WO2020122609A1 - Novel compound and pharmaceutical composition comprising same for treating neurological disorders - Google Patents

Novel compound and pharmaceutical composition comprising same for treating neurological disorders Download PDF

Info

Publication number
WO2020122609A1
WO2020122609A1 PCT/KR2019/017519 KR2019017519W WO2020122609A1 WO 2020122609 A1 WO2020122609 A1 WO 2020122609A1 KR 2019017519 W KR2019017519 W KR 2019017519W WO 2020122609 A1 WO2020122609 A1 WO 2020122609A1
Authority
WO
WIPO (PCT)
Prior art keywords
deoxo
demethyl
formula
dihydro
represented
Prior art date
Application number
PCT/KR2019/017519
Other languages
French (fr)
Korean (ko)
Inventor
윤여준
정은지
반용선
윤성준
강상현
전수연
권안성
유영지
송명종
범지윤
정진아
이수정
황보아름
이경태
Original Assignee
주식회사 인트론바이오테크놀로지
이화여자대학교 산학협력단
연세대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020190086647A external-priority patent/KR102134782B1/en
Application filed by 주식회사 인트론바이오테크놀로지, 이화여자대학교 산학협력단, 연세대학교 산학협력단 filed Critical 주식회사 인트론바이오테크놀로지
Publication of WO2020122609A1 publication Critical patent/WO2020122609A1/en

Links

Images

Classifications

    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin

Definitions

  • the present invention is a novel compound 9-deoxo-31- O- demethyl-prolyl-FK506 (9-deoxo-31- O -dimethyl-prolyl-) that can be utilized as a main component of a pharmaceutical composition for preventing or treating neurological diseases FK506), 9-deoxo-36,37-dihydro-FK506 (9-deoxo-36,37-dihydro-FK506), 31- O- demethyl-36,37-dihydro-FK506 (31- O -dimethyl- 36,37- dihydro -FK506), 9-deoxo-31- O -demethyl-36,37-dihydro-FK506 (9- Deoxo -31- O - dimethyl -36,37- dihydro -FK506), 9 -deoxo-FK520 (9-deoxo-FK520), 31- O- demethyl-FK520 (
  • Nerve damage can occur in a variety of nervous systems, such as the brain, spine, and peripheral nerves.
  • the resulting diseases are neurodegenerative disease, peripheral nerve injury, and traumatic brain injury. brain injury) and cerebral infarction.
  • Degenerative brain diseases caused by nerve damage in the central nervous system may be dementia, Parkinson's disease, Huntington's disease, and Amyotrophic lateral sclerosis.
  • Parkinson's disease and Alzheimer's disease make up the majority
  • Huntington's disease and amyotrophic lateral sclerosis have relatively few patients.
  • Peripheral nerve disorders are caused by damage to the peripheral nervous system by various causes, and 60% of the causes are complications caused by diabetes and often get peripheral nerve disorders during chemotherapy to treat cancer.
  • Traumatic brain injury is a disease in which brain tissue is damaged due to trauma such as a traffic accident or a fall.
  • Korean Patent Publication No. 10-2005-0071491 name of the invention: the use of a tacrolimus derivative in combination with a beta2-agonist for the treatment of asthma
  • a tacrolimus derivative in combination with a beta2-agonist for the treatment of asthma
  • novel use of FK506 derivatives and beta2-agonists for the manufacture of a medicament for separate or sequential use has been disclosed, no FK506 derivative, FK520 derivative or FK523 derivative has been applied to treat neurological diseases.
  • a novel compound 9-deoxo-31- O- demethyl-prolyl-FK506, 9-deoxo-36 which can be used as a main component of a pharmaceutical composition for preventing or treating neurological diseases 37-dihydro-FK506, 31- O- demethyl-36,37-dihydro-FK506, 9-deoxo-31- O- demethyl-36,37-dihydro-FK506, 9-deoxo-FK520, 31- O- demethyl- FK520, 9-deoxo-31- O- demethyl-FK520, 9-deoxo-FK523, and 9-deoxo-31- O- demethyl-FK523 (hereinafter collectively referred to as '9 new compounds') promote the effect of promoting nerve cell growth.
  • One object of the present invention is to provide the above nine new compounds, isomers thereof, or pharmaceutically acceptable salts thereof.
  • An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a nervous system disease comprising at least one selected from nine new compounds as an active ingredient.
  • the pharmaceutical composition for preventing or treating a nervous system disease means a pharmaceutical composition for preventing or treating a nervous system disease or a composition for promoting nerve regeneration.
  • Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurological diseases comprising at least one selected from isomers or pharmaceutically acceptable salts of nine new compounds as an active ingredient.
  • the pharmaceutical composition may be utilized as a composition for promoting nerve regeneration.
  • Another object of the present invention is to provide a biological production method for each of the nine new compounds.
  • Another object of the present invention is a production strain that can be used in the biological manufacturing process of nine new compounds, Streptomyces kanamyceticus ⁇ fkbD-fkbM (Accession No. KCTC13581BP), Streptomyces kanamyceticus ⁇ fkbD,tcsD (Accession number KCTC13580BP), Streptomyces kanamyceticus ⁇ fkbM,tcsD (Accession number KCTC13584BP), Streptomyces kanamyceticus ⁇ fkbD-fkbM,tcsD (Accession number KCTC13585BP), Streptomyces kanamyset B (Accession No.
  • KCTC13579BP Streptomyces kanamyceticus ⁇ fkbM,tcsB (Accession No. KCTC13583BP), and Streptomyces kanamyceticus ⁇ fkbD-fkbM,tcsB (Accession No. KCTC13582BP).
  • the pharmaceutical composition for the prevention or treatment of a nervous system disease comprising at least one selected from the nine new compounds according to the present invention as an active ingredient can be effectively utilized in the treatment of a nervous system disease without a suitable effective drug. Compared to the existing drug treatment, a more fundamental treatment effect can be expected.
  • HMBC-NMR nuclear magnetic resonance analysis
  • HMBC-NMR nuclear magnetic resonance analysis
  • FIG. 17 shows the results of nuclear magnetic resonance analysis (HSQC-NMR) for 31- O- demethyl-36,37-dihydro-FK506.
  • HMBC-NMR nuclear magnetic resonance analysis
  • HMBC-NMR nuclear magnetic resonance analysis
  • 25 is a high-performance liquid chromatography analysis results for 9-deoxo-FK520.
  • HMBC-NMR nuclear magnetic resonance analysis
  • FIG. 31 shows the results of high performance liquid chromatography analysis for 31- O- demethyl-FK520.
  • FIG. 32 shows the results of nuclear magnetic resonance analysis ( 1 H-NMR) for 31- O- demethyl-FK520.
  • FIG. 33 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 31- O- demethyl-FK520.
  • FIG. 34 is a nuclear magnetic resonance analysis (COSY-NMR) result for 31- O- demethyl-FK520.
  • HMBC-NMR nuclear magnetic resonance analysis
  • FIG. 39 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 9-deoxo-31- O- demethyl-FK520.
  • HMBC-NMR nuclear magnetic resonance analysis
  • FIG. 44 shows the results of nuclear magnetic resonance analysis ( 1 H-NMR) for 9-deoxo-FK523.
  • FIG. 45 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 9-deoxo-FK523.
  • FIG. 50 shows the results of nuclear magnetic resonance analysis ( 1 H-NMR) for 9-deoxo-31- O- demethyl-FK523.
  • FIG. 51 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 9-deoxo-31- O- demethyl-FK523.
  • FIG. 54 shows the results of nuclear magnetic resonance analysis (HMBC-NMR) for 9-deoxo-31- O- demethyl-FK523.
  • Figure 56 shows the results of investigating the nerve cell growth promoting ability (in vitro) of 9 novel compounds of the present invention.
  • Figure 57 shows the results of investigating the nerve cell growth promoting ability (in vivo) of 9 new compounds of the present invention.
  • Figure 58 is a result of investigating the ability to treat neurological diseases of nine new compounds of the present invention (Rotarod test, open field test).
  • 61 is a result of investigating the ability to treat neurological diseases of nine new compounds of the present invention (BBB test).
  • Fig. 62 shows the results of investigating the treatment ability of 9 new compounds of the present invention for nervous system diseases (grid work test).
  • the present invention is a pharmaceutical composition for the prevention or treatment of a nervous system disease, including a manufacturing process of nine new compounds, each new compound prepared using the manufacturing process, and the prevention of this nervous system disease Or it provides a treatment method for a nervous system disease using a therapeutic pharmaceutical composition.
  • the present invention is a new compound, 9-deoxo-31- O- demethyl-prolyl-FK506 represented by the following [Formula 1], represented by the following [Formula 2] 9-deoxo-36,37-dihydro-FK506, 31- O- demethyl-36,37-dihydro-FK506 represented by the following [Formula 3], 9-deoxo-31- O -represented by the following [Formula 4] demethyl-36,37-dihydro-FK506, 9-deoxo-FK520 represented by the following [Formula 5], 31- O- demethyl-FK520 represented by the following [Formula 6], 9- represented by the following [Formula 7] deoxo-31- O- demethyl-FK520, 9-deoxo-FK523 represented by the following [Formula 8], 9-deoxo-31- O- demethyl-FK5
  • FK506, tacrolimus (Tacrolimus) or fujimycin (fujimycin) is a substance having the first immunosuppressive activity isolated from the various Streptomyces tsukubaensis ( Streptomyces tsukubaensis ) 23-won ( 23-Membered Polyketide macrolide. Immunosuppressive action is stronger than cyclosporine and is known to be used to suppress rejection, especially when transplanting the liver during organ transplantation.
  • the FK506 can be synthesized by the PKS/NRPS (Polyketide synthase/nonribosomal peptide synthetase) complex system, but the FK506 derivative of the present invention is a novel compound produced through a novel strain produced through a biosynthetic gene defect in Streptomyces genus. Can be
  • FK520 or ascomycin is also an immunosuppressant, a compound of 23 macro-carbon macrolead structures, an ethyl analogue of the C21 position of FK506.
  • FK523 is the methyl analog of the C21 position of FK506.
  • the compounds of the present invention may include isomers or pharmaceutically acceptable salts thereof.
  • Isomer means the relationship of compounds having the same chemical formula but not the same, and may include, for example, structural isomers, geometric isomers, optical isomers (enantiomers), stereoisomers, diastereomers.
  • Pharmaceutically acceptable salts are concentrations that have a relatively non-toxic and harmless effect on the patient, and can mean any and all organic or inorganic addition salts whose side effects caused by the salts do not diminish the beneficial efficacy of the parent compound.
  • the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid.
  • Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile.
  • the salt may be a pharmaceutically acceptable metal salt prepared using a base.
  • the compounds of the present invention may be in the form of solvates or pro-drugs, which are included within the scope of the present invention.
  • Solvates may preferably include hydrates and ethanolates.
  • the pharmaceutical composition of the present invention can also be used as a single agent, and can be prepared and used as a combination preparation by further including a drug known to have an approved neurological disease treatment effect, and is formulated by using a pharmaceutically acceptable carrier or excipient. It can be made in unit dose form or can be made by incorporating into a multi-dose container.
  • the term "pharmaceutically acceptable carrier” may mean a carrier or diluent that does not inhibit the biological activity and properties of the compound being injected without stimulating the organism.
  • the type of the carrier that can be used in the present invention is not particularly limited, and any carrier that is commonly used in the art and pharmaceutically acceptable may be used.
  • Non-limiting examples of the carrier transcutol (Transcutol), polyethylene glycol (Polyethylene glycol), triacetin (Triacetin) and co-surfactants exemplified by mixtures thereof (Co-surfactant); Cremophor, Tween, Myrj, Poloxamer, Pluronic, Lutrol, Imwitor, Span, Labra Surfactant, which can be illustrated alone or in a mixture such as Labrafil; Oils which can be exemplified alone or in mixtures such as Miglyol, Captex, and Ethyl oleate; And organic acids that can be exemplified singly or in mixtures such as erythorobic acid and citric acid. These may be used alone or in combination of two or more.
  • additives such as antioxidants, buffers, and/or bacteriostatic agents can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to form a solution such as aqueous solutions, suspensions, emulsions, etc. It can be formulated and used in dosage forms, pills, capsules, granules or tablets.
  • the present invention provides a method of treating a nervous system disease, comprising administering the pharmaceutical composition to a subject.
  • the term "individual” may mean any animal that has or is likely to develop a neurological disorder.
  • the pharmaceutical composition of the present invention may include one or more selected from nine new compounds in a pharmaceutically effective amount, or isomers or salts thereof.
  • pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 It may be administered in an amount of 200 to 200 mg/kg, more preferably 0.1 to 100 mg/kg once to several times a day.
