Benzoates, Chlorobenzoates, Methylbenzoates, Dinitrobenzoates, Hydroxybenzoates , Methoxybenzoate, phthalate, terephthalate , Benzenesulfonate, toluenesulfon
  • Acid addition salts according to the present invention can be prepared by a conventional method, for example, the 8-oxoprotoberberine derivative represented by the formula (1), such as methane, ethanol, acetone, dichloromethane, acetonitrile, etc.
  • the precipitate produced by dissolving in an organic solvent and adding an organic acid or an inorganic acid may be prepared by filtration and drying, or the solvent and excess acid are distilled under reduced pressure and dried to crystallize under an organic solvent.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained by, for example, dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate.
  • it is pharmaceutically suitable to prepare a natrim, potassium or calcium salt as the metal salt.
  • the salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (for example, silver nitrate).
  • the present invention includes not only the 8-oxoprotoberberine derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof, but also all solvates and hydrates prepared therefrom.
  • the novel 8-oxoprotoberberine derivatives according to the present invention are excellent in inhibiting the activity of NFAT5, and in particular, it can be seen that the derivatives of Examples 1, 3, 6 and 10 are excellent in inhibiting the activity of NFAT5. (See Experimental Examples 2 and 3).
  • Experimental Examples 2 and 3 See Experimental Examples 2 and 3.
  • the symptoms were alleviated, and histological staining also showed that the symptoms of arthritis recovered significantly (see Experimental Example 10).
  • the derivative according to the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of NFAT5 activity-related diseases.
  • the present invention as shown in the following reaction formula 1,
  • step 1 Reacting the compound represented by the formula (2) with the compound represented by the formula (3) to obtain a compound represented by the formula (4) (step 1);
  • step 2 Chemical formula comprising the step (step 2) to obtain a compound represented by formula 1 by reacting the compound represented by formula 4 obtained in step 1 under basic conditions
  • step 1 is a step of obtaining a compound represented by the formula (4) by reacting the compound represented by the formula (2) and the compound represented by the formula (3).
  • the compound represented by the formula (3) is preferably used in 1.2 to 2 equivalents of the compound represented by the formula (2).
  • a catalyst such as sodium iodide, potassium iodide, sodium chloride or potassium chloride may be used, and it is more preferable to use sodium iodide. desirable.
  • the solvent usable in step 1 is acetonitrile, chloroform, dimethylformamide, dimethylsulfoxide, methane, ethanol, propanol, butane, tetrahydrofuran, dioxane, methylene chloride, 1,2-dimethoxyethane and the like. It could be used alone or in combination, it is preferable to use acetonitrile or the like.
  • step 2 is a step of obtaining a compound represented by Chemical Formula 1 by reacting the compound represented by Chemical Formula 4 obtained in Step 1 under basic conditions.
  • the base can be used without limitation as long as it is inexpensive, it is preferable to use a base such as potassium hydroxide, sodium hydroxide, calcium hydroxide, strontium hydroxide, more preferably using potassium hydroxide or sodium hydroxide as an aqueous solution desirable.
  • a base such as potassium hydroxide, sodium hydroxide, calcium hydroxide, strontium hydroxide, more preferably using potassium hydroxide or sodium hydroxide as an aqueous solution desirable.
  • step 2 is as shown in the following reaction formula 2,
  • Said step A by the formula (5) compound represented by the all-banung oxide obtained in step (step B) to obtain a compound represented by Formula 1; may be a step that includes:,
  • step A is a step of obtaining a compound represented by the formula (5) by reducing the compound represented by the formula (4).
  • the reducing agent that can be used may be sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, pyridine borohydride, zinc borohydride and the like, sodium cyanoborohydride, sodium borohydride It is preferable to use a lide, sodium triacetoxyborohydride and the like.
  • a base may be used as a catalyst, and any base can be used without limitation, but it is preferable to use a base such as potassium hydroxide, sodium hydroxide, calcium hydroxide, strontium hydroxide, and an aqueous solution of potassium hydroxide or sodium hydroxide. It is more preferable to use.
  • step B is a step of obtaining a compound represented by the formula (1) by oxidizing the compound represented by the formula (5) obtained in the step A.
  • manganese dioxide hydrogen peroxide, potassium dichromate, or the like may be used, and manganese dioxide is preferably used.
  • usable solvents include ether solvents such as methylene chloride, 1,2-dimethicethane, dimethylformamide, dimethyl sulfoxide and the like.
  • the present invention provides a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5, which contains a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an effective phase.
  • the activity-related diseases of NFAT5 may be arthritis, autoimmune diseases, and the like.
  • the arthritis disease is characterized in that rheumatoid arthritis.
  • the autoimmune disease is at least one selected from systemic scleroderma, lupus erythematosus, atopic dermatitis, Beche's disease, Sjogren's syndrome, multiple sclerosis and Graves' hyperthyroidism.
  • the novel 8-oxoprotoberberine derivatives are excellent in inhibiting the activity of NFAT5, and in particular, it can be seen that the derivatives of Examples 1, 3, 6 and 10 are excellent in inhibiting the activity of NFAT5 (experimental) See examples 2 and 3).
  • the symptoms of arthritis were alleviated when the joints of the mouse legs were visually observed in animal experiments, and the arthritis symptoms were recovered significantly through histological staining (see Experimental Example 11).
  • the compounds of the present invention inhibited the increase of LPS-induced iNOS gene expression (see Table 2), inhibited NFAT5 transcriptional activity (see Table 3, and FIG. 1) —another transcription factors, NF-kB, NFATc, It was also confirmed that the transcriptional activity of CREB and ELK was also inhibited (see FIG. 2).
  • the compounds of the present invention showed no inhibitory effect on R0S induction, p38 activity, and C0X1 and C0X2 (see FIGS. 3 and 6, Tables 4 and 5), and showed little toxicity to cells, and inflammatory It was confirmed that the secretion of cytokines was inhibited (see Table 6 and FIG. 6).
  • Compounds of the present invention in collagen-induced rheumatoid arthritis mice recovered arthritis symptoms (Fig. 7 acupuncture), and were found to reduce the expression of inflammatory cytokines (TNF- ⁇ , IL-6) (see Fig. 8). .
  • the compounds of the present invention showed increased oral absorption (see Table 7), and inflammatory cytokines in human mononuclear blood cells. Secretion was inhibited (see FIG. 11), and it was confirmed to inhibit differentiation from T cells to Thl7 cells (see FIG. I 2 ).
  • novel 8-oxoprotoberberine derivatives of the present invention or pharmaceutically acceptable salts thereof have significantly improved oral absorption rate than the conventional protoberberine due to the improvement of physical properties and do not pass through p38, which is an upstream stage of NFAT5.
  • p38 which is an upstream stage of NFAT5.
  • directly inhibiting the transcription of NFAT5 inhibiting NFAT5 activity and inflammatory cytokine secretion and reducing the expression of NFAT5 in rheumatoid arthritis mice, thereby preventing or preventing activity-related diseases of NFAT5, particularly rheumatoid arthritis or inflammatory diseases. It can be usefully used as a therapeutic pharmaceutical composition.
  • the 8'oxoprotoberberine derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof may be administered in a variety of oral and parenteral formulations for clinical administration.
  • it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, wherein the solid preparations are at least one 8 ⁇ oxoprotoberberine derivative represented by Formula 1 of the present invention, or a pharmaceutical thereof.
  • It may be formulated by mixing at least one excipient, for example starch, calcium carbonate, sucrose or lactose or gelatin, etc. with a generally acceptable salt.
  • excipients for example starch, calcium carbonate, sucrose or lactose or gelatin, etc.
  • lubricants such as stearic acid magnesium talc and the like may also be used.
  • Liquid preparations for oral administration include suspensions, solvents, emulsions or syrups.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. Can be.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • non-aqueous solvents and suspensions propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.
  • wi tepsol, macrogol, tvveen 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used. See also .
  • the dosage of the 8-oxoprotoberberine derivative represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, Based on an adult patient weighing 70 Kg, typically 0.1-1000 mg / day, preferably 1-500 mg / day, and also 1 day at regular time intervals as determined by the physician or pharmacist 1 time. It may be administered in several divided doses.
  • the pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of arthritis or autoimmune diseases.
  • the present invention also provides a nutraceutical composition for preventing or ameliorating the activity-related diseases of NFAT5 containing a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the derivative according to the present invention has an excellent effect of inhibiting the activity of NFAT5 can be usefully used as a health functional food composition for the prevention or improvement of NFAT5 activity-related diseases.
  • the disease associated with activity of NFAT5 may be arthritis or autoimmune disease.
  • the arthritis is characterized in that rheumatoid arthritis.
  • the autoimmune disease is at least one selected from systemic scleroderma, lupus erythematosus, atopic dermatitis, Beche's disease, Sjogren's syndrome, multiple sclerosis, and Graves' hyperthyroidism.
  • the novel 8-oxoprotoberberine derivatives of the present invention or pharmaceutically acceptable salts thereof have significantly improved oral absorption rate than the conventional protoberberine due to the improvement of physical properties and do not pass through p38, which is an upstream stage of NFAT5.
  • NFAT5 By directly inhibiting the transcription of NFAT5, inhibiting NFAT5 activity and inflammatory cytokine secretion and reducing the expression of NFAT5 in rheumatoid arthritis mice, thereby preventing or preventing activity-related diseases of NFAT5, particularly rheumatoid arthritis or inflammatory diseases. It can be usefully used as a dietary supplement for improvement. .
  • Examples of the food to which the substance may be added include drink, meat, sausage, bread, biscuit, rice cake, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, and 3 ⁇ 4.
  • Dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and includes all the health functional foods in the ordinary sense.
  • 8 ⁇ oxoprotoberberine derivatives represented by the formula (1) according to the present invention can be used as it is or added to other foods or food ingredients as it is, and may be appropriately used according to conventional methods.
  • the combined amount of the active ingredient can be suitably determined depending on the purpose of use (prevention or improvement).
  • the amount of the compound in the health food can be added from 0.1 to 90 parts by weight of the total food weight.
  • the amount may be below the above range, and the active ingredient is in the above range because there is no problem in terms of safety.
  • the above amounts may also be used.
  • the health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have.
  • natural carbohydrates examples include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as polysaccharides, for example, conventional sugars such as textine, cyclotextine and the like, and xylyl, sorbitol, and erytri Colour
  • natural flavoring agents tactin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.
  • 8-oxoprotoberberine derivatives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, coloring and neutralizing agents (cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.
  • the 8-oxoprotoberberine derivatives of the present invention may contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages.
  • the present invention also provides a method for preventing, ameliorating or treating an activity-related disease of NFAT5 comprising administering to a subject an effective amount of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof.
  • the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof for use as a prophylactic or therapeutic agent for a disease related to activity of NFAT5 is provided.
  • the 8-oxoprotoberberine derivatives or pharmaceutically acceptable salts thereof for use as a dietary supplement for the prevention or improvement of NFAT5 activity-related diseases is provided.
  • the derivative according to the present invention can be usefully used as a preventive or therapeutic agent for the activity-related diseases of NFAT5, and a dietary supplement for improvement.
  • the present invention will be described in detail by Examples and Experimental Examples.
  • Berberine hydrochloride (5 g, 13.45 ⁇ ol) was added to a 5N aqueous sodium hydroxide solution (23 mL) by a known method, and after stirring to 0 o C, acetone (5 mL, 67.23 ⁇ ol) was slowly added dropwise. After stirring for 1 hour at real silver, the resulting solid was filtered, washed twice with 40 mL of 80% methane, and dried to obtain the target compound (4.65g, 89%).
  • Example 1 Step 1 and Example 1 except that 2-methylbenzyl bromide was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1 In the same manner, the target compound (28%) was prepared.
  • the target compound (89%) was prepared by the same method as Step 1 of Example 1, except that 2-methoxybenzyl bromide was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1.
  • the target compound (59 «) was prepared in the same manner as in Example 1, Step 1 except that 2-chloromethylbenzoamidazole was used instead of 2-poloroben 3/4 bromide in Step 1 of Example 1.
  • Example 4 9-10-dimethoxy— 13- (2-methylbenzyl) -5, 6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline- 13- (1H-benzo [d] imidazol-2-yl) methyl) -9, 10-dimethoxy-5, 6-dihydro- [1,3] diox obtained in step 1 instead of 7-umbromide
  • the target compound (8%) was obtained by the same method as Step 2 of Example 4, except that Solo [4 ′ 5-g] isoquinolino [3,2-a] isoquinoline-7-iumbromide was used. .
  • the target compound (51%) was prepared in the same manner as in Step 1 of Example 1, except that 2 ⁇ chloro-5-chloromethylthiazole was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1. Prepared.
  • Step 2 13-((2-chlorothiazol-5-yl) meryl) -9,10-dimethoxy-5,6-dihydro-VII, 3] dioxolo [4, 5-g] isoquinolino [3 Al 2-alisoquinoline-8 (6H) -silver
  • the target compound (55%) was prepared in the same manner as in Step 1 of Example 1, except that ethyl chloroformate was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1.
  • the compound according to the present invention has an effect of inhibiting iNOS inducing activity.
  • the derivatives according to the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of iNOS-induced activity-related diseases.
  • NFAT5 reporter search was performed. Remove the CMV promoter sequence from the backbone of pEGFP-Nl and bind the sequence of NFAT5 (TGGAAAATTACCG) partial transduced plasmid vector
  • Raw264.7 macrophages were cultured in 10% FBS-RPMr medium (containing 500 g / ml of G418). The cells were dispensed in a 96-well plate at a density of 3 X 10 3 cells / well and further incubated for 2 hours. After treatment with LPS at a concentration of 1 yg / ml for 48 hours, the cells were fixed and the expression of GFP was measured by HCS fluorescence. Measured by image (Thermo,
  • FIG. 1 is a graph showing the NFAT5 transcriptional activity inhibitory effect of the derivative according to the present invention.
  • the derivatives according to the present invention is excellent in the effect of inhibiting the activity of NFAT5, especially in Example 1, 3, 6 and 10 It can be seen that the derivative is excellent in the effect of inhibiting the activity of NFAT5. Therefore, the derivatives according to the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of NFAT5 activity-related diseases.
  • NF- ⁇ transcriptional activity was measured by culturing THPl-Lucia NF- ⁇ reporter cells in 10% FBS-RPMI medium (containing 100 ug / ml Zeocin). Cells were incubated in a 96-well plate at a density of 2 ⁇ 10 4 cells / well and incubated for 24 hours with the addition of 1 g / ml LPS and the compound. Luminescence was measured by adding 50 ⁇ 13 ⁇ 4 of a fluorescence assay reagent to 10 ⁇ of the supernatant.
  • NFAT transcription activity evaluation was measured by incubating THPl-XBhie-MD2-CD14 cells in 10% FBS-RPMI medium (containing 200 g / ml Zeocin, 25 Mg ml ⁇ ] G418). First, the cells were incubated in a 96-well plate at a density of 2 X 10 4 cells / well, incubated for 24 hours with LPS 1 (Jg / ml and the compound added), and secreted embryonic alkaline phosphatase (SEAP) detect ion medium in 20 ⁇ of supernatant. Add 180 ⁇ and incubate for 2 hours
  • CREB and EL 1 transcriptional activity was evaluated using CHO / CREB-luc, HEK293 / CREB-luc, and HL / ELK1 cells.
  • Cells were cultured in 10% FBS-F12 (containing 100 pg / ml hygromycin), 10% FBS-DMEM (containing 100 g / ml hygromycin), and 10% FBS-DMEM (containing 200 ug / ml G418).
  • FIG. 2 is a graph showing the effect of the derivative according to the invention on the transcriptional activity other than NFAT transcription factor, specifically, A is NF- ⁇ , B is NFATc, C is
  • CREB CHO-Kl
  • D CREB (HEK293)
  • E is a graph showing the inhibition of transcriptional activity against ELK.
  • the protoberberine derivatives according to the present invention showed a slight effect on other transcription factors, unlike inhibition of NFAT5 at 1 ⁇ M or more than 803 ⁇ 4>. It can be seen that the derivatives of Examples 1 and 3 according to the present invention slightly inhibit NF- ⁇ and NFAT promoter activity at 10 ⁇ M.
  • the protoberberine derivatives according to the present invention show selective inhibition of the transcription factor activity of NFAT5.
  • FIG. 1 is an image of the Western blot expressing the p38 protein and active state p38 protein (phosphorylation) of the derivative according to the present invention. As shown in Figure 3, the group treated with the derivative according to the present invention and There was no difference in the degree of p38 protein (phosphorylation) expression in the untreated controls.
  • R 264.7 (10% FBS-RPMI 1640 medium) was incubated at a density of 1.5 x 10 5 cells / well in a 12 well plate and stabilized for one day.
  • the compound of Example 1 according to the present invention was pretreated at a concentration of ⁇ for one hour, cells were stimulated with LPS for 12 hours.
  • mRNA was isolated from cells to synthesize cDNA, and mRNA expression of NFAT5 was measured by real-time polymerase reaction.
  • NFAT5 a transcriptional regulator
  • Figure 4 is a graph showing the effect of the derivative according to the invention on the transcriptional step and nuclear migration.
  • the expression of NFAT5 was increased in the protein isolated from the nucleus than in the protein isolated from the cytoplasm due to the treatment of LPS, and the LPS treatment when the compound of Example 1 according to the present invention was pretreated. Decreased protein levels due to
  • Example 1 specifically inhibits NFAT5, which is increased by LPS, from the transcript stage, thereby inducing the inhibition of NFAT5 protein expression.
  • Raw264.7 cells (1OT FBS-RPMI 1640 medium) were incubated at a density of 1.5 ⁇ 10 5 cells / well in 6-well plates to stabilize. After treating the compound of Example 1 ( ⁇ ) an hour ago, NaCK45 mM) was stimulated for 3 hours, cDNA was synthesized from the extracted mRNA, and the NFAT5 transcript was confirmed by real-time polymerase reaction. Indicated.
  • BGT1 is a graph showing the effect of the derivative according to the present invention on the transcript expression of NFAT5 increased by sodium chloride, specifically A is BGT1, B is a graph of the transcript of AR.
  • BGT1 and AR which are transcripts of NFAT5
  • FIG. 5 the expression of BGT1 and AR, which are transcripts of NFAT5 was increased when NaCl was treated, and the increased transcripts were different even when treated with the compound of Example 1 according to the present invention. Did not look.
  • Inhibition of C0X enzyme is a representative anti-inflammatory mechanism.
  • the inhibition degree of Example 1 was compared with berberine according to the present invention.
  • Example 1 according to the present invention can be seen that the C0X1 and C0X2 enzyme inhibitory effect is insignificant.
  • the derivative according to the present invention had a minimal toxicity to cells at 10 ⁇ or less.
  • GM-CSF2, MCP-1 and IL-6 are target genes of NFAT5.
  • Raw264.7 cells were incubated in a 10% FBS-RPMI medium at a density of 1.5 x 10 6 cells / well in a 12-well plate, and the LPS (lpg / ml) was added the following day after pretreatment of the compound of Example 1 with ⁇ . Treatment was for 6 hours. After 6 hours of treatment, the supernatants of the wells were determined by ELISA for GM-CSF, MCP-1, and IL-6, and are shown in FIG. 6. 6 is a graph showing the inflammatory cytokine inhibitory effect of the derivatives according to the present invention. Specifically, A represents GM-CSF2, B represents MCP-1, and C represents IL-6.
  • the symptoms of arthritis were alleviated when the joints of the mouse legs of the group treated with the compound of Example 1 of the present invention were visually observed as compared with the negative control group.
  • histological staining showed that the symptoms of arthritis recovered significantly.
  • the derivative according to the present invention for preventing or treating arthritis diseases It will be appreciated that it may be useful as a pharmaceutical composition.
  • arthritis-induced mice were used as collagen used in Experimental Example 10. After the cells were extracted from the spleen of the mouse, the cells were stabilized by culturing the spleen cells in a 12 well plate. After stimulating with LPS for 12 hours, expression of inflammatory cytokines (TNF- ⁇ IL-6) was measured by ELISA.
  • TNF- ⁇ IL-6 inflammatory cytokines
  • TNF-a inflammatory cytokines
  • IL-6 inflammatory cytokines
  • NFAT protein In order to measure the expression of NFAT protein in rheumatoid arthritis mice, the following experiment was performed. Specifically, after stimulating LPS from splenocytes of arthritis induced by collagen-induced mouse splenocytes in the same manner as in ⁇ Experimental Example 11>, the protein was extracted from the cells, and NFAT5 was confirmed.
  • Table 7 was obtained by measuring the amount of the compound remaining after each compound was incubated for 30 minutes with rat and human liver microsome fractions.
  • male pharmacokinetic profiles administered at 5 mg / kg by intravenous (i v ) and oral (po) rats were obtained as shown in FIGS. 10A and 10B.
  • PBMC monocytes
  • the capsule was prepared by filling in gelatin capsule according to a conventional method for preparing capsules.
  • the compound according to the invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to 3.5 using dilute hydrochloric acid BP, and the volume was adjusted and sufficiently mixed with injection of sodium chloride BP.
  • the solution was filled into a 5 type I ampoule of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 0 C for at least 15 minutes to prepare an injection solution.
  • Vitamin B2 0. 15 nig
  • Vitamin B6 0.5 nig
  • composition ratio of the vitamin and mineral mixture mentioned above is relatively dry
  • the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method, and then granules are prepared, It can be used to prepare possible product compositions.
  • Purified water is added to the total 900
  • the resulting solution is filtered and obtained in a sterilized container, sealed and sterilized and then stored in a steamed beverage Used to prepare the composition.
  • composition ratio is a composition that is relatively suitable for a preferred beverage in a preferred embodiment
  • the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

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Abstract

The present invention relates to a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof, a preparation method therefor, and a pharmaceutical composition for preventing or treating diseases associated with the activity of NFAT5, containing the same as an active ingredient. The novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof according to the present invention can be useful in a pharmaceutical composition for preventing or treating diseases associated with the activity of NFAT5, and particularly, rheumatoid arthritis or inflammatory diseases since it is ascertained that the derivative or a pharmaceutically acceptable salt has remarkably increased oral absorption compared with known protoberberine due to an improvement in the properties thereof, and inhibits the activity of NFAT5 and the secretion of inflammatory cytokines and reduces the expression of NFAT5 in mice with rheumatoid arthritis by directly inhibiting the transcription of NFAT5.

Description

【명세세  [Specifications
【발명의 명칭】  [Name of invention]
신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염 이의 제조방법 및 이를 유효성분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물  Novel 8-oxoprotoberberine derivatives or pharmaceutically acceptable salts thereof preparations thereof and pharmaceutical compositions for the prevention or treatment of diseases related to the activity of NFAT5 containing the same as an active ingredient
【기술분야】 Technical Field
본 발명은 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염, 이의 제조방법 및 이를 유효성분으로 함유하는 NFAT5와 활성 관련 .질환의 예방 또는 치료용 약학적 조성물에 관한 것이다.  The present invention relates to a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof, a method for preparing the same, and a pharmaceutical composition for preventing or treating NFAT5 and an activity-related disease containing the same as an active ingredient.
【배경가술】 [Background Art]
자가면역은 몸에서 자신의 가관이나 조직을 외부에서 유래한 항원으로 인식， 면역 반응을 일으키는 증상을 말한다. 원래의 면역 반웅은 병원균 같은 외부의 항원에 대비해서 만들어진 체계이다. 하지만 자가면역으로 인해 스스로의 기관이나 조직을 공격하여 여러가지 질병들이 유발된다. 류머티스성 관절염， 전신성 경피증, 흥반성 낭창， 아토피 피부염， 베체씨병, 쇼그렌 증후군，- 다발성 경화증， 그레이브씨 갑상선 항진증 등이 자가면역질환에 속한다.  Autoimmunity refers to a condition in which the body recognizes its speculum or tissue as an externally derived antigen and causes an immune response. The original immune response is a system made against external antigens such as pathogens. However, autoimmunity attacks their own organs or tissues and causes various diseases. Autoimmune diseases include rheumatoid arthritis, systemic scleroderma, thrush lupus, atopic dermatitis, Beche's disease, Sjogren's syndrome, multiple sclerosis, and Grave's hyperthyroidism.
류마티스 관절염은 자가면역질환으로 많은 조직과 장기에 만성적인 잔신적 염증을 일으키나, 주로 유연한 활막 (synovi al )이 공격을 당하여 통증올 느끼고 변형되게 되며 적절히 치료되지 않으면 기능과 운동성을 상실하게 되는 질환이다ᅳ 또한 병의 진행에 따라 다량의 활막액으로 관절이 붓고 활막안의 섬유조직 (pannus)이 발달하며 관절연골의 파괴와 관절의 강직 (ankylos i s) , 폐 , 심막 (per icardium) , 늑
Figure imgf000003_0001
, 눈의 공막 (sclera) 등으로의 염증 확산이 일어나지만 아직 류마티스관절염을 유발하는 자가면역의 원인은 명확히 규명되지 않고 있다. 대략 0.6%의 미국 성인은 류마티스 관절염을 앓고 있고, 여성이 남성보다 2-3배 더 많다 (비특허문헌 1) . 류마티스 관절염의 치료는 비약리적인 물리치료， 정형술 (orthoses), 작업요법 (occupational therapy) 및 영양치료와 진통제， 항염증제 등의 약리치료가 있고， 약리치료에 사용되는 약물은 비스테로이드성 항염증약물 (non-steroidal anti-inflammatory drug: NSAID) 또는 항류마티스약물 (disease-modified ant i -rheumatic drug, DMARD)을 사용할 수 있다. 항염증약물로는 주로 염증의 중요한 매개자인 시클로옥시게나아제 (C0X) 효소 활성을 억제하여 프로스타글란딘 합성을 저해함으로써 항염증 작용을 나타내는 항염증 약물， 예를 들어 디클로페낙 (diclofenac), 피톡시캄 (piroxicam)， 인도메타신 ( indomethacin)， 멜록入 1캄 (meloxicam) , 쎄리콕시브 (celecoxib), 로페콕시브 (rofecoxib) , 루미라콕시브 (kimiracoxib) 둥에서 선택하여 사용할 수 있으나 관절의 손상진행을 멈추게 할 수는 없다. 질병의 진행을 느리게 하거나 멈추게 할 수 있는 항류마티스약물로는 메토트렉세이트 (methotrexate), 레플루노마이드 ( lef hmomide)，Rheumatoid arthritis is an autoimmune disease that causes chronic remnant inflammation in many tissues and organs, but is mainly a symptomatic synovial attack, which causes pain and deformation and loss of function and mobility if not treated properly. ᅳ Also, as the disease progresses, the joint swells with a large amount of synovial fluid, the fibrous tissue (pannus) develops in the synovial membrane, and the destruction of articular cartilage (ankylos is), the lungs, the pericardium, and the ribs.