  • a specific therapeutically effective amount for a particular patient may include a specific composition, the age, weight, general health status of the patient, including the type and extent of the response to be achieved, and, in some cases, whether other agents are used. It is desirable to apply differently depending on various factors including sex and diet, time of administration, route of administration and composition, secretion rate of treatment, duration of treatment, and drugs used with or concurrently with the specific composition and similar factors well known in the pharmaceutical field.
  • the frequency of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered once a day or divided into doses several times.
  • the dosage amount of the pharmaceutical composition of the present invention may be 0.001 mg/kg to 1000 mg/kg, specifically 0.05 mg/kg to 200 mg/kg, 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg It may be 20 mg/kg, but is not limited thereto.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount while minimizing the induction of side effects, and can be easily determined by those skilled in the art.
  • the term "administration" refers to the introduction of the pharmaceutical composition of the present invention to a patient in any suitable way, the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.
  • the method of administration of the pharmaceutical composition according to the present invention is not particularly limited, and may be in accordance with a method commonly used in the art.
  • the pharmaceutical composition may be administered by oral administration or parenteral administration.
  • the pharmaceutical composition according to the present invention may be prepared in various dosage forms depending on the desired mode of administration.
  • the pharmaceutical composition for preventing or treating diseases of the nervous system of the present invention may be used for the purpose of treating diseases of the nervous system.
  • neurological diseases include neurological diseases, and neurological diseases include neurodegenerative diseases, peripheral nerve injury, traumatic brain injury, and strokes caused by cerebrovascular disorders. (Cerebral infarction) may be exemplified, but is not limited thereto.
  • the degenerative neurological disease refers to a disease that causes various symptoms while degenerative changes appear in neurons of the central nervous system.
  • dementia Alzheimer's disease, Parkinson's disease, progressive Progressive supranuclear palsy, Multiple system strophy, Olive-pond-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatum-black matter degeneration (Striatonigral) degeneration), Huntington's disease, Amyotrophic lateral sclerosis (ALS), Essential tremor, Corticobasal ganlionic degeneration, Diffuse Lewy body disease ), Parkinson-ALS-dementia complex of Guam or Pick's disease.
  • ALS Amyotrophic lateral sclerosis
  • Essential tremor Corticobasal ganlionic degeneration
  • Diffuse Lewy body disease Diffuse Lewy body disease
  • Parkinson-ALS-dementia complex of Guam or Pick's disease Parkinson-ALS-dementia complex of Guam or Pick's disease.
  • the neurological damage disease may include epilepsy, stroke, stroke, ischemic brain disease, spinal cord injury disease, peripheral nerve disease, behavioral disorder, developmental disorder, mental retardation, Down syndrome or schizophrenia, but is not limited thereto. no.
  • the nerve injury disease may be a disease caused by nerve cell damage or cell death.
  • prevention means that the pharmaceutical composition of the present invention is administered to an individual who has a possible or suspected condition or condition of developing a nervous system disease, thereby lowering the likelihood of developing a nervous system disease or It refers to the act of alleviating symptoms or conditions.
  • treatment refers to all actions to improve or benefit the symptoms of a nervous system disease by administering the pharmaceutical composition of the present invention to a suspected neurological disease development subject.
  • the immunosuppressive activity effect of the nine new compounds was confirmed by an in vitro T cell activity assay to confirm the reduced immunosuppressive activity than FK506.
  • the neuroaxon-producing effect of the nine new compounds was confirmed to confirm the ability to stimulate neuronal growth.
  • the BBB test and the grid walk test were used to confirm the therapeutic effects of the 9 new compounds on nervous system diseases.
  • the present invention is 9 new compounds, 9-deoxo-31- O- demethyl-prolyl-FK506, 9-deoxo-36,37-dihydro-FK506, 31- O -demethyl-36,37-dihydro-FK506, 9-deoxo-31- O -demethyl-36,37-dihydro-FK506, 9-deoxo-FK520, 31- O -demethyl-FK520, 9-deoxo-31- Provides biological manufacturing processes of O- demethyl-FK520, 9-deoxo-FK523, and 9-deoxo-31- O- demethyl-FK523.
  • the culture temperature generally employed in the culture process of Streptomyces genus is used.
  • a culture temperature suitable for the implementation of the present invention preferably, 23-30°C may be applied, and more preferably, a culture temperature of 25-28°C may be applied.
  • the pH of the culture process is maintained between 6.5 and 9, preferably, the culture pH is maintained at 7-8.
  • the dissolved oxygen level in the culture medium high.
  • the dissolved oxygen level at the beginning of the culture is 100%, it is important to maintain the dissolved oxygen level until the end of the culture at 30% or more. In order to implement this, it is generally desirable to stir at a level of 800-1,500 rpm.
  • the extraction of the 9 new compounds produced from the cultured cell body in the above manufacturing process is achieved through the implementation of the primary extraction process, the secondary extraction process and the tertiary extraction process.
  • the organic solvent extraction method is used as the primary extraction process.
  • ethyl acetate, methanol, acetone, and the like may be used as the solvent, but ethyl acetate or methanol is preferred.
  • silica gel chromatography is used as a secondary extraction process.
  • a solvent that can be used methanol and methylene chloride are preferable.
  • chromatography is used as the third extraction process.
  • acetonitrile ammonium acetate buffer, acetic acid, formic acid, etc. may be used as the solvent, but acetonitrile is preferred.
  • the application of this method facilitates the recovery of 9 new compounds and also increases the yield.
  • the present invention is a production strain that can be used for the production of 9 new compounds, Streptomyces kanamyceticus ⁇ fkbD-fkbM (Accession No. KCTC13581BP), Streptomyces Kanamyceticus ⁇ fkbD,tcsD (Accession No. KCTC13580BP), Streptomyces kanamyceticus ⁇ fkbM,tcsD (Accession No. KCTC13584BP), Streptomyces kanamyceticus ⁇ fkbD-fkbM,tcsD (Accession No.
  • KTCTCD 85BP CTC Kanamyceticus ⁇ fkbD,tcsB (Accession No. KCTC13579BP), Streptomyces kanamyceticus ⁇ fkbM,tcsB (Accession No. KCTC13583BP), and Streptomyces kanamyceticus ⁇ fkbD-fkbM,tcsB (Accession No. .
  • Example 1 9-deoxo-31- O -demethyl-prolyl-FK506 manufacture
  • each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 in order to construct a mutant of the fkbD and fkbM genes in a strain of Streptomyces kanamyceticus producing FK506, and then transferred to a conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)).
  • the produced fkbD-fkbM gene-deficient strain Streptomyces kanamyceticus ⁇ fkbD-fkbM, was deposited with the Korea Institute of Bioscience and Biotechnology (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession No. KCTC13581BP ).
  • 9-deoxo-31- O- demethyl-prolyl-FK506 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ⁇ fkbD-fkbM (Accession No. KCTC13581BP). Specifically, it is as follows.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed.
  • the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration.
  • the extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used.
  • fraction 3 9-deoxo-31- O- demethyl-prolyl-FK506 was identified.
  • the obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 in order to construct a mutant of the fkbD and tcsD genes in a strain of Streptomyces kanamyceticus producing FK506, and then transferred to a conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbDLF/FkbDLR of the left-neighbor segment and the primer pair FkbDRF/FkbDRR of the right-neighbor segment were designed, and for the deletion of the tcsD gene, the primer pair of the left-neighbor segment TcsDLF/TcsDLR, right- Primer pairs TcsDRF/TcsDRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • the plasmid to remove C9 hydroxylase (Hydroxylase), p ⁇ fkbD, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to delete the target gene by homologous recombination.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected as a culture of a transmycin-resistant transconjugant.
  • Colonies secured thereafter were allowed to multiply three times without selection at 28°C to allow the second crossover.
  • Two achieved double crossover mutations, ⁇ fkbD were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
  • the prepared fkbD gene was deleted by introducing p ⁇ tcsD into ⁇ fkbD of Streptomyces kanamyceticus deficient, and the tcsD gene was deleted using the same method as the fkbD gene deletion method.
  • ⁇ fkbD,tcsD was selected as apramycin-sensitive expression trait, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
  • 9-deoxo-36,37-dihydro-FK506 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ⁇ fkbD,tcsD (Accession No. KCTC13580BP). Specifically, it is as follows.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used.
  • fraction 3 9-deoxo-36,37-dihydro-FK506 was identified.
  • the obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 to produce a deletion mutant of the fkbM and tcsD genes in the Streptomyces kanamyceticus strain producing FK506, and then transferred to the conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbMLF/FkbMLR of the left-neighbor segment and the primer pair FkbMRF/FkbMRR of the right-neighbor segment were designed, and for the deletion of the tcsD gene, the primer pair of the left-neighbor segment TcsDLF/TcsDLR, right- Primer pairs TcsDRF/TcsDRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • the plasmid for removing 31- O -methyltransferase, p ⁇ fkbM, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation, thereby deleting the target gene by homologous recombination.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant.
  • the prepared fkbM gene was introduced into p ⁇ tcsD in ⁇ fkbM of Streptomyces kanamyceticus deficient, and the tcsD gene was deleted using the same method as the fkbM gene deletion method.
  • ⁇ fkbM,tcsD was selected as the expression trait of apramycin-sensitivity, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
  • 31- O- demethyl-36,37-dihydro-FK506 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ⁇ fkbM,tcsD (Accession No. KCTC13584BP). Specifically, it is as follows.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used.
  • fraction 3 31- O- demethyl-36,37-dihydro-FK506 was identified.
  • the obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002, in order to prepare a deletion mutant of the fkbD-fkbM and tcsD genes in the Streptomyces kanamyceticus strain producing FK506, and then transferred to Escherichia coli ET12567/pUZ8002, It was transformed into the FK506 production strain Streptomyces kanamyceticus through conjugation.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed, and the left-adjacent for deletion of the tcsD gene.
  • the primer pair TcsDLF/TcsDLR of the fragment and the primer pair TcsDRF/TcsDRR of the right-adjacent fragment were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)).
  • the prepared fkbD-fkbM gene was deleted by introducing p ⁇ tcsD into ⁇ fkbD-fkbM of Streptomyces kanamyceticus, and the tcsD gene was deleted using the same method as the fkbD-fkbM gene deletion method.
  • ⁇ fkbD-fkbM,tcsD was selected as an apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
  • 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 was prepared through cultivation of the produced strain S. pneumoniae streptomyces kanamyceticus ⁇ fkbD-fkbM,tcsD (Accession No. KCTC13585BP). Specifically, it is as follows.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used.
  • fraction 3 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 was identified.
  • the obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • each gene was cloned into a pKC1139 vector and Escherichia coli in order to produce a deletion mutant of the fkbD and tcsB genes or a deletion mutant of the fkbD and tcsD genes in the Streptomyces kanamyceticus strain producing FK506. After transfer to ET12567/pUZ8002, it was transformed into FK506 production strain Streptomyces kanamyceticus through conjugation.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbDLF/FkbDLR of the left-adjacent segment and the primer pair FkbDRF/FkbDRR of the right-adjacent segment were designed, and for the deletion of the tcsB gene, the primer pair of the left-adjacent segment TcsBLF/TcsBLR, right- Primer pairs TcsBRF/TcsBRR of adjacent sections were designed.
  • the primer pair TcsDLF/TcsDLR of the left-adjacent segment and the primer pair TcsDRF/TcsDRR of the right-adjacent segment were designed.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • the plasmid to remove C9 hydroxylase (Hydroxylase), p ⁇ fkbD, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to delete the target gene by homologous recombination.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected as a culture of a transmycin-resistant transconjugant.
  • Colonies secured thereafter were allowed to multiply three times without selection at 28°C to allow the second crossover.
  • Two achieved double crossover mutations, ⁇ fkbD were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
  • the prepared fkbD gene was introduced into p ⁇ tcsB or p ⁇ tcsD in ⁇ fkbD of Streptomyces kanamyceticus deficient, and the tcsB gene or tcsD gene was deleted using the same method as the fkbD gene deletion method.
  • ⁇ fkbD, tcsB or ⁇ fkbD, tcsD was selected as apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
  • 9-deoxo-FK520 was prepared by culturing the production strain of Streptomyces kanamyceticus ⁇ fkbD,tcsB (Accession No. KCTC13579BP) or Streptomyces kanamyceticus ⁇ fkbD,tcsD (Accession No. KCTC13580BP). Specifically, it is as follows.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 9-deoxo-FK520 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • each gene was cloned into a pKC1139 vector and Escherichia coli in order to produce a deletion mutant of the fkbM and tcsB genes or a deletion mutant of the fkbM and tcsD genes in the Streptomyces kanamyceticus strain producing FK506. After transfer to ET12567/pUZ8002, it was transformed into FK506 production strain Streptomyces kanamyceticus through conjugation.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • a primer pair FkbMLF/FkbMLR of the left-neighbor segment and a primer pair FkbMRF/FkbMRR of the right-neighbor segment were designed, and for deletion of the tcsB gene, a primer pair of the left-neighbor segment TcsBLF/TcsBLR, right- Primer pairs TcsBRF/TcsBRR of adjacent sections were designed.