Figure imgf000003_0001
Inflammation spreads to the sclera of the eye, but the cause of autoimmunity that causes rheumatoid arthritis is not clear. Approximately 0.6% of American adults suffer from rheumatoid arthritis, with 2-3 times more women than men (Non-Patent Document 1). The treatment of rheumatoid arthritis includes nonpharmacological physiotherapy, orthoses, occupational therapy, nutritional therapy, and pharmacological treatments such as analgesics and anti-inflammatory drugs. The drugs used for pharmacological treatment are nonsteroidal anti-inflammatory drugs ( Non-steroidal anti-inflammatory drugs (NSAIDs) or anti-rheumatic drugs (disease-modified ant i -rheumatic drugs, DMARD) can be used. Anti-inflammatory drugs are mainly anti-inflammatory drugs that exhibit anti-inflammatory action by inhibiting the synthesis of prostaglandins by inhibiting cyclooxygenase (C0X) enzyme activity, an important mediator of inflammation, for example, diclofenac, piroxicam ), Indomethacin, meloloc 1 meloxicam, celecoxib, rofecoxib, and lumiracoxib, which can be used to stop the progression of joints. You can't. Antirheumatic drugs that can slow or stop disease progression include methotrexate, lef hmomide,
(hydroxychloroquine), . 설파살라진 (sul fasalazine)， 아자티오프린 (azathioprine), 사이클로포스파미드 (cyclophosphamide) , 사이클로스포린 A cyclosporine A) 등의 약물과 TNF-α에 대한 항체치료제가 있고 2012년에 JAK3 저해제인 Xeljanz (Tofacitinib, Pfizer)가 저분자 치료제로서는 처음으로 시장에 진출하였으나 아직도 좀래의 약물로 치료되지 않는 무반옹 환자들이 많으므로 의료적 미충족 수요가 높고 새로운 기전의 치료제의 개발이 요구되고 있다. (hydroxychloroquine),. Drugs such as sul fasalazine, azathioprine, cyclophosphamide, and cyclosporine A, and antibodies to TNF-α are available.In 2012, JAK3 inhibitor Xeljanz (Tofacitinib, Pfizer ) Is the first to enter the market as a low-molecular medicine, but there are many non-reflective patients who are not yet treated with medicinal drugs.
NFAT(nuclear factor of activated T cells)은 세포질에 존재하는 단백질로서 Ca 2+ 이동 (mobilization)과 커플된 세포 표면의 수용체의 자극에 의해서 활성화 된다. NFAT 단백질은 Ca 2+ 에 의해 활성화되는 포스파타제 (phosphatase)즉， 칼시네우린 (calcineurin)에 의해서 탈인산화되며, 탈인산화된 NFAT는 핵으로 이동하여 T 세포의 활성화에 필요한 IL-2 유전자를 포함한 여러 사이토카인 (cytokine)유전자의 전사를 유도한다. 특히， NFAT5 (nuclear factor of activated T cells 5)는 TonEBP (tonicity enhancer binding protein), OREBP, NFATLl, NFATz등으로 불리우며 Rel family 중 가장 길이가 긴 전사조절인자로서 다른 NFAT 전사인자들 WFAT1 (NFATp, NFATc2), NFAT2 (NFATc, NFATcl), NFAT3 (NFATc4), NFAT4 (NFATx, NFATc3)}과는 구조적， 기능적으로 뚜렷한 차이를 가지고 있다 (비특허문헌 2 및 3). NFAT5는 약 1，500개의 아미노산으로 되어 있고 거의 모든 조직에서 발현되며 특히 대사와 증식이 활발한 태아의 신장， 폐, 뇌하수체， 태반， 고환， 흉선 등에서 높은 발현을 보인다 (비특허문헌 4). 구조적으로 NFAT5는 calcineurin도메인을 가지고 있지 않기 때문에 칼슘농도에 의해 직접적인 영향을 받지 않으며 삼투압이 증가된 고장액에서 osmostress에 의해 활성화되어 항상성을 유지하는 역할을 한다. 또한， T세포에서 활성화된 NFAT5는 CD24의 promoter에 결합하여 CD24의 전사를 증가시키며 이에 의해 T세포의 증폭이 가능해지도록 하는 것으로 알려져 있다. NFAT5의 상위단계에서 관련된 인자로는 ROS reactive oxygen species)및 p38 MAPK가 있고， R0S는 TLR(Toll-like receptor)로 유도된 NFAT5 활성에 관련되어 있다는 내용이 보고되었다 (비특허문헌 5). R0S의 일종인 NO nitric oxide)는 iNOS lnducible nitric oxide synthase)에 의해 생성된다. iNOS에 의해 유도된 NO는 여러 질환， 특히 염증 반웅에 중요한 역할을 하는 것으로 알려져 있으며， iNOS의 발현은 여저 질환에 관련되어 있는 것으로 보고되었다 (비특허문헌 6). 최근에 NFAT5는 자가면역 질환인 류마티스 환자의 관절활액에서 많이 발현되고 염증성 사이토카인인 IL-Ιβ, TNF-α 등의 분비를 증가시킨다는 것이 보고되었다 (비특허문헌 7). 또한 siRNA를 주입하여 NFAT5를 knockdown 시킨 활액세포나 혈관세포인 HUVEC등에서 세포의 증식아나 혈관신생， 세포이동 등이 현저히 감소되는 것이 관찰되며 NFAT5(+/-)인 실험쥐에서는 관절의 염증이 현저히 줄어들었으므로 NFAT5의 활성을 저해하는 화합물은 류마티스관절염을 개선시키는 새로운 치료제로 개발될 수 있을 것으로 판단된다. NFAT (nuclear factor of activated T cells) is a protein in the cytoplasm that is activated by stimulation of receptors on the cell surface coupled with Ca 2+ mobilization. NFAT protein is dephosphorylated by phosphatase, which is activated by Ca 2+ , or calcineurin, and dephosphorylated NFAT migrates to the nucleus and contains several IL-2 genes required for T cell activation. Induces transcription of cytokine genes In particular, NFAT5 (nuclear factor of activated T cells 5) is called TonEBP (tonicity enhancer binding protein), OREBP, NFATLl, NFATz, and is the longest transcription regulator in the Rel family. ), NFAT2 (NFATc, NFATcl), NFAT3 (NFATc4), NFAT4 (NFATx, NFATc3)} and structurally and functionally distinct differences (non-patent documents 2 and 3). NFAT5 is composed of approximately 1,500 amino acids and is expressed in almost all tissues. In particular, NFAT5 is highly expressed in the kidneys, lungs, pituitary gland, placenta, testicles, thymus, etc., which are actively metabolized and proliferated (Non-Patent Document 4). Structurally, NFAT5 does not have a calcineurin domain, which is not directly affected by calcium concentration, and is activated by osmostress in hypertonic fluid with increased osmotic pressure to maintain homeostasis. In addition, NFAT5 activated in T cells is known to bind to the promoter of CD24 to increase transcription of CD24, thereby enabling amplification of T cells. Relevant factors at higher levels of NFAT5 include ROS reactive oxygen species and p38 MAPK, and it has been reported that R0S is related to NFAT5 activity induced by TLR (Toll-like receptor) (Non-Patent Document 5). NO nitric oxide, a type of R0S, is produced by iNOS lnducible nitric oxide synthase. NO induced by iNOS is known to play an important role in various diseases, particularly inflammatory reactions, and the expression of iNOS has been reported to be related to various diseases (Non-Patent Document 6). Recently, NFAT5 has been reported to be highly expressed in joint synovial fluid of rheumatoid patients with autoimmune diseases and to increase the secretion of inflammatory cytokines IL-Ιβ, TNF-α, and the like (Non-Patent Document 7). In addition, it was observed that proliferation, angiogenesis, and cell migration were significantly reduced in synovial cells or HUVECs that knocked down NFAT5 by siRNA injection. Inflammation of joints was observed in NFAT5 (+/-) mice. Significantly diminished, compounds that inhibit NFAT5 activity could be developed as novel therapeutics to improve rheumatoid arthritis.
베르베린은 이소퀴놀린 알카로이드의 4차 암모늄염으로서 황련에 함유된. 생약제제로 잘 알려져 있고 항암작용， 항비만 및 당뇨치료효과 등이 보고되어 있다. '또한， 베르베린은 시판되는 찬연물 정장제인 정로환의 주성분으로 장내 세균에 대한 억제작용， 진정진경작용， 동맥경화 예방작용， 소염작용， 이담작용， 췌장액분비촉진작용 둥을 가지고 있다. 기존에 베르베린의 13위치 (프로토베르베린 고리)에 다양한 치환기가 도입된 유도체로서 베르베린보다 월등히 향상된 NFAT5 저해효능과 C0X 효소 저해효과를 보이는 프로토베르베린이 관절염동물모델에서 우수한 염증억제효과를 나타낸다고 알려진 바 있으나, 상기 프로토베르베린은 용해도가 낮아서 경구투여시 흡수가 되지않아 경구투여제로서의 개발이 어려운 단점이 있다. 이에, 본 발명자들은 관절염이나 자가면역질환은 지속적인 치료가 필요한 질환으로 약제내성에 대비하기 위한 새로운 기전의 약물이 요구되며 주사제보다는 경구제제의 개발이 필요함을 인식하고， NFAT5 저해제의 효과를 나타내며 경구투여제로 사용할 수 있는 화합물을 개발하던 중, 본 발명에 따른 샨규한 8-옥소프로토베르베린 유도체가 NFAT5 저해 효과가 우수하고， 약동력 연구를 통해 물성을 개선하고 경구흡수율을 높여 경구투여 형태의 류마티스 관절염 치료제로 유용할 수 있다는 것올 알아내어 본 발명을 완성하였다. Berberine is a quaternary ammonium salt of isoquinoline alkaloids contained in sulfur. It is well known as a herbal medicine and anti-cancer, anti-obesity and diabetes treatment effects have been reported. In addition, berberine is the main component of the commercial cold-form preparation Jeong, Ro-hwan, which has an inhibitory effect on intestinal bacteria, sedative nervous system, arteriosclerosis prevention, anti-inflammatory effect, biliary tract, and pancreatic fluid secretion. Previously, a derivative having various substituents at the 13 position (protoberberine ring) of berberine, a protoberberine having a significantly improved NFAT5 inhibitory effect and a C0X enzyme inhibitory effect than berberine, has been known to exhibit excellent inflammation inhibitory effect in arthritis animal models. The protoberberine has a disadvantage in that it is difficult to develop as an oral administration agent because the solubility is not soaked upon oral administration. Accordingly, the present inventors recognize that arthritis or autoimmune disease is a disease that requires continuous treatment, and requires a new mechanism for the preparation of drug resistance and requires the development of an oral preparation rather than an injection, and shows the effect of an NFAT5 inhibitor. During the development of a compound that can be used as a zero, the shangyu 8-oxoprotoberberine derivative according to the present invention has an excellent effect of inhibiting NFAT5, and has improved the physical properties and the oral absorption rate through pharmacokinetic studies, thereby treating oral administration of rheumatoid arthritis The present invention has been completed by finding out that it can be useful.
【선행기술문헌】 Prior Art Documents
【비특허문헌】  [Non-patent literature]
[비특허문헌 l]Handout on Health: Rheumatoid Arthr i t i s . Nat ional Inst i tute of Arthr i t i s and Musculoskeletal and Skin Di seases , Apr i l 2013； [비특허문헌 2]Biochem. Pharm. 72 (2006) 1597-1604； [비특허문헌 3]Nuc lei c Acids Res . 33 (2005) 3845-3854； [비특허문헌 4]J . nmunol . 165 (2000) 4884-4894; [Non-Patent Document 1] Handout on Health: Rheumatoid Arthr itis. Nat ional Inst i tute of Arthr itis and Musculoskeletal and Skin Di seases, Apr il 2013; [Non-Patent Document 2] Biochem. Pharm. 72 (2006) 1597-1604; [Non-Patent Document 3] Nuc lei c Acids Res. 33 (2005) 3845-3854; [Non-Patent Document 4] J. nmunol. 165 (2000) 4884-4894;
[비특허문헌 5] Eur J I隱 unol , 44 (2014) 2721-2736.  [Non-Patent Document 5] Eur J I'unol, 44 (2014) 2721-2736.
[비특허문헌 6]Semin Cancer Biol . 15 (2005) 277-289.  [Non-Patent Document 6] Semin Cancer Biol. 15 (2005) 277-289.
[비특허문헌 7]Arthr i t i s and Rheumat i sm. 63 (2011) 1843-1852.  [Non-Patent Document 7] Arthr i t i s and Rheumat i sm. 63 (2011) 1843-1852.
[발명의 상세한 설명】 Detailed description of the invention
[기술적 과제】 본 발명의 목적은 신규한 8-옥소프로토베르베린 유도체 또는 、이의 약학적으로 허용가능한 염을 제공하는데 있다.  TECHNICAL PROBLEM An object of the present invention is to provide a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof.
본 발명의 다른 목적은 상기 8-옥소프로토베르베린 유도체의 제조방법을 제공하는데 있다.  Another object of the present invention is to provide a method for preparing the 8-oxoprotoberberine derivative.
본 발명의 다른 목적은 상기 8—옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 약학적 조성물을 제공하는데 있다.  Another object of the present invention is to provide a pharmaceutical composition containing the 8—oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물을 제공하는데 있다.  Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 다른 목적은 상기 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 억제제를 제공하는데 있다.  Another object of the present invention to provide an activity-related inhibitor of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 다른 목적은 상기 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 개선용 건강기능식품 조성물올 제공하는데 있다.  Another object of the present invention is to provide a dietary supplement for the prevention or improvement of NFAT5 activity-related diseases containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 또 다른 목적은 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효한 양으로 개체에 투여하는 것을 포함하는 NFTA5 관련질환 예방, 개선 또는 치료방법을 제공하는데 있다. 본 발명의 다른 목적은 NFTA5관련질환 예방， 개선 또는 치료용 조성물을 사용하기 위한 8-옥소프로토베.르베린 유도체 또는 이의 약학적으로 허용가능한 염의 용도를 제공하는데 있다. 【기술적 해결방법】 Another object of the present invention is to provide a method for preventing, ameliorating or treating NFTA5 related diseases, which comprises administering to a subject an effective amount of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof. It is another object of the present invention to provide the use of an 8-oxoprotobe.verberine derivative or a pharmaceutically acceptable salt thereof for use in the composition for preventing, ameliorating or treating NFTA5-related diseases. Technical Solution
상기 목적을 달성하기 위하여， 본 발명은 하기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 제공한다:  In order to achieve the above object, the present invention provides an 8-oxoprotoberberine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
Figure imgf000008_0001
Figure imgf000008_0001
상기 화학식 1에서，  In Chemical Formula 1,
A는 d-6알킬렌, -C(O)- 또는 -NHCO0)-이고; A is d- 6 alkylene, -C (O)-or -NHCO0)-;
R은 하이드록시， 아미노， 직쇄 또는 측쇄의 d-6알콕시 , C6-12아릴 또는 질소 (N) , 산소 (0) 및 황 (S)으로 이루어진 군으로부터 선택되는 동일 또는 상이한 해테로 원자를 1종 이상 포함하는 5내지 8원자 단일 고리 또는 8내지 11 원자 이중 고리의 해테로아릴이고， 여기서, 상기 아릴 또는 헤테로아릴은 비치환 또는 할로겐 및 직쇄 또는 측쇄의 Ci-6알킬 또는 d-6알콕시로 이루어지는 군으로부터 1종 이상으로 치환된다. R is hydroxy, amino, straight or branched d-6 alkoxy, C 6 - 12 aryl or a nitrogen (N), oxygen (O) and sulfur (S) be the same or different selected from the group consisting of an interrogating atoms 1 Heteroaryl of 5 to 8 membered single ring or 8 to 11 membered double ring containing at least one species, wherein the aryl or heteroaryl is unsubstituted or halogen and straight or branched Ci-6 alkyl or d- 6 alkoxy. It is substituted by 1 or more types from the group which consists of.
또 h 본 발명은 하기 반웅식 1에 나타낸 바와 같이，  In addition, the present invention, as shown in the following reaction formula 1,
화학식 2로 표시되는 화합물과 화학식 3으로 표시되는 화합물을 반웅시켜 화학식 4로 표시되는 화합물을 얻는 단계 (단계 1) ;  Reacting the compound represented by the formula (2) with the compound represented by the formula (3) to obtain a compound represented by the formula (4) (step 1);
상기 단계 1에서 얻은 화학식 4로 표시되는 화합물을 염기 조건 하에서 반웅시켜 화학식 1로 표시되는 화합물을 얻는 단계 (단계 2)를 포함하는 화학식 1로 표시되는 제 1항의 8-옥소프로토베르베린 유도체의 제조방법을 제공한다:  Method of preparing the 8-oxoprotoberberine derivative of claim 1 represented by the formula (1) comprising the step (step 2) to obtain a compound represented by the formula (1) by reacting the compound represented by the formula (4) obtained in the step 1 under basic conditions Provides:
[반웅식 1]
Figure imgf000009_0001
[Banungsik 1]
Figure imgf000009_0001
4  4
상기 반움식 1에서， A 및 R은 상기에서 정의한 바와 같고, X1 및 X2는 할로겐이다. 본 발명은 상기 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 약학적 조성물을 제공한다. 또한， 본 발명은 상기 8-옥소프로토베르베린 유도체 또는 이의 약학작으로 허용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다. In the above formula 1, A and R are as defined above, and X 1 and X 2 are halogen. The present invention provides a pharmaceutical composition containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명은 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물을 제공한다. 본 발명은 상기 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 억제제를 제공한다. 나아가， 본 발명은 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 ^용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. 나아가， 본 발명은 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효한 양으로 개체에 투여하는 것을 포함하는 NFAT5의 활성 관련 질환의 예방， 개선 또는 치료 방법을 제공한다. 본 발명은 NFTA5 관련질환 예방, 개선 또는 치료용 조성물을 사용하기 위한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염의 용도를 제공한다. The present invention provides a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5, which contains a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention provides an inhibitor related to the activity of NFAT5 containing the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient. Furthermore, the present invention provides a nutraceutical composition for preventing or ameliorating the activity-related diseases of NFAT5 containing a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient. Furthermore, the present invention provides an activity of NFAT5 comprising administering to a subject an effective amount of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof Provides methods for preventing, ameliorating or treating related diseases. The present invention provides the use of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof for use in the composition for preventing, ameliorating or treating NFTA5-related diseases.
【유리한 효과】 Advantageous Effects
본 발명에 따른 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염은 물성 개선으로 경구 흡수율이 기존의 프로토베르베린보다 현저히 증가되었고, 또한 NFAT5 활성 및 염증성 사이토카인의 분비를 억제하고 류마티스 관절염 마우스에서 NFAT5의 발현을 감소시킴을 확인함으로써， NFAT5의 활성 관련 질환，특히 류마티스 관절염 또는 염증 질환의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다. [도면의 간단한 설명】  The novel 8-oxoprotoberberine derivatives or pharmaceutically acceptable salts thereof according to the present invention have significantly improved oral absorption rate compared to conventional protoberberine due to the improvement of physical properties, and also inhibit the secretion of NFAT5 activity and inflammatory cytokines and rheumatoid arthritis. By confirming the reduced expression of NFAT5 in the mouse, it can be usefully used as a pharmaceutical composition for the prevention or treatment of NFAT5 activity-related diseases, particularly rheumatoid arthritis or inflammatory diseases. [Brief Description of Drawings]
도 1은 본 발명에 따른 유도체의 NFAT5 전사활성 억제효과를 나타낸 그래프이고;  1 is a graph showing the inhibitory effect of NFAT5 transcriptional activity of the derivatives according to the present invention;
도 2는 본 발명에 따른 유도체가 NFAT ¾ί사인자 이외의 전사활성에 미치는 영향을 나타낸 그래프로서， 구체적으로 Α는 NF-κΒ , B는 NFATc , C는 C EB(CHO-Kl) , D는 CREBCHEK293)， E는 ELK에 대한 전사활성의 억제를 나타낸 그래프이고;  Figure 2 is a graph showing the effect of the derivative according to the invention on the transcriptional activity other than NFAT ¾ί factor, specifically, A is NF-κΒ, B is NFATc, C is C EB (CHO-Kl), D is CREBCHEK293 ), E is a graph showing the inhibition of transcriptional activity against ELK;
도 3은 본 발명에 따른 유도체의 p38 단백질과 활성 상태 p38 단백질 (인산화)의 발현 웨스턴 블롯의 이미지이고;  3 is an image of a Western blot expressing p38 protein and active state p38 protein (phosphorylation) of the derivatives according to the invention;
도 4는 본 발명에 따른 유도체가 전사단계 및 핵 이동에 미치는 영향을 나타낸 그래프이고;  4 is a graph showing the effect of the derivative according to the invention on the transcriptional step and nuclear migration;
도 5의 본 발명에 따른 유도체가 염화나트륨에 의해 증가되는 NFAT5의 전사체 발현에 미치는 영향을 나타낸 그래프이고， 구체적으로 A는 BGTl , B는 AR의 전사체에 관한 그래프이고; 5 is a graph showing the effect of the derivative according to the invention on the transcript expression of NFAT5 increased by sodium chloride, specifically A is BGTl, B is A graph of the transcript of AR;
도 6은 본 발명에 따른 유도체가 염증성 사이토카인 억제 효과를 나타낸 그래프로서, 구체적으로 A는 GM-CSF2, B는 MCP-1 및 C는 IL-6를 나타내고; 및  6 is a graph showing the inflammatory cytokine inhibitory effect of the derivatives according to the present invention, specifically, A represents GM-CSF2, B represents MCP-1 and C represents IL-6; And
도 7의 본 발명에 따른 유도체의 마우스의 면역 반웅에 미치는 영향을 나타낸 그래프이고, 마우스 다리의 관절 부위의 대조군 및 본 발명에 따른 유도체를주입한 후의 사진이다.  Figure 7 is a graph showing the effect on the immune response of the mouse according to the derivative of the present invention, a control of the joint region of the mouse leg and a photograph after the injection of the derivative according to the present invention.
도 8은 실시예 1의 화합물에 의해 콜라겐으로 유도된 류마티스 관절염 마우스에서 LPS로 유도된 염증성 사이토카인 (TNF-α, IL-6)의 발현 억제를 나타낸 도이다.  8 is a diagram showing the inhibition of the expression of inflammatory cytokines (TNF-α, IL-6) induced by LPS in collagen-induced rheumatoid arthritis mice by the compound of Example 1. FIG.
도 9는 실시예 1의 화합물에 의해 콜라겐으로 유도된 류마티스 관절염 마우스에서 LPS로 유도된 NFAT의 발현 억제를 나타낸 도이다.  9 is a diagram showing the inhibition of LPS-induced NFAT expression in collagen-induced rheumatoid arthritis mice by the compound of Example 1. FIG.
도 10a 도 10b는 실시예 1 및 실시예 3의 화합물에 의한 간마이크로좀에서 대사안정성 및 약물동태를 나타낸 도이다.  Figure 10a Figure 10b is a diagram showing metabolic stability and pharmacokinetics in hepatic microsomes by the compounds of Examples 1 and 3.
도 11은 인간 단핵혈액세포에서 실시예 1의 화합물에 의한 염증성 사이토카인 분비 억제 효과를 나타낸 도이다.  11 is a diagram showing the inhibitory effect of inflammatory cytokine secretion by the compound of Example 1 in human mononuclear blood cells.
도 12는 실시예 1의 화합물에 의한 Thl7 세포로의 분화 억제를 나타낸 도이다.  12 is a diagram showing inhibition of differentiation into Thl7 cells by the compound of Example 1. FIG.
【발명의 실시를 위한 최선의 형태】 [Best form for implementation of the invention]
이하， 본 발명을 상세히 설명한다. 본 발명은 하기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 제공한다:  Hereinafter, the present invention will be described in detail. The present invention provides an 8-oxoprotoberberine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
【화학식 1】
Figure imgf000012_0001
[Formula 1]
Figure imgf000012_0001
상기 화학식 1에서,  In Chemical Formula 1,
A는 d— 6알킬렌， -CO0)- 또는 -NHC(=0)-이고; A is d- 6 alkylene, -CO0)-or -NHC (= 0)-;
R은 하이드록시, 아미노， 직쇄 또는 측쇄의 d-6알콕시， C6— 12아릴 또는 질소 (N) , 산소 (0) 및 황 (S)으로 이루어진 군으로부터 선택되는 동일 또는 상이한 헤테로 원자를 1종 이상 포함하는 5내지 8원자 단일 고리 또는 8내지 11 원자 이중 고리의 헤테로아릴이고， 여기서， 상기 아릴 또는 헤테로아릴은 비치환 또는 할로겐 및 직쇄 또는 측쇄의 6알킬 또는 d-6알콕시로 이투어지는 군으로부터 1종 이상으로 치환된다. 또한, 상기 A는 바람직하게는 d-4알킬렌， -C(=0)- 또는 -NHC(=0)-이고， 보다 바람직하게는 Ci-2알킬렌， -C(=0)- 또는 -NHCO0)-이다. R is one or more of the same or different hetero atoms selected from the group consisting of hydroxy, amino, straight or branched d- 6 alkoxy, C 6 — 12 aryl or nitrogen (N), oxygen (0) and sulfur (S) A heteroaryl of 5 to 8 membered single ring or 8 to 11 membered double ring, wherein the aryl or heteroaryl is unsubstituted or substituted from halogen and straight or branched 6 alkyl or d- 6 alkoxy. It is substituted by 1 or more types. In addition, A is preferably d- 4 alkylene, -C (= 0)-or -NHC (= 0)-, more preferably Ci-2 alkylene, -C (= 0)-or- NHCO0)-.
상기 R은 바람직하게는 하이드록시， 아미노， 직쇄 또는 측쇄의 Ci-4알콕시， C6-10아릴 또는 질소 (N) , 산소 (0) 및 황 (S)으로 이루어진 군으로부터 선택되는 동일 또는 상이한 헤테로 원자를 1종 아상 포함하는 5 내지 8 원자 단일 고리 또는 8내지 11원자 이증 고리의 헤테로아릴이고,여기서，상기 아릴 또는 헤테로아릴은 비치환 또는 할로겐 및 직쇄 또는 측쇄의 d-6알킬 또는 알콕시로 이루어지는 군으로부터 1종 이상으로 치환되고, 보다 바람직하게는 하이드록시 ,아미노， 직쇄 또는 측쇄의 d-2알콕시，페닐，피리디닐， 티아졸릴 및 벤조이미다졸릴로 이루어진 군으로부터 선택되는 1종이고, 여기서, 상기 페닐， 피리디닐， 티아졸릴 또는 벤조이미다졸릴은 비치환 또는 할로겐 및 직쇄 또는 측쇄의 Cw알킬 또는 d-4알콕시로 이루어지는 군으로부터 1종 이상으로 치환된다. 나아가， 상기 A가 d-4알킬렌인 경우， R은 비치환 또는 할로겐, 메틸 및 메톡시로 이루어진 군으로부터 선택되는 1종 이상의 치환기로 치환된 페닐, 피리디닐， 티아졸릴 및 벤조이미다졸릴로 이루어진 군으로부터 선택되는Wherein R is preferably a hydroxy, amino, linear or Ci-4 alkoxy side chains, C 6 - 10 aryl or a nitrogen (N), oxygen (0) and the same sulfur is selected from the group consisting of (S) or different hetero A heteroaryl of 5 to 8 membered single ring or 8 to 11 membered dicyclic ring containing one subphase, wherein the aryl or heteroaryl consists of unsubstituted or halogen and linear or branched d- 6 alkyl or alkoxy. One or more selected from the group, more preferably one selected from the group consisting of hydroxy, amino, straight or branched d- 2 alkoxy, phenyl, pyridinyl, thiazolyl and benzoimidazolyl, wherein , with said phenyl, pyridinyl, thiazolyl or benzo-imidazolyl are unsubstituted or halogen, and straight chain or the group consisting of Cw alkyl or alkoxyalkyl side chain d- 4 Site is substituted with at least one member. Furthermore, when A is d- 4 alkylene, R is unsubstituted or halogen, methyl and Selected from the group consisting of phenyl, pyridinyl, thiazolyl and benzoimidazolyl substituted with one or more substituents selected from the group consisting of methoxy
1종이고; . 1 type; .
상기 A가 -C(=0)-인 경우， R은 하이드록시， 아미노, 직쇄 또는 측쇄의 d-2알콕시 또는 비치환 또는 할로겐으로 하나 이상 치환된 페닐이고; 및 상기 A가 -NHC(=0)-인 경우， R은 비치환 또는 할로겐으로 하나 이상 치환 ¾ 페닐이다. 또한， 상기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체를 보다 구체적으로 예시하면 하기와 같다: ~ When A is -C (= 0)-, R is hydroxy, amino, straight or branched d- 2 alkoxy or phenyl substituted one or more by unsubstituted or halogen; And when A is -NHC (= 0)-, R is unsubstituted or halogen substituted one or more ¾ phenyl. Further, Specific examples of 8-oxo than the protocol berberine derivatives of the formula (1) as follows: -
1)  One)
13-(2-플루오로벤질 )-9，10-디메톡시-51]-[1ᅳ3]디옥솔로[4,5- 이소퀴놀리노[3ᅳ2 -a]이소퀴놀린 -8(6H)-은;  13- (2-fluorobenzyl) -9,10-dimethoxy-51]-[1 ᅳ 3] dioxolo [4,5-isoquinolino [3 ᅳ 2-a] isoquinoline-8 (6H)- silver;
2)  2)
13-(2, 4-디플루오로벤질 )-9， 10-디메톡시 -5H- [ 1， 3]디옥솔로 [4 , 5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온; 13- (2,4-difluorobenzyl) -9,10-dimethoxy-5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-8 (6H) -one;
3)  3)
13-(2,6-디晉루오로벤질) -9,10-디메톡시-511—[1，3]디옥솔로[4，5- ]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온;  13- (2,6-Difluorolubenzyl) -9,10-dimethoxy-511— [1,3] dioxolo [4,5-] isoquinolino [3,2-a] isoquinoline-8 (6H) -one;
4)  4)
9， 10-디메톡시 -13-(2-메틸벤질 )-5， 6-디하이드로- [ 1， 3]디옥솔로 [4 , 5-g]이소퀴놀 리노 [3,2-a]이소퀴놀린 -8(6H)-온;  9,10-dimethoxy-13- (2-methylbenzyl) -5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline- 8 (6H) -one;
5)  5)
9，10-디메특시-13-(2-메톡시벤질)-5，6-디하이드로-[1，3]디옥솔로[4,5기이소퀴 놀리노 [3,2-a]이소퀴놀린 -8(6H)-온;  9,10-dimethoxy-13- (2-methoxybenzyl) -5,6-dihydro- [1,3] dioxolo [4,5giisoquinolino [3,2-a] isoquinoline -8 (6H) -one;
6)  6)
9，1으디메톡시 -13- (피리딘 -2-일메틸 )-5, 6-디하이드로 -[ 1,3]디옥솔로 [4, 5-g]이 소퀴놀리노 [3,2-a]이소퀴놀린ᅳ 8(6H)-온; 7) 9,1-dimethoxy-13- (pyridin-2-ylmethyl) -5, 6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] Isoquinolin ᅳ 8 (6H) -one; 7)
13- C1H-벤조 [d]이미다졸 -2-일)메틸) -9, 10-디메톡시 -5ᅳ 6-디하이드로 -[1,3]디옥 솔로 [4,5ᅳ8]이소퀴놀라노[3,2-3]이소퀴놀린-8(6¾-은； 13- C1H- benzo [d] imidazol-2-yl) methyl) -9, 10-dimethoxy-eu -5 6-dihydro [1, 3] dioxolo [4,5 eu 8] isoquinolinyl no surprise [3,2-3] isoquinoline-8 (6¾-silver;
. 8)  . 8)
13 ( (2-클로로티아졸ᅳ 5-일 )메틸 )-9， 10-디메톡시 -5， 6-디하이드로- [ 1 , 3]디옥솔로 [4,5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온; 13 ((2-chlorothiazol- 5-yl) methyl) -9, 10-dimethoxy-5, 6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3, 2-a] isoquinolin-8 (6H) -one;
9)  9)
13-(2ᅳ플루오로벤조일) -9, 10-디메특시 -5H-U, 3]디옥솔로 [4,5-g]이소퀴놀리노 [3 ,2-a]이소퀴놀린 -8(6H)-온;  13- (2 ᅳ fluorobenzoyl) -9, 10-dimethoxy-5H-U, 3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-8 (6H) -On;
10) ' 10) '
13-에틸 -9， 10-디메특시 -8-옥소 -6 , 8-디하이드로 -5H-[ 1， 3]디옥솔로 [4 , 5-g]이소퀴 놀리노 [3， 2-a]이소퀴놀린— 13-카르복실레이트; 13-ethyl-9, 10-dimethoxy-8-oxo-6,8-dihydro-5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] Isoquinoline—13-carboxylate;
11)  11)
9， 10-디메특시 -8-옥소 -6， 8-디하이드로— 5H- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀리노 [3 ,2-a]이소퀴놀린 -13-카르복실산;  9,10-dimethoxy-8-oxo-6,8-dihydro—5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-13- Carboxylic acid;
12)  12)
9， 10-디메톡시 -8-옥소ᅳ 6， 8-디하이드로— 5H- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀리노 [3 ,2-a]이소퀴놀린 -13-카르복사마이드; 및  9,10-dimethoxy-8-oxosulfone 6,8-dihydro—5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-13-car Copyamide; And
13)  13)
N-(2-플루오로페닐 )— 9 , 10-디메톡시 -8-옥소 -6， 8-디하이드로 -5H-[ 1 , 3]디옥솔로 [4 ， 5-g]이소퀴놀리노 [3 ,2-a]이소퀴놀린 -13-카르복사마이드. 본 발명에 따른 상기 화학식 1로 표시되는 8-옥소프로토베르베린의 바람직한 구조를 하기 표 1에 나타내었다. N- (2-fluorophenyl) —9,10-dimethoxy-8-oxo-6,8-dihydro-5H- [1,3] dioxolo [4,5-g] isoquinolino [3, 2-a] isoquinoline-13-carboxamide. The preferred structure of 8-oxoprotoberberine represented by the formula (1) according to the present invention is shown in Table 1 below.
[표 1】 실시예 구조 실시예 구조 Table 1 Example Structure Example Structure
Figure imgf000015_0001
S0CT0/M0ZaM/X3d 08CZ0l/ST0Z OAV
Figure imgf000016_0001
Figure imgf000015_0001
S0CT0 / M0ZaM / X3d 08CZ0l / ST0Z OAV
Figure imgf000016_0001
발명의 상기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체는 약학적으로 허용가능한 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용가능한 유리산 ( free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산 질산， 인산， 황산 브름화수소산， 요오드화수소산， 아질산, 아인산 등과 같은 무기산류， 지방족 모노 및 다이카르복실레이트, 페닐ᅳ치환된 알카노에이트， 하이드록시 알카노에이트 및 알칸디오에이트， 방향족 산류, 지방족 및 방향족 설폰산류 등과 같은 무독성 유기산， 아세트산， 안식향산， 구연산， 젖산, 말레인산, 글루콘산， 메탄설폰산， 4-를루엔설폰산， 주석산， 푸마르산등과 같은 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염의 종류로는 설페이트， 피로설페이트， 바이설페이트， 설파이트， 바이설파이트, 나이트레이트, 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， 핵산 -1 , 6ᅳ다이오에이트, 벤조에이트， 클로로벤조에이트， 메틸벤조에이트, 디니트로 벤조에이트， 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트， 테레프탈레이트， 벤젠설포네이트， 를루엔설포네이트， 클로로벤젠설포네이트， 크실렌설포네이트 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트， 락테이트， β-하이드톡시부티레이트, 글리콜레이트， 말레이트， 타트레이트， 메탄설포네이트， 프로판설포네이트， 나프탈렌 -1-설포네이트， 나프탈렌 -2-설포네이트, 만델레이트 둥을 포함한다. 본 발명에 따른 산 부가염은 통상의 방법으로 제조할 수 있으며， 예를 들면 상기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체를 메탄을， 에탄훌， 아세톤， 다이클로로메탄, 아세토나이트릴 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， 염기를 사용하여 약학적으로 허용가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중쎄 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이때， 금속염으로는 나트룸， 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다.또한， 이에 대웅하는 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염 (예, 질산은)과 반웅시켜 얻는다. 나아가, 본 발명은 상기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체 또는 이와 약학적으로 허용가능한 염뿐만 아니라， 이로부터 제조될 수 있는 용매화물， 수화물 둥을 모두 포함한다. 본 발명에 따른 신규한 8-옥소프로토베르베린 유도체는 NFAT5의 활성을 저해하는 효과가 우수하고 특히， 실시예 1， 3 , 6 및 10의 유도체가 NFAT5의 활성을 저해하는 효과가 우수다는 것을 알 수 있다 (실험예 2및 3참조) . 또한, 동물실험에서 마우스 다리의 관절 부위를 육안으로 관찰하였올 경우 관절염의 증상이 완화된 것을 확인할 수 있었고, 조직학적 염색을 통해서도 관절염 증상이 월등히 회복된 것을 확인할 수 있다 (실험예 10 참조) . The 8-oxoprotoberberine derivative represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. . Acid addition salts include inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono and dicarboxylates, phenylsubstituted alkanoates, hydroxy alkanoates and alkanedioates. And non-toxic organic acids such as aromatic acids, aliphatic and aromatic sulfonic acids, acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-luluenesulfonic acid, tartaric acid, fumaric acid and the like. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, eye Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, ' heptanoate, propiolate, oxalate, malonate, succinate, Suberates, Sebacates, Fumarates, Maleates, Butyne-1, 4-Diates, Nucleic Acids-1, 6 ᅳ dioates, Benzoates, Chlorobenzoates, Methylbenzoates, Dinitrobenzoates, Hydroxybenzoates , Methoxybenzoate, phthalate, terephthalate , Benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate phenylacetate, phenylpropionate, phenylbutyrate, Citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate dung. Acid addition salts according to the present invention can be prepared by a conventional method, for example, the 8-oxoprotoberberine derivative represented by the formula (1), such as methane, ethanol, acetone, dichloromethane, acetonitrile, etc. The precipitate produced by dissolving in an organic solvent and adding an organic acid or an inorganic acid may be prepared by filtration and drying, or the solvent and excess acid are distilled under reduced pressure and dried to crystallize under an organic solvent. Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained by, for example, dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a natrim, potassium or calcium salt as the metal salt. In addition, the salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (for example, silver nitrate). Furthermore, the present invention includes not only the 8-oxoprotoberberine derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof, but also all solvates and hydrates prepared therefrom. The novel 8-oxoprotoberberine derivatives according to the present invention are excellent in inhibiting the activity of NFAT5, and in particular, it can be seen that the derivatives of Examples 1, 3, 6 and 10 are excellent in inhibiting the activity of NFAT5. (See Experimental Examples 2 and 3). In addition, in the animal experiments, when the joints of the mouse legs were visually observed, The symptoms were alleviated, and histological staining also showed that the symptoms of arthritis recovered significantly (see Experimental Example 10).
따라서， 본 발명에 따른 유도체는 NFAT5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다. 또한， 본 발명은 하기 반웅식 1에 나타낸 바와 같이，  Therefore, the derivative according to the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of NFAT5 activity-related diseases. In addition, the present invention, as shown in the following reaction formula 1,
화학식 2로 표시되는 화합물과 화학식 3으로 표시되는 화합물을 반웅시켜 화학식 4로 표시되는 화합물을 얻는 단계 (단계 1)；  Reacting the compound represented by the formula (2) with the compound represented by the formula (3) to obtain a compound represented by the formula (4) (step 1);
상기 단계 1에서 얻은 화학식 4로 표시되는 화합물을 염기 조건 하에서 반웅시켜 화학식 1로 표시되는 화합물을 얻는 단계 (단계 2)를 포함하는 화학식 Chemical formula comprising the step (step 2) to obtain a compound represented by formula 1 by reacting the compound represented by formula 4 obtained in step 1 under basic conditions
1로 표시되는 상기 8-옥소프로토베르베린 유도체의 제조방법을 제공한다: Provided is a method for preparing the 8-oxoprotoberberine derivative represented by 1:
Figure imgf000018_0001
Figure imgf000018_0001
2 4 1  2 4 1
상기 반웅식 1에서, A 및 R은 상기에서 정의한 바와 같고， X1 및 X2는 할로겐이다. 이하, 상기 제조방법을 단계별로 구체적으로 설명한다. 본 발명에 따른 제조방법에 있어서， 단계 1은 화학식 2로 표시되는 화합물과 화학식 3으로 표시되는 화합물을 반웅시켜 화학식 4로 표시되는 화합물을 얻는 단계이다. In Reaction Formula 1, A and R are as defined above, and X 1 and X 2 are halogen. Hereinafter, the manufacturing method will be described in detail step by step. In the preparation method according to the present invention, step 1 is a step of obtaining a compound represented by the formula (4) by reacting the compound represented by the formula (2) and the compound represented by the formula (3).
이때， 화학식 3으로 표시되는 화합물은 화학식 2로 표시되는 화합물의 1.2 내지 2 당량으로 사용하는 것이 바람직하다. 또한， 상기 반웅성을 향상시키기 위하여 요오드화 나트륨， 요오드화칼륨， 염화나트륨 염화칼륨 등과 같은 촉매를 사용할 수 있고, 요오드화나트륨을 사용하는 것이 보다 바람직하다. 상기 단계 1에서 사용가능한 용매는 아세토나이트릴， 클로로포름, 디메틸포름아마이드, 디메틸설폭사이드， 메탄을， 에탄올， 프로판올， 부탄을， 테트라하이드로퓨란， 디옥산, 메틸렌 클로라이드， 1，2ᅳ디메톡시에탄 등을 단독 또는 흔합하여 사용할 수 았고， 아세토나이트릴 등을 사용하는 것이 바람직하다. At this time, the compound represented by the formula (3) is preferably used in 1.2 to 2 equivalents of the compound represented by the formula (2). In addition, in order to improve the reaction properties, a catalyst such as sodium iodide, potassium iodide, sodium chloride or potassium chloride may be used, and it is more preferable to use sodium iodide. desirable. The solvent usable in step 1 is acetonitrile, chloroform, dimethylformamide, dimethylsulfoxide, methane, ethanol, propanol, butane, tetrahydrofuran, dioxane, methylene chloride, 1,2-dimethoxyethane and the like. It could be used alone or in combination, it is preferable to use acetonitrile or the like.
본 발명에 따른 제조방법에 있어서， 단계 2는 상기 단계 1에서 얻은 화학식 4로 표시되는 화합물을 염기 조건 하에서 반웅시켜 화학식 1로 표시되는 화합물을 얻는 단계이다. In the preparation method according to the present invention, step 2 is a step of obtaining a compound represented by Chemical Formula 1 by reacting the compound represented by Chemical Formula 4 obtained in Step 1 under basic conditions.
이때， 상기 염기는 통상적으로 사용하는 염가아면 제한없이 사용가능하나， 수산화칼륨, 수산화나트륨， 수산화칼슘， 수산화스트로튬 등의 염기를 사용하는 것이 바람직하고， 수산화칼륨 또는 수산화나트륨을 수용액으로 사용하는 것이 보다 바람직하다.  At this time, the base can be used without limitation as long as it is inexpensive, it is preferable to use a base such as potassium hydroxide, sodium hydroxide, calcium hydroxide, strontium hydroxide, more preferably using potassium hydroxide or sodium hydroxide as an aqueous solution desirable.
또한， 상기 단계 2는 하기 반웅식 2에 나타난바와 같이， In addition, the step 2 is as shown in the following reaction formula 2,
화학식 4로 표시되는 화합물을 환원반웅시켜 화학식 5로 표시되는 화합물을 얻는 단계 (단계 A) ; 및  Reducing the compound represented by the formula (4) to obtain a compound represented by the formula (5) (step A); And
상기 단계 A에서 얻은 화학식 5로 표시되는 화합물올 산화반웅시켜 화학식 1로 표시되는 화합물을 얻는 단계 (단계 B) ;를 포함하는 단계일 수 있다: , Said step A by the formula (5) compound represented by the all-banung oxide obtained in step (step B) to obtain a compound represented by Formula 1; may be a step that includes:,
Figure imgf000019_0001
Figure imgf000019_0001
(상기 반웅식 2에서， A 및 R은 상기에서 정의한 바와 같고， X2는 할로겐이다) . 구체적으로，상기 단계 A는 화학식 4로 표시되는 화합물을 환원반웅시켜 화학식 5로 표시되는 화합물을 얻는 단계이다. (In the above formula 2, A and R are as defined above, and X 2 is halogen). Specifically, step A is a step of obtaining a compound represented by the formula (5) by reducing the compound represented by the formula (4).
이때， 사용가능한 환원제는 소듐 보로하이드라이드， 소듐 시아노보로하이드라이드，소듐트리아세톡시보로하이드라이드, 피리딘 보로착제， 징크 보로하이드라이드 등을 사용할 수 있고, 소듐 시아노보로하이드라이드, 소듐 보로하이드라이드, 소듐트리아세톡시보로하이드라이드 등을 사용하는 것이 바람직하다.  In this case, the reducing agent that can be used may be sodium borohydride, sodium cyanoborohydride, sodium triacetoxyborohydride, pyridine borohydride, zinc borohydride and the like, sodium cyanoborohydride, sodium borohydride It is preferable to use a lide, sodium triacetoxyborohydride and the like.
또한， 염기를 촉매로 사용할 수 있으며， 통상적으로 사용하는 염기이면 제한없이 사용가능하나,수산화칼륨，수산화나트륨,수산화칼슘，수산화스트로튬 등의 염기를 사용하는 것이 바람직하고， 수산화칼륨 또는 수산화나트륨을 수용액으로 사용하는 것이 보다 바람직하다.  In addition, a base may be used as a catalyst, and any base can be used without limitation, but it is preferable to use a base such as potassium hydroxide, sodium hydroxide, calcium hydroxide, strontium hydroxide, and an aqueous solution of potassium hydroxide or sodium hydroxide. It is more preferable to use.
나아가，사용가능한 용매는 메탄올， 에탄올，프로판올， 부탄올 등과 같은 알코올계 용매， 테트라하이드로퓨란， 디옥산, 메틸렌 클로라이드 1 , 2-디메톡시에탄과 같은 에테르계 용매, 디메틸포름아마이드, 디메틸설폭사이드 등을 단독 또는 흔합하여 사용 ¾ 수 있다. 다음으로 상기 단계 B는 상기 단계 A에서 얻은 화학식 5로 표시되는 화합물을 산화반웅시켜 화학식 1로 표시되는 화합물을 얻는 단계이다.  Furthermore, usable solvents include alcohol solvents such as methanol, ethanol, propanol and butanol, ether solvents such as tetrahydrofuran, dioxane, methylene chloride 1 and 2-dimethoxyethane, dimethylformamide and dimethyl sulfoxide. Can be used alone or in combination. Next, step B is a step of obtaining a compound represented by the formula (1) by oxidizing the compound represented by the formula (5) obtained in the step A.
이때， 사용가능한 산화제로는 이산화망간， 과산화수소수， 중크롬산칼륨 등을 사용할 수 있고, 이산화망간을 사용하는 것이 바람직하다.  In this case, as the oxidizing agent that can be used, manganese dioxide, hydrogen peroxide, potassium dichromate, or the like may be used, and manganese dioxide is preferably used.
또한， 사용가능한 용매는쩨틸렌 클로라이드， 1 , 2-디메특시에탄과 같은 에테르계 용매， 디메틸포름아마이드， 디메틸설폭사이드 등을 사용할 수 있다ᅳ  In addition, usable solvents include ether solvents such as methylene chloride, 1,2-dimethicethane, dimethylformamide, dimethyl sulfoxide and the like.
상기 반웅을 수행한 후， 유기용매로 추출, 건조, 여과 및 감압 증류하는 과정을 수행하고 추가적으로 컬럼크로마토그래피 또는 재결정을 수행하여 제조할 수 있다. 나아가， 본 발명은 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효상분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물올 제공한다. After performing the reaction, extraction, drying, filtration, and distillation under reduced pressure with an organic solvent may be performed, and further, may be prepared by performing column chromatography or recrystallization. Furthermore, the present invention provides a pharmaceutical composition for preventing or treating an activity-related disease of NFAT5, which contains a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an effective phase.
여기서， 상기 NFAT5의 활성 관련 질환은 관절염， 자가면역 질환 등일 수 있다. Here, the activity-related diseases of NFAT5 may be arthritis, autoimmune diseases, and the like.
· 상기 관절염 질환은 류마티스 관절염인 것을 특징으로 한다. The arthritis disease is characterized in that rheumatoid arthritis.
상기 자가면역질환은 전신성 경피증， 흥반성 낭창， 아토피 피부염， 베체씨병， 쇼그렌 증후군, 다발성 경화증 및 그레이브씨 갑상선 항진증으로부터 선택되는 어느 하나 이상인 것을 특징으로 한다ᅳ 본 발명의 구체적인 실시예에서， 본 발명에 따른 신규한 8-옥소프로토베르베린 유도체는 NFAT5의 활성을 저해하는 효과가 우수하고 특히， 실시예 1， 3 , 6 및 10의 유도체가 NFAT5의 활성을 저해하는 효과가 우수다는 것을 알 수 있다 (실험예 2 및 3 참조) . 또한, 동물실험에서 마우스 다리의 관절 부위를 육안으로 관찰하였을 경우 관절염의 증상이 완화된 것을 확인할 수 있었고， 조직학적 염색을 통해서도 관절염 증상이 월등히 회복된 것을 확인할 수 있다 (실험예 11 참조) . 또한， 본 발명의 화합물은 LPS로 유도된 iNOS 유전자 발현증가를 억제하였고 (표 2 참조)， NFAT5 전사활성을 억제하였으며 (표 3， 및 도 1참조)ᅳ다른 전사인자인 NF-kB , NFATc , CREB, ELK의 전사활성도 억제함을 확인하였다 (도 2참조) . 또한 본 발명의 화합물은 R0S 유도, p38의 활성， 및 C0X1과 C0X2에 대해 저해효과를 나타내지 않았으며 (도 3 및 도 6 , 표 4 및 표 5 참조) , 세포에는 거의 독성을 나타내지 않았으며， 염증성 사이토카인의 분비를 억제함을 확인하였다 (표 6 및 도 6 참조) . 콜라겐으로 유도된 류마티스 관절염 마우스에서 본 발명의 화합물은 관절염 증상을 회복시켰고 (도 7 침조)， 염증상 사이토카인 (TNF-α , IL-6)의 발현을 감소시킴을 확인하였다 (도 8 참조) . 아을러， 본 발명의 화합물은 증가된 경구흡수율을 나타냈고 (표 7 참조)， 인간 단핵혈액세포의 염증성 사이토카인 분비를 억제하였으며 (도 11참조) , T세포로부터 Thl7세포로의 분화를 억제함을 확인하였다 (도 I2 참조) . 따라서， 본 발명의 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염은 물성 개선으로 경구 흡수율이 기존의 프로토베르베린보다 현저히 증가되었고， 또한 NFAT5의 상위 (upstream) 단계인 p38을 경유하지 않고， 직접적으로 NFAT5의 전사를 억제시켜 NFAT5 활성 및 염증성 사이토카인의 분비를 억제하고 류마티스 관절염 마우스에서 NFAT5의 발현을 감소시킴을 확인함으로써， NFAT5의 활성 관련 질환， 특히 류마티스 관절염 또는 염증 질환의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다. 본 발명에 따른 약학적 조성물에 있어서, 상기 화학식 1로 표시돠는 8ᅳ옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염은 임상 투여시에 경구 및 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. 경구투여를 위한 고형 제제에는 정제， 환제， 산제， 과립제， 캡슐제， 트로키제 등이 포함되며， 이러한 고형 제제는 하나 이상의 본 발명의 상기 화학식 1로 표시되는 8ᅳ옥소프로토베르베린 유도체, 또는 이의 약학적으로 허용가능한 염에 적어도 하나 이상의 부형제 예를 들면， 전분， 탄산칼슘, 수크로오스 (sucrose)또는 락토오스 ( lactose)또는 젤라틴 등을 섞어 조제될 수 있다. 또한， 단순한 부형제 외에 스테아린산 마그네습 탈크 등과 같은 윤활제들도 사용될 수 있다. 경구 투여를 위한 액상 제제로는 현탁제， 내용액제， 유제 또는 시럽제 등이 해당되는데， 흔히 사용되는 단순 희석제인 물， 리퀴드 파라핀 이외에 여러 가지 부형제， 예를 들면 습윤제， 감미제, 방향제， 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액， 비수성용제, 현탁용제， 유제， 동결건조제제， 좌제가 포함된다. 비수성용제， 현탁용제로는 프로필렌글리콜， 폴리에틸렌 글리콜， 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 둥이 사용될 수 있다. 좌제의 기제로는 위텝솔 (wi tepsol ) , 마크로골， 트원 ( tvveen) 61， 카카오지， 라우린지, 글리세를， 젤라틴 등이 사용될 수 있다. 또한 본 .발명의 상기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염의 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 투여할 수도 있다. 본 발명의 약학적 조성물은 관절염의 또는 자가면역 질환의 예방 또는 치료를 위하여 단독으로, 또는 수술， 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다. 또한, 본 발명은 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 NFAT5의 활성 관련 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. 본 발명에 따른 유도체는 NFAT5의 활성을 억제하는 효과가 우수하여 NFAT5의 활성 관련 질환의 예방 또는 개선용 건강기능식품 조성물로 유용하게 사용될 수 있다. The autoimmune disease is at least one selected from systemic scleroderma, lupus erythematosus, atopic dermatitis, Beche's disease, Sjogren's syndrome, multiple sclerosis and Graves' hyperthyroidism. The novel 8-oxoprotoberberine derivatives are excellent in inhibiting the activity of NFAT5, and in particular, it can be seen that the derivatives of Examples 1, 3, 6 and 10 are excellent in inhibiting the activity of NFAT5 (experimental) See examples 2 and 3). In addition, it was confirmed that the symptoms of arthritis were alleviated when the joints of the mouse legs were visually observed in animal experiments, and the arthritis symptoms were recovered significantly through histological staining (see Experimental Example 11). In addition, the compounds of the present invention inhibited the increase of LPS-induced iNOS gene expression (see Table 2), inhibited NFAT5 transcriptional activity (see Table 3, and FIG. 1) —another transcription factors, NF-kB, NFATc, It was also confirmed that the transcriptional activity of CREB and ELK was also inhibited (see FIG. 2). In addition, the compounds of the present invention showed no inhibitory effect on R0S induction, p38 activity, and C0X1 and C0X2 (see FIGS. 3 and 6, Tables 4 and 5), and showed little toxicity to cells, and inflammatory It was confirmed that the secretion of cytokines was inhibited (see Table 6 and FIG. 6). Compounds of the present invention in collagen-induced rheumatoid arthritis mice recovered arthritis symptoms (Fig. 7 acupuncture), and were found to reduce the expression of inflammatory cytokines (TNF-α, IL-6) (see Fig. 8). . In addition, the compounds of the present invention showed increased oral absorption (see Table 7), and inflammatory cytokines in human mononuclear blood cells. Secretion was inhibited (see FIG. 11), and it was confirmed to inhibit differentiation from T cells to Thl7 cells (see FIG. I 2 ). Therefore, the novel 8-oxoprotoberberine derivatives of the present invention or pharmaceutically acceptable salts thereof have significantly improved oral absorption rate than the conventional protoberberine due to the improvement of physical properties and do not pass through p38, which is an upstream stage of NFAT5. By directly inhibiting the transcription of NFAT5, inhibiting NFAT5 activity and inflammatory cytokine secretion and reducing the expression of NFAT5 in rheumatoid arthritis mice, thereby preventing or preventing activity-related diseases of NFAT5, particularly rheumatoid arthritis or inflammatory diseases. It can be usefully used as a therapeutic pharmaceutical composition. In the pharmaceutical composition according to the present invention, the 8'oxoprotoberberine derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof may be administered in a variety of oral and parenteral formulations for clinical administration. In this case, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, and the like, wherein the solid preparations are at least one 8 ᅳ oxoprotoberberine derivative represented by Formula 1 of the present invention, or a pharmaceutical thereof. It may be formulated by mixing at least one excipient, for example starch, calcium carbonate, sucrose or lactose or gelatin, etc. with a generally acceptable salt. In addition to the simple excipients, lubricants such as stearic acid magnesium talc and the like may also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions or syrups.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, wi tepsol, macrogol, tvveen 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used. See also . The dosage of the 8-oxoprotoberberine derivative represented by Formula 1 of the present invention or a pharmaceutically acceptable salt thereof to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient, Based on an adult patient weighing 70 Kg, typically 0.1-1000 mg / day, preferably 1-500 mg / day, and also 1 day at regular time intervals as determined by the physician or pharmacist 1 time. It may be administered in several divided doses. The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of arthritis or autoimmune diseases. The present invention also provides a nutraceutical composition for preventing or ameliorating the activity-related diseases of NFAT5 containing a novel 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof as an active ingredient. The derivative according to the present invention has an excellent effect of inhibiting the activity of NFAT5 can be usefully used as a health functional food composition for the prevention or improvement of NFAT5 activity-related diseases.