  • the primer pair TcsDLF/TcsDLR of the left-adjacent segment and the primer pair TcsDRF/TcsDRR of the right-adjacent segment were designed.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • the plasmid for removing 31- O -methyltransferase, p ⁇ fkbM, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation, thereby deleting the target gene by homologous recombination.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant.
  • the prepared fkbM gene was introduced into p ⁇ tcsB or p ⁇ tcsD in ⁇ fkbM of Streptomyces kanamyceticus deficient, and the tcsB gene or tcsD gene was deleted using the same method as the fkbM gene deletion method.
  • ⁇ fkbM,tcsB or ⁇ fkbM,tcsD was selected as apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
  • 31- O- demethyl-FK520 was prepared by culturing the above-produced production strain Streptomyces kanamyceticus ⁇ fkbM,tcsB (Accession No. KCTC13583BP) or Streptomyces kanamyceticus ⁇ fkbM,tcsD (Accession No. KCTC13584BP). . Specifically, it is as follows.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 31- O- demethyl-FK520 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed.
  • the primer pair TcsBLF/TcsBLR of the left-neighbor segment and the primer pair TcsBRF/TcsBRR of the right-neighbor segment were designed, and for the deletion of the tcsD gene, the primer pair TcsDLF/TcsDLR of the left-neighbor segment, right- Primer pairs TcsDRF/TcsDRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)).
  • the prepared fkbD-fkbM gene was deleted by introducing p ⁇ tcsB or p ⁇ tcsD into ⁇ fkbD-fkbM of Streptomyces kanamyceticus, and the tcsB gene or tcsD gene was deleted using the same method as the fkbD-fkbM gene deletion method. .
  • ⁇ fkbD-fkbM,tcsB or ⁇ fkbD-fkbM,tcsD was selected as an apramycin-sensitive expression trait, and then confirmed by PCR, and optionally by Southern blot analysis.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used.
  • fraction 3 9-deoxo-31- O- demethyl-FK520 was identified.
  • the obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 in order to construct a mutant of the fkbD and tcsB genes in a strain of Streptomyces kanamyceticus producing FK506, and then transferred to a conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbDLF/FkbDLR of the left-adjacent segment and the primer pair FkbDRF/FkbDRR of the right-adjacent segment were designed, and for the deletion of the tcsB gene, the primer pair of the left-adjacent segment TcsBLF/TcsBLR, right- Primer pairs TcsBRF/TcsBRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • the plasmid to remove C9 hydroxylase (Hydroxylase), p ⁇ fkbD, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to delete the target gene by homologous recombination.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected as a culture of a transmycin-resistant transconjugant.
  • Colonies secured thereafter were allowed to multiply three times without selection at 28°C to allow the second crossover.
  • Two achieved double crossover mutations, ⁇ fkbD were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
  • tcsB By introducing a p ⁇ tcsB the produced fkbD gene defect Streptomyces Kana Mai Shetty kusu ⁇ fkbD, using the same method as fkbD gene-deficient deletion method was the tcsB gene.
  • ⁇ fkbD,tcsB was selected as the expression trait of apramycin-sensitivity, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
  • 9-deoxo-FK523 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ⁇ fkbD,tcsB (Accession No. KCTC13579BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inocul
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used.
  • 9-deoxo-FK523 was identified in aliquot 3. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • 9-deoxo-31- O- demethyl-FK520 has the same molecular weight as the molecular weight, and from 1 H-NMR, 13 C-NMR and gHSQC data, a total of 42 carbon atoms was confirmed in a very similar form, but 9-deoxo-31 -Unlike O- demethyl-FK520, methoxy ( ⁇ H 3.41, ⁇ C 56.81) was additionally observed, and it was confirmed that one of the CH 2 functional groups was a structural isomer with less observation.
  • each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002, in order to produce a deletion mutant of the fkbD-fkbM and tcsB genes in the Streptomyces kanamyceticus strain producing FK506, and then transferred to Escherichia coli ET12567/pUZ8002, It was transformed into the FK506 production strain Streptomyces kanamyceticus through conjugation.
  • the method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
  • the production of the in-frame gene deletion plasmid E. coli - Streptomyces shuttle
  • the vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out.
  • the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed.
  • tcsB gene For deletion of the tcsB gene, a primer pair TcsBLF/TcsBLR of the left-adjacent segment and a primer pair TcsBRF/TcsBRR of the right-adjacent segment were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
  • the plasmid used to construct the gene deletion strain is summarized in Table 1.
  • the plasmid for removing C9 hydroxylase (Hydroxylase) and 31- O -methyltransferase together, p ⁇ fkbD-fkbM, is transferred to Escherichia coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to target genes. It was deleted by homologous recombination.
  • Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)).
  • the prepared fkbD-fkbM gene was introduced into p ⁇ tcsB into ⁇ fkbD-fkbM of Streptomyces kanamyceticus deficient, and the tcsB gene was deleted using the same method as the fkbD-fkbM gene deletion method.
  • ⁇ fkbD-fkbM,tcsB was selected as an apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
  • 9-deoxo-31- O- demethyl-FK523 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ⁇ fkbD-fkbM,tcsB (Accession No. KCTC13582BP). Specifically, it is as follows.
  • R2YE medium sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask
  • the first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel.
  • the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used.
  • fraction 3 9-deoxo-31- O- demethyl-FK523 was identified.
  • the obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
  • the degree of immunosuppressive activity reduction of 9 novel compounds was investigated using a conventional in vitro T-cell activity assay (J. Immunol. 143: 718-726, 1989). Cleavage of CD4+ T cells is an indicator that an immune response is taking place. CD4+ T cells are stained with Cell Trace TM Violet (CTV), and when cells divide according to the immune response, CTV of each cell proliferates. As a phenomenon in which the retention amount decreases, the degree of immunosuppressive activity was investigated using this as an indicator.
  • CTV Cell Trace TM Violet
  • CD4+ T cells Single cells were detached from spleens of 6-8 weeks old B6J experimental mice and CD4+ T cells were separated using MagniSort ® Mouse CD4 T cell Enrichment Kit (eBioscience). CD4+ T cells were stained with Cell Trace TM Violet (CTV) Cell Proliferation Kit (Molecular Probes) and FK506 or 9 new compounds were 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng /ml, added to a concentration of 1000 ng/ml, and then incubated for 72 hours. Dynabeads ® Mouse T-Activator CD3/CD28 (Gibco) was used for T cell activation. As a control, inactivated T cells were used. After cultivation, CTV intensity was analyzed by flow cytometry.
  • CTV Cell Trace TM Violet
  • Table 12 and FIG. 55 show the degree of T cell proliferation as measured by CTV intensity using a flow cytometer, and show the degree of immunosuppressive activity of FK506 and nine new compounds. As shown in Table 12 and FIG. 55, all the new compounds proposed in the present invention showed reduced immunosuppressive activity than FK506.
  • Example 11 Investigation of nerve cell growth promoting activity of 9 new compounds ( In vitro )
  • the ability to promote neuronal growth of 9 new compounds was investigated according to the method reported by Mo, SJ et al. (J. Am. Chem. Soc. 133: 976-985, 2011) using rat PC12 cells (Pheochromocytoma cells). . Specifically, the PC12 cells were treated with neural growth factor (NGF; KOMA Biotech; 10 ng/ml) for inducing neurite outgrowth for 96 hours, during which treatment either FK506 or one of 9 new compounds was treated together. (Treatment concentration: 1 ng/ml, 10 ng/ml), the control group did not give any treatment. The length of the neurites was measured according to a previously reported method (J. Pharmacol. Exp. Ther. 302: 1278-1285, 2002) using a printed photograph.
  • NGF neural growth factor
  • Example 12 Investigation of the neuronal growth promoting activity of new compounds ( In vivo )
  • a cannula injection system was used to treat FK506 or two new compounds in the ventricles of awake experimental animals (treatment concentration: 1 mg/kg), and saline was treated in the control group.
  • the black matter-pars compacta the beginning of the black matter-progenitor neuropathy In
  • the number of dopaminergic neuronal cell bodies was measured, and the density change of nerve fibers extending from the black matter was quantified in the striatum, the distal end of the neuropath.
  • Degenerative neurological diseases were identified using the MPTP-induced Parkinson's disease mouse model, and a basic evaluation was performed on changes in gait and motor function in the lower extremities following treatment with FK506 or new compounds.
  • the experimental animal was placed on a stick rotating at a speed of 10 rpm, and the time taken to drop was measured to confirm changes in walking and athletic ability of the lower extremities.
  • the change in basic motor function was compared and analyzed by measuring the total travel distance of the experimental animal for 10 minutes in an empty space surrounded by walls.
  • the results of the rotarod test and open field test are presented in FIG. 58.
  • the results are compared to the group treated with a mixture of 9-deoxo-31- O- demethyl-FK520 and 9-deoxo-31- O -demethyl-FK523 among 9 new compounds of the present invention, compared to the group treated with physiological saline. Indicates a significant improvement in gait and motor skills.
  • the group treated with the mixture of 9-deoxo-31- O- demethyl-FK520 and 9-deoxo-31- O- demethyl-FK523 showed higher mobility compared to the group treated with physiological saline.
  • a pharmaceutical composition for preventing or treating neurological diseases comprising at least one selected from 9 new compounds of the present invention as an active ingredient is effective in treating degenerative neurological diseases.
  • the functional synapse-forming activity of the novel compound was analyzed using D. Park. In addition, it was investigated according to the method reported by (Sci. Rep. 7: 7260, 2017). Specifically, exposure to FK506 or a new compound (processing concentration: 1 ng/ml) was performed for patch-fixed recording on hippocampal neurons from the first to 10th to 14th days of primary culture to measure the frequency and amplitude of excitatory synaptic currents. .
  • the frequency of the excitatory synaptic current is used as an index for confirming a change in the number of synapses, and the amplitude is an index for synaptic plasticity, and it is possible to grasp the qualitative change of synapses by a new compound.
  • the results showing the frequency of the measured excitatory synaptic current are presented in FIG. 59.
  • the frequency of excitatory synaptic currents in the group treated with a mixture of 9-deoxo-31- O- demethyl-FK520 and 9-deoxo-31- O -demethyl-FK520 among 9 new compounds of the present invention It was confirmed that it increased significantly. This suggests that the in vivo neuron growth promoting activity and the treatment effect of degenerative neurological diseases shown by the groups treated with the novel compounds in Examples 12 and 13 may be attributable to an increase in functional synapse formation.
  • Figure 60 is a graph showing the amplitude of the excitatory synaptic current, whereas the group treated with physiological saline and FK506 showed similar amplitude, 9-deoxo-31- O- demethyl-FK520 and 9-deoxo- out of 9 new compounds The average amplitude was observed to increase in the group treated with the mixture of 31- O- demethyl-FK520.
  • a pharmaceutical composition for preventing or treating a nervous system disease comprising at least one selected from the nine new compounds as an active ingredient is involved in the action of enhancing synaptic plasticity, more than simply promoting growth of nerve cells.
  • the rats were placed on a warm sawdust to maintain body temperature, and the contents of the bladder were discharged by massaging the lower abdomen 3 to 4 times daily for about 7 days until autonomic bladder control was fully restored.
  • 5 mg of antibiotic cephalexin per 100 g of body weight was injected intramuscularly daily.
  • the BBB test and grid walk test for confirming the effect of treating neurological diseases were performed 14 days before the nerve injury surgery, and a basic evaluation of behavior and motor function was performed 3 days before the operation, and the results were compared and analyzed after the administration of 9 new compounds.
  • the rats were placed in a transparent plexiglass box with a rough surface, and observed for 4-5 minutes to score according to criteria such as joint exercise, weight bearing, forelimb-hindlimb coordination, and tail position.
  • the maximum 21 points were defined as having normal walking ability.
  • the rat In the grid walk test, the rat walked a 1 m long metal parallel rod to evaluate the regular step ability, and the regulating ability of the hind leg when descending an oblique rod was counted as the number of steps, so the rat made 10 mistakes regularly. In addition to not being able to walk, it indicates that the legs cannot be controlled spontaneously, and mistakes from 0 to 1 are defined as being intact and indicating normal motor performance.
  • G1 daily oral administration of excipients
  • G2 9-deoxo-31- O- demethyl-prolyl-FK506 daily oral dose at 5 mg/kg dose
  • G3 9-deoxo-36,37-dihydro-FK506 daily oral dose at 5 mg/kg dose
  • G4 5 mg 31- O- demethyl-36,37-dihydro-FK506 daily oral dose at /kg dose
  • G5 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 daily oral dose at 5 mg/kg dose
  • G6 9-deoxo-FK520 daily oral dose at 5 mg/kg dose
  • G7 31- O- demethyl-FK520 daily oral dose at 5 mg/kg dose
  • G8 9-deoxo-31-
  • FIGS. 57 and 58 The results of performing the BBB test and the grid walk test at intervals of one week from the third day after the nerve injury surgery are presented in FIGS. 57 and 58.
  • the result was superior to that of the excipient-administered group, and the score of the nine new compound-administered groups was superior, and from this result, the pharmaceutical composition for preventing or treating a neurological disease comprising at least one selected from the nine new compounds as an active ingredient was a nervous system disease. It was confirmed that the treatment was effective.

Abstract

The present invention relates to the production of a novel compound usable as a main ingredient of a pharmaceutical composition for preventing or treating neurological disorders, the compound being 9-deoxo-31-O-demethyl-prolyl-FK506, 9-deoxo-36,37-dihydro-FK506, 31-O-demethyl-36,37-dihydro-FK506, 9-deoxo-31-O-demethyl-36,37-dihydro-FK506, 9-deoxo-FK520, 31-O-demethyl-FK520, 9-deoxo-31-O-demethyl-FK520, 9-deoxo-FK523, or 9-deoxo-31-O-demethyl-FK523. In addition, the present invention relates to a use of same in the prevention or treatment of neurological disorders.

Description

신규 화합물 및 이를 포함하는 신경계 질환 치료용 약학적 조성물 Novel compounds and pharmaceutical compositions for treating neurological diseases
본 발명은 신경계 질환의 예방 또는 치료용 약학적 조성물의 주요 성분으로 활용이 가능한 신규 화합물 9-deoxo-31-O-demethyl-prolyl-FK506 (9-데옥소-31-O-디메틸-프롤릴-FK506), 9-deoxo-36,37-dihydro-FK506 (9-데옥소-36,37-디히드로-FK506), 31-O-demethyl-36,37-dihydro-FK506 (31-O-디메틸-36,37-디히드로-FK506), 9-deoxo-31-O-demethyl-36,37-dihydro-FK506 (9-데옥소-31-O-디메틸-36,37-디히드로-FK506), 9-deoxo-FK520 (9-데옥소-FK520), 31-O-demethyl-FK520 (31-O-디메틸-FK520), 9-deoxo-31-O-demethyl-FK520 (9-데옥소-31-O-디메틸-FK520), 또는 9-deoxo-FK523 (9-데옥소-FK523), 9-deoxo-31-O-demethyl-FK523 (9-데옥소-31-O-디메틸-FK523)의 제조 및 활용에 관한 것으로, 구체적으로는 상기 신규 화합물의 제조방법, 및 상기 신규 화합물을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다. The present invention is a novel compound 9-deoxo-31- O- demethyl-prolyl-FK506 (9-deoxo-31- O -dimethyl-prolyl-) that can be utilized as a main component of a pharmaceutical composition for preventing or treating neurological diseases FK506), 9-deoxo-36,37-dihydro-FK506 (9-deoxo-36,37-dihydro-FK506), 31- O- demethyl-36,37-dihydro-FK506 (31- O -dimethyl- 36,37- dihydro -FK506), 9-deoxo-31- O -demethyl-36,37-dihydro-FK506 (9- Deoxo -31- O - dimethyl -36,37- dihydro -FK506), 9 -deoxo-FK520 (9-deoxo-FK520), 31- O- demethyl-FK520 (31- O -dimethyl-FK520), 9-deoxo-31- O -demethyl-FK520 (9-deoxo-31- O Preparation and utilization of -dimethyl-FK520), or 9-deoxo-FK523 (9-deoxo-FK523), 9-deoxo-31- O- demethyl-FK523 (9-deoxo-31- O -dimethyl-FK523) In particular, it relates to a method for preparing the novel compound, and a pharmaceutical composition for the prevention or treatment of a nervous system disease comprising the novel compound as an active ingredient.
신경손상은 뇌, 척추, 말초신경 등 다양한 신경계에서 나타날 수 있는데, 그에 따른 질병으로는 신경세포의 손실로 나타나는 퇴행성 신경질환 (Neurodegenerative disease), 말초신경장애 (Peripheral nerve injury), 외상성뇌손상 (Traumatic brain injury)과 뇌혈관장애로 나타나는 뇌졸중 (Cerebral infarction) 등이 있다. 중추신경계의 신경손상에 의해 나타나는 퇴행성 뇌질환은 치매, 파킨슨병, 헌팅턴병, 근위축성 측색 경화증 (Amyotrophic lateral sclerosis)이 대표적이라 할 수 있다. 퇴행성 신경질환 중에서는 파킨슨병과 알츠하이머병이 대부분을 차지하며, 헌팅턴병과 근위축성 측색경화증은 발생 환자수가 비교적 적다. 말초신경장애는 여러 가지 원인에 의해 말초신경계의 손상으로 발병되는데, 원인의 60%는 당뇨병에 의한 합병증이고 암을 치료하기 위한 화학요법 (Chemotherapy) 중에 말초신경장애 질환을 얻는 경우도 많다. 외상성뇌손상은 교통사고나 낙상 등의 외상으로 인해 뇌조직이 손상을 입은 것으로, 사망환자가 많이 발생되고 신체적 장애가 높은 질환이라 할 수 있다. Nerve damage can occur in a variety of nervous systems, such as the brain, spine, and peripheral nerves. The resulting diseases are neurodegenerative disease, peripheral nerve injury, and traumatic brain injury. brain injury) and cerebral infarction. Degenerative brain diseases caused by nerve damage in the central nervous system may be dementia, Parkinson's disease, Huntington's disease, and Amyotrophic lateral sclerosis. Among degenerative neurological diseases, Parkinson's disease and Alzheimer's disease make up the majority, and Huntington's disease and amyotrophic lateral sclerosis have relatively few patients. Peripheral nerve disorders are caused by damage to the peripheral nervous system by various causes, and 60% of the causes are complications caused by diabetes and often get peripheral nerve disorders during chemotherapy to treat cancer. Traumatic brain injury is a disease in which brain tissue is damaged due to trauma such as a traffic accident or a fall.
현재, 이들 신경손상 질환들에 대하여 손상된 신경을 회복시키고 신경세포를 재생시키는 등의 근본적 치료가 실시되지 못하고 통증 완화나 악화 완화 등을 목적으로 한 치료가 행하여지고 있다. 이는 신경손상을 지연시키는 효과를 넘어 신경손상 질환의 치료에 사용될 수준의 신경재생 및 회복 효과를 제공해 주는 약물이 없었기 때문이다. 신경손상에 의한 장애가 발생한 환자는 사회 및 가정에서의 역할에 크게 제한을 받게 되고, 이로 인한 개인적 손실 및 삶의 질 악화 등을 고려할 때, 신경손상에 관련되는 다양한 질환들에 단독 또는 병용 요법으로 활용될 수 있는 신경재생의 효과를 제공해 줄 수 있는 약물의 개발이 절실하다 할 수 있다. Currently, the basic treatments such as restoring damaged nerves and regenerating nerve cells are not carried out for these neurological damage diseases, and treatments aimed at alleviating pain or alleviating exacerbations are being performed. This is because there is no drug that provides a level of nerve regeneration and recovery effect to be used in the treatment of neuroinjury diseases beyond the effect of delaying the nerve damage. Patients with neurological impairment are greatly limited by their role in society and at home, and considering personal loss and deterioration in quality of life due to this, they can be used alone or in combination therapy for various diseases related to neurological damage. It can be said that the development of drugs that can provide the effect of nerve regeneration can be urgent.
또한, 한국공개특허 제10-2005-0071491호 (발명의 명칭: 천식의 치료를 위한 베타2-작용제와 배합된 타크롤리무스 유도체의 용도)는 급성 또는 만성 천식을 치료 또는 예방하기 위해 동시적, 분리적 또는 순차적으로 사용하기 위한 의약의 제조를 위한 FK506 유도체 및 베타2-작용제의 신규한 용도를 개시하고 있으나, FK506 유도체, FK520 유도체 또는 FK523 유도체를 신경계 질환을 치료하는 데 적용한 사례는 없다.In addition, Korean Patent Publication No. 10-2005-0071491 (name of the invention: the use of a tacrolimus derivative in combination with a beta2-agonist for the treatment of asthma) is simultaneous to treat or prevent acute or chronic asthma, Although novel use of FK506 derivatives and beta2-agonists for the manufacture of a medicament for separate or sequential use has been disclosed, no FK506 derivative, FK520 derivative or FK523 derivative has been applied to treat neurological diseases.
이에, 본 발명자들은 다양한 노력을 한 결과로 신경계 질환의 예방 또는 치료용 약학적 조성물의 주요 성분으로 활용이 가능한 신규 화합물 9-deoxo-31-O-demethyl-prolyl-FK506, 9-deoxo-36,37-dihydro-FK506, 31-O-demethyl-36,37-dihydro-FK506, 9-deoxo-31-O-demethyl-36,37-dihydro-FK506, 9-deoxo-FK520, 31-O-demethyl-FK520, 9-deoxo-31-O-demethyl-FK520, 9-deoxo-FK523, 및 9-deoxo-31-O-demethyl-FK523 (이하 '9종 신규 화합물'로 통칭)의 신경세포 성장 촉진 효과를 규명하고, 이들의 제조공정을 개발하고, 더 나아가 이들을 유효성분으로 활용한 신경계 질환의 예방 또는 치료용 약학적 조성물을 개발하고, 이 조성물이 신경계 질환의 예방 또는 치료용 약학적 조성물로서 효과적으로 활용될 수 있음을 확인함으로써 본 발명을 완성하였다. Accordingly, the present inventors, as a result of various efforts, a novel compound 9-deoxo-31- O- demethyl-prolyl-FK506, 9-deoxo-36, which can be used as a main component of a pharmaceutical composition for preventing or treating neurological diseases 37-dihydro-FK506, 31- O- demethyl-36,37-dihydro-FK506, 9-deoxo-31- O- demethyl-36,37-dihydro-FK506, 9-deoxo-FK520, 31- O- demethyl- FK520, 9-deoxo-31- O- demethyl-FK520, 9-deoxo-FK523, and 9-deoxo-31- O- demethyl-FK523 (hereinafter collectively referred to as '9 new compounds') promote the effect of promoting nerve cell growth. Identifying, developing their manufacturing process, and further developing a pharmaceutical composition for the prevention or treatment of neurological diseases utilizing them as an active ingredient, and the composition can be effectively utilized as a pharmaceutical composition for the prevention or treatment of neurological diseases The present invention has been completed by confirming that it is possible.
본 발명의 하나의 목적은 상기 9종 신규 화합물, 이의 이성질체, 또는 이의 약제학적으로 허용 가능한 염을 제공하는 것이다.One object of the present invention is to provide the above nine new compounds, isomers thereof, or pharmaceutically acceptable salts thereof.
본 발명의 목적은 9종 신규 화합물 중 선택된 하나 이상을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다. 여기서 신경계 질환의 예방 또는 치료용 약학적 조성물은 신경계 질환의 예방 또는 치료용 약학 조성물 또는 신경 재생 촉진용 조성물을 의미한다.An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a nervous system disease comprising at least one selected from nine new compounds as an active ingredient. Here, the pharmaceutical composition for preventing or treating a nervous system disease means a pharmaceutical composition for preventing or treating a nervous system disease or a composition for promoting nerve regeneration.
본 발명의 또 다른 목적은 9종 신규 화합물의 이성질체 또는 약제학적으로 허용 가능한 염 중 선택된 하나 이상을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다. 여기서 상기 약학적 조성물은 신경 재생 촉진용 조성물로 활용될 수 있다.Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurological diseases comprising at least one selected from isomers or pharmaceutically acceptable salts of nine new compounds as an active ingredient. Here, the pharmaceutical composition may be utilized as a composition for promoting nerve regeneration.
본 발명의 또 다른 목적은 9종 신규 화합물 각각의 생물학적 제조 방법을 제공하는 것이다.Another object of the present invention is to provide a biological production method for each of the nine new compounds.
본 발명의 또 다른 목적은 9종의 신규 화합물의 생물학적 제조공정에 이용될 수 있는 생산 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM (수탁번호 KCTC13581BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsB (수탁번호 KCTC13583BP), 및 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP)를 제공하는 것이다. Another object of the present invention is a production strain that can be used in the biological manufacturing process of nine new compounds, Streptomyces kanamyceticus ΔfkbD-fkbM (Accession No. KCTC13581BP), Streptomyces kanamyceticus ΔfkbD,tcsD (Accession number KCTC13580BP), Streptomyces kanamyceticus ΔfkbM,tcsD (Accession number KCTC13584BP), Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD (Accession number KCTC13585BP), Streptomyces kanamyset B (Accession No. KCTC13579BP), Streptomyces kanamyceticus ΔfkbM,tcsB (Accession No. KCTC13583BP), and Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB (Accession No. KCTC13582BP).