상기 NFAT5의 활성 관련 질환은 관절염 또는 자가면역 질환 등일 수 있다. 상기 관절염은 류마티스 관절염인 것을 특징으로 한다. 상기 자가면역 질환은 전신성 경피증， 흥반성 낭창， 아토피 피부염， 베체씨병， 쇼그렌 증후군， 다발성 경화증, 및 그레이브씨 갑상선 항진증으로부터 선택되는 어느 하나 이상인 것을 특징으로 한다. The disease associated with activity of NFAT5 may be arthritis or autoimmune disease. The arthritis is characterized in that rheumatoid arthritis. The autoimmune disease is at least one selected from systemic scleroderma, lupus erythematosus, atopic dermatitis, Beche's disease, Sjogren's syndrome, multiple sclerosis, and Graves' hyperthyroidism.
따라서， 본 발명의 신규한 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염은 물성 개선으로 경구 흡수율이 기존의 프로토베르베린보다 현저히 증가되었고， 또한 NFAT5의 상위 (upstream) 단계인 p38을 경유하지 않고， 직접적으로 NFAT5의 전사를 억제시켜 NFAT5 활성 및 염증성 사이토카인의 분비를 억제하고 류마티스 관절염 마우스에서 NFAT5의 발현을 감소시킴을 확인함으로써， NFAT5의 활성 관련 질환， 특히 류마티스 관절염 또는 염증 질환의 예방 또는 개선용 건강기능식품으로 유용하게 사용될 수 있다. . 상기 식품의 종류에는 특별한 제한은 없다.상기 물질을 첨가할 수 있는 식품의 예로는드링크제，육류，소시지，빵,비스킷，떡,초콜릿,캔디류，스낵류， 과자류， 피자， 라면， 기타 면류， ¾류， 아이스크림류를 포함한 낙농제품， 각종 스프， 음료수， 알코을 음료 및 비타민 복합제， 유제품 및 유가공 제품 등이 있으며， 통상적인 의미에서의 건강기능식품을 모두 포함한다. 본 발명에 따른 상기 화학식 1로 표시되는 8ᅳ옥소프로토베르베린 유도체는 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함깨 사용될 수 있고， 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 흔합량은 그의 사용 목적 (예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로， 건강식품 중의 상기 화합물의 양은 전체 식품 중량의 0. 1 내지 90 중량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. 또한， 본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 화합물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드， 예를 들어， 포도당， 과당 등; 디사카라이드， 예를 들어 말토스， 슈크로스 등; 및 폴리사카라이드， 예를 들어 텍스트린， 시클로텍스트린 등과 같은 통상적인 당， 및 자일리를, 소르비를， 에리트리 » 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제 (타우마틴 , 스테비아 추출물 (예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제 (사카린， 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 g당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다. 나아가， 상기 외에 본 발명에 따른 화학식 1로 표시되는Therefore, the novel 8-oxoprotoberberine derivatives of the present invention or pharmaceutically acceptable salts thereof have significantly improved oral absorption rate than the conventional protoberberine due to the improvement of physical properties and do not pass through p38, which is an upstream stage of NFAT5. By directly inhibiting the transcription of NFAT5, inhibiting NFAT5 activity and inflammatory cytokine secretion and reducing the expression of NFAT5 in rheumatoid arthritis mice, thereby preventing or preventing activity-related diseases of NFAT5, particularly rheumatoid arthritis or inflammatory diseases. It can be usefully used as a dietary supplement for improvement. . There is no particular limitation on the type of food. Examples of the food to which the substance may be added include drink, meat, sausage, bread, biscuit, rice cake, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, and ¾. , Dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, and includes all the health functional foods in the ordinary sense. 8 ᅳ oxoprotoberberine derivatives represented by the formula (1) according to the present invention can be used as it is or added to other foods or food ingredients as it is, and may be appropriately used according to conventional methods. The combined amount of the active ingredient can be suitably determined depending on the purpose of use (prevention or improvement). In general, the amount of the compound in the health food can be added from 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient is in the above range because there is no problem in terms of safety. The above amounts may also be used. In addition, the health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as polysaccharides, for example, conventional sugars such as textine, cyclotextine and the like, and xylyl, sorbitol, and erytri ». As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.
8-옥소프로토베르베린 유도체는 여러 가지 영양제， 비타민， 광물 (전해질)， 합성 풍미제 및 천연 풍미제 등의 풍미제， 착색제 및 중진제 (치즈, 초콜릿 둥)， 펙트산 및 그의 염, 알긴산 및 그의 염， 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제， 방부제， 글리세린， 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 8-옥소프로토베르베린 유도체는 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 8-oxoprotoberberine derivatives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, coloring and neutralizing agents (cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition, the 8-oxoprotoberberine derivatives of the present invention may contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages.
또한， 본 발명은 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효한 양으로 개체에 투여하는 것을 포함하는 NFAT5의 활성 관련 질환의 예방, 개선 또는 치료 방법을 제공한다.  The present invention also provides a method for preventing, ameliorating or treating an activity-related disease of NFAT5 comprising administering to a subject an effective amount of an 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof.
아울러， NFAT5의 활성 관련 질환의 예방 또는 치료제로 사용하기 위한 상기 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 제공한다. 또한， NFAT5의 활성 관련 질환의 예방 또는 개선용 건강기능식품으로 사용하가 위한 상기 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 제공한다. In addition, the 8-oxoprotoberberine derivative or a pharmaceutically acceptable salt thereof for use as a prophylactic or therapeutic agent for a disease related to activity of NFAT5 is provided. In addition, the 8-oxoprotoberberine derivatives or pharmaceutically acceptable salts thereof for use as a dietary supplement for the prevention or improvement of NFAT5 activity-related diseases.
따라서， 본 발명쎄 따른 유도체는 NFAT5의 활성 관련 질환의 예방 또는 치료제， 및 개선용 건강기능식품으로 유용하게 사용될 수 있다. 이하, 본 발명올 실시예 및 실험예에 의해 상세히 설명한다.  Therefore, the derivative according to the present invention can be usefully used as a preventive or therapeutic agent for the activity-related diseases of NFAT5, and a dietary supplement for improvement. Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐， 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다.  However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.
<제조예 1> 8-아세토닐디히드로베르베린 Preparation Example 1 8-acetonyldihydroberberine
Figure imgf000026_0001
Figure imgf000026_0001
공지의 방법으로 염산베르베린 (5 g, 13.45 麵 ol)을 5N 수산화나트륨 수용액 (23 mL)에 가하고 0oC로 넁각 후 아세톤 (5 mL, 67.23 隱 ol)를 천천히 적가하였다. 실은에서 1시간 동안 교반한 후 생성된 고체를 여과하고 80% 메탄을 40 mL로 2회 세척한 다음 건조하여 목적 화합물 (4.65g, 89%)을 얻었다. Berberine hydrochloride (5 g, 13.45 麵 ol) was added to a 5N aqueous sodium hydroxide solution (23 mL) by a known method, and after stirring to 0 o C, acetone (5 mL, 67.23 隱 ol) was slowly added dropwise. After stirring for 1 hour at real silver, the resulting solid was filtered, washed twice with 40 mL of 80% methane, and dried to obtain the target compound (4.65g, 89%).
¾ NMR (300 MHz, CDC13) δ 2.04 (s, 3H), 2.37-2.44 (ddᅳ J = 3.0， 15.0¾ NMR (300 MHz, CDC1 3 ) δ 2.04 (s, 3H), 2.37-2.44 (dd ᅳ J = 3.0, 15.0
Hz, 1H), 2.76-2.84 (m, 2H), 3.08-3.11 (dd, = 6.0， 15.0 Hz, 1H), 3.30-3.36 (m, 2H), 3.83 (s, 3H) , 3.89 (s, 3H) , 5.30-5.34 (dd, 7=3.0, 6.0Hz, 1H)， 5.89(s, 1H), 5.93-5.94 (d, 3.0 Hz, 1H) , 6.57 (s, 1H) , 6.76-6.79 (m, 2H), 7.13 (s, 1H). Hz, 1H), 2.76-2.84 (m, 2H), 3.08-3.11 (dd, = 6.0, 15.0 Hz, 1H), 3.30-3.36 (m, 2H), 3.83 (s, 3H), 3.89 (s, 3H ), 5.30-5.34 (dd, 7 = 3.0, 6.0 Hz, 1H), 5.89 (s, 1H), 5.93-5.94 (d, 3.0 Hz, 1H), 6.57 (s, 1H), 6.76-6.79 (m, 2H), 7.13 (s, 1 H).
<실시예 1> <Example 1>
13-(2-플루오로벤질) -9 , 10-디메특시 -5Η- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀리 노 [3,2-a]이소퀴놀린 -8(6H)-은 13- (2-fluorobenzyl) -9,10-dimethoxy--5Η- [1, 3] dioxolo [4, 5-g] isoquinoli No [3,2-a] isoquinoline-8 (6H) -silver
Figure imgf000027_0001
Figure imgf000027_0001
단계 1:  Step 1:
13-(2-플루오로벤질 )-9，10-디메톡시-511-[1,3]디옥솔로[4,5-8]이소퀴놀리노[3,2 -a]이소퀴놀린 -7-이움브로마이드  13- (2-fluorobenzyl) -9,10-dimethoxy-511- [1,3] dioxolo [4,5-8] isoquinolino [3,2-a] isoquinoline-7-imbromide
상기 제조예 1에서 얻은 8-아세토닐디히드로베르베린 (lg)을 아세토나이트릴 (20 mL)에 녹인 용액에 2-플루오로벤질브로마이드 (2당량) 및 요오드 나트륨 (0.54g, 3.55 國 ol, 1.2당량)올 가하고 80oC에서 4시간 동안 교반하였다. 반웅 종료 후 생성물을 여과한 후 염화메틸렌 (3 X 20 mL)으로 세척하여 제거하고 유기층은 증류수 (5 mL)로 세척한 다음 황산 나트륨으로 수분을 제거 후 농축하였다. 잔류물을 플래시 칼럼크로마토 그래피로 (염화메틸렌: 메탄을 = 20： 1 to 10:1) 분리하여 노란색 고체의 목적 화합물 (45%)을 얻었다. In the solution of 8-acetonyldihydroberberine (lg) obtained in Preparation Example 1 in acetonitrile (20 mL), 2-fluorobenzylbromide (2 equivalents) and sodium iodine (0.54 g, 3.55 phenol ol, 1.2 equivalents) ) Was added and stirred at 80 ° C. for 4 hours. After completion of the reaction, the product was filtered and washed with methylene chloride (3 X 20 mL), and the organic layer was washed with distilled water (5 mL), followed by removing water with sodium sulfate and concentrating. The residue was separated by flash column chromatography (methylene chloride: methane = 20: 1 to 10: 1) to give the title compound (45%) as a yellow solid.
¾ NMR (300 MHz, CDC13) δ 3.27-3.31 (t, J= 6.0 Hz, 2H), 4.02 (s, 3H), 4.43 (s, 3H), 4.63 (s, 2H), 5.21 (s, 2H)， 6.02 (s, 2H), 6.76-6.81 On' 1H) , 6.88-6.89 (111， 2H) , 7.02-7.07 (m, 1H) ' 7.19-7.25 (m, 1H)， 7.29-7.34 (m, 1H) , 7.54-7.57 (dᅳ J 9.0 Hz, 1H)， 7.69-772 (d, J= 9.0 Hz, 1H), 10.49 (s, 1H). 단계 2: ¾ NMR (300 MHz, CDC1 3 ) δ 3.27-3.31 (t, J = 6.0 Hz, 2H), 4.02 (s, 3H), 4.43 (s, 3H), 4.63 (s, 2H), 5.21 (s, 2H ), 6.02 (s, 2H), 6.76-6.81 On '1H), 6.88-6.89 (111, 2H), 7.02-7.07 (m, 1H)' 7.19-7.25 (m, 1H), 7.29-7.34 (m, 1H), 7.54-7.57 (d ᅳ J 9.0 Hz, 1H), 7.69-772 (d, J = 9.0 Hz, 1H), 10.49 (s, 1H). Step 2:
13ᅳ(2-플루오로벤질 )-9,10-디메특시-511-[1,3]디옥솔로[4,5- 이소퀴놀 리노 [3,2-a]이소퀴놀린 -8(6H)-온  13 '(2-fluorobenzyl) -9,10-dimethoxy-511- [1,3] dioxolo [4,5-isoquinolino [3,2-a] isoquinoline-8 (6H)- On
상기 단계 1에서 얻은 Obtained in step 1 above
13ᅳ (2-플루오로벤질 )-9,10-디메톡시-511-[1，3]디옥솔로[4，5- 이소퀴놀리노[3，2 -a]이소퀴놀린 -7-이움브로마이드를 50% 테트라하이드로퓨란 수용액에 분산시킨 용액에 포타슘 페리시아나이드 (10 당량)을' 10% 수산화칼륨에 녹인 용액을 8-KTC에서 가하고 30분 동안 교반 후 실온에서 10시간 동안 교반하였다. 상기 반웅물을 디클로로메탄으로 추출한 다음 황산나트륨으로 수분을 제거하고 여과하여 감압농축하였다. 상기 잔류물올 플래시 칼럼크로마토그래피로 분리하여 목적 화합물 (28%)을 앋었다. 13 '(2-fluorobenzyl) -9,10-dimethoxy-511- [1,3] dioxolo [4,5-isoquinolino [3,2-a] isoquinoline-7-imumbromide Dispersed in aqueous tetrahydrofuran solution Solution in potassium ferry when added to a solution of cyanide (10 eq.) In the "10% potassium hydroxide in 8-KTC and stirred at stirred for 30 minutes at room temperature for 10 hours. The reaction product was extracted with dichloromethane, and then dried with sodium sulfate, filtered and concentrated under reduced pressure. The residue was separated by flash column chromatography to give the title compound (28%).
¾ NMR (300 MHz, CDC13) δ 2.82-2.86 (t, /= 6.0 Hz, 2H) , 3.89 (s, 3H),.¾ NMR (300 MHz, CDC1 3 ) δ 2.82-2.86 (t, / = 6.0 Hz, 2H), 3.89 (s, 3H) ,.
4.03 (s, 3H), 4.31 (s, 2H) , 5.92 (s, 2H), 6.75 (s, 1H) , 6.78 (s, 1H), 6.92-7.03 (m, 2H), 7.11-7.27 (m, 4H). 4.03 (s, 3H), 4.31 (s, 2H), 5.92 (s, 2H), 6.75 (s, 1H), 6.78 (s, 1H), 6.92-7.03 (m, 2H), 7.11-7.27 (m, 4H).
<실시예 2> <Example 2>
13-(2 , 4-디플루오로벤질 )-9 , 10-디메톡시 -5Η-[ 1 ,3]디옥솔로 [4 , 5-g]이소퀴 놀리노 -a]이소퀴놀린 ^8(6H)-은  13- (2,4-difluorobenzyl) -9, 10-dimethoxy-55- [1, 3] dioxolo [4, 5-g] isoquinnolino-a] isoquinoline ^ 8 (6H) -silver
Figure imgf000028_0001
Figure imgf000028_0001
단계 1:  Step 1:
13-(2,4-디플루오로벤질) -9,10-디메특시-^-[1,3]디옥솔로[4,5기이소 퀴놀리노 [3， 2-a]이소퀴놀린 -7-이움브로마이드  13- (2,4-difluorobenzyl) -9,10-dimethoxy-^-[1,3] dioxolo [4,5isoisoquinolino [3,2-a] isoquinoline-7- Ium bromide
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 2，4-디플루오로벤질 브로마이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (55%)을 제조하였다. ： ¾NMR (300 MHz, CDC13) δ 3.27 (s, 2H), 4.03 (s, 3H) , 4.42 (s, 3H), 4.58Preparation of the target compound (55%) in the same manner as in Step 1 of Example 1, except that 2,4-difluorobenzyl bromide was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1 It was. ： ¾NMR (300 MHz, CDC1 3 ) δ 3.27 (s, 2H), 4.03 (s, 3H), 4.42 (s, 3H), 4.58
(s, 2H), 5.28 (s, 2H), 6.03 (s, 2H) , 6.72-6.82 (m, 3H), 6.89 (s, 1H), 6.96-7.02 (m, 1H), 7.50-7.53 (d, /= 9.0 Hz, 1H) , 7.70-77(d, /= 9.0 Hz, 1H), 1.0.68 (s, 1H). 단계 2: (s, 2H), 5.28 (s, 2H), 6.03 (s, 2H), 6.72-6.82 (m, 3H), 6.89 (s, 1H), 6.96-7.02 (m, 1H), 7.50-7.53 (d , / = 9.0 Hz, 1H), 7.70-77 (d, / = 9.0 Hz, 1H), 1.0.68 (s, 1H). Step 2:
13-(2，4-디플루오로벤짐)-9，10-디메톡시-^-[1，3]디옥솔로[4，5- 이소 퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온  13- (2,4-difluorobenzyl) -9,10-dimethoxy-^-[1,3] dioxolo [4,5-isoquinolino [3,2-a] isoquinoline-8 (6H )-On
상기 실시예 1의 단계 2에서 13-(2-플루오로벤질 )-9，10-디메록시-511-[1，3]디옥솔로[4,5- 이소퀴놀리노[3，2 -a]이소퀴놀린 -7-이움브로마이드 대신에 상기 실시예 2의 단계 1에서 얻은 13-(2， 4-디플루오로벤질 )-9， 10-디메톡시 -5H- [ 1， 3]디옥솔로 [4 , 5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -7-이움브로마이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 2와 동일한 방법으로 목적 화합물 (25%)을 얻었다. ¾賺 (300 MHz, CDCls) δ 2.84 (s, 2H)， 3.91 (s, 3H) , 4.03 (s, 3Η), 4.25 13- (2-fluorobenzyl) -9,10-dimethoxy-511- [1,3] dioxolo [4,5-isoquinolino [3,2-a] iso in step 2 of Example 1 13- (2,4-difluorobenzyl) -9, 10-dimethoxy-5H- [1,3] dioxolo [4, 5 obtained in step 1 of Example 2 instead of quinoline-7-iumbromide -g] Isoquinolino [3,2-a] isoquinoline-7-umbromide was used in the same manner as in Step 2 of Example 1, except that the target compound (25%) was obtained. ¾ 賺 (300 MHz, CDCls) δ 2.84 (s, 2H), 3.91 (s, 3H), 4.03 (s, 3Η), 4.25
(s, 2H), 5.94 (s, 2H), 6.73-6.76 (m, 3H), 6.86-6.91 (m, 2H)， 7.09-7.12 (d, J= 9.0, 1H), 7.20-7.23 (d, /= 9.0, 1H). (s, 2H), 5.94 (s, 2H), 6.73-6.76 (m, 3H), 6.86-6.91 (m, 2H), 7.09-7.12 (d, J = 9.0, 1H), 7.20-7.23 (d, / = 9.0, 1H).
<실시예 3> <Example 3>
13-(2， 6-디플루오로벤질) -9 , 10-디메톡시 -5Η- [ 1， 3]디옥솔로 [4， 5-g]이소퀴 놀리노 [3 , 2-a]이소퀴놀린 -δ(6Η)-온  13- (2, 6-difluorobenzyl) -9, 10-dimethoxy-5 5- [1, 3] dioxolo [4, 5-g] isoquinolino [3, 2-a] isoquinoline- δ (6Η) -on
Figure imgf000029_0001
Figure imgf000029_0001
단계 1:  Step 1:
13-(2 , 6-디플루오로벤질) -9， 10-디메톡시 -5Η- [ 1 , 3]디옥솔로 [4 , 5-g]이소 퀴놀리노 [3， 2-a]아소퀴놀린— 7-이움브로마이드  13- (2, 6-difluorobenzyl) -9, 10-dimethoxy-5 5- [1, 3] dioxolo [4, 5-g] isoquinolino [3, 2-a] isoquinoline— 7 Ium bromide
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 2,6—디플루오로벤질 브로마이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (27%)을 제조하였다.  Preparation of the target compound (27%) in the same manner as in Step 1 of Example 1, except that 2,6—difluorobenzyl bromide was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1 It was.
¾ NMR (300 MHz, CDCI3) δ 3.24-3.28 ， J = 6.0 Hz, 2H)， 4.02 (sᅳ 3H)， 4.37 (s 3H), 4.74 (s, 2H) , 5.21 (s, 2H) , 6.07 (s, 2H)， 6.83-6.88 (m, 2H) , 6.93 (s, 1H), 7.05 (s, 1H), 7.17-7.25 (m, 1H), 7.73 (s, 2H), 10.45 (s, 1H) . 단계 2: ¾ NMR (300 MHz, CDCI 3 ) δ 3.24-3.28, J = 6.0 Hz, 2H, 4.02 (s ᅳ 3H), 4.37 (s 3H), 4.74 (s, 2H), 5.21 (s, 2H), 6.07 (s, 2H), 6.83-6.88 (m, 2H), 6.93 (s, 1H), 7.05 (s, 1H), 7.17-7.25 (m, 1 H), 7.73 (s, 2 H), 10.45 (s, 1 H). Step 2:
13-C2 , 6-디플루오로벤질 )-9， 10-디메톡시 -5H-[ 1， 3]디옥솔로 [4 , 5-g]이소 퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온  13-C2, 6-difluorobenzyl) -9, 10-dimethoxy-5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-8 ( 6H) -on
상기 실시예 1의 단계 2에서 In step 2 of Example 1
13-(2-플루오로벤질 )-9,10-디메특시-511-[1，3]디옥솔로[4，5- 이소퀴놀리노[3,2 -a]이소퀴놀린 -7-이움브로마이드 대신에 상기 실시예 3의 단계 1에서 얻은 13— (2， 6-디플루오로벤질 )-9， 10-디메특시 5H- [ 1， 3]디옥솔로 [4 , 5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -7-이움브로마이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 2와 동일한 방법으로 목적 화합물 (26%)을 얻었다. Instead of 13- (2-fluorobenzyl) -9,10-dimethoxy-511- [1,3] dioxolo [4,5-isoquinolino [3,2-a] isoquinoline-7-isobromide 13— (2, 6-difluorobenzyl) -9, 10-dimethoxy 5H- [1,3] dioxolo [4,5-g] isoquinolino [ A target compound (26%) was obtained by the same method as Step 2 of Example 1, except that 3,2-a] isoquinoline-7-iumbromide was used.
¾賺 (300 MHz, CDCl3)5=2.85(s,2H),3.89 (s, 3H) , 3.99 (s, 3Η), 4.22¾ 賺 (300 MHz, CDCl 3 ) 5 = 2.85 (s, 2H), 3.89 (s, 3H), 3.99 (s, 3Η), 4.22
(s, 2H), 4.42 (s, 2H), 6.75-6.79 (m, 3H), 6.96 (s, 1H) , 7.08-7.13 (m, 1H) , 7.21-7.24 (d, J=9.0 , 1H) , 7.35-7.38(d , 7=9.0 , 1H) (s, 2H), 4.42 (s, 2H), 6.75-6.79 (m, 3H), 6.96 (s, 1H), 7.08-7.13 (m, 1H), 7.21-7.24 (d, J = 9.0, 1H) , 7.35-7.38 (d, 7 = 9.0, 1H)
<실시예 4> <Example 4>
9, 10-디메록시-13-(2-메틸벤질)-5，.&"디ᅳ하이드로ᅳ[1，3]디옥솔로[4,5-8]이 소퀴놀 노 [3,2-a]이소퀴놀린 -8(6H)-온  9,10-dimethoxy-13- (2-methylbenzyl) -5, " &quot; dihydrohydroquinol [1,3] dioxolo [4,5-8] isoquinol furnace [3,2-a] Isoquinoline-8 (6H) -one
Figure imgf000030_0001
Figure imgf000030_0001
단계 1:  Step 1:
9 ,10-디메톡시 -13-(2-메틸벤질) -5， 6-디하이드로 -[1,3]디옥솔로 [4， 5-g] 이소퀴놀리노 [3,2-a]이소퀴놀린— 7-이움브로마이드  9,10-dimethoxy-13- (2-methylbenzyl) -5, 6-dihydro- [1,3] dioxolo [4, 5-g] isoquinolino [3,2-a] isoquinoline— 7-ium bromide
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 2-메틸벤질 브로마이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (28%)을 제조하였다. Example 1 Step 1 and Example 1 except that 2-methylbenzyl bromide was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1 In the same manner, the target compound (28%) was prepared.