본 발명에 따른 9종 신규 화합물 중 선택된 하나 이상을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물은 적합한 유효 약물이 없었던 신경계 질환의 치료에 효과적으로 활용될 수 있다. 기존 약물 치료에 비교하여 보다 근본적인 치료 효과를 기대할 수 있다. The pharmaceutical composition for the prevention or treatment of a nervous system disease comprising at least one selected from the nine new compounds according to the present invention as an active ingredient can be effectively utilized in the treatment of a nervous system disease without a suitable effective drug. Compared to the existing drug treatment, a more fundamental treatment effect can be expected.
도 1은 9-deoxo-31-O-demethyl-prolyl-FK506에 대한 고성능액체크로마토그래피 분석 결과이다.1 is a high-performance liquid chromatography analysis results for 9-deoxo-31- O- demethyl-prolyl-FK506.
도 2는 9-deoxo-31-O-demethyl-prolyl-FK506에 대한 핵자기공명 분석 (1H-NMR) 결과이다.2 is a nuclear magnetic resonance analysis ( 1 H-NMR) results for 9-deoxo-31- O- demethyl-prolyl-FK506.
도 3은 9-deoxo-31-O-demethyl-prolyl-FK506에 대한 핵자기공명 분석 (13C-NMR) 결과이다.3 is a nuclear magnetic resonance analysis ( 13 C-NMR) results for 9-deoxo-31- O- demethyl-prolyl-FK506.
도 4는 9-deoxo-31-O-demethyl-prolyl-FK506에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.4 is a nuclear magnetic resonance analysis (COSY-NMR) results for 9-deoxo-31- O- demethyl-prolyl-FK506.
도 5는 9-deoxo-31-O-demethyl-prolyl-FK506에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.5 is a nuclear magnetic resonance analysis (HSQC-NMR) results for 9-deoxo-31- O- demethyl-prolyl-FK506.
도 6은 9-deoxo-31-O-demethyl-prolyl-FK506에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.6 is a nuclear magnetic resonance analysis (HMBC-NMR) results for 9-deoxo-31- O- demethyl-prolyl-FK506.
도 7은 9-deoxo-36,37-dihydro-FK506에 대한 고성능액체크로마토그래피 분석 결과이다.7 is a high-performance liquid chromatography analysis results for 9-deoxo-36,37-dihydro-FK506.
도 8은 9-deoxo-36,37-dihydro-FK506에 대한 핵자기공명 분석 (1H-NMR) 결과이다.8 is a nuclear magnetic resonance analysis ( 1 H-NMR) results for 9-deoxo-36,37-dihydro-FK506.
도 9는 9-deoxo-36,37-dihydro-FK506에 대한 핵자기공명 분석 (13C-NMR) 결과이다.9 is a nuclear magnetic resonance analysis ( 13 C-NMR) results for 9-deoxo-36,37-dihydro-FK506.
도 10은 9-deoxo-36,37-dihydro-FK506에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.10 is a nuclear magnetic resonance analysis (COSY-NMR) results for 9-deoxo-36,37-dihydro-FK506.
도 11은 9-deoxo-36,37-dihydro-FK506에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.11 is a nuclear magnetic resonance analysis (HSQC-NMR) results for 9-deoxo-36,37-dihydro-FK506.
도 12는 9-deoxo-36,37-dihydro-FK506에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.12 is a nuclear magnetic resonance analysis (HMBC-NMR) results for 9-deoxo-36,37-dihydro-FK506.
도 13은 31-O-demethyl-36,37-dihydro-FK506에 대한 고성능액체크로마토그래피 분석 결과이다.13 is a high-performance liquid chromatography analysis results for 31- O- demethyl-36,37-dihydro-FK506.
도 14는 31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (1H-NMR) 결과이다.14 is a nuclear magnetic resonance analysis ( 1 H-NMR) results for 31- O- demethyl-36,37-dihydro-FK506.
도 15는 31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (13C-NMR) 결과이다.15 is a nuclear magnetic resonance analysis ( 13 C-NMR) results for 31- O- demethyl-36,37-dihydro-FK506.
도 16은 31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.16 is a nuclear magnetic resonance analysis (COSY-NMR) results for 31- O- demethyl-36,37-dihydro-FK506.
도 17은 31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.FIG. 17 shows the results of nuclear magnetic resonance analysis (HSQC-NMR) for 31- O- demethyl-36,37-dihydro-FK506.
도 18은 31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.18 is a nuclear magnetic resonance analysis (HMBC-NMR) results for 31- O- demethyl-36,37-dihydro-FK506.
도 19는 9-deoxo-31-O-demethyl-36,37-dihydro-FK506에 대한 고성능액체크로마토그래피 분석 결과이다.19 is a high-performance liquid chromatography analysis results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506.
도 20은 9-deoxo-31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (1H-NMR) 결과이다.20 is a nuclear magnetic resonance analysis ( 1 H-NMR) results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506.
도 21은 9-deoxo-31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (13C-NMR) 결과이다.21 is a nuclear magnetic resonance analysis ( 13 C-NMR) results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506.
도 22는 9-deoxo-31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.22 is a nuclear magnetic resonance analysis (COSY-NMR) results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506.
도 23은 9-deoxo-31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.23 is a nuclear magnetic resonance analysis (HSQC-NMR) results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506.
도 24는 9-deoxo-31-O-demethyl-36,37-dihydro-FK506에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.24 is a nuclear magnetic resonance analysis (HMBC-NMR) results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506.
도 25는 9-deoxo-FK520에 대한 고성능액체크로마토그래피 분석 결과이다.25 is a high-performance liquid chromatography analysis results for 9-deoxo-FK520.
도 26은 9-deoxo-FK520에 대한 핵자기공명 분석 (1H-NMR) 결과이다.26 is a nuclear magnetic resonance analysis ( 1 H-NMR) results for 9-deoxo-FK520.
도 27은 9-deoxo-FK520에 대한 핵자기공명 분석 (13C-NMR) 결과이다.27 is a nuclear magnetic resonance analysis ( 13 C-NMR) results for 9-deoxo-FK520.
도 28은 9-deoxo-FK520에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.28 is a nuclear magnetic resonance analysis (COSY-NMR) results for 9-deoxo-FK520.
도 29는 9-deoxo-FK520에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.29 is a nuclear magnetic resonance analysis (HSQC-NMR) results for 9-deoxo-FK520.
도 30은 9-deoxo-FK520에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.30 is a nuclear magnetic resonance analysis (HMBC-NMR) results for 9-deoxo-FK520.
도 31은 31-O-demethyl-FK520에 대한 고성능액체크로마토그래피 분석 결과이다.FIG. 31 shows the results of high performance liquid chromatography analysis for 31- O- demethyl-FK520.
도 32는 31-O-demethyl-FK520에 대한 핵자기공명 분석 (1H-NMR) 결과이다.FIG. 32 shows the results of nuclear magnetic resonance analysis ( 1 H-NMR) for 31- O- demethyl-FK520.
도 33은 31-O-demethyl-FK520에 대한 핵자기공명 분석 (13C-NMR) 결과이다.FIG. 33 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 31- O- demethyl-FK520.
도 34는 31-O-demethyl-FK520에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.FIG. 34 is a nuclear magnetic resonance analysis (COSY-NMR) result for 31- O- demethyl-FK520.
도 35는 31-O-demethyl-FK520에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.35 shows nuclear magnetic resonance analysis (HSQC-NMR) results for 31- O- demethyl-FK520.
도 36은 31-O-demethyl-FK520에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.36 is a nuclear magnetic resonance analysis (HMBC-NMR) results for 31- O- demethyl-FK520.
도 37은 9-deoxo-31-O-demethyl-FK520에 대한 고성능액체크로마토그래피 분석 결과이다.37 is a high-performance liquid chromatography analysis results for 9-deoxo-31- O- demethyl-FK520.
도 38은 9-deoxo-31-O-demethyl-FK520에 대한 핵자기공명 분석 (1H-NMR) 결과이다.38 is a nuclear magnetic resonance analysis ( 1 H-NMR) results for 9-deoxo-31- O- demethyl-FK520.
도 39는 9-deoxo-31-O-demethyl-FK520에 대한 핵자기공명 분석 (13C-NMR) 결과이다.FIG. 39 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 9-deoxo-31- O- demethyl-FK520.
도 40은 9-deoxo-31-O-demethyl-FK520에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.40 shows nuclear magnetic resonance analysis (COSY-NMR) results for 9-deoxo-31- O- demethyl-FK520.
도 41은 9-deoxo-31-O-demethyl-FK520에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.41 shows nuclear magnetic resonance analysis (HSQC-NMR) results for 9-deoxo-31- O- demethyl-FK520.
도 42는 9-deoxo-31-O-demethyl-FK520에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.42 is a nuclear magnetic resonance analysis (HMBC-NMR) results for 9-deoxo-31- O- demethyl-FK520.
도 43은 9-deoxo-FK523에 대한 고성능액체크로마토그래피 분석 결과이다.43 is a high-performance liquid chromatography analysis results for 9-deoxo-FK523.
도 44는 9-deoxo-FK523에 대한 핵자기공명 분석 (1H-NMR) 결과이다.FIG. 44 shows the results of nuclear magnetic resonance analysis ( 1 H-NMR) for 9-deoxo-FK523.
도 45는 9-deoxo-FK523에 대한 핵자기공명 분석 (13C-NMR) 결과이다.FIG. 45 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 9-deoxo-FK523.
도 46은 9-deoxo-FK523에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.46 shows nuclear magnetic resonance analysis (COSY-NMR) results for 9-deoxo-FK523.
도 47은 9-deoxo-FK523에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.47 is a nuclear magnetic resonance analysis (HSQC-NMR) results for 9-deoxo-FK523.
도 48은 9-deoxo-FK523에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.48 shows nuclear magnetic resonance analysis (HMBC-NMR) results for 9-deoxo-FK523.
도 49는 9-deoxo-31-O-demethyl-FK523에 대한 고성능액체크로마토그래피 분석 결과이다.49 is a high-performance liquid chromatography analysis results for 9-deoxo-31- O- demethyl-FK523.
도 50은 9-deoxo-31-O-demethyl-FK523에 대한 핵자기공명 분석 (1H-NMR) 결과이다.FIG. 50 shows the results of nuclear magnetic resonance analysis ( 1 H-NMR) for 9-deoxo-31- O- demethyl-FK523.
도 51은 9-deoxo-31-O-demethyl-FK523에 대한 핵자기공명 분석 (13C-NMR) 결과이다.FIG. 51 shows the results of nuclear magnetic resonance analysis ( 13 C-NMR) for 9-deoxo-31- O- demethyl-FK523.
도 52는 9-deoxo-31-O-demethyl-FK523에 대한 핵자기공명 분석 (COSY-NMR) 결과이다.52 shows nuclear magnetic resonance analysis (COSY-NMR) results for 9-deoxo-31- O- demethyl-FK523.
도 53은 9-deoxo-31-O-demethyl-FK523에 대한 핵자기공명 분석 (HSQC-NMR) 결과이다.53 shows the results of nuclear magnetic resonance analysis (HSQC-NMR) for 9-deoxo-31- O- demethyl-FK523.
도 54는 9-deoxo-31-O-demethyl-FK523에 대한 핵자기공명 분석 (HMBC-NMR) 결과이다.FIG. 54 shows the results of nuclear magnetic resonance analysis (HMBC-NMR) for 9-deoxo-31- O- demethyl-FK523.
도 55는 본 발명의 신규 화합물 9종의 면역억제활성 감소 정도를 조사한 결과이다.55 is a result of investigating the degree of reduction in immunosuppressive activity of 9 novel compounds of the present invention.
도 56은 본 발명의 신규 화합물 9종의 신경세포 성장 촉진능(in vitro)을 조사한 결과이다. Figure 56 shows the results of investigating the nerve cell growth promoting ability (in vitro) of 9 novel compounds of the present invention.
도 57은 본 발명의 신규 화합물 9종의 신경세포 성장 촉진능(in vivo)을 조사한 결과이다. Figure 57 shows the results of investigating the nerve cell growth promoting ability (in vivo) of 9 new compounds of the present invention.
도 58은 본 발명의 신규 화합물 9종의 신경계 질환 치료능을 조사한 결과이다 (로타로드 테스트, 오픈필드 테스트).Figure 58 is a result of investigating the ability to treat neurological diseases of nine new compounds of the present invention (Rotarod test, open field test).
도 59은 본 발명의 신규 화합물 9종의 기능성 시냅스 형성 활성을 조사한 결과이다 (시냅스 전류의 빈도 확인). 59 is a result of investigating the functional synapse-forming activity of 9 novel compounds of the present invention (check the frequency of synaptic current).
도 60은 본 발명의 신규 화합물 9종의 기능성 시냅스 형성 활성을 조사한 결과이다 (시냅스 전류의 진폭 확인). 60 is a result of investigating the functional synapse-forming activity of 9 novel compounds of the present invention (amplification of synaptic current).
도 61은 본 발명의 신규 화합물 9종의 신경계 질환 치료능을 조사한 결과이다 (BBB 테스트). 61 is a result of investigating the ability to treat neurological diseases of nine new compounds of the present invention (BBB test).
도 62은 본 발명의 신규 화합물 9종의 신경계 질환 치료능을 조사한 결과이다 (그리드 워크 테스트). Fig. 62 shows the results of investigating the treatment ability of 9 new compounds of the present invention for nervous system diseases (grid work test).
이하에서는, 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
한편, 본원에서 개시되는 각각의 설명 및 실시 형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본원에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술되는 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 할 수 없다.Meanwhile, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.
또한, 당해 기술 분야의 통상의 지식을 가진 자는 통상의 실험만을 사용하여 본 출원에 기재된 본 발명의 특정 양태에 대한 다수의 등가물을 인지하거나 확인할 수 있다. 또한, 이러한 등가물은 본 발명에 포함되는 것으로 의도된다. In addition, one of ordinary skill in the art can recognize or confirm a number of equivalents to certain aspects of the invention described in this application using only routine experimentation. In addition, such equivalents are intended to be included in the present invention.
상기와 같은 과제를 해결하기 위하여, 본 발명은 9종 신규 화합물의 제조공정, 제조공정을 이용하여 제조한 각 신규 화합물을 포함하는 신경계 질환 의 예방 또는 치료용 약학적 조성물, 및 이 신경계 질환의 예방 또는 치료용 약학적 조성물을 이용한 신경계 질환에 대한 치료방법을 제공한다. In order to solve the problems as described above, the present invention is a pharmaceutical composition for the prevention or treatment of a nervous system disease, including a manufacturing process of nine new compounds, each new compound prepared using the manufacturing process, and the prevention of this nervous system disease Or it provides a treatment method for a nervous system disease using a therapeutic pharmaceutical composition.
상기의 목적을 달성하기 위한 하나의 양태로서, 본 발명은 9종 신규 화합물, 하기 [화학식 1]로 표시되는 9-deoxo-31-O-demethyl-prolyl-FK506, 하기 [화학식 2]로 표시되는 9-deoxo-36,37-dihydro-FK506, 하기 [화학식 3]으로 표시되는 31-O-demethyl-36,37-dihydro-FK506, 하기 [화학식 4]로 표시되는 9-deoxo-31-O-demethyl-36,37-dihydro-FK506, 하기 [화학식 5]로 표시되는 9-deoxo-FK520, 하기 [화학식 6]으로 표시되는 31-O-demethyl-FK520, 하기 [화학식 7]로 표시되는 9-deoxo-31-O-demethyl-FK520, 하기 [화학식 8]로 표시되는 9-deoxo-FK523, 하기 [화학식 9]로 표시되는 9-deoxo-31-O-demethyl-FK523, 그의 이성질체 또는 약제학적으로 허용가능한 염을 제공한다.As one aspect for achieving the above object, the present invention is a new compound, 9-deoxo-31- O- demethyl-prolyl-FK506 represented by the following [Formula 1], represented by the following [Formula 2] 9-deoxo-36,37-dihydro-FK506, 31- O- demethyl-36,37-dihydro-FK506 represented by the following [Formula 3], 9-deoxo-31- O -represented by the following [Formula 4] demethyl-36,37-dihydro-FK506, 9-deoxo-FK520 represented by the following [Formula 5], 31- O- demethyl-FK520 represented by the following [Formula 6], 9- represented by the following [Formula 7] deoxo-31- O- demethyl-FK520, 9-deoxo-FK523 represented by the following [Formula 8], 9-deoxo-31- O- demethyl-FK523 represented by the following [Formula 9], isomers or pharmaceuticals thereof An acceptable salt is provided.
[화학식 1][Formula 1]
Figure PCTKR2019017519-appb-I000001
Figure PCTKR2019017519-appb-I000001
[화학식 2][Formula 2]
Figure PCTKR2019017519-appb-I000002
Figure PCTKR2019017519-appb-I000002
[화학식 3][Formula 3]
Figure PCTKR2019017519-appb-I000003
Figure PCTKR2019017519-appb-I000003
[화학식 4][Formula 4]
Figure PCTKR2019017519-appb-I000004
Figure PCTKR2019017519-appb-I000004
[화학식 5][Formula 5]
Figure PCTKR2019017519-appb-I000005
Figure PCTKR2019017519-appb-I000005
[화학식 6][Formula 6]
Figure PCTKR2019017519-appb-I000006
Figure PCTKR2019017519-appb-I000006
[화학식 7][Formula 7]
Figure PCTKR2019017519-appb-I000007
Figure PCTKR2019017519-appb-I000007
[화학식 8][Formula 8]
Figure PCTKR2019017519-appb-I000008
Figure PCTKR2019017519-appb-I000008
[화학식 9][Formula 9]
Figure PCTKR2019017519-appb-I000009
Figure PCTKR2019017519-appb-I000009
본 발명에서 사용된 용어, "FK506, 타크롤리무스 (Tacrolimus) 또는 푸지마이신 (fujimycin)"은 다양한 스트렙토마이세스 쯔꾸바엔시스 (Streptomyces tsukubaensis)로 부터 최초로 분리된 면역억제활성을 갖는 물질로 23-원 (23-Membered) 폴리케티드 (Polyketide) 매크롤리드이다. 면역억제작용은 사이클로스포린보다도 강하고, 장기이식 시 특히 간을 이식할 때 거부반응을 억제시키기 위해 사용하는 것으로 알려져 있다. 상기 FK506은 PKS/NRPS (Polyketide synthase/nonribosomal peptide synthetase) 복합 시스템에 의해 합성될 수 있으나, 본 발명의 FK506 유도체는 스트렙토마이세스 속의 생합성 유전자 결손을 통해 제조된 신규한 균주를 통해 생산된 신규한 화합물일 수 있다.As used herein, the term "FK506, tacrolimus (Tacrolimus) or fujimycin (fujimycin)" is a substance having the first immunosuppressive activity isolated from the various Streptomyces tsukubaensis ( Streptomyces tsukubaensis ) 23-won ( 23-Membered Polyketide macrolide. Immunosuppressive action is stronger than cyclosporine and is known to be used to suppress rejection, especially when transplanting the liver during organ transplantation. The FK506 can be synthesized by the PKS/NRPS (Polyketide synthase/nonribosomal peptide synthetase) complex system, but the FK506 derivative of the present invention is a novel compound produced through a novel strain produced through a biosynthetic gene defect in Streptomyces genus. Can be
본 발명에서 사용된 용어, "FK520 또는 아스코마이신 (Ascomycin)"은 역시 면역억제제로서, 23개의 탄소로 형성되는 매크로리드 구조의 화합물이며, FK506의 C21 위치의 에틸 유사체이다. The term "FK520 or ascomycin", as used herein, is also an immunosuppressant, a compound of 23 macro-carbon macrolead structures, an ethyl analogue of the C21 position of FK506.
본 발명에서 사용된 용어, "FK523"는 FK506의 C21 위치의 메틸 유사체이다. As used herein, the term "FK523" is the methyl analog of the C21 position of FK506.
하나의 구체예로서, 본 발명의 화합물은 그의 이성질체 또는 약제학적으로 허용가능한 염을 포함할 수 있다.In one embodiment, the compounds of the present invention may include isomers or pharmaceutically acceptable salts thereof.
이성질체란 화학식은 같으나 동일하지는 않은 화합물의 관계를 의미하며, 예를 들어 구조 이성질체, 기하 이성질체, 광학 이성질체 (거울상 이성질체), 입체 이성질체, 부분 입체 이성질체를 포함할 수 있다.Isomer means the relationship of compounds having the same chemical formula but not the same, and may include, for example, structural isomers, geometric isomers, optical isomers (enantiomers), stereoisomers, diastereomers.
약제학적으로 허용가능한 염은 환자에게 비교적 비독성이고 무해한 유효작용을 갖는 농도로서, 이 염에 기인한 부작용이 모체 화합물의 이로운 효능을 저하시키지 않는 임의의 모든 유기 또는 무기 부가염을 의미할 수 있다. 예를 들어 상기 염은 약학적으로 허용가능한 유리산 (Free acid)에 의해 형성된 산부가염일 수 있다. 산부가염은 통상의 방법, 예를 들어 화합물을 과량의 산 수용액에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들어 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 동 몰량의 화합물 및 물중의 산 또는 알코올 (예, 글리콜 모노메틸에테르)을 가열하고, 이어서 상기 혼합물을 증발시켜 건조시키거나, 또는 석출된 염을 흡인 여과시켜 제조된 것일 수 있다. 이때, 유리산으로는 유기산과 무기산을 사용할 수 있다. 상기 염은 염기를 사용하여 제조된 약학적으로 허용가능한 금속염일 수 있다.Pharmaceutically acceptable salts are concentrations that have a relatively non-toxic and harmless effect on the patient, and can mean any and all organic or inorganic addition salts whose side effects caused by the salts do not diminish the beneficial efficacy of the parent compound. . For example, the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excess aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It may be prepared by heating an equal molar amount of the compound and an acid or alcohol (eg, glycol monomethyl ether) in water, followed by evaporation of the mixture to dryness or suction filtration of the precipitated salt. At this time, organic and inorganic acids can be used as the free acid. The salt may be a pharmaceutically acceptable metal salt prepared using a base.
또 하나의 구체예로서, 본 발명의 화합물은 용매화물 또는 전구약물 (Pro-drug) 형태일 수 있으며, 이는 본 발명의 범위 내에 포함된다. 용매화물은 바람직하게는 수화물 및 에탄올화물을 포함할 수 있다.In another embodiment, the compounds of the present invention may be in the form of solvates or pro-drugs, which are included within the scope of the present invention. Solvates may preferably include hydrates and ethanolates.
본 발명의 약학적 조성물은 단일제제로도 사용할 수 있고, 공인된 신경계 질환 치료 효과를 가진다고 알려진 약물을 추가로 포함하여 복합제제로 제조하여 사용할 수 있으며, 약제학적으로 허용되는 담체 또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 다용량 용기 내에 내입시켜 제조될 수 있다. The pharmaceutical composition of the present invention can also be used as a single agent, and can be prepared and used as a combination preparation by further including a drug known to have an approved neurological disease treatment effect, and is formulated by using a pharmaceutically acceptable carrier or excipient. It can be made in unit dose form or can be made by incorporating into a multi-dose container.
본 발명에서 사용되는 용어, "약학적으로 허용 가능한 담체"란 생물체를 자극하지 않으면서, 주입되는 화합물의 생물학적 활성 및 특성을 저해하지 않는 담체 또는 희석제를 의미할 수 있다. 본 발명에 사용 가능한 상기 담체의 종류는 특별히 제한되지 아니하며 당해 기술 분야에서 통상적으로 사용되고 약학적으로 허용되는 담체라면 어느 것이든 사용할 수 있다. 상기 담체의 비제한적인 예로는, 트란스큐톨 (Transcutol), 폴리에틸렌 글리콜 (Polyethylene glycol), 트리아세틴 (Triacetin) 및 이들의 혼합물로 예시할 수 있는 공계면활성제 (Co-surfactant); 크레모포어 (Cremophor), 트윈 (Tween), 미리즈 (Myrj), 폴록사머 (Poloxamer), 플루로닉 (Pluronic), 루트롤 (Lutrol), 임비토르 (Imwitor), 스판 (Span), 라브라필 (Labrafil) 등의 단독 또는 혼합물로 예시할 수 있는 계면활성제 (Surfactant); 미그리올 (Miglyol), 캅텍스 (Captex), 에틸 올레이트 (Ethyl oleate) 등의 단독 또는 혼합물로 예시할 수 있는 오일 (Oil); 및, 에리소르빈산 (Erythorobic acid), 구연산 (Citric acid) 등의 단독 또는 혼합물로 예시할 수 있는 유기산류 등을 들 수 있다. 이들은 단독으로 사용되거나 2 종 이상을 혼합하여 사용될 수 있다.As used in the present invention, the term "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not inhibit the biological activity and properties of the compound being injected without stimulating the organism. The type of the carrier that can be used in the present invention is not particularly limited, and any carrier that is commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier, transcutol (Transcutol), polyethylene glycol (Polyethylene glycol), triacetin (Triacetin) and co-surfactants exemplified by mixtures thereof (Co-surfactant); Cremophor, Tween, Myrj, Poloxamer, Pluronic, Lutrol, Imwitor, Span, Labra Surfactant, which can be illustrated alone or in a mixture such as Labrafil; Oils which can be exemplified alone or in mixtures such as Miglyol, Captex, and Ethyl oleate; And organic acids that can be exemplified singly or in mixtures such as erythorobic acid and citric acid. These may be used alone or in combination of two or more.