賺 (300MHz, CDCI3) 62.46 (s, 3H), 3.29-3.33 (t, J=6.0Hz, 2H),4.02300 (300 MHz, CDCI3) 62.46 (s, 3H), 3.29-3.33 (t, J = 6.0 Hz, 2H), 4.02
(s, 3H), 4.42 (s, 3H), 4,46 (s, 2H), 5.19 (s, 2H), 5.99 (s, 2H) , 6.63-6.66 (d, J=9.0Hz, IH), 6.83-6.87 (m, 2H) , 7.07-7.10 (d, J=9.0Hz, IH), 7.22-7.27 (m, IH), 7.36-7.38 (m, IH) , 7.51-7.54 (m, IH), 7.68-7.71 (m, IH), 10.44 (s, IH). 단계 2: (s, 3H), 4.42 (s, 3H), 4,46 (s, 2H), 5.19 (s, 2H), 5.99 (s, 2H), 6.63-6.66 (d, J = 9.0 Hz, IH), 6.83-6.87 (m, 2H), 7.07-7.10 (d, J = 9.0 Hz, IH), 7.22-7.27 (m, IH), 7.36-7.38 (m, IH), 7.51-7.54 (m, IH), 7.68-7.71 (m, I H), 10.44 (s, I H). Step 2:
9,10-디메톡시-13-(2-메틸벤질)-5，6-디하이드로-[1，3]디옥솔로[4，5- 이소퀴놀리노 [3 , 2-a]아소퀴놀린 -8(6H)-은  9,10-dimethoxy-13- (2-methylbenzyl) -5,6-dihydro- [1,3] dioxolo [4,5-isoquinolino [3,2-a] isoquinoline-8 ( 6H) -silver
상기 단계 1에서 얻은 Obtained in step 1 above
9， 10-디메톡시 -13 (2-메틸벤질 )-5 , 6-디하이드로- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀 리노 [3,2-a]이소퀴놀린 -7-이움브로마이드를 20% 수산화 칼륨 수용액에 가한 후 24 시간 동안 교반환류하였다. 반웅 종결 후， 상기 반응물을 디클로로메탄으로 추출한 다음 황산나트륨으로 쑤분을 제거하고 여과하여 감압농축하였다. 상기 잔류물을 플래시 칼뜀크로마토그패,피로 분리하여 목적 화합물 (12%)을 얻었다. 9,10-dimethoxy-13 (2-methylbenzyl) -5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-7 -Ium bromide was added to 20% aqueous potassium hydroxide solution and stirred under reflux for 24 hours. After completion of reaction, the reaction mixture was extracted with dichloromethane and concentrated under reduced pressure by removing flakes with sodium sulfate and filtering. The residue was separated by flash runaway chromatography, blood to give the title compound (12%).
¾ NMR (300 MHz, CDC13) δ 2.40 (s, 3H) , 2.81-2.85 (t, J= 6.0 Hz, 2H) , 3.89 (s, 3H), 4.04 (s, 3H) , 4.15 (s, 2H) , 5.89 (s, 2H) , 6.73 (s, IH), 6.76 (s, IH), 6.90-6.92 (d, J = 6.0 Hz, IH) , 7.04-7.20(m , 4H) , 7.26-7.30(m , IH) . ¾ NMR (300 MHz, CDC1 3 ) δ 2.40 (s, 3H), 2.81-2.85 (t, J = 6.0 Hz, 2H), 3.89 (s, 3H), 4.04 (s, 3H), 4.15 (s, 2H ), 5.89 (s, 2H), 6.73 (s, IH), 6.76 (s, IH), 6.90-6.92 (d, J = 6.0 Hz, IH), 7.04-7.20 (m, 4H), 7.26-7.30 ( m, IH).
<실시예 5> Example 5
9 , 10-다메톡시 -13-( 2-메톡시벤질) -5， 6-디하이드로- [1,3]다옥솔로 [4 , 5-g] 이소퀴놀리노 [3，2-3]이소퀴놀린-8(6¾-온
Figure imgf000032_0001
9,10-damethoxy-13- (2-methoxybenzyl) -5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-3] isoquinoline -8 (6¾-on
Figure imgf000032_0001
단계 1:  Step 1:
9，10-디메톡시-13-(2-메톡시벤질)—5，6-디하이드로-[1,3]디옥솔로[4，5- ]이소퀴놀리노 [3 , 2-a]이소퀴놀린 -7-이움브로마이드  9,10-dimethoxy-13- (2-methoxybenzyl) —5,6-dihydro- [1,3] dioxolo [4,5-] isoquinolino [3,2-a] isoquinoline- 7-ium bromide
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 2-메특시벤질 브로마이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (89%)을 제조하였다.  The target compound (89%) was prepared by the same method as Step 1 of Example 1, except that 2-methoxybenzyl bromide was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1.
Ή NMR (300 MHz, CDC13)53.26-3.30 (t, /= 6.0 Hz, 2H),3.96 (s, 3H)， 4.02 (s, 3H), 4.41 (s, 3H), 4.52 (s, 2H), 5.22 (s, 2H), 5.99 (s, 2H) , 6.63-6.68 (m," 2H), 6.78-6.92 (m, 3H) , 7.01-7.04 (m, 1H), 7.56-7.71 (m, 2H) , 10.43 (s, 1H). 단계 2: NMR (300 MHz, CDC1 3 ) 53.26-3.30 (t, / = 6.0 Hz, 2H), 3.96 (s, 3H), 4.02 (s, 3H), 4.41 (s, 3H), 4.52 (s, 2H) , 5.22 (s, 2H), 5.99 (s, 2H), 6.63-6.68 (m, " 2H), 6.78-6.92 (m, 3H), 7.01-7.04 (m, 1H), 7.56-7.71 (m, 2H ), 10.43 (s, 1H) .Step 2:
9,10-디메톡시—13-(2-메록시벤질)-5,6-디하이드로-[1，3]디옥솔로[4，5- ]이소퀴놀리노 [3，2-3]이소퀴놀린-8(611)-온  9,10-dimethoxy—13- (2-methoxybenzyl) -5,6-dihydro- [1,3] dioxolo [4,5-] isoquinolino [3,2-3] isoquinoline- 8 (611) -on
상기 실시예 4에서 In Example 4 above
9 , 10-디메특시 -13ᅳ(2-메틸벤질 )-5， 6—디하이드로- [ 1， 3]디옥솔로 [4ᅳ 5-g]이소퀴놀 리노 [3ᅳ 2ᅳ a]이소퀴놀린 -7-이움브로마이드 대신에 상기 단계 1에서 얻은 9， 10-디메록시 -13-(2-메특시벤질 )-5， 6ᅳ디하이드로 -[ 1， 3]디옥솔로 [4ᅳ 5-g]이소퀴 놀리노 [3, 2-a]이소퀴놀린 -7-이움브로마이드를 사용한 것을 제외하고는 상기 실시예 4의 단계 2와 동일한 방법으로 목적 화합물 (14%)을 얻었다. 9,10-dimethoxy-13 '(2-methylbenzyl) -5, 6-dihydro- [1,3] dioxolo [4'5-g] isoquinolino [3'2'a] isoquinoline 9, 10-dimethoxy-13- (2-methoxybenzyl) -5, 6 ᅳ dihydro- [1,3] dioxolo [4 ᅳ 5-g] iso, obtained in step 1 above in place of -7-umbromide The target compound (14%) was obtained by the same method as Step 2 of Example 4, except that Quinolino [3, 2-a] isoquinoline-7-iumbromide was used.
¾ NMR (300丽 z, CDC13) δ 2.81-2.85 (t, J = 6.0 Hz, 2H),3.89 (s, 3H)，¾ NMR (300 ley z, CDC1 3 ) δ 2.81-2.85 (t, J = 6.0 Hz, 2H), 3.89 (s, 3H) ，
3.96 (s, 3H), 4.03 (s, 3H), 4.21 (s, 2H)， 5.90 (s, 2H) , 6.73 (s, 1H)， 6.79-6.89 (m, 3H), 6.95-6.98 (d, / = 9.0 Hz, 1H) ,7.17-7.3 (m, 3H). <실시예 6> 3.96 (s, 3H), 4.03 (s, 3H), 4.21 (s, 2H), 5.90 (s, 2H), 6.73 (s, 1H), 6.79-6.89 (m, 3H), 6.95-6.98 (d, / = 9.0 Hz, 1H), 7.17-7.3 (m, 3H). <Example 6>
9,10-디메특시 -13- (피리딘 -2-일메틸 )-5,6-디하이드로 -[1,3]디옥솔로 [4，5 -g]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)_온  9,10-dimethoxy-13- (pyridin-2-ylmethyl) -5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] Isoquinoline-8 (6H) _On
Figure imgf000033_0001
Figure imgf000033_0001
단계 1:  Step 1:
9， 10-디메톡시 -13- (피리딘 -2-일메틸) -5 , 6-디하이드로 -[ 1 , 3]다옥솔로 [4 , 5-g]이소퀴놀리노 [3 , 2-a]이소퀴놀린 -7-이움브로마어드  9,10-dimethoxy-13- (pyridin-2-ylmethyl) -5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] iso Quinoline-7-Iumbromare
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 2-시아노벤질 브로마'이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (7%)을 제조하였다. And it is produced of the desired compound (7%) in the same manner as in Step 1 of Example 1, except for benzyl bromide instead of 2-fluoro-in step 1 of Example 1 was used for 2-cyano-benzyl Bromma Eid It was.
¾匿 (300 MHz, CDC13) 63.27 (s, 1H)， 3.99 (s, 3H), 4.30 (s, 3H) , 4.86¾ 匿 (300 MHz, CDC1 3 ) 63.27 (s, 1H), 3.99 (s, 3H), 4.30 (s, 3H), 4.86
(s, 2H), 5.24 (s, 2H), 6.01 (s, 2H)， 6.89 (s, 1H>, 7.23-7.31 (m, 1H), 7.36 (s, 1H), 7.48-7.50 Cm, 1H), 7.58—7.61 (d, J= 9.0 Hz, 1H)， 7.71-7.74 (d, J = 9.0 Hz, 1H), 7.77-7.82 (m, 1H) , 8.48—8.49 (d, J= 3.0 Hz, 1H) , 10.47 (s, 1H). 단계 2: (s, 2H), 5.24 (s, 2H), 6.01 (s, 2H), 6.89 (s, 1H>, 7.23-7.31 (m, 1H), 7.36 (s, 1H), 7.48-7.50 Cm, 1H , 7.58—7.61 (d, J = 9.0 Hz, 1H), 7.71-7.74 (d, J = 9.0 Hz, 1H), 7.77-7.82 (m, 1H), 8.48—8.49 (d, J = 3.0 Hz, 1H ), 10.47 (s, 1 H). Step 2:
9, 10-디메톡시 -13- (피리딘— 2-일메틸 )-5, 6-디하이드로 -[1,3]디옥솔로 [4， 5-g]이소퀴놀리노 [3,2-3]이소퀴놀린-8(611)-온  9,10-dimethoxy-13- (pyridin— 2-ylmethyl) -5, 6-dihydro- [1,3] dioxolo [4, 5-g] isoquinolino [3,2-3] iso Quinolin-8 (611) -on
상기 실시예 4에서 In Example 4 above
9, 10-디메록시 -13-(2-메틸벤질) -5, 6-디하이드로 -[1,3]디옥솔로 [4， 5-g]이소퀴놀 리노 [3,2— a]이소퀴놀린 -7-이움브로마이드 대신에 상기 단계 1에서 얻은 9， 10-디메특시 -13-(피리딘 -2-일메틸 )-5， 6-디하이드로- [ 1， 3]디옥솔로 [4， 5-g]이 소퀴놀리노 [3,2— a]이소퀴놀린 -7—이움브로마이드를 사용한 것을 제외하고는 상기 실시예 4의 단계 2와 동일한 방법으로 목적 화합물 (15%)을 얻었다. 9,10-dimethoxy-13- (2-methylbenzyl) -5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2—a] isoquinoline- 9, 10-dimethoxy-13- (pyridin-2-ylmethyl) -5, 6-dihydro- [1,3] dioxolo [4,5-g] obtained in step 1 in place of 7-umbromide ] Except using this soquinolino [3,2—a] isoquinoline-7-umombromide In the same manner as in Step 2 of Example 4, the target compound (15%) was obtained.
¾ NMR (300 MHz, CDC13) δ 2.83-2.87 (t, J= 6.0 Hz, 2H), 3.89 (s, 3H) ,¾ NMR (300 MHz, CDC1 3 ) δ 2.83-2.87 (t, J = 6.0 Hz, 2H), 3.89 (s, 3H),
4.01 (s, 3H), 4.53 (s, 2H)， 5.92 (s, 2H) , 6.76 (s, 1H), 7.00 (s, 1H)， 7.11-7.20 On, 4H), 7.59-7.64 (m, 1H), 8.63-8.65 (d, / = 6.0 Hz, 1H). 4.01 (s, 3H), 4.53 (s, 2H), 5.92 (s, 2H), 6.76 (s, 1H), 7.00 (s, 1H), 7.11-7.20 On, 4H), 7.59-7.64 (m, 1H ), 8.63-8.65 (d, / = 6.0 Hz, 1H).
<실시예 > <Example>
13-( (1H-벤조 [d]이미다졸 -2-일)메틸) -9,10-디메톡시 -5,6-디하이드로 -[1， 3]디옥 로 [4 , 5-g]이소퀴놀리노 [3， 2-a]이소퀴놀린 -8(6H)-온  13-((1H-benzo [d] imidazol-2-yl) methyl) -9,10-dimethoxy-5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinol Lino [3,2-a] isoquinolin-8 (6H) -one
Figure imgf000034_0001
Figure imgf000034_0001
단계 1:  Step 1:
13- ( ( 1H-벤조 [ d ]이미다졸 -2-일)메틸)—9 , 10-디메록시 -5， 6—디하이드로 -[ 1 ， 3]디옥솔로 [4 , 5-g]이소퀴놀리노 [3， 2-a]이소퀴놀린 -그이움브로마이드  13-((1H-benzo [d] imidazol-2-yl) methyl) —9,10-dimethoxy-5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinol Reno [3，2-a] isoquinoline-Iumbromide
상기 실시예 1의 단계 1에서 2-폴루오로벤 ¾ 브로마이드 대신에 2-클로로메틸벤조아미다졸을 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (59«을 제조하였다.  The target compound (59 «) was prepared in the same manner as in Example 1, Step 1 except that 2-chloromethylbenzoamidazole was used instead of 2-poloroben 3/4 bromide in Step 1 of Example 1.
¾NMR (300 MHz, CDC13) δ 3.31 (s, 2Η), 3.80 (s, 3H)， 4.14 (s, 3H), 4.87¾NMR (300 MHz, CDC1 3 ) δ 3.31 (s, 2Η), 3.80 (s, 3H), 4.14 (s, 3H), 4.87
(s, 2H), 5.11 (s, 2H), 5.99 (s, 2H) , 6.83 (s, 1H) , 7.16-7.19 (m, 2H), 7.46-7.49 (d, J= 9.0 Hz, 1H), 7.60-7.63 (m, 2H) , 7.72 (s, 1H), 7.80-7.83 (d, J= 9.0 Hz, 1H), 9.83 (s, 1H). 단계 2: (s, 2H), 5.11 (s, 2H), 5.99 (s, 2H), 6.83 (s, 1H), 7.16-7.19 (m, 2H), 7.46-7.49 (d, J = 9.0 Hz, 1H), 7.60-7.63 (m, 2H), 7.72 (s, 1H), 7.80-7.83 (d, J = 9.0 Hz, 1H), 9.83 (s, 1H). Step 2:
13- (( 1H-벤조 [d]이미다졸 -2-일)메틸 )-9， 10-디메톡시 -5 , 6-디하이드로 -[ 1 ,3]디옥솔로 [4,5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온  13-((1H-benzo [d] imidazol-2-yl) methyl) -9,10-dimethoxy-5,6-dihydro- [1,3] dioxolo [4,5-g] isoquinol Reno [3,2-a] isoquinolin-8 (6H) -one
상기 실시예 4에서 9， 10-디메톡시— 13-(2-메틸벤질 )-5， 6-디하이드로- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀 리노 [3,2-a]이소퀴놀린 -7-이움브로마이드 대신에 상기 단계 1에서 얻은 13- ( 1H-벤조 [d]이미다졸 -2-일)메틸) -9， 10-디메록시 -5， 6-디하이드로- [ 1， 3]디옥 솔로 [4ᅳ 5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -7-이움브로마이드를 사용한 것을 제외하고는 상기 실시예 4의 단계 2와 동일한 방법으로 목적 화합물 (8%)을 얻었다. In Example 4 above 9,10-dimethoxy— 13- (2-methylbenzyl) -5, 6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline- 13- (1H-benzo [d] imidazol-2-yl) methyl) -9, 10-dimethoxy-5, 6-dihydro- [1,3] diox obtained in step 1 instead of 7-umbromide The target compound (8%) was obtained by the same method as Step 2 of Example 4, except that Solo [4 ′ 5-g] isoquinolino [3,2-a] isoquinoline-7-iumbromide was used. .
¾ NMR (300 MHz, CDC13) δ 2.81-2.85 (t, J= 6.0 Hz, 2H) , 3.81 (s, 3H),¾ NMR (300 MHz, CDC1 3 ) δ 2.81-2.85 (t, J = 6.0 Hz, 2H), 3.81 (s, 3H),
3.85 (s, 3H), 3.98 (s, 3H) , 4.22-4.24 (m, 2H) , 4.45 (s, 2H)， 5.90 (s, 2H), 6.77 (s, 1H), 7.09-7.38 (m, 6H) , 7.72-7.74 (d, J = 6.0 Hz, 1H). 3.85 (s, 3H), 3.98 (s, 3H), 4.22-4.24 (m, 2H), 4.45 (s, 2H), 5.90 (s, 2H), 6.77 (s, 1H), 7.09-7.38 (m, 6H), 7.72-7.74 (d, J = 6.0 Hz, 1H).
<실시예 8> <Example 8>
13-( (2-클로로티아졸 -5-일 )메 )-9 , 10-디메톡시 5 , 6-다하이드로- [ 1， 3]디 옥솔로 [45-g]이소퀴놀리노 [3 , 2-a]이소퀴놀린 -8(6Η)-온  13-((2-chlorothiazol-5-yl) meth) -9,10-dimethoxy 5,6-dihydro- [1,3] dioxolo [45-g] isoquinolino [3,2 -a] isoquinoline-8 (6Η) -one
Figure imgf000035_0001
Figure imgf000035_0001
단계 1:  Step 1:
13-( (2-클로로티아졸 -5-일 )메틸 )-9， 10-디메록시 -5 ' 6-디하이드로 -[ 1 ,3] 디옥솔로 [4 , 5-g]이소퀴놀리노 [3， 2-a]이소퀴놀린 -그이움브로마이드 13-((2-chlorothiazol-5-yl) methyl) -9,10-dimethoxy-5 ' 6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3 , 2-a] isoquinoline-Iumbromide
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 2ᅳ클로로 -5-클로로메틸티아졸을 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (51%)을 제조하였다.  The target compound (51%) was prepared in the same manner as in Step 1 of Example 1, except that 2 ᅳ chloro-5-chloromethylthiazole was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1. Prepared.
¾證 (300 MHz, CDCI3) δ 3.31 (s, 1H), 4.06 (s, 3H) , 4.40 (s, 3H), 4.71¾ 證 (300 MHz, CDCI 3 ) δ 3.31 (s, 1H), 4.06 (s, 3H), 4.40 (s, 3H), 4.71
(s, 2H), 5.12 (s, 2H) , 6.07 (s, 2H) , 6.91 (s, 1H)， 6.98 (s, 1H)， 7.09 (s, 1H)， 7.71-7.75 (d, J= 9.0 Hz, IH), 7.80-7.83 (d, J= 9.0 Hz, 1H) , 10.39 (s, 1H). 단계 2: 13-((2-클로로티아졸 -5-일)메릴 )-9,10-디메톡시-5,6-디하이드로-卜1,3] 디옥솔로 [4 , 5-g]이소퀴놀리노 [3ᅳ 2-al이소퀴놀린 -8(6H)-은 (s, 2H), 5.12 (s, 2H), 6.07 (s, 2H), 6.91 (s, 1H), 6.98 (s, 1H), 7.09 (s, 1H), 7.71-7.75 (d, J = 9.0 Hz, IH), 7.80-7.83 (d, J = 9.0 Hz, 1H), 10.39 (s, 1H). Step 2: 13-((2-chlorothiazol-5-yl) meryl) -9,10-dimethoxy-5,6-dihydro-VII, 3] dioxolo [4, 5-g] isoquinolino [3 Al 2-alisoquinoline-8 (6H) -silver
상기 실시예 4에서 In Example 4 above
9， 10-디메톡시 -13-(2-메틸벤질 )-5， 6-디하이드로- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀 리노 [3, 2-a]이소퀴놀린 -그이움브로마이드 대신에 상기 단계 1에서 얻은 13- ( (2-클로로티아졸 -5-일 )메틸 )-9， 10-디메톡시 -5, 6-디하이드로 -[ 1， 31디옥솔로 [4,5-g]이소퀴놀리노 [3,2-a]이소퀴놀린-그이움브로마이드를 사용한 것을 제외하 ί는 상기 실시예 4의 단계 2와 동일한 방법으로 목적 화합물 (5%)을 얻었다ᅳ 9, 10-dimethoxy-13- (2-methylbenzyl) -5, 6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline- Instead of ium bromide, 13-((2-chlorothiazol-5-yl) methyl) -9, 10-dimethoxy-5, 6-dihydro- [1,31dioxolo [4, Except for using 5-g] isoquinolino [3,2-a] isoquinoline-iumum bromide, ί obtained the target compound (5%) in the same manner as in Step 2 of Example 4
¾鍾 (300 MHz, CDCI3) 52.87-2.91 (t, J= 6.0， 2H), 3.95 (s, 3H), 4.01¾ 鍾 (300 MHz, CDCI 3 ) 52.87-2.91 (t, J = 6.0, 2H), 3.95 (s, 3H), 4.01
(s, 3H), 4.27-4.31 (t, J= 6.0 Hz, 2H) , 6.01 (s, 2H), 6.70— 6.72 (m, 2H) , 7.22 (s, 1H), 7.26-7.33 (m, 2H). (s, 3H), 4.27-4.31 (t, J = 6.0 Hz, 2H), 6.01 (s, 2H), 6.70— 6.72 (m, 2H), 7.22 (s, 1H), 7.26-7.33 (m, 2H ).
<실시예 9> Example 9
13-(2-플루오로벤조일 ) 9 , 10-디메톡시 -5Η- [ 1 , 3]디옥솔로 [4 , 5-g]이소퀴놀 리노 [3 , -a]이소퀴놀린 -8(6H)-은  13- (2-fluorobenzoyl) 9,10-dimethoxy-5 5- [1, 3] dioxolo [4,5-g] isoquinolino [3, -a] isoquinoline-8 (6H) -silver
Figure imgf000036_0001
Figure imgf000036_0001
단계 1:  Step 1:
13-(2-폴루오로벤조일) -9，10-디메톡시-511-[1,3]디옥솔로[4,5- 이소퀴 놀리노 [3, 2-a]이소퀴놀린-이움클로라이드  13- (2-polourobenzoyl) -9,10-dimethoxy-511- [1,3] dioxolo [4,5-isoquinolino [3,2-a] isoquinoline-iumchloride
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 2-플루오로벤조일 클로라이드를 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (60%)을 제조하였다.  Except for using 2-fluorobenzoyl chloride instead of 2-fluorobenzyl bromide in Step 1 of Example 1 to prepare the target compound (60%) in the same manner as in Step 1 of Example 1.
¾賺 (300 MHz, CDCI3) δ 3.18-3.47 (m, 2H), 4.04(s, 3H), 4.06(s, 3H), 4.40(s, 2H), 6.08 (s, 2H), 6.59(s, 1H)， 6.95(s, 1H) , 7.03-7.13 (m, 1H) , 7.18-7.20(m, 1H), 7.42-7.60(m, 2H), 7.69-7.77 (m, 1H), 7.82-7.87(m, 1H). 단계 2: . ¾ 賺 (300 MHz, CDCI 3 ) δ 3.18-3.47 (m, 2H), 4.04 (s, 3H), 4.06 (s, 3H), 4.40 (s, 2H), 6.08 (s, 2H), 6.59 (s , 1H), 6.95 (s, 1H), 7.03-7.13 (m, 1H), 7.18-7.20 (m, 1 H), 7.42-7.60 (m, 2 H), 7.69-7.77 (m, 1 H), 7.82-7.87 (m, 1 H). Step 2:.
13-(2-플루오로벤조일) -9,10-디메톡시-511-[1,3]디옥솔로[4,5- 이소퀴 놀리노 [3,2-a]이소퀴놀린 -8(6H)-온  13- (2-fluorobenzoyl) -9,10-dimethoxy-511- [1,3] dioxolo [4,5-isoquinolino [3,2-a] isoquinoline-8 (6H)- On
상기 실시예 4에서 In Example 4 above
9， 10-디메톡시- ^-(.2ᅳ메틸벤질) -5，6-디하이드로 -[1,3]디옥솔로 [4， 5-g]이소퀴놀 리노 [3,2-a]이소퀴놀린 -7-이움브로마이드 대신에 상기 단계 1에서 얻은 13-(2-플루오로벤조일)-9,10-디메특시 -5H- [ 1 , 3 ]디옥솔로 [ 4， 5-g ]이소퀴놀리노 [ 3 ,2-a]이소퀴놀린ᅳ이움클로라이드를 사용한 것을 제외하고는 상기 실시예 4의 단계 2와 동일한 방법으로 목적 화합물 (15%)을 얻었다. 9, 10-dimethoxy - ^ - (2 eu-methylbenzyl) -5,6-dihydro [1, 3] dioxolo [4, 5-g] iso-1,2,3,4 [3,2-a] isoquinoline 13- (2-fluorobenzoyl) -9,10-dimethoxy-5H- [1,3] dioxolo [4,5-g] isoquinolino [ A target compound (15%) was obtained by the same method as Step 2 of Example 4, except that 3,2-a] isoquinolinedium chloride was used.