또한, 필요한 경우 항산화제, 완충액 및/또는 정균제 등 다른 통상의 첨가제를 첨가하여 사용할 수 있으며, 희석제, 분산제, 계면 활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제 등으로 제제화하여 사용할 수 있다.In addition, if necessary, other conventional additives such as antioxidants, buffers, and/or bacteriostatic agents can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to form a solution such as aqueous solutions, suspensions, emulsions, etc. It can be formulated and used in dosage forms, pills, capsules, granules or tablets.
상기의 목적을 달성하기 위한 또 하나의 양태로서, 본 발명은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는 신경계 질환의 치료 방법을 제공한다. As another aspect for achieving the above object, the present invention provides a method of treating a nervous system disease, comprising administering the pharmaceutical composition to a subject.
본 발명에서 사용되는 용어, "개체"란, 신경계 질환이 발병되었거나 발병할 가능성이 있는 모든 동물을 의미할 수 있다. As used in the present invention, the term "individual" may mean any animal that has or is likely to develop a neurological disorder.
본 발명의 약학적 조성물은 약제학적으로 유효한 양의 9종 신규 화합물 중 선택된 하나 이상, 또는 이것의 이성질체나 염을 포함할 수 있다. 본 발명에서 용어, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 일반적으로 0.001 내지 1000 mg/kg의 양, 바람직하게는 0.05 내지 200 mg/kg, 보다 바람직하게는 0.1 내지 100 mg/kg의 양을 일일 1회 내지 수회로 나누어 투여할 수 있다. 그러나 본 발명의 목적상, 특정 환자에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 조성물, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다. The pharmaceutical composition of the present invention may include one or more selected from nine new compounds in a pharmaceutically effective amount, or isomers or salts thereof. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 It may be administered in an amount of 200 to 200 mg/kg, more preferably 0.1 to 100 mg/kg once to several times a day. However, for the purposes of the present invention, a specific therapeutically effective amount for a particular patient may include a specific composition, the age, weight, general health status of the patient, including the type and extent of the response to be achieved, and, in some cases, whether other agents are used. It is desirable to apply differently depending on various factors including sex and diet, time of administration, route of administration and composition, secretion rate of treatment, duration of treatment, and drugs used with or concurrently with the specific composition and similar factors well known in the pharmaceutical field.
본 발명의 약학적 조성물의 투여 빈도는 특별히 이에 제한되지 않으나, 1일 1회 투여하거나 또는 용량을 분할하여 수회 투여할 수 있다.The frequency of administration of the pharmaceutical composition of the present invention is not particularly limited, but may be administered once a day or divided into doses several times.
본 발명의 약학적 조성물의 투여 용량은 0.001 mg/kg 내지 1000 mg/kg일 수 있으며, 구체적으로는 0.05 mg/kg 내지 200 mg/kg, 0.1 mg/kg 내지 100 mg/kg, 0.1 mg/kg 내지 20 mg/kg 일 수 있으나 이에 제한 되는 것은 아니다. The dosage amount of the pharmaceutical composition of the present invention may be 0.001 mg/kg to 1000 mg/kg, specifically 0.05 mg/kg to 200 mg/kg, 0.1 mg/kg to 100 mg/kg, 0.1 mg/kg It may be 20 mg/kg, but is not limited thereto.
본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여할 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 유발을 최소화 하면서 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount while minimizing the induction of side effects, and can be easily determined by those skilled in the art.
본 발명에서 사용된 용어, "투여"는 어떠한 적절한 방법으로 환자에게 본 발명의 약학적 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 경구 또는 비경구의 다양한 경로를 통하여 투여될 수 있다.As used herein, the term "administration" refers to the introduction of the pharmaceutical composition of the present invention to a patient in any suitable way, the route of administration of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through a variety of routes.
본 발명에 따른 약학적 조성물의 투여 방식은 특별히 제한되지 아니하며, 당해 기술 분야에서 통상적으로 사용하는 방식에 따를 수 있다. 상기 투여 방식의 비제한적인 예로, 약학적 조성물을 경구 투여 또는 비경구 투여 방식으로 투여할 수 있다. 본 발명에 따른 약학적 조성물은 목적하는 투여 방식에 따라 다양한 제형으로 제작될 수 있다. The method of administration of the pharmaceutical composition according to the present invention is not particularly limited, and may be in accordance with a method commonly used in the art. As a non-limiting example of the above administration method, the pharmaceutical composition may be administered by oral administration or parenteral administration. The pharmaceutical composition according to the present invention may be prepared in various dosage forms depending on the desired mode of administration.
본 발명의 신경계 질환의 예방 또는 치료용 약학적 조성물은 신경계 질환의 치료 목적으로 사용될 수 있다.The pharmaceutical composition for preventing or treating diseases of the nervous system of the present invention may be used for the purpose of treating diseases of the nervous system.
본 발명에서 신경계 질환에는 신경손상질환이 포함되며, 신경손상질환으로는 퇴행성 신경질환 (Neurodegenerative disease), 말초신경장애 (Peripheral nerve injury), 외상성뇌손상 (Traumatic brain injury)과 뇌혈관장애로 나타나는 뇌졸중 (Cerebral infarction) 등이 예시될 수 있으나, 이에 국한되지 않음은 자명하다.In the present invention, neurological diseases include neurological diseases, and neurological diseases include neurodegenerative diseases, peripheral nerve injury, traumatic brain injury, and strokes caused by cerebrovascular disorders. (Cerebral infarction) may be exemplified, but is not limited thereto.
일 예로 상기 퇴행성 신경질환은 중추신경계의 신경세포에 퇴행성 변화가 나타나면서 여러 가지 증상을 유발하는 질환을 의미하는 것으로, 예를 들어 치매, 알츠하이머병 (Alzheimer's disease), 파킨슨 병 (Parkinson's disease), 진행성 핵상마비 (Progressive supranuclear palsy), 다계통 위축증 (Multiple system strophy), 감람핵-뇌교-소뇌 위축증 (Olivopontocerebellar atrophy: OPCA), 샤이-드래거 증후군 (Shy-Drager syndrome), 선조체-흑질 퇴행증 (Striatonigral degeneration), 헌팅톤병 (Huntington's disease), 근위축성 측색 경화증 (Amyotrophic lateral sclerosis;ALS), 본태성 진전증 (Essential tremor), 피질-기저핵 퇴행증 (Corticobasal ganlionic degeneration), 미만성 루이 소체 질환 (Diffuse Lewy body disease), 파킨스-ALS-치매 복합증 (Parkinson-ALS-dementia complex of Guam) 또는 픽병 (Pick's disease)일 수 있다.For example, the degenerative neurological disease refers to a disease that causes various symptoms while degenerative changes appear in neurons of the central nervous system. For example, dementia, Alzheimer's disease, Parkinson's disease, progressive Progressive supranuclear palsy, Multiple system strophy, Olive-pond-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatum-black matter degeneration (Striatonigral) degeneration), Huntington's disease, Amyotrophic lateral sclerosis (ALS), Essential tremor, Corticobasal ganlionic degeneration, Diffuse Lewy body disease ), Parkinson-ALS-dementia complex of Guam or Pick's disease.
다른 일 예로 상기 신경손상질환은 간질, 중풍, 뇌졸중, 허혈성 뇌질환, 척수 손상 질환, 말초신경질환, 행동 장애, 발달장애, 정신 지체, 다운증후군 또는 정신분열증을 포함할 수 있으나, 이에 제한되는 것은 아니다.As another example, the neurological damage disease may include epilepsy, stroke, stroke, ischemic brain disease, spinal cord injury disease, peripheral nerve disease, behavioral disorder, developmental disorder, mental retardation, Down syndrome or schizophrenia, but is not limited thereto. no.
또 다른 일 예로 상기 신경손상질환은 신경세포 손상 또는 세포 사멸 등을 원인으로 하는 질환일 수 있다.As another example, the nerve injury disease may be a disease caused by nerve cell damage or cell death.
본 발명에서 사용되는 용어, "예방"이란, 본 발명의 상기 약학적 조성물을 신경계 질환의 발병 가능성이 있거나 상기 질환이 의심되는 증상 또는 상태를 가진 개체에게 투여하여 신경계 질환의 발병 가능성을 낮추거나 상기 증상 또는 상태를 완화하는 행위를 의미한다.As used in the present invention, the term "prevention" means that the pharmaceutical composition of the present invention is administered to an individual who has a possible or suspected condition or condition of developing a nervous system disease, thereby lowering the likelihood of developing a nervous system disease or It refers to the act of alleviating symptoms or conditions.
본 발명에서 사용되는 용어, "치료"란, 본 발명의 상기 약학적 조성물을 신경계 질환 발병 의심 개체에 투여하여 신경계 질환의 증세가 호전되도록 하거나 이롭게 되도록 하는 모든 행위를 의미한다.As used in the present invention, the term "treatment" refers to all actions to improve or benefit the symptoms of a nervous system disease by administering the pharmaceutical composition of the present invention to a suspected neurological disease development subject.
본 발명의 일 구체예에서는 상기 9종 신규 화합물의 면역억제활성 효과를 인비트로 T세포 활성 분석법으로 확인하여 FK506 보다 감소된 면역억제활성을 확인할 수 있었다. In one embodiment of the present invention, the immunosuppressive activity effect of the nine new compounds was confirmed by an in vitro T cell activity assay to confirm the reduced immunosuppressive activity than FK506.
또한, 본 발명의 다른 구체예에서는 상기 9종 신규화합물의 신경 축삭 생성효과를 확인하여 신경세포 성장촉진 활성능을 확인하였다. In addition, in another embodiment of the present invention, the neuroaxon-producing effect of the nine new compounds was confirmed to confirm the ability to stimulate neuronal growth.
본 발명의 또 다른 구체예에서는 신경을 손상시킨 마우스의 9종 신규화합물을 투여 후 BBB 테스트 및 그리드 워크 테스트에 의하여 상기 9종 신규화합물의 신경계 질환 치료효과를 확인할 수 있었다. In another embodiment of the present invention, after administering 9 new compounds of a mouse with nerve damage, the BBB test and the grid walk test were used to confirm the therapeutic effects of the 9 new compounds on nervous system diseases.
상기 결과는 상기 9종 신규화합물이 면역억제활성에 기인한 부작용 없는 신경계 질환의 치료효과를 확인한 바, 신경예 질환의 예방 또는 치료에 유용하게 사용될 수 있음을 시사하는 것이다. The above results suggest that the nine new compounds can be usefully used for the prevention or treatment of neurological diseases, confirming the therapeutic effect of neurological diseases without side effects due to immunosuppressive activity.
상기의 목적을 달성하기 위한 또 다른 하나의 양태로서, 본 발명은 9종 신규 화합물인 9-deoxo-31-O-demethyl-prolyl-FK506, 9-deoxo-36,37-dihydro-FK506, 31-O-demethyl-36,37-dihydro-FK506, 9-deoxo-31-O-demethyl-36,37-dihydro-FK506, 9-deoxo-FK520, 31-O-demethyl-FK520, 9-deoxo-31-O-demethyl-FK520, 9-deoxo-FK523, 9-deoxo-31-O-demethyl-FK523의 생물학적 제조공정을 제공한다. As another aspect for achieving the above object, the present invention is 9 new compounds, 9-deoxo-31- O- demethyl-prolyl-FK506, 9-deoxo-36,37-dihydro-FK506, 31- O -demethyl-36,37-dihydro-FK506, 9-deoxo-31- O -demethyl-36,37-dihydro-FK506, 9-deoxo-FK520, 31- O -demethyl-FK520, 9-deoxo-31- Provides biological manufacturing processes of O- demethyl-FK520, 9-deoxo-FK523, and 9-deoxo-31- O- demethyl-FK523.
상기 생물학적 제조공정에서는 일반적으로 스트렙토마이세스 속 배양공정에서 채택되는 배양 온도를 사용한다. 본 발명의 구현에 적합한 배양 온도로는 바람직하게는 23-30℃를 적용할 수 있고, 보다 바람직하게는 25-28℃의 배양 온도를 적용할 수 있다. In the biological manufacturing process, the culture temperature generally employed in the culture process of Streptomyces genus is used. As a culture temperature suitable for the implementation of the present invention, preferably, 23-30°C may be applied, and more preferably, a culture temperature of 25-28°C may be applied.
또한, 상기 제조공정에서는 배양공정의 pH를 6.5-9 사이로 유지하는데, 바람직하게는 배양 pH를 7-8로 유지한다. In addition, in the manufacturing process, the pH of the culture process is maintained between 6.5 and 9, preferably, the culture pH is maintained at 7-8.