¾ MR (300 MHz, CDC13) 62.80 (m, 2H), 3.94 (s, 3H) , 4.03 (s, 3H)， 4.24(m¾ MR (300 MHz, CDC1 3 ) 62.80 (m, 2H), 3.94 (s, 3H), 4.03 (s, 3H), 4.24 (m
2H), 5.88 (s, 2H), 6.59 (s, 1H) , 6.866.92 (m, 1H)， 6.97 (s, 1H) , 7.02-7.07 (m, 1H), 7.29 (d, J= 9.0 Hz, 1H), 7.34=7.39 (m, 1H)， 7.49 (d, J = 9.0 Hz, 1H), 7.63-7.68 (m, 1H) 2H), 5.88 (s, 2H), 6.59 (s, 1H), 6.866.92 (m, 1H), 6.97 (s, 1H), 7.02-7.07 (m, 1H), 7.29 (d, J = 9.0 Hz , 1H), 7.34 = 7.39 (m, 1H), 7.49 (d, J = 9.0 Hz, 1H), 7.63-7.68 (m, 1H)
<실시예 10> <Example 10>
에틸 9 , 10-디메특시 8-옥소 -6 , 8-디하이드로 -5Η- [ 1， 3]디옥솔로 [4 , 5-g]이소 퀴놀리노 -a]이소퀴놀린 -13-카르복실레이트  Ethyl 9,10-dimethoxy 8-oxo-6,8-dihydro-55- [1,3] dioxolo [4,5-g] isoquinolino-a] isoquinoline-13-carboxylate
Figure imgf000037_0001
Figure imgf000037_0001
단계 1:  Step 1:
13- (에톡시카르보닐) -9, 10-디메톡시 -5 ,6-디하이드로 -[1,3]디옥솔로 [4, 5 -g]이소퀴놀리노 [3, 2-a]이소퀴놀린 -7-이움클로라이드 13- (ethoxycarbonyl) -9,10-dimethoxy-5,6-dihydro- [1,3] dioxolo [4, 5 -g] isoquinolino [3, 2-a] isoquinoline-7-ium chloride
상기 실시예 1의 단계 1에서 2-플루오로벤질 브로마이드 대신에 에틸 클로로포메이트를 사용한 것을 제외하고는 상기 실시예 1의 단계 1과 동일한 방법으로 목적화합물 (55%)을 제조하였다. ¾ NMR (300 MHz, CDC13) δ 1.31 (t, J = 6.9 Hz, 3H) , 3.26-3.29 (m, 2H),The target compound (55%) was prepared in the same manner as in Step 1 of Example 1, except that ethyl chloroformate was used instead of 2-fluorobenzyl bromide in Step 1 of Example 1. ¾ NMR (300 MHz, CDC1 3 ) δ 1.31 (t, J = 6.9 Hz, 3H), 3.26-3.29 (m, 2H),
4.08(s, 3H), 4.38(s, 3H)， 4.47 (q, J = 6.9 Hz, 2H), 5.40 (m, 2H), 6.09 (s, 2H), 6.89 (s, 1H), 7.14 (s, 1H)， 7.74 (d, J = 9.3 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 11.01 (s, 1H) 단계 2: 4.08 (s, 3H), 4.38 (s, 3H), 4.47 (q, J = 6.9 Hz, 2H), 5.40 (m, 2H), 6.09 (s, 2H), 6.89 (s, 1H), 7.14 (s , 1H), 7.74 (d, J = 9.3 Hz, 1H), 7.88 (d, J = 9.0 Hz, 1H), 11.01 (s, 1H) Step 2:
에틸 9 , 10-디메톡시 -6 , 8二디하이드로- [ 1 , 3]디옥솔로 [4， 5-g]이소퀴놀리노  Ethyl 9,10-dimethoxy-6,82-dihydro- [1,3] dioxolo [4,5-g] isoquinolino
[3， 2-a]이소퀴놀린 -13-카르복실레이트 [3, 2-a] isoquinoline-13-carboxylate
상기 단계 1에서 얻은 Obtained in step 1 above
13- (에톡시카르보닐 )-9， 10-디메톡시 -5， 6-디하이드로 -[ 1， 3]디옥솔로 [4， 5-g]이소 퀴놀리노 [3, 2-a]이소퀴놀린 -7-이움클로라이드 (0.84g, 1.89 mmol)을 메탄올 (50 mL)에 녹인 용액에 소듐보로하이드라이드 (54mg, 1.42 mmol)를 5¾ 수산화나트륨 13- (ethoxycarbonyl) -9, 10-dimethoxy-5, 6-dihydro- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline- Sodium borohydride (54 mg, 1.42 mmol) was dissolved in 5¾ sodium hydroxide in a solution of 7-ium chloride (0.84 g, 1.89 mmol) in methanol (50 mL).
5 mL에 녹인 수용액을 0oC에서 천천히 적가한 후 1시간 동안 교반하였다. 반응물을 에틸 아세테이트로 추출 후 황산나트륨으로 수분을 제거하고 농축 후 플래시 칼럼크로마토그래피 (에틸아세테이트: 핵산 =1:3)로 분리하여 목적 화합물 (93%)을 얻었다. Aqueous solution dissolved in 5 mL was slowly added dropwise at 0 ° C. and stirred for 1 hour. The reaction mixture was extracted with ethyl acetate, dried over sodium sulfate, concentrated, and separated by flash column chromatography (ethyl acetate: nucleic acid = 1: 3) to obtain the title compound (93%).
¾ NMR (300 MHz, CDCI3) δ 1.14 (t, J = 7.2 Hz, 3H)， 2.80-2.84 (m, 2H) ,¾ NMR (300 MHz, CDCI 3 ) δ 1.14 (t, J = 7.2 Hz, 3H), 2.80-2.84 (m, 2H),
3.23-3.26 (m, 2H), 3.81 (s, 3H), 3.84 (s, 3H) , 4.14 (q, J = 7.2 Hz, 2H) , 4.51 (s, 2H), 5.93 (s, 2H), 6.62 (s, 1H), 6.93 (s, 1H) , 6.81 (d, J = 9.0 Hz, 1H) , 7.38 (d, J = 8.7 Hz, 1H) 단계 3: 3.23-3.26 (m, 2H), 3.81 (s, 3H), 3.84 (s, 3H), 4.14 (q, J = 7.2 Hz, 2H), 4.51 (s, 2H), 5.93 (s, 2H), 6.62 (s, 1H), 6.93 (s, 1H), 6.81 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H) Step 3:
에틸 9 , 10-디메톡시 -8-옥소 -6 , 8-디하이드로 -5Η-[ 1 , 3]디옥솔로 [4 , 5-g]이 소퀴놀리노 [3 , 2-a]이소퀴놀린 -13-카르복실레이트 Ethyl 9,10-dimethoxy-8-oxo-6,8-dihydro-5Η- [1,3] dioxolo [4,5-g] Soquinolino [3, 2-a] isoquinoline-13-carboxylate
상기 단계 2에서 얻은 에틸 9 ,10-디메톡시 -6, 8-디하이드로 -[ 1,3]디옥솔로 [4, 5-g]이소퀴놀리노  Ethyl 9,10-dimethoxy-6,8-dihydro- [1,3] dioxolo [4,5-g] isoquinolino obtained in step 2 above
[3,2-a]이소퀴놀린 -13-카르복실레이트 (0.8g, 1.95 隱 ol )를디클로로메탄 (300 mL)에 녹인 용액에 이산화망간 (2g, 23 mmol)을 가하고 15시간 동안 교반환류하였다. 반웅 종료 후 과량의 이산화망간를 여과하여 제거하고 농축 후 플래시 칼럼크로마토그래피로 분리하여 ^적화합물 (1 )을 얻었다. · Manganese dioxide (2 g, 23 mmol) was added to a solution of [3,2-a] isoquinoline-13-carboxylate (0.8 g, 1.95 隱 ol) in dichloromethane (300 mL), followed by stirring under reflux for 15 hours. After completion of reaction, excess manganese dioxide was filtered off, concentrated and separated by flash column chromatography to obtain ^ red compound (1). ·
¾ NMR (300 MHz, CDC13) δ 1.23-1.28 (t, J= 6.0 Hz, 3H), 2.86-2.90 (t,¾ NMR (300 MHz, CDC1 3 ) δ 1.23-1.28 (t, J = 6.0 Hz, 3H), 2.86-2.90 (t,
J= 6.0 Hz, 2H),3.95 (s, 3H), 3.99 (s, 3H), 4.20-4.24 (t, /= 6.0 Hz, 2H) , 4.30-3.37 (q, J = 6.0 hz, 2H), 6.00 (s, 2H), 6.75 (s, 1H), 7.08 (s, 1H), 7.32-7.35 (d, J = 9.0 Hz, 1H) , 7.43-7.46 (d, J = 9.0 Hz, 1H). J = 6.0 Hz, 2H), 3.95 (s, 3H), 3.99 (s, 3H), 4.20-4.24 (t, / = 6.0 Hz, 2H), 4.30-3.37 (q, J = 6.0 hz, 2H), 6.00 (s, 2H), 6.75 (s, 1H), 7.08 (s, 1H), 7.32-7.35 (d, J = 9.0 Hz, 1H), 7.43-7.46 (d, J = 9.0 Hz, 1H).
<실시예 11> <Example 11>
9 , 10-디메톡시 -8-옥소 -6 , 8-디하이드로 -5Η- [ 1 , 3]디옥솔로 [4 , 5-g]이소퀴놀 리노 [3, -a]이소퀴놀린 -13-카르복실산  9,10-dimethoxy-8-oxo-6,8-dihydro-55- [1,3] dioxolo [4,5-g] isoquinolino [3, -a] isoquinoline-13-carboxyl mountain
Figure imgf000039_0001
Figure imgf000039_0001
상기 실시예 10에서 얻은 에틸 9， 10-디메톡시 -8ᅳ옥소 -6， 8-디하이드로 -5H- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀리 노 [3,2-a]이소퀴놀린 -13-카르복실레이트 (0.106g, 0.25 mmol)을 4N수산화나트륨 수용액 (50 mL)을 가하고 80oC에서 36시간 동안 교반하였다. 반웅물을 감압 농축 후 증류수 (20 mL)를 가하고 1N 염산 수용액으로 pH 4로 조절한 후 에틸 아세테이트로 추출하고 황산나트륨으로 수분을 제거하여 농축 후 풀래시 칼럼크로마토그래피로 분리하여 (디클로로메탄: 메탄을 -10:1)목적화합물 (61 mg, 61%)을 얻었다. ¾ NMR (300 MHz, DMSO-d6) 5 2.81 (s, 2H), 3.76 (s, 3H), 3.88 (s, 3H),Ethyl 9, 10-dimethoxy-8oxo-6,8-dihydro-5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2- obtained in Example 10 above a] isoquinoline-13-carboxylate (0.106 g, 0.25 mmol) was added to 4N aqueous sodium hydroxide solution (50 mL) and stirred at 80 ° C. for 36 hours. The reaction mixture was concentrated under reduced pressure, distilled water (20 mL) was added thereto, and the mixture was adjusted to pH 4 with 1N aqueous hydrochloric acid solution. The reaction mixture was extracted with ethyl acetate, dried with sodium sulfate, concentrated and separated by full-flash column chromatography (dichloromethane: methane. -10: 1) The target compound (61 mg, 61%) was obtained. ¾ NMR (300 MHz, DMSO-d 6 ) 5 2.81 (s, 2H), 3.76 (s, 3H), 3.88 (s, 3H),
4.03 (s, 2H); 6.08 (s, 2H), 6.98 (s, 1H), 7.29 (s, 1H) , 7.36-7.39 (d, J= 9.0 Hz, 1H), 7.55-7.58 (d, J = 9.0 Hz, 1H). <실시예 12> 4.03 (s, 2 H); 6.08 (s, 2H), 6.98 (s, 1H), 7.29 (s, 1H), 7.36-7.39 (d, J = 9.0 Hz, 1H), 7.55-7.58 (d, J = 9.0 Hz, 1H). <Example 12>
9 , 10-디메특시 -8-옥소 -6， 8-디하이드로 -5H- [ 1 , 3]디옥솔로 [4， 5-g]이소퀴놀 리노 [3， 2-a]이소퀴놀린 -13-카르복사마이드  9,10-dimethoxy-8-oxo-6,8-dihydro-5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-13- Carboxamide
Figure imgf000040_0001
Figure imgf000040_0001
9 , 10-디메톡시 -8-옥소 -6， 8-디하이드로 -5H- [ 1 , 3]디옥솔로 [4， 5-g]이소퀴놀리노 [3 , 2-a]이소퀴놀린 -13-카르복실산 (15 mg, 37.9 umol)을 디클로로메탄 (10 mL)에 녹인 후 옥살산 염화물 (48mg, 379 umol)을 가한 후 실온에서 2시간 동안 교반하였다. 반응물을 농축 후 테트라하이드로 퓨란 (10 mL)에 녹인 후 0oC로 냉각하고 암모니아 수용액 (28%， 5 mL)을 가하고 1시간 동안 교반하였다. 반웅물을 에틸 아세테이트로 추출하고 황산나트륨으로 수분을 제거하고 농축 후 플래시 칼럼크로마토그래피로 분리하여 (디클로로메탄: 메탄을 =20:1) 목적화합물 (7 mgᅳ 47%)을 얻었다. 9,10-dimethoxy-8-oxo-6,8-dihydro-5H- [1,3] dioxolo [4,5-g] isoquinolino [3,2-a] isoquinoline-13-car Acid (15 mg, 37.9 umol) was dissolved in dichloromethane (10 mL), then oxalic acid chloride (48 mg, 379 umol) was added and stirred at room temperature for 2 hours. The reaction was concentrated and then dissolved in tetrahydrofuran (10 mL), cooled to 0 o C, an aqueous ammonia solution (28%, 5 mL) was added and stirred for 1 hour. The reaction product was extracted with ethyl acetate, water was removed with sodium sulfate, concentrated, and separated by flash column chromatography (dichloromethane: methane = 20: 1) to obtain a target compound (7 mg ᅳ 47%).
¾ NMR (300 MHz, CDC13) δ 2.79-2.83 (t, J= 6.0 Hz, 2H), 3.92 (s, 3H) ,¾ NMR (300 MHz, CDC1 3 ) δ 2.79-2.83 (t, J = 6.0 Hz, 2H), 3.92 (s, 3H),
3.95 (s, 3H), 4.15 (s, 2H), 5.89 (s, 1H) , 6.01 (s, 2H) , 6.07 (s, 1H) , 6.72 (s, 1H), 7.30-7.33 (d, / = 9.0 Hz, 1H), 7.56-7.58 (m, 2H) . 3.95 (s, 3H), 4.15 (s, 2H), 5.89 (s, 1H), 6.01 (s, 2H), 6.07 (s, 1H), 6.72 (s, 1H), 7.30-7.33 (d, / = 9.0 Hz, 1H), 7.56-7.58 (m, 2H).
<실시예 13> Example 13
N-(2-플루오로페닐) -9， 10-디메톡시 -8-옥소 -6， 8-디하이드로 -5H-[ 1 , 3]디옥 솔로 [4 , 5-g]이소퀴놀리노 [3 , 2-a]이소퀴놀린 -13-카르복사마이드 N- (2-fluorophenyl) -9, 10-dimethoxy-8-oxo-6,8-dihydro-5H- [1,3] dioxolo [4, 5-g] isoquinolino [3, 2-a] isoquinoline-13-carboxamide
Figure imgf000041_0001
Figure imgf000041_0001
상기 실시예 12에서 얻은 Obtained in Example 12.
9 , 10-디메톡시 -8-옥소 -6 , 8-디하이드로 -5H- [ 1 , 3]디옥솔로 [4 , 5-g]이소퀴놀리노 [3 9,10-dimethoxy-8-oxo-6,8-dihydro-5H- [1,3] dioxolo [4,5-g] isoquinolino [3
,2-a]이소퀴놀린 -13-카르복사마이드 (15 mg, 37.9 (jmol)을 디메틸포름아미드 (3 mL)에 녹인 용액에 2-풀루오르아닐린 (5 μί, 45 μπιοΐ), 1-하이드록시벤조트리아졸 (8 mg, 57 mol), 디이소프로필에틸아민 (16 L, 95 μηιοΐ), EDC (11 mg, 57 μπιοΐ)을 순서대로 가하고 48시간 동안 교반하였다. 반웅물에 증류수 (5 mL)를 가하고 에틸 아세테이트로 추출하여 황산나트륨으로 수분을 제거하고 농축 후 플래시 칼럼크로마토그래피로 분리하여 (에틸아세테이트: 핵산 = 1:1) 목적화합물 (5 mg, 27%)을 얻었다. , 2-a] Isoquinoline-13-carboxamide (15 mg, 37.9 (jmol) in dimethylformamide (3 mL) in 2-fluluaniline (5 μί, 45 μπιοΐ), 1-hydroxy benzotriazole (8 mg, 57 mol), diisopropylethylamine (16 L, 95 μ η ιοΐ ), EDC (11 mg, 57 μπιοΐ) was added sequentially and the mixture was stirred for 48 hours. distilled water (5 to banung water mL) was added, extracted with ethyl acetate, water was removed with sodium sulfate, concentrated, and separated by flash column chromatography (ethyl acetate: nucleic acid = 1: 1) to obtain the target compound (5 mg, 27%).
¾ NMR (300 MHz, CDC13) 52.96-3.00 (t, /= 6.0 Hz, 2H) , 3.99 (s, 3Η) ,¾ NMR (300 MHz, CDC1 3 ) 52.96-3.00 (t, / = 6.0 Hz, 2H), 3.99 (s, 3Η),
4.04 (s, 3Η), 4.28-4.32 (t, J= 6.0 Hz, 2H) , 6.13 (s, 2H), 6.85 (s, 1H) , 7.08 (s, 1H), 7.32-7.57 (m, 5H),.7.73-7.76 (d, J = 9.0 Hz, 1H) , 8.07-8.10 (d, J = 9.0 Hz, 1H). 4.04 (s, 3Η), 4.28-4.32 (t, J = 6.0 Hz, 2H), 6.13 (s, 2H), 6.85 (s, 1H), 7.08 (s, 1H), 7.32-7.57 (m, 5H) , .7.73-7.76 (d, J = 9.0 Hz, 1H), 8.07-8.10 (d, J = 9.0 Hz, 1H).
<실험예 1> iNOS유전자 발현증가의 억제효과측정 Experimental Example 1 Measurement of Inhibitory Effect of Increased iNOS Gene Expression
본 발명에 따른 유도체가 iNOS유도를 저해하는 효과가 있는지 알아보기 위하여， 먼저 마우스의 대식세포인 Raw264.7세포를 384 웰플레이트에 3 x 104 세포 /웰의 밀도로 분주하고 100 ng/ml 농도의 LPS를 처리하여 37°C에서 24시간 동안 배양하고 배지에 증가되는 Nitrate를 Griess 시약을 사용하여 측정하였다. 활성 저해율은감지된 신호를 하기 수학식 1에 의하여 계산하여， 그 결과를 하기 표 2에 나타내었다. [수학식 1]
Figure imgf000042_0001
In order to determine whether the derivative according to the present invention has an effect of inhibiting iNOS induction, first, raw264.7 cells, which are macrophages of mouse, are divided into 384 well plates at a density of 3 x 10 4 cells / well and 100 ng / ml concentration. LPS was incubated for 24 hours at 37 ° C and increased Nitrate in the medium was measured using Griess reagent. Activity inhibition rate was calculated by the following equation 1, the results are shown in Table 2 below. [Equation 1]
Figure imgf000042_0001
【표 2】 Table 2
Figure imgf000042_0002
Figure imgf000042_0002
상기 표 2에 나타난 바와 같이， 본 발명에 따른 화합물은 iNOS 유도 활성을 저해하는 효과가 있다는 것을 알 수 있다.  As shown in Table 2, it can be seen that the compound according to the present invention has an effect of inhibiting iNOS inducing activity.
따라서， 본 발명에 따른 유도체들은 iNOS와유도활성 관련 질환의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다.  Therefore, the derivatives according to the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of iNOS-induced activity-related diseases.
<실험예 2> NFAT5전사활성 억제효과조사 Experimental Example 2 Investigation of Inhibitory Effect on NFAT5 Transcription Activity
본 발명에 따른 유도체가 NFAT5 전사활성 억제효과가 있는지 알아보기 위하여 NFAT5 리포터검색을 실시하였다. pEGFP-Nl에 backbone 중에서 CMV promoter sequence 부분을 제거하고 NFAT5의 binding si te sequence (TGGAAAATTACCG) 부분올 넣어 제작된 플라스미드 백터를 형질도입시킨 In order to find out whether the derivative according to the present invention has an inhibitory effect on NFAT5 transcription activity, NFAT5 reporter search was performed. Remove the CMV promoter sequence from the backbone of pEGFP-Nl and bind the sequence of NFAT5 (TGGAAAATTACCG) partial transduced plasmid vector
Raw264.7 대식 세포를 10% FBS-RPMr 배지 (500 g/ml의 G418 함유)에서 배양하였다. 세포를 96-웰플레이트에 3 X 103 세포 /웰의 밀도로 분주하고 하투 동안 더 배양한 후에 LPS를 1 yg/ml의 농도로 48시간 처리한 후 세포를 고정시키고 GFP의 발현정도를 HCS 형광이미지로 측정하였다 (Thermo ,Raw264.7 macrophages were cultured in 10% FBS-RPMr medium (containing 500 g / ml of G418). The cells were dispensed in a 96-well plate at a density of 3 X 10 3 cells / well and further incubated for 2 hours. After treatment with LPS at a concentration of 1 yg / ml for 48 hours, the cells were fixed and the expression of GFP was measured by HCS fluorescence. Measured by image (Thermo,
ArrayScanvl ) . 상기 실험결과를 하기 표 3 및 도 1에 나타내었다. 도 1은 본 발명에 따른 유도체의 NFAT5 전사활성 억제효과를 나타낸 그래프이다. ArrayScan vl ). The experimental results are shown in Table 3 and FIG. 1. 1 is a graph showing the NFAT5 transcriptional activity inhibitory effect of the derivative according to the present invention.
【표 3】 Table 3
Figure imgf000043_0001
Figure imgf000043_0001
상기 표 3 및 도 1에 나타난 바와 같이， 본 발명에 따른 유도체들은 NFAT5의 활성을 저해하는 효과가 우수하고 특히ᅳ 실시예 1 , 3 , 6 및 10의 유도체가 NFAT5의 활성을 저해하는 효과가 우수다는 것을 알 수 있다. 따라서， 본 발명에 따른 유도체들은 NFAT5의 활성 관련 질환의 예방 또는 치료용 약학적 조성물로 유용하게 사용될 수 있다. As shown in Table 3 and Figure 1, the derivatives according to the present invention is excellent in the effect of inhibiting the activity of NFAT5, especially in Example 1, 3, 6 and 10 It can be seen that the derivative is excellent in the effect of inhibiting the activity of NFAT5. Therefore, the derivatives according to the present invention can be usefully used as a pharmaceutical composition for the prevention or treatment of NFAT5 activity-related diseases.
<실험예 3> F-κΒ, FATC, CREB, ELK전사활성에 대한 영향평가 본 발명에 따른 화합물들이 NFAT 전사인자 이외의 NF-κΒ, NFATC, CREB, ELK 전사활성에 미치는 영향을 알아보기 위하여 다음과 같이 실험하였다. 구체적으로, NF— κΒ 전사활성은 THPl-Lucia NF-κΒ 리포터세포를 10% FBS-RPMI 배지 (100 ug/ml의 Zeocin 함유)에서 배양하여 측정하였다. 세포를 96-웰플레이트에 2 X 104 세포 /웰의 밀도로 배양하고， LPS 1 g/ml과 화합물을 가하여 24시간 배양한. 뒤 상등액 10 μΐ에 형광 분석 시약 50 μ1¾ 가하여 발광을 측정하였다. Experimental Example 3 Evaluation of Effects on F-κΒ, FATC, CREB, and ELK Transcriptional Activities In order to examine the effects of the compounds according to the present invention on NF-κΒ, NFATC, CREB, and ELK transcriptional activity other than NFAT transcription factors, Experiment was as follows. Specifically, NF-κΒ transcriptional activity was measured by culturing THPl-Lucia NF-κΒ reporter cells in 10% FBS-RPMI medium (containing 100 ug / ml Zeocin). Cells were incubated in a 96-well plate at a density of 2 × 10 4 cells / well and incubated for 24 hours with the addition of 1 g / ml LPS and the compound. Luminescence was measured by adding 50 μ1¾ of a fluorescence assay reagent to 10 μΐ of the supernatant.
또한， NFAT 전사활성 평가는 THPl-XBhie-MD2-CD14 세포를 10% FBS-RPMI 배지 (200 g/ml의 Zeocin, 25 Mg ml≤] G418 함유)에서 배양하여 측정하였다. 먼저 세포를 96-웰플레이트에 2 X 104.세포 /웰의 밀도로 배양하고， LPS 1 (Jg/ml과 화합물을 가하여 24시간 배양한 뒤 상등액 20 μΐ에 secreted embryonic alkalinephosphatase(SEAP) detect ion medium 180 μΐ를 가하여 2시간 배양한 뒤In addition, NFAT transcription activity evaluation was measured by incubating THPl-XBhie-MD2-CD14 cells in 10% FBS-RPMI medium (containing 200 g / ml Zeocin, 25 Mg ml≤] G418). First, the cells were incubated in a 96-well plate at a density of 2 X 10 4 cells / well, incubated for 24 hours with LPS 1 (Jg / ml and the compound added), and secreted embryonic alkaline phosphatase (SEAP) detect ion medium in 20 μΐ of supernatant. Add 180 μΐ and incubate for 2 hours
655 nm에서 흡광도를 측정하였다. Absorbance was measured at 655 nm.
나아가， CREB 및 EL 1 전사활성 평가는 CHO/CREB-luc, HEK293/CREB-luc, HL /ELK1 세포를 이용하여 실시하였다. 세포는 각각 10% FBS-F12 (100 pg/ml의 hygromycin함유)， 10% FBS-DMEM (100 g/ml의 hygromycin함유), 10% FBS-DMEM (200 ug/ml의 G418 함유)에서 배양하여 96-웰플레이트에 1 x 104 세포 /웰의 밀도로 배양하고 하루 동안 더 배양한 후， 10 μΜ의 Forskolin 또는 1 g/ml의 PMA를 24시간 처리한 세포를 분쇄시키고 발광검색시약 25 μ ΐ를 가하여 발광을 측정하였다. 상기 실험결과들을 도 2에 나타내었다. 도 2는 본 발명에 따른 유도체가 NFAT 전사인자 이외의 전사활성에 미치는 영향을 나타낸 그래 ¾로서， 구체적으로 Α는 NF-κΒ, B는 NFATc , C는Furthermore, CREB and EL 1 transcriptional activity was evaluated using CHO / CREB-luc, HEK293 / CREB-luc, and HL / ELK1 cells. Cells were cultured in 10% FBS-F12 (containing 100 pg / ml hygromycin), 10% FBS-DMEM (containing 100 g / ml hygromycin), and 10% FBS-DMEM (containing 200 ug / ml G418). Incubate in a 96-well plate at a density of 1 x 10 4 cells / well and incubate for another day, then 10 μΜ of Forskolin or 1 g / ml Cells treated with PMA for 24 hours were pulverized and luminescence was measured by adding 25 μΐ of a luminescent detection reagent. The experimental results are shown in FIG. 2. Figure 2 is a graph showing the effect of the derivative according to the invention on the transcriptional activity other than NFAT transcription factor, specifically, A is NF-κΒ, B is NFATc, C is
CREB(CHO-Kl) , D는 CREB(HEK293) , E는 ELK에 대한 전사활성의 억제를 나타낸 그래프이다. 도 2에 나타난 바와 같이， 본 발명에 따른 프로토베르베린 유도체는 NFAT5를 1 μΜ에서 80¾>이상 저해하는 것과는 달리 다른 전사인자들에 대한 효과는 미미하게 나타났다. 본 발명에 따른 실시예 1 및 3의 ,유도체는 10 μΜ에서 NF-κΒ 및 NFAT프로모터활성을 약간 저해하는 것을 알 수 있다. CREB (CHO-Kl), D is CREB (HEK293), E is a graph showing the inhibition of transcriptional activity against ELK. As shown in Figure 2, the protoberberine derivatives according to the present invention showed a slight effect on other transcription factors, unlike inhibition of NFAT5 at 1 μM or more than 80¾>. It can be seen that the derivatives of Examples 1 and 3 according to the present invention slightly inhibit NF-κΒ and NFAT promoter activity at 10 μM.
따라서, 본 발명에 따른 프로토베르베린 유도체는 NFAT5의 전사인자 활성에 대하여 선택적인 억제를 보이는 것을 알 수 있다.  Therefore, it can be seen that the protoberberine derivatives according to the present invention show selective inhibition of the transcription factor activity of NFAT5.
<실험예 4> p38 전사활성에 태한명향평가 Experimental Example 4 Preference Evaluation on p38 Transcriptional Activity
본 발명에 따른 유도체와 p38의 인산화를 확인하기 위해서 12웰 플레이트에 RAW264.7 ( 10¾ FBS-RPMI 1640배지)을 1.5 x l05의 밀도로 배양하였다. 실시예 1( 1μΜ)을 한 시간 전 처리하거나 처리하지 않은 뒤 LPS ( lpg/ml )로 자극하여 시간 ( 15， 30， 60분)에 따른 p38 단백질과 활성 상태 p38 단백질 (인산화)의 발현 차이를 웨스턴 블롯 (western Mot )으로 확인하였다. 도 3은 본 발명에 따른 유도체의 p38 단백질과 활성 상태 p38 단백질 (인산화)의 발현 웨스턴 블롯의 이미지이다. 도 3에 나타난 바와 같이, 본 발명에 따른 유도체를 처리한 군과 처리하지 않은 대조군에서 p38 단백질 (인산화) 발현 정도의 차이가 없었다. In order to confirm the phosphorylation of the derivative and p38 according to the present invention, RAW264.7 (10¾ FBS-RPMI 1640 medium) was incubated at a density of 1.5 × 10 5 in a 12-well plate. Example 1 (1 μM) was treated with or without an hour before and then stimulated with LPS (lpg / ml) to determine the difference in expression of p38 protein and active p38 protein (phosphorylation) over time (15, 30, 60 minutes). Confirmed by Western Blot. Figure 3 is an image of the Western blot expressing the p38 protein and active state p38 protein (phosphorylation) of the derivative according to the present invention. As shown in Figure 3, the group treated with the derivative according to the present invention and There was no difference in the degree of p38 protein (phosphorylation) expression in the untreated controls.
<실험예 5> NFAT5의 전사단계 및 핵 이동 조사 Experimental Example 5 Investigation of Transcriptional Steps and Nuclear Migration of NFAT5
본 발명에 따른 유도체가 NFAT5의 mRNA발현에 영향을 미치는지 확인해 보았다.  It was confirmed whether the derivative according to the present invention affects the mRNA expression of NFAT5.
구체적으로， 12웰 플레이트에 R 264.7( 10% FBS-RPMI 1640배지)을 1.5 x 105 세포 /웰의 밀도로 배양한 뒤 하루 동안 안정화를 시켰다. 본 발명에 따른 실시예 1의 화합물을 ΙμΜ 의 농도로 한 시간 전처리 한 후， LPS로 12시간 동안 세포에 자극 시켰다. 12시간 후 세포에서 mRNA를 분리하여 cDNA를 합성한 후， 실시간 중합 효소반웅을 통해서 NFAT5의 mRNA 발현 양을 측정하였다. 또한， 전사 조절 물질인 NFAT5가 세포질에서 핵으로 이동하는 장도를 확인하였다. 6 웰 플레이트에 R 264.7( 10% FBS- PMI 1640 배지)의 6 x 105 세포 /웰의 밀도로 배양한 뒤 ᅳ 화합물 6( 1μΜ)을 한 시간 전처리 하고， LPS( lpg/ml )를 24시간 척리하였다. 자극된 세포에서 세포질과 핵의 단백질 분리를 통해서， NFAT5와 NF-κΒ의 서브유닛 ( subuni t )인 p65의 단백질을 확안하여 도 4에 나타내었다. Specifically, R 264.7 (10% FBS-RPMI 1640 medium) was incubated at a density of 1.5 x 10 5 cells / well in a 12 well plate and stabilized for one day. After the compound of Example 1 according to the present invention was pretreated at a concentration of ΙμΜ for one hour, cells were stimulated with LPS for 12 hours. After 12 hours, mRNA was isolated from cells to synthesize cDNA, and mRNA expression of NFAT5 was measured by real-time polymerase reaction. In addition, we confirmed the longevity of NFAT5, a transcriptional regulator, migrates from the cytoplasm to the nucleus. Incubate in 6 well plates at a density of 6 x 10 5 cells / well of R 264.7 (10% FBS-PMI 1640 medium) and pretreatment with Compound 6 (1μΜ) for 1 hour and LPS (lpg / ml) for 24 hours. Quenched. The protein of p65, a subunit of NFAT5 and NF-κΒ, was identified through FIG.
도 4는 본 발명에 따른 유도체가 전사단계 및 핵 이동에 미치는 영향을 나타낸 그래프이다. 도 4에 나타난 바와 같이, LPS의 처리로 인해 NFAT5의 발현은 세포질로부터 분리한 단백질에서 보다 핵에서 분리한 단백질에서 증가한 것을 알 수 있었고， 본 발명에 따른 실시예 1의 화합물을 전처리하였을 경우 LPS 처리로 인해 증가했던 단백질 양이 감소하는 양상을 보였다/ Figure 4 is a graph showing the effect of the derivative according to the invention on the transcriptional step and nuclear migration. As shown in FIG. 4, the expression of NFAT5 was increased in the protein isolated from the nucleus than in the protein isolated from the cytoplasm due to the treatment of LPS, and the LPS treatment when the compound of Example 1 according to the present invention was pretreated. Decreased protein levels due to
따라서， LPS에 의해 발현이 증가된 NFAT5을 실시예 1의 화합물이 특이적으로 전사체 단계부터 억제시켜 NFAT5의 단백질 발현의 억제를 유도할 수 있다는 것을 알 수 있었다. <실험예 6>고장성에서 반웅하는 인자들에 대한 효과조사 고장성 조건에서 NFAT5가 조절하는 인자들이 실시예 1에 의해서 영향을 미치는지 확인해 보았다. 6웰 플레이트에 Raw264.7 세포를 ( 1OT FBS-RPMI 1640 배지) 1.5 X 105 세포 /웰의 밀도로 배양하여 안정화를 시켰다. 실시예 .1의 화합물 ( ΙμΜ)을 한 시간 전 처리한 후， NaCK45 mM)을 3시간 자극해 준 뒤, 추출한 mRNA에서 cDNA를 합성하여 NFAT5 전사체를 실시간 중합효소 반웅^로 확인하여 도 5에 나타내었다. 도 5의 본 발명에 따른 유도체가 염화나트롬에 의해 증가되는 NFAT5의 전사체 발현에 미치는 영향을 나타낸 그래.프이고， 구체적으로 A는 BGT1 , B는 AR의 전사체에 관한 그래프이다. 도 5에 나타난 바와 같이 NaCl을 처리하였을 때 NFAT5의 전사체인 BGT1 및 AR의 발현이 증가되는 것을 확인할 수 있었고， 이렇게 증가된 상기 전사체는 본 발명에 따른 실시예 1의 화합물로 처리한 경우에도 차이를 보이지 않았다. Therefore, it was found that the compound of Example 1 specifically inhibits NFAT5, which is increased by LPS, from the transcript stage, thereby inducing the inhibition of NFAT5 protein expression. Experimental Example 6 Investigation of Effects on Factors Responding to Trouble In the hypertonic conditions, it was confirmed whether the factors controlled by NFAT5 influenced by Example 1. Raw264.7 cells (1OT FBS-RPMI 1640 medium) were incubated at a density of 1.5 × 10 5 cells / well in 6-well plates to stabilize. After treating the compound of Example 1 (ΙμΜ) an hour ago, NaCK45 mM) was stimulated for 3 hours, cDNA was synthesized from the extracted mRNA, and the NFAT5 transcript was confirmed by real-time polymerase reaction. Indicated. 5 is a graph showing the effect of the derivative according to the present invention on the transcript expression of NFAT5 increased by sodium chloride, specifically A is BGT1, B is a graph of the transcript of AR. As shown in FIG. 5, the expression of BGT1 and AR, which are transcripts of NFAT5, was increased when NaCl was treated, and the increased transcripts were different even when treated with the compound of Example 1 according to the present invention. Did not look.
<실험예 7> C0X.-효소저해_효과 -조사 Experimental Example 7 C0X.-Enzyme Inhibition Effect
. C0X 효소 저해는 대표적인 항염증 기전으로서 선별된 화합물이 C0X1 및 C0X2 효소에 대한 저해효과를 보이는지 확인하기 위하여 본 발명에 따론 실시예 1의 저해도를 베르베린과 비교하여 조사하였다. . Inhibition of C0X enzyme is a representative anti-inflammatory mechanism. In order to confirm whether the selected compound exhibits inhibitory effect on C0X1 and C0X2 enzymes, the inhibition degree of Example 1 was compared with berberine according to the present invention.
구체적으로, Cayman사의 COX assay ki t (Cat# 760111)를 사용하였다. 완충액 69 μ ΐ와 Heme 5 μ ΐ， 효소 5 μ ΐ를 실시예 1 μ ΐ와 섞은 후 상은에서 5분간 미리 반웅시키고 기질 10 μ ΐ와 아라키도닉산 10 μΐ를 넣어 빛이 없는 상태에서 10분간 반웅을 시킨 후， 590 nm에서 흡광을 측정하여， 그 결과를 하기 표 4 및 표 5에 나타내었다. 【표 4] Specifically, Cayman's COX assay kit (Cat # 760111) was used. After mixing 69 μΐ, Heme 5μΐ, and 5μΐ, enzyme with 1μΐ in Example, react the mixture for 5 minutes in the silver and add 10μΐ of substrate and 10μΐ of arachidonic acid. After measuring the absorbance at 590 nm, the results are shown in Tables 4 and 5 below. [Table 4]
Figure imgf000048_0001
Figure imgf000048_0001
【표 5] [Table 5]
Figure imgf000048_0002
Figure imgf000048_0002
상기 표 4및 5에 나타난바와 같이， 본 발명에 따른 실시예 1은 C0X1과 C0X2 효소저해효과가 미미함을 알 수 있다.  As shown in Tables 4 and 5, Example 1 according to the present invention can be seen that the C0X1 and C0X2 enzyme inhibitory effect is insignificant.
<실험예 8>세포독성조사 Experimental Example 8 Cytotoxicity Study
본 발명에 따른 유도체의 세포 독성을 알아보기 위하여， 구체적으로 5종의' 세포 (VERO: African green monkey kidney cell line, HFL—l: human embryonic lung cell line, L929: NCTC clone 929, mouse fibroblast cell line, NIH 3T3: mouse embryonic fibroblast cell line, CHO-Kl： Chinese hamster ovary cell line)를 1 x 104개 /웰의 밀도로 96-웰플레이트에 분주하여 화합물을 24시간동안 처리하고 수용성의 WST-8이라는 테트라아조늄염을 가한 후 살아있는 세포에 존재하는 탈수소효소에 의해 분해되어 오렌지색의 포르마잔 염료를 흡광에서 읽은 후 비.선형회귀분석 (nonlinear regression)을 하고 50% 성장저해농도 (Growth Inhibition)를 결정하여， 그 결과를 표 6에 나타내었다. In order to examine the cytotoxicity of the derivatives according to the present invention, five kinds of cells (VERO: African green monkey kidney cell line, HFL—l: human Embryonic lung cell line, L929: NCTC clone 929, mouse fibroblast cell line, NIH 3T3: mouse embryonic fibroblast cell line, CHO-Kl: Chinese hamster ovary cell line) 96-well plate at a density of 1 x 10 4 / well The compound was treated for 24 hours, treated with a water-soluble tetraazonium salt called WST-8, decomposed by a dehydrogenase present in living cells, and the orange formazan dye was read by absorption, followed by non-linear regression analysis (nonlinear). regression) and 50% growth inhibition (Growth Inhibition) was determined, and the results are shown in Table 6.
【표 6】 Table 6
Figure imgf000049_0001
Figure imgf000049_0001
표 6에 나타난 바와 같이， 본 발명에 따른 유도체는 10 μΜ 이하에서 세포에 미치는 독성은 미미한 수준이었다.  As shown in Table 6, the derivative according to the present invention had a minimal toxicity to cells at 10 μΜ or less.
<실험예 9> 염증성사이토카인의 분비억제효과조사 Experimental Example 9 Inhibition of Inflammatory Cytokine Secretion
본 발명에 따른 유도체가 NFAT5의 표적 유전자인 GM-CSF2, MCP-1 및 IL-6,의 발현에 영향을 미치는지 보기 위하여， Real time PCR, ELISA, Western blot을 수행하였다. Raw264.7 세포를 12-웰플레이트에 1.5 x 106 세포 /웰의 밀도로 10%FBS-RPMI배지에 배양을 하고 다음날 실시예 1의 화합물을 ΙμΜ로 한 시간 전처리 후에 LPS (lpg/ml)를 6시간 동안 처리하였다. 6시간 처리 후에 웰의 상층액을 따서 GM-CSF, MCP-1 및 IL-6을 ELISA로 측정하여 도 6에 나타내었다. 도 6은 본 발명에 따른 유도체가 염증성 사이토카인 억제 효과를 나타낸 그래프로서， 구체적으로 A는 GM-CSF2 , B는 MCP-1 및 C는 IL-6를 나타낸다. Real time PCR, ELISA and Western blot were performed to determine whether the derivatives of the present invention affect the expression of GM-CSF2, MCP-1 and IL-6, which are target genes of NFAT5. Raw264.7 cells were incubated in a 10% FBS-RPMI medium at a density of 1.5 x 10 6 cells / well in a 12-well plate, and the LPS (lpg / ml) was added the following day after pretreatment of the compound of Example 1 with ΙμΜ. Treatment was for 6 hours. After 6 hours of treatment, the supernatants of the wells were determined by ELISA for GM-CSF, MCP-1, and IL-6, and are shown in FIG. 6. 6 is a graph showing the inflammatory cytokine inhibitory effect of the derivatives according to the present invention. Specifically, A represents GM-CSF2, B represents MCP-1, and C represents IL-6.
도 6에 나타낸 바와 같이， LPS자극으로 인해 증가된 GM-CSF2 , MCP-1 및 IL-6의 단백질 발현이 본 발명에 따른 유도체의 처리로 인해 현저하게 감소하는 것을 확인할 수 있었다. As shown in Figure 6, it was confirmed that the protein expression of GM-CSF2, MCP-1 and IL-6 increased due to LPS stimulation is significantly reduced due to the treatment of the derivative according to the present invention.
<실험예 10>콜라겐으로유도된 류마티스관절염 마우스모델시험 Experimental Example 10 Rheumatoid Arthritis Mouse Model Test Induced by Collagen
본 발명에 따론 실시예 1의 화합물이 생체 내 ( / vivo)상에서 관절염의 유도를 억제시킬 수 있는지 조사하기 위하여, 하기와 같은 실험을 수행하였다. 구체적으로， 그 8주령의 수컷 DBA/1 마우스 (Jackson Laborator i es , Bar Harbor , ME)를 bovine 타입 I I 콜라겐 (CI I : Chondrex , Redmond , WA)으로 면역반응을 유도한 뒤， 3주 후부터 매일 5주 동안 일주일에 3번 씩 실시예 1( 15 mg/kg , 60 mg/kg)을 경구투여 하였다. 다음과 같은 관절염 증상 정도를 기준으로 (0점은 정상, 1점=미약함， 2점=약함， 3점=보통， 4점=심함) 점수를 측정하였다. 실시예 1을 주입하는 5주 동안 마우스 다리의 관절염 정도를 평가하여 도 7에 나타냈다.  In order to investigate whether the compound of Example 1 according to the present invention can inhibit the induction of arthritis in vivo (/ vivo), the following experiment was performed. Specifically, the 8-week-old male DBA / 1 mouse (Jackson Laboratories, Bar Harbor, ME) was induced an immune response with bovine type II collagen (CI I: Chondrex, Redmond, WA), followed by daily Example 1 (15 mg / kg, 60 mg / kg) was orally administered three times a week for 5 weeks. Based on the degree of arthritis symptoms as follows (0 points are normal, 1 point = weak, 2 points = weak, 3 points = normal, 4 points = severe). The degree of arthritis of the mouse leg was evaluated during 5 weeks of injecting Example 1, and is shown in FIG. 7.
[수학식 2] mi le scare' ¾ ^mn ci2|≤j @가¾수 si mm ca& [Equation 2] mi le scare ' ¾ ^ mn ci2 | ≤ j @ ¾ number si mm ca &
時우 수 그 결과， 도 7에 나타난 바와 같이， 음성 대조군과 비교하면 본 발명에 따른 실시예 1의 화합물을 처리한 군의 마우스 다리의 관절 부위가 육안으로 관찰하였을 경우 관절염의 증상이 완화된 것올 확인할 수 있었고， 조직학적 염색을 통해서도 관절염 증상이 월등히 회복된 것을 확인할 수 있었다.  As a result, as shown in FIG. 7, the symptoms of arthritis were alleviated when the joints of the mouse legs of the group treated with the compound of Example 1 of the present invention were visually observed as compared with the negative control group. In addition, histological staining showed that the symptoms of arthritis recovered significantly.
따라서， 본 발명에 따른 유도체는 관절염 질환을 예방 또는 치료용 약학적 조성물로 유용할 수 있다는 것을 알 수 있다. Therefore, the derivative according to the present invention for preventing or treating arthritis diseases It will be appreciated that it may be useful as a pharmaceutical composition.
<실험예 11> 콜라겐으로 유도된 류마티스관절염 마우스에서 염증성 사이토카인의 발현 확인 Experimental Example 11 Expression of Inflammatory Cytokines in Collagen-Induced Rheumatoid Arthritis Mice
류마티스관절염 마우스에서 염증성사이토카인의 발현 여부를 측정하기 위하여， 하기와 같은 실험을 수행하였다ᅳ  In order to measure the expression of inflammatory cytokines in rheumatoid arthritis mice, the following experiment was performed.
구체적으로, 상기 <실험예 10>에서 사용한 콜라겐으로 관절염이 유도된 마우스를 이용하였다. 상기 마우스의 비장 (spleen)에서 세포를 추출한 뒤， 12 웰 플레이트에 비장 세포를 배양하여 안정화를 시켰다. LPS로 12시간 동안 자 극을 시켜준 후， 염증성사이토카인 (TNF- α IL-6)의 발현을 ELISA로 측정을 하 였다.  Specifically, arthritis-induced mice were used as collagen used in Experimental Example 10. After the cells were extracted from the spleen of the mouse, the cells were stabilized by culturing the spleen cells in a 12 well plate. After stimulating with LPS for 12 hours, expression of inflammatory cytokines (TNF-α IL-6) was measured by ELISA.
그 결과， 도 8에 나타낸 바와 같이 관절염이 유도된 마우스의 바장 세 포를 LPS로 유도하였을 때， 염증성 사이토카인 (TNF- a , IL-6)이 배지 (medium) 를 처리한 군에 비해 증가하는 것을 나타냈다. TNF- a는 530 (pg/ml ) , IL-6는 400 (pg/ml )를 발현시킴을 확인하였다. 그러나, 실시예 1의 화합물을 각각 15， 60 (mg/kg) 처리했을 때 TNF- a 및 IL-6의 발현의 양이 감소하는 것을 확인하였 다.  As a result, as shown in FIG. 8, when Ljang cells of arthritis-induced mice were induced with LPS, inflammatory cytokines (TNF-a, IL-6) were increased compared to the group treated with medium. Indicated that. It was confirmed that TNF-a expresses 530 (pg / ml) and IL-6 expresses 400 (pg / ml). However, when the compounds of Example 1 were treated with 15, 60 (mg / kg), respectively, it was confirmed that the amount of expression of TNF-a and IL-6 decreased.
<실험예 12> 콜라겐으로 유도된 류마티스관절염 -마우스에서 NFAT5의 발현 확인 Experimental Example 12 Expression of NFAT5 in Collagen-Induced Rheumatoid Arthritis-Mouse
류마티스관절염 마우스에서 NFAT 단백질의 발현 여부를 측정하기 위하 여， 하기와 같은 실험을 수행하였다. 구체적으로， 상기 <실험예 11>의 동일한 방법으로 콜라겐에 의해서 관절염이 유도된 마우스의 비장세포에서 LPS를 자극을 시켜준 후， 세포에서 단백질을 추출하여， NFAT5의 단빡질을 확인하였다.  In order to measure the expression of NFAT protein in rheumatoid arthritis mice, the following experiment was performed. Specifically, after stimulating LPS from splenocytes of arthritis induced by collagen-induced mouse splenocytes in the same manner as in <Experimental Example 11>, the protein was extracted from the cells, and NFAT5 was confirmed.
그 결과， 도 9에 나타낸 바와 같이 CIA Col lagen Induced Arthr i st i s)에서 LPS로 유도된 군에서 NFAT5의 발현이 증가 되었으나 실시예 1 의 화합물을 처리하였을 때 증가되었던 NFAT5의 발현량이 감소띔을 확인하였다. As a result, as shown in FIG. 9, the expression of NFAT5 was increased in the LPS-induced group in CIA Col lagen Induced Arthr i st is), but the amount of NFAT5 expression increased when the compound of Example 1 was treated was decreased. Confirmed.
<실험예 13>간마이크로좀에서의 대사안정성 및 약물동태 조사 Experimental Example 13 Investigation of Metabolic Stability and Pharmacokinetics in Liver Microsomes
실시예 . 1 및 실시예 3의 화합물의 대사안정성 및 약물동태를 조사하기 위하여， 하기와 같은 실험을 수행하였다.  EXAMPLE In order to investigate the metabolic stability and pharmacokinetics of the compounds of Example 1 and Example 3, the following experiment was performed.
구체적으로, 각각의 화합물을 래트 (rat)와 인간의 간마이크로좀 분획과 함께 30분 동안 배양한 후에 남아있는 화합물의 양을 측정하여 표 7을 얻었다. 또한 래트의 정맥 (iv) 및 경구 (po)로 5 mg/kg투여한 수 약물동태프로파일을 도 10a 및 10b와 같이 얻었다. Specifically, Table 7 was obtained by measuring the amount of the compound remaining after each compound was incubated for 30 minutes with rat and human liver microsome fractions. In addition, male pharmacokinetic profiles administered at 5 mg / kg by intravenous (i v ) and oral (po) rats were obtained as shown in FIGS. 10A and 10B.
그 결과， 표 7, 도 10a 및 10b에 나타낸 바와 같이 8-옥소 유도체의 물성개선으로 경구 흡수율이 프로토베르베린 유도체에 비해 현저히 증가됨을 확인할 수 있었다.  As a result, as shown in Table 7, 10a and 10b it was confirmed that the oral absorption rate is significantly increased compared to the protoberberine derivatives by physical properties of the 8-oxo derivatives.
【표 7】  Table 7
Figure imgf000052_0001
Figure imgf000052_0001
<실험예 14> 화합불에 의한 인간 단핵혈액세포의 염증성사이토카인 분 비억제효과 인간 단핵혈액세포에서 화합물이 염증성사이토카인 분비를 억제하는지 를 조사하기 위하여 하가와 같은 실험을수행하였다. Experimental Example 14 Inhibitory Effect of Inflammatory Cytokine Secretion on Human Mononuclear Blood Cells by Incompatibility In order to investigate whether the compound inhibits the secretion of inflammatory cytokines in human mononuclear blood cells, the same experiment was performed.
구체적으로, 건강한 인간의 혈액에서 분리한 단핵세포 (PBMC)에 Specifically, monocytes (PBMC) isolated from healthy human blood
LPS 1 j«g/ml)를 처리하여 유도되는 염증성사이토카인의 발현증가를 화합물이 얼마나 잘 억제하는지 화합물을 1 g/ml로 함께 처리하고 6시간동안 배양한 후 세포의 RNA를 분리하여 IL-6와 TNF-α 의 유전자 전사량을 실시간 PCR 로 분석하여 도. 11에 표시하였다 (NC는 음성대조군， PC는 양성대조군임). . - 그 결과, 도 11에 나타낸 바와 같이 NFAT5 저해제인 실시예 1의 화합 물은 사이토카인의 발현을 억제하는 것을 확인하였다. How well the compound inhibits the expression of inflammatory cytokines induced by treatment with LPS 1 j «g / ml) and treated with 1 g / ml and incubated for 6 hours After the RNA of the cell is isolated and the amount of gene transcription of IL-6 and TNF-α is analyzed by real-time PCR. 11 is indicated (NC is negative control, PC is positive control). . As a result, as shown in FIG. 11, it was confirmed that the compound of Example 1, which is an NFAT5 inhibitor, inhibits the expression of cytokines.
<실험예 15> 마우스 T 세포의 Thl7 세포로의 분화 억제능 확인 실시예 1의 화합물이 Thl7 세포 분화를 억제하는지를 알아보기 위하여, 하기와같은 실험을 수행하였다. 구체적으로, 형질전환마우스 (Rag2 KO/OVA-TCRtg)의 원천 (naiVe) T 세포를 T 헬퍼 (helper) 17 세포로의 분화조건 (APC세포와 함께 ILᅳ 6, ILᅳ 1 (3， TGF- β를 처리)에서 3일간쎄양하였다. Thl7 분화 억제제로 알려져 있는 베 르베린과 비교하여 NFAT5 저해제가 Thl7으로의 분화 억제 정도를 분비되는 사이토카인 단백질 정량으로 조사하였다. 또한, 동일한 조건에서 세포생존율을 CCK— 8으로 측정하였다. 그 결과， 도 12에 나타낸 바와 같이 실시예 1의 화합물은 베르베린과 유사한 수준의 Thl7 분화억제능을 나타냄을 확인하였다. Experimental Example 15 Confirmation of Inhibition of Differentiation of Mouse T Cells into Thl7 Cells To determine whether the compound of Example 1 inhibited Thl7 cell differentiation, the following experiment was performed. Specifically, differentiation conditions of naiVe T cells of transgenic mice (Rag2 KO / OVA-TCRtg) into T helper 17 cells (in combination with APC cells, IL ᅳ 6, IL ᅳ 1 (3, TGF- β treatment) was examined for three days.The degree of inhibition of differentiation of NFAT5 inhibitors into Thl7 compared to berberine, known as a Thl7 differentiation inhibitor, was investigated by quantitating cytokine protein secreted under the same conditions. It was measured by CCK-8. As a result, it was confirmed that the compound of Example 1 exhibits a similar level of Thl7 differentiation inhibitory ability to berberine as shown in FIG.
<제제예 1> 약학적 제제의 제조 Preparation Example 1 Preparation of Pharmaceutical Formulation
<1-1>산제의 제조 <1-1> Preparation of powder
화학식 1로 표시되는 유도체 유당 1 g 상기의 성분을 흔합한 후， 기밀포에 충진하여 산제를 제조하였다.  1 g of the derivative lactose represented by the formula (1) After mixing the above components, and filled into an airtight cloth to prepare a powder.
<1-2>정제의 제조 <1-2> Preparation of the tablet
화학식 1로 표시되는 유도체 100 nig 옥수수전분 100 nig 유 당 100 nig 스테아린산 마그네슘 2 nig 상기의 성분을 흔합한 후， 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다. 100 nig of derivative represented by formula (1) Corn starch 100 nig Lactose 100 nig Magnesium stearate 2 nig After the above components were mixed, tablets were prepared by tableting according to a conventional method for preparing tablets.
<1-3>캡술제의 제조 <1-3> Preparation of the capsule
화학식 1로 표시되는 유도체 100 nig 옥수수전분 100 rag 유 당 100 mg 스테아린산 마그네슘 2 nig  100 nig of corn starch 100 rag Lactose 100 mg magnesium stearate 2 nig
상기의 성분을 흔합한 후， 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡술에 충전하여 캡슐제를 제조하였다.  After mixing the above components, the capsule was prepared by filling in gelatin capsule according to a conventional method for preparing capsules.
<1-4>주사액제의 제조 <1-4> Preparation of the injection solution
화학식 1로 표시되는 유도체 10 ug/me  Derivative represented by Formula 1 10 ug / me
묽은 염산 BP 3.5로 될 때까지 주사용 염화나트륨 BP 최대 1  Sodium chloride BP for injection up to 1 in dilute hydrochloric acid BP 3.5
적당한 용적의 주사용 염화나트륨 BP 중에 본 발명에 따른 화합물을 용해시키고， 생성된 용액의 pH를 묽은 염산 BP를 사용하여 3.5로 조절하고， 주사용 염화나트륨 BP를 사용하여 용적을 조절하고 충분히 흔합하였다. 용액을 투명 유리로 된 5 타입 I 앰플 중에 충전시키고， 유리를 용해시킴으로써 공기의 상부 격자하에 봉입시키고， 120 0C에서 15 분 이상 오토클래이브시켜 살균하여 주사액제를 제조하였다. The compound according to the invention was dissolved in an appropriate volume of sodium chloride BP for injection, the pH of the resulting solution was adjusted to 3.5 using dilute hydrochloric acid BP, and the volume was adjusted and sufficiently mixed with injection of sodium chloride BP. The solution was filled into a 5 type I ampoule of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 0 C for at least 15 minutes to prepare an injection solution.
<제조예 2> 건강기능식품의 제조 Preparation Example 2 Preparation of Health Functional Food
화학식 1로 표시되는 유도체 500 ng 비타민 흔합물 적량 비타민 A 아세테이트 70 μ& 500 ng of derivative represented by formula (1) Vitamin Mixes Proper Vitamin A Acetate 70 μ &
비타민 E 1.0 nig  Vitamin E 1.0 nig
비타민 , 0. 13 nig  Vitamin, 0. 13 nig
비타민 B2 0. 15 nig  Vitamin B2 0. 15 nig
비타민 B6 0.5 nig  Vitamin B6 0.5 nig
비타민 B12 0.2 fig  Vitamin B12 0.2 fig
비타민 C 10 mg  Vitamin C 10 mg
비오틴 10 β  Biotin 10 β
니코틴산아미드 1.7 nig  Nicotinamide 1.7 nig
엽산 50 .nig  Folic acid 50 .nig
'판토텐산 칼슘 0.5 nig 'Calcium pantothenate 0.5 nig
무기질 흔합물 적량  Mineral Mixtures Proper
황산제 1철 1.75 nig  Ferrous Sulfate 1.75 nig
산화아연 0.82 nig  Zinc Oxide 0.82 nig
탄산마그네슘 25.3 nig  Magnesium carbonate 25.3 nig
제 1인산칼륨 15 nig - 제 2인산칼슘 55 nig  15 nig potassium monophosphate-55 nig dicalcium phosphate
구연산칼륨 90 nig  Potassium Citrate 90 nig
탄산칼슴 100 nig  100 grams of carbonic acid deer
염화마그네슘 24.8 nig  Magnesium chloride 24.8 nig
상기의 비타민 및 미네랄 흔합물의 조성비는 비교적 건  The composition ratio of the vitamin and mineral mixture mentioned above is relatively dry
적합한 성분을 바람직한 실시예로 흔합 조성하였지만， 그 배합비를 임의로 변형 실시하여도 무방하며， 통상의 건강기능식품 제조방법에 따라 상기의 성분올 흔합한 다음, 과립을 제조하고， 통상의 방법에 따라 건강가능싀품 조성물 제조에 사용할 수 있다. Although a suitable composition is mixed in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method, and then granules are prepared, It can be used to prepare possible product compositions.
<제제예 3> 건강음료의 제조 화학식 1로 표시되는 유도체 500 ng 구연산 1000 nig Preparation Example 3 Preparation of Health Beverage Derivative represented by the formula (1) 500 ng citric acid 1000 nig
올리고당 100 g  100 g oligosaccharides
매실농축액 2 g  Plum concentrate 2 g
타우린 . 1 g  Taurine. 1 g
정제수를 가하여 전체 900  Purified water is added to the total 900
통상의 건강 음료 제조방법에 따라 상기의 성분을 흔합한 다음， 약 1시간 동안 850C에서 교반 가열한 후， 만들어진 용액을 여과하여 멸균된 용기에 취득하여 밀봉 멸균한 뒤 넁장 보관한 다음 건강 음료 조성물 제조에 사용하였다. After mixing the above ingredients according to the usual method of preparing a healthy beverage, and stirring and heating at 85 0 C for about 1 hour, the resulting solution is filtered and obtained in a sterilized container, sealed and sterilized and then stored in a steamed beverage Used to prepare the composition.
상기 조성비는 비교적 기호 음료에 적합한 성분을 바람직한 실시예로 흔합 조성하였지만 수요계층이나, 수요국가， 사용용도 등 지역적， 민족적 기호 도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.  Although the composition ratio is a composition that is relatively suitable for a preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Claims

【청구의 범위】 【Scope of Claim】
【청구항 1】 【Claim 1】
하기 화학식 1로 표시되는 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염 : 8-oxoprotoberberine derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
Figure imgf000057_0001
Figure imgf000057_0001
(상기 화학식 1에서， (In Formula 1 above,
A는 d-6알킬렌， -C(=0)_ 또는 -NHC( ))-이고; A is d- 6 alkylene, -C(=0)_ or -NHC())-;
R은 하이드록시， 아미노， 직쇄 또는 측쇄의 Cl-6알콕시， C6— 12아릴 또는 질소 (N) , 산소 (0) 및 황 (S)으로 이루어진 군으로부터 선택되는 동일 또는 상이한 헤테로 원자를 1종 이상 포함하는 5내지 8원자 단일 고리 또는 8내지 11 원자 이중 고리의 해테로아릴이고， 여기서， 상기 아릴 또는 헤테로아릴은 비치환 또는 할로겐 및 직쇄 또는 측쇄의 Ci— 6알킬 또는 d-6알콕시로 이루어지는 군으로부터 1종 이상으로 치환된다) . R is hydroxy, amino, straight or branched C l -6 alkoxy, C 6 — 12 aryl, or the same or different heteroatom selected from the group consisting of nitrogen (N), oxygen (0) and sulfur (S). It is a heteroaryl of a 5 to 8-membered single ring or an 8 to 11-membered double ring containing more than one species, wherein the aryl or heteroaryl is unsubstituted or halogen and straight or branched Ci- 6 alkyl or d-6 alkoxy. is substituted with one or more types from the group consisting of).
[청구항 2】 [Claim 2]
제 1항에 있어서， In clause 1,
상기 A는 d-4알킬렌， -C(=0)- 또는 -NHC(=0)—인 것을 특징으로 하는 8-옥소프로토쎄르베린 유도체 . The A is an 8-oxoprotocerberine derivative, characterized in that d- 4 alkylene, -C (=0)-, or -NHC (=0)—.
[청구항 3】 [Claim 3]
제 1항에 있어서， In clause 1,
상기 A는 d-2알킬렌， -C(O)- 또는 -NHC(=0)-인 것을 특징으로 하는 -옥소프로토베르베린 유도체. The A is an -oxoprotoberberine derivative, characterized in that d- 2 alkylene, -C(O)-, or -NHC(=0)-.
【청구항 4】 【Claim 4】
제 1항에 있어서， In clause 1,
R은 하이드록시， 아미노, 직쇄 또는 측쇄의 Cl-4알콕시， C6-10아릴 또는 질소 (N) , 산소 (0) 및 황 (S)으로 이루어진 군으로부터 선택되는 동일 또는 상이한 해테로 원자를 1종 이상 포함하는 5내지 8원자 단일 고리 또는 8내지 11 원자 이중 고리의 헤테로아릴이고， 여기서， 상기 아릴 또는 헤테로아릴은 비치환 또는 할로겐 및 직쇄 또는 측쇄의 d-6알킬 또는 d-6알콕시로 이루어지는 군으로부터 1종 이상으로 치환되는 것을 특징으로 하는 8-옥소프로토베르베린 유도체. - R is hydroxy, amino, straight or branched Cl - 4 alkoxy, C 6-10 aryl or 1 same or different heteroatom selected from the group consisting of nitrogen (N), oxygen (0) and sulfur (S). It is a heteroaryl of a 5 to 8-membered single ring or an 8 to 11-membered double ring containing at least one species, wherein the aryl or heteroaryl is unsubstituted or halogen and straight or branched d- 6 alkyl or d- 6 alkoxy. An 8-oxoprotoberberine derivative characterized by being substituted with one or more types from the group. -
【청구항 5】 【Claim 5】
제 1항에 있어서， In clause 1,
상기 R은 하이드록시， 아미노， 직쇄 또는 측쇄의 d-2알콕시， 페닐, 피리디닐， 티아졸릴 및 벤조이미다졸릴로 이루어진 군으로부터 선택되는 1종이고, 여기서，상기 페닐，피리디닐，티아졸릴 또는 벤조이미다졸릴은 비치환 또는 할로겐 및 직쇄 또는 측쇄의 d-4알킬 또는 d-4알콕시로 이루어지는 군으로부터 1종 이상으로 치환되는 것을 특징으로 하는 8-옥소프로토베르베린 유도체. The R is hydroxy, amino, linear or branched d -2 alkoxy, phenyl, pyridinyl, thiazolyl, and benzoimidazolyl, wherein the phenyl, pyridinyl, thiazolyl or Benzoimidazolyl is an 8-oxoprotoberberine derivative characterized in that it is unsubstituted or substituted with one or more types from the group consisting of halogen and straight or branched d- 4 alkyl or d- 4 alkoxy.
【청구항 6】 【Claim 6】
제 1항에 있어서， In clause 1,
상기 A가 ( 4알킬렌인 경우， If A is ( 4 alkylene),
R은 비치환 또는 할로겐, 메틸 및 메록시로 이루어진 군으로부터 선택되는 1종 이상의 치환기로 치환된 페닐， 피리디닐, 티아졸릴 및 벤조이미다졸릴로 이루어진 군으로부터 선택되는 1종인 것을 특징으로 하는 8-옥소프로토베르베린 유도체. ~ R is phenyl, unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, methyl and meroxy, pyridinyl, thiazolyl and benzoimidazolyl, characterized in that 8- Oxoprotoberberine derivative. ~
【청구항 7] 계 1항에 있어서， [Claim 7] In paragraph 1,
상기 A가 -C(=0)-인 경우， If A is -C(=0)-,
R은 하이드록시， 아미노， 직쇄 또는 측쇄의 Ci-2알콕시 또는 비치환 또는 할로겐으로 하나 이상 치환된 페닐인 것을 특징으로 하는 8-옥소프로토베르베린 유도체. R is hydroxy, amino, straight or branched Ci-2 alkoxy or phenyl unsubstituted or substituted with one or more halogens. 8-oxoprotoberberine derivative.
【청구항 8】 【Claim 8】
제 1항에 있어서， In clause 1,
상기 A가 -NHC(=0)ᅳ인 경우， If A is -NHC(=0)ᅳ,
R은 비치환 또는 할로겐으로 하나 이상 치환된 페닐인 것을 특징으로 하는 8-옥소프로토베르베린 유도체. R is an 8-oxoprotoberberine derivative, characterized in that phenyl is unsubstituted or substituted with one or more halogens.
【청구항 9】 【Claim 9】
저 U항에 있어서， In that U port,
상기 8-옥소프로토베르베린 유도체는: The 8-oxoprotoberberine derivative is:
1) One)
13-(2-플루오로벤질 )-9，10-디메록시-511-[1，3]디옥솔로[4，5- 이소퀴놀리노[3，2 -a]이소퀴놀린 -8(6H)_은; 13-(2-fluorobenzyl)-9,10-dimeroxy-511-[1,3]dioxolo[4,5-isoquinolino[3,2-a]isoquinoline-8(6H)_is ;
2) 2)
13-(2，4-디폴루오로벤질) -9，10-디메톡시-511-[1,3]디옥솔로[4，5 ]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온; 13-(2,4-dipoluorobenzyl)-9,10-dimethoxy-511-[1,3]dioxolo[4,5]isoquinolino [3,2-a]isoquinoline-8(6H )-on;
3) 3)
13-(2， 6-디€루오로벤질 )-9， 10-디메톡시 -5H- [ 1， 3]디옥솔로 [4， 5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온; 13-(2, 6-di€luorobenzyl)-9, 10-dimethoxy -5H- [1, 3] dioxolo [4, 5-g] isoquinolino [3,2-a] isoquinoline - 8(6H)-on;
4) 4)
9，1으디메록시 -13-(2-메틸벤질) -5,6ᅳ디하이드로-[1，3]디옥솔로[4,5- ]이소퀴놀 리노 [3,2-a]이소퀴놀린 -8(6H)—은; 9,1dimeroxy -13-(2-methylbenzyl) -5,6ᅳdihydro-[1,3]dioxolo[4,5-]isoquinol lino [3,2-a]isoquinoline -8( 6H)—silver;
5) 9，10ᅳ디메록시-13-(2-메톡시벤질)-5，6-디하이드로-[1，3]디옥솔로[4，5- 이소퀴 놀리노 [3,2-a]이소퀴놀린 -8(6H)-온; 5) 9,10-dimeroxy-13-(2-methoxybenzyl)-5,6-dihydro-[1,3]dioxolo[4,5-isoquinolino [3,2-a]isoquinoline - 8(6H)-on;
6) 6)
9, 10-디메록시— 13— (피리딘 -2—일메틸 )-5,6-디하이드로 -[1,3]디옥솔로 [4,5-g]이 소퀴놀리노 [3,2— a]이소퀴놀린 -8(6H)-온; 9, 10-dimeroxy— 13— (pyridin-2—ylmethyl)-5,6-dihydro-[1,3]dioxolo [4,5-g] is soquinolino [3,2— a] Isoquinoline-8(6H)-one;
7) 7)
13- (( 1H-벤조 [d]이미다졸— 2-일 )메틸) -9， 10-디메록시 -5， 6-디하이드로- [ 1， 3]디옥 솔로 [4,5-g]이소퀴놀리노 [3,2-a]이소퀴놀린 -8(6H)-온; 13- (( 1H-benzo [d]imidazol— 2-yl)methyl) -9，10-dimeroxy-5，6-dihydro- [1，3]dioxolo [4,5-g]isoquinol Lino [3,2-a]isoquinoline-8(6H)-one;
8) 8)
13-( (2-클로로티아졸 -5-일 )메틸 )ᅳ9， 10-디메록시 -5， 6-디하이드로- [ 1， 3]디옥솔로 [4，5ᅳ^이소퀴놀리노[3.2-3]이소퀴놀린-8(611)-온； 13-( (2-chlorothiazol-5-yl)methyl)ᅳ9，10-dimeroxy-5，6-dihydro- [1，3]dioxolo [4，5ᅳ^isoquinolino[3.2- 3] Isoquinoline-8(611)-one;
9) 9)
13-(2-플루오로벤조일) -9，10-디메록시-511-[1，3]디옥솔로[4，5-8]이소퀴놀리노[3 ,2-a]이소퀴놀린 -8(6H)-온 13-(2-fluorobenzoyl)-9,10-dimeroxy-511-[1,3]dioxolo[ 4,5-8 ]isoquinolino[3,2-a]isoquinoline-8(6H) -on
- 10) - 10)
13-에틸 -9， 10-디메특시 -8-옥소 -6， 8-디하이드로 -5H-[ 1 , 3]디옥솔로 [4， 5-g]이소퀴 놀리노 [3， 2-a]이소퀴놀린 -13-카르복실레이트； 13-ethyl-9，10-dimethoxy-8-oxo-6，8-dihydro-5H-[1, 3]dioxolo [4， 5-g]isoquinolino [3， 2-a] Isoquinoline -13-carboxylate;
11) 11)
9， 10-디메톡시 -8-옥소 -6， 8-디하이드로 -5H-[ 1， 3]디옥솔로 [4 , 5-g]이소퀴놀리노 [3 ,2-a]이소퀴놀린 -13-카르복실산; 9, 10-dimethoxy -8-oxo -6, 8-dihydro -5H-[ 1, 3] dioxolo [4, 5-g] isoquinolino [3, 2-a] isoquinoline -13-car boxylic acid;
12) 12)
9， 10-디메톡시 -8-옥소 -6， 8一디하이드로 -5H-[ 1， 3]디옥솔로 [4 , 5-g]이소퀴놀리노 [3 ,2-a]이소퀴놀린 -13-카르복사마이드; 및 9, 10-dimethoxy -8-oxo -6, 8 1 dihydro -5H-[ 1, 3] dioxolo [4, 5-g] isoquinolino [3, 2-a] isoquinoline -13-car boxamid; and
13) 13)
N-(2-플루오로페닐 )-9, N-(2-fluorophenyl)-9,
10-디메톡시 -8ᅳ옥소 -6,8-디하이드로 -5H-[ 1,3]디옥솔로 [4 ， 5-g]이소퀴놀리노 [3， 2-a]이소퀴놀린 -13-카르복사마이드로 이루어진 군으로부터 선택되는 1종인 것을 특징으로 하는 8-옥소프로토베르베린 유도체. 【청구항 10】 10-dimethoxy -8-oxo -6,8-dihydro -5H-[ 1,3] dioxolo [4, 5-g] isoquinolino [3, 2-a] isoquinoline -13-carboxamide An 8-oxoprotoberberine derivative, characterized in that it is one type selected from the group consisting of. 【Claim 10】
하기 반웅식 1에 나타낸 바와 같이， As shown in reaction equation 1 below,
화학식 2로 표시되는 화합물과 화학식 3으로 표시되는 화합물을 반웅시켜 화학식 4로 표시되는 화합물을 얻는 단계 (단계 1) ; Obtaining a compound represented by Formula 4 by reacting the compound represented by Formula 2 with the compound represented by Formula 3 (Step 1);
상기 단계 1에서 얻은 화학식 4로 표시되는 화합물을 염기 조건 하에서 반웅시켜 화학식 1로 표시되는 화합물을 얻는 단계 (단계 2)를 포함하는 화학식 1로 표시되는 제 1항의 8-옥소프로토베르베린 유도체의 제조방법: A method for producing the 8-oxoprotoberberine derivative of claim 1, represented by Formula 1, comprising the step (step 2) of obtaining a compound represented by Formula 1 by reacting the compound represented by Formula 4 obtained in Step 1 under basic conditions. :
. .
Figure imgf000061_0001
(상기 반웅식 1에서， A 및 R은 제 1항에서 정의한 바와 같고， X1 및 X2는 할로겐이다) .
Figure imgf000061_0001
(In reaction formula 1, A and R are as defined in paragraph 1, and X 1 and X 2 are halogen).
[청구항 11】 [Claim 11]
제 10항에 있어서， In clause 10,
상기 단계 2는 하기 반응식 2에 나타난 바와 같이ᅳ Step 2 is as shown in Scheme 2 below:
화학식 4로 표시되는 화합물을 환원반웅시켜 화학식 5로 표시되는 화합물을 얻는 단계 (단계 A) ; 및 Reducing the compound represented by Formula 4 to obtain a compound represented by Formula 5 (Step A); and
상기 단계 A에서 얻은 화학식 5로 표시되는 화합물을 산화반웅시켜 화학식 1로 표시되는 화합물을 얻는 단계 (단계 B) ;를 포함하는 단계인 것을 특장으로 하는 화학식 1로 표시되는 제 1항의 8-옥소프로토베르베린 유도체의 쎄조방법: The 8-oxoproto of claim 1, represented by Formula 1, comprising a step (Step B) of obtaining a compound represented by Formula 1 by oxidizing the compound represented by Formula 5 obtained in Step A. Sejo method of berberine derivatives:
[반웅식 2]
Figure imgf000062_0001
 [Banwoongsik 2]
Figure imgf000062_0001
(상기 반웅식 2에서, A 및 R은 제 1항에서 정의한 바와 같고， X2는 할로겐이다) . (In reaction formula 2, A and R are as defined in paragraph 1, and X 2 is halogen).
【청구항 12】 . 【Claim 12】.
제 1항의 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 멈을 유효성분으로 함유하는 NFAT5 활성 관련 질환의 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for the prevention or treatment of diseases related to NFAT5 activity, containing the 8-oxoprotoberberine derivative of claim 1 or a pharmaceutically acceptable derivative thereof as an active ingredient.
【청구항 13】 【Claim 13】
제 12항에 있어서， In clause 12,
상기 NFAT5 활성 관련 질환은 관절염 또는 자가면역질환인 것을 특징으로 하는 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for prevention or treatment, wherein the disease related to NFAT5 activity is arthritis or an autoimmune disease.
【청구항 14】 【Claim 14】
제 13항에 있어서, According to clause 13,
상기 관절염은 류마티스 관절염인 것을 특징으로 하는 NFAT5 활성 관련 질환의 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for preventing or treating a disease related to NFAT5 activity, wherein the arthritis is rheumatoid arthritis.
【청구항 15】 【Claim 15】
제 13항에 있어서, According to clause 13,
상기 자가면역질환은 전신성 경피증， 홍반성 낭창， 아토피 피부염, 베체씨병， 쇼그렌 증후군， 다발성 경화증， 및 그레이브씨 갑상선 항진증으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 NFAT5 활성 관련 질환의 예방 또는 치료용 약학적 조성물. The autoimmune disease is a pharmaceutical for the prevention or treatment of diseases related to NFAT5 activity, characterized in that at least one selected from systemic scleroderma, lupus erythematosus, atopic dermatitis, Behçet's disease, Sjögren's syndrome, multiple sclerosis, and Grave's hyperthyroidism. Composition.
【청구항 16】 【Claim 16】
제 1항의 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 함유하는 NFAT5 활성 관련 질환의 예방 또는 개선용 건강기능식품 조성물. A health functional food composition for preventing or improving diseases related to NFAT5 activity, containing the 8-oxoprotoberberine derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
【청구항 17】 【Claim 17】
제 16항에 있어서， In clause 16,
상기 NFAT5 활성 관련 질환은 관절염 또는 자가면역질환인 것을 특징으로 하는 예방 또는 개선용 건강기능식품 조성물. A health functional food composition for preventing or improving the disease, wherein the disease related to NFAT5 activity is arthritis or an autoimmune disease.
【청구항 18】 【Claim 18】
제 1항의 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염을 유효한 양으로 개체에 투여하는 것을 포함하는 NFAT5 활성 관련 질환의 예방， 개선 또는 치료 방법. A method for preventing, improving or treating diseases related to NFAT5 activity, comprising administering to an individual an effective amount of the 8-oxoprotoberberine derivative of claim 1 or a pharmaceutically acceptable salt thereof.
【청구항 19】 【Claim 19】
NFAT5 활성 관련 질환의 예방 또는 치료제로 사용하기 위한 제 1항의 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 염 . The 8-oxoprotoberberine derivative of claim 1 or a pharmaceutically acceptable salt thereof for use as a preventive or therapeutic agent for diseases related to NFAT5 activity.
[청구항 20】 [Claim 20]
NFAT5 활성 관련 질환의 예방 또는 개선용 건강기능식품으로 사용하기 위한 제 1항의 8-옥소프로토베르베린 유도체 또는 이의 약학적으로 허용가능한 The 8-oxoprotoberberine derivative of Article 1 or a pharmaceutically acceptable product thereof for use as a health functional food for preventing or improving diseases related to NFAT5 activity.
PCT/KR2014/013059 2013-12-31 2014-12-30 Novel 8-oxoprotoberberine derivative or pharmaceutically acceptable salt thereof, preparation method therefor and pharmaceutical composition for preventing or treating diseases associated with activity of nfat5, containing same as active ingredient WO2015102380A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105130981A (en) * 2015-07-27 2015-12-09 西南大学 Berberine benzimidazole compounds, and preparation method and application thereof
CN105218537A (en) * 2015-10-14 2016-01-06 西南大学 Berberine benzimidazoles compound or its pharmacologically acceptable salt and its preparation method and application

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070095279A (en) * 2004-09-17 2007-09-28 인스티튜트 오브 메디시날 바이오테크놀로지 차이니즈 아카데미 오브 메디칼 사이언시즈 Methods and compositions for the treatment of hyperlipidemia
US20100286396A1 (en) * 2009-05-08 2010-11-11 Li Chen Novel isoquinoline derivatives
US20130237556A1 (en) * 2012-03-12 2013-09-12 Hong Kong Baptist University Berberine alkaloid as a medicament for prevention and treatment of neural disease

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20070095279A (en) * 2004-09-17 2007-09-28 인스티튜트 오브 메디시날 바이오테크놀로지 차이니즈 아카데미 오브 메디칼 사이언시즈 Methods and compositions for the treatment of hyperlipidemia
US20100286396A1 (en) * 2009-05-08 2010-11-11 Li Chen Novel isoquinoline derivatives
US20130237556A1 (en) * 2012-03-12 2013-09-12 Hong Kong Baptist University Berberine alkaloid as a medicament for prevention and treatment of neural disease

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HONG-YING CHEN ET AL., FITOTERAPIA, vol. 83, 2012, pages 67 - 73 *
JANA SKOPALOVA ET AL., BIOELECTROCHEMISTRY, vol. 87, 2012, pages 15 - 20 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105130981A (en) * 2015-07-27 2015-12-09 西南大学 Berberine benzimidazole compounds, and preparation method and application thereof
CN105218537A (en) * 2015-10-14 2016-01-06 西南大学 Berberine benzimidazoles compound or its pharmacologically acceptable salt and its preparation method and application
CN105218537B (en) * 2015-10-14 2017-01-11 西南大学 Berberine benzimidazole compounds or pharmaceutically acceptable salts thereof as well as preparation methods and applications of berberine benzimidazole compounds or pharmaceutically acceptable salts of berberine benzimidazole compounds

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