한편, 상기 제조공정에서는 배양액에서의 용존산소 수준을 높게 유지하는 것이 중요한데, 배양 초기의 용존산소 수준을 100%로 하였을 때 배양 종료 시점까지의 용존산소 수준을 30% 이상으로 유지하는 것이 중요하다. 이를 구현하기 위해서는 통상적으로 800-1,500 rpm 수준으로 교반해 주는 것이 바람직하다. On the other hand, in the manufacturing process, it is important to keep the dissolved oxygen level in the culture medium high. When the dissolved oxygen level at the beginning of the culture is 100%, it is important to maintain the dissolved oxygen level until the end of the culture at 30% or more. In order to implement this, it is generally desirable to stir at a level of 800-1,500 rpm.
상기 제조공정에서의 배양 세포체로부터의 생산된 9종 신규 화합물의 추출은 1차 추출공정, 2차 추출공정 및 3차 추출공정의 실시를 통하여 달성되는데, 본 발명에서는 1차 추출공정으로 유기용매 추출법을 사용하는데, 이때 사용할 수 있는 용매로는 에틸 아세테이트, 메탄올, 아세톤 등이 있을 수 있으나, 에틸 아세테이트 또는 메탄올의 사용이 바람직하다. 그리고 2차 추출공정으로 실리카겔 크로마토그래피 (Silica gel chromatography)를 사용하는데, 이때 사용할 수 있는 용매로는 메탄올 (Methanol), 메틸렌 클로라이드 (Methylene chloride)가 바람직하다. 그리고 3차 추출공정으로 크로마토그래피를 사용하는데, 이때 사용할 수 있는 용매로는 아세토니트릴, 아세트산암모늄 버퍼, 아세트산, 개미산 등이 있을 수 있으나, 아세토니트릴의 사용이 바람직하다. 이러한 방법의 적용은 9종 신규 화합물의 회수를 용이하게 하며, 또한 수율도 높게 해 준다.The extraction of the 9 new compounds produced from the cultured cell body in the above manufacturing process is achieved through the implementation of the primary extraction process, the secondary extraction process and the tertiary extraction process. In the present invention, the organic solvent extraction method is used as the primary extraction process. In this case, ethyl acetate, methanol, acetone, and the like may be used as the solvent, but ethyl acetate or methanol is preferred. In addition, silica gel chromatography is used as a secondary extraction process. As a solvent that can be used, methanol and methylene chloride are preferable. In addition, chromatography is used as the third extraction process. At this time, acetonitrile, ammonium acetate buffer, acetic acid, formic acid, etc. may be used as the solvent, but acetonitrile is preferred. The application of this method facilitates the recovery of 9 new compounds and also increases the yield.
상기의 목적을 달성하기 위한 또 다른 하나의 양태로서, 본 발명은 9종 신규 화합물의 제조에 이용될 수 있는 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM (수탁번호 KCTC13581BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP), 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsB (수탁번호 KCTC13583BP), 및 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP)를 제공한다.As another aspect for achieving the above object, the present invention is a production strain that can be used for the production of 9 new compounds, Streptomyces kanamyceticus ΔfkbD-fkbM (Accession No. KCTC13581BP), Streptomyces Kanamyceticus ΔfkbD,tcsD (Accession No. KCTC13580BP), Streptomyces kanamyceticus ΔfkbM,tcsD (Accession No. KCTC13584BP), Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD (Accession No. KTCTCD, 85BP CTC Kanamyceticus ΔfkbD,tcsB (Accession No. KCTC13579BP), Streptomyces kanamyceticus ΔfkbM,tcsB (Accession No. KCTC13583BP), and Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB (Accession No. .
이하, 하기 실시예에 의하여 본 발명을 보다 상세하게 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들로 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only for illustrating the present invention and the scope of the present invention is not limited to them.
실시예 1: 9-deoxo-31-Example 1: 9-deoxo-31- OO -demethyl-prolyl-FK506 제조-demethyl-prolyl-FK506 manufacture
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbD-fkbM 유전자의 비활성화를 초래하여 9-deoxo-31-O-demethyl-prolyl-FK506의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM (수탁번호 KCTC13581BP)을 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 ), resulting in inactivation of the fkbD-fkbM gene using the in-frame deletion method by Streptomyces kanamyceticus ΔfkbD, a production strain of 9-deoxo-31- O- demethyl-prolyl-FK506 -fkbM (Accession No. KCTC13581BP) was produced.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbD와 fkbM 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 in order to construct a mutant of the fkbD and fkbM genes in a strain of Streptomyces kanamyceticus producing FK506, and then transferred to a conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbD-fkbM 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbD-MLF/FkbD-MLR, 오른쪽-인접 절편의 프라이머 쌍 FkbD-MRF/FkbD-MRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 하기 표 1과 표 2에 제시하였다. The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbD-fkbM gene, the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example are presented in Tables 1 and 2 below.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. C9 수산화효소 (Hydroxylase)와 31-Ｏ-메틸변환효소를 함께 제거하기 위한 플라스미드, p△fkbD-fkbM는 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbD-fkbM를 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid for removing C9 hydroxylase (Hydroxylase) and 31- O -methyltransferase together, pΔfkbD-fkbM, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to target genes. It was deleted by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant. The colonies thus secured were allowed to multiply three times without selection at 28°C to allow the second crossover. Two achieved double crossover mutations, ΔfkbD-fkbM, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD-fkbM 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM은 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13581BP).The produced fkbD-fkbM gene-deficient strain, Streptomyces kanamyceticus ΔfkbD-fkbM, was deposited with the Korea Institute of Bioscience and Biotechnology (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession No. KCTC13581BP ).
Figure PCTKR2019017519-appb-T000001
Figure PCTKR2019017519-appb-T000001
Figure PCTKR2019017519-appb-I000010
Figure PCTKR2019017519-appb-I000010
Figure PCTKR2019017519-appb-T000002
Figure PCTKR2019017519-appb-T000002
Figure PCTKR2019017519-appb-I000011
Figure PCTKR2019017519-appb-I000011
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM (수탁번호 KCTC13581BP)의 배양을 통하여 9-deoxo-31-O-demethyl-prolyl-FK506을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 9-deoxo-31-O-demethyl-prolyl-FK506을 추출하였다. 1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 9-deoxo-31- O- demethyl-prolyl-FK506 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ΔfkbD-fkbM (Accession No. KCTC13581BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 9-deoxo-31- O- demethyl-prolyl-FK506 produced through the primary extraction process was extracted. The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration.
1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 9-deoxo-31-O-demethyl-prolyl-FK506이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 9-deoxo-31- O- demethyl-prolyl-FK506 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 1]로 표시되는 물질인 9-deoxo-31-O-demethyl-prolyl-FK506을 얻을 수 있었다.This was lyophilized to obtain 9-deoxo-31- O- demethyl-prolyl-FK506, a substance represented by [Formula 1] in powder form.
제조한 9-deoxo-31-O-demethyl-prolyl-FK506의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 9-deoxo-31-O-demethyl-prolyl-FK506에 대한 분석 결과는 표 3과 도 1 내지 도 6으로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM으로부터 9-deoxo-31-O-demethyl-prolyl-FK506이 생산됨을 확인할 수 있었다.Confirmation of the prepared 9-deoxo-31- O- demethyl-prolyl-FK506 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The analysis results for 9-deoxo-31- O- demethyl-prolyl-FK506 are summarized in Table 3 and FIGS. 1 to 6, and the production strain Strepttomyces canaaceticus produced from these results was obtained from ΔfkbD-fkbM. It was confirmed that -deoxo-31- O- demethyl-prolyl-FK506 was produced.
9-deoxo-31-O-demethyl-prolyl-FK506 (분자식 C41H57NO11, 분자량 749.4714)에 대한 분석 결과를 하기의 표 3에 나타내었다.The analysis results for 9-deoxo-31- O- demethyl-prolyl-FK506 (molecular formula C 41 H 57 NO 11 , molecular weight 749.4714) are shown in Table 3 below.
Figure PCTKR2019017519-appb-T000003
Figure PCTKR2019017519-appb-T000003
Figure PCTKR2019017519-appb-I000012
Figure PCTKR2019017519-appb-I000012
Figure PCTKR2019017519-appb-I000013
Figure PCTKR2019017519-appb-I000013
Figure PCTKR2019017519-appb-I000014
Figure PCTKR2019017519-appb-I000014
1H-NMR 및 13C-NMR로부터 특징적인 작용기로써 1개의 ketone 탄소 (δC 214.00)와 2개의 carbonyl 탄소 (δC 171.99, 170.09), exomehtylene 골격 (δC 116.84, 135.80)과 2개의 olefine 골격 (δC 141.22, 122.04;δC 132.64, 129.79)이 확인되었고, dioxygenated 4급탄소 (δC 98.73), oxygenated 메틴탄소 7개 (δC 78.28, 77.39, 75.28, 75.81, 74.73, 71.14, 69.23), 2개의 메톡시 탄소 (δC 57.98, 56.48)가 관측되었으며, 5개의 메틸탄소 (δC 19.10, 17.20, 15.90, 14.41, 10.13)가 관측되었으며, 탄소는 모두 42개로 이루어진 FK506 유도체로 관측되었다.Characteristic functional groups from 1 H-NMR and 13 C-NMR, 1 ketone carbon (δ C 214.00) and 2 carbonyl carbons (δ C 171.99, 170.09), exomehtylene skeleton (δ C 116.84, 135.80) and 2 olefine skeletons (δ C 141.22, 122.04; δ C 132.64, 129.79) were identified, dioxygenated quaternary carbon (δ C 98.73), 7 oxygenated methine carbons (δ C 78.28, 77.39, 75.28, 75.81, 74.73, 71.14, 69.23), Two methoxy carbons (δ C 57.98, 56.48) were observed, five methyl carbons (δ C 19.10, 17.20, 15.90, 14.41, 10.13) were observed, and all carbons were observed as 42 FK506 derivatives.
정확한 구조를 확인하기 위하여 2D-NMR을 확인하였다. gCOSY로부터 proton의 연결을 확인한 결과, H-2~H-4사이의 coupling으로부터 본 화합물은 pipecolyl 골격의 FK506이 아닌, CH2작용기 1개가 없는 prolyl 골격임을 확인하였다. gHMBC 데이터로부터 H-9 (δH 2.56, 2.63)이 C-8 (δC 171.99), C-10 (δC 98.73)과 correlation으로부터 본 화합물은 C-9에 ketone이 아닌 CH2로 환원된 골격임을 확인하였다. 이와 함께, 2개의 메톡시 작용기가 C-13, C-15에 결합되어 있어, C-31에는 메톡시가 존재하지 않는 구조임을 확인하였다. 이를 종합하여, 본 화합물은 9-deoxo-31-O-demethyl-prolyl-FK506임을 확인하였다.2D-NMR was confirmed to confirm the correct structure. As a result of confirming the connection of the proton from gCOSY, it was confirmed from the coupling between H-2 and H-4 that this compound was not the FK506 of the pipecolyl skeleton, but a prolyl skeleton without one CH 2 functional group. From the gHMBC data, H-9 (δ H 2.56, 2.63) and C-8 (δ C 171.99), C-10 (δ C 98.73) and the correlation of this compound are reduced to C-9 instead of ketone, CH 2 Was confirmed. Along with this, two methoxy functional groups were attached to C-13 and C-15, confirming that C-31 had no methoxy structure. Overall, it was confirmed that the present compound was 9-deoxo-31- O- demethyl-prolyl-FK506.
실시예 2: 9-deoxo-36,37-dihydro-FK506 제조Example 2: Preparation of 9-deoxo-36,37-dihydro-FK506
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbD 및 tcsD 유전자의 비활성화를 초래하여 9-deoxo-36,37-dihydro-FK506의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP)를 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 Streptomyces kanamyceticus ΔfkbD,tcsD, a production strain of 9-deoxo-36,37-dihydro-FK506, resulting in inactivation of the fkbD and tcsD genes using the in-frame deletion method by (Accession No. KCTC13580BP) was produced.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbD 및 tcsD 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 in order to construct a mutant of the fkbD and tcsD genes in a strain of Streptomyces kanamyceticus producing FK506, and then transferred to a conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbDLF/FkbDLR, 오른쪽-인접 절편의 프라이머 쌍 FkbDRF/FkbDRR을 설계하였고, tcsD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsDLF/TcsDLR, 오른쪽-인접 절편의 프라이머 쌍 TcsDRF/TcsDRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다. The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbD gene, the primer pair FkbDLF/FkbDLR of the left-neighbor segment and the primer pair FkbDRF/FkbDRR of the right-neighbor segment were designed, and for the deletion of the tcsD gene, the primer pair of the left-neighbor segment TcsDLF/TcsDLR, right- Primer pairs TcsDRF/TcsDRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. C9 수산화효소 (Hydroxylase)를 제거하기 위한 플라스미드, p△fkbD는 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbD를 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid to remove C9 hydroxylase (Hydroxylase), pΔfkbD, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to delete the target gene by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected as a culture of a transmycin-resistant transconjugant. Colonies secured thereafter were allowed to multiply three times without selection at 28°C to allow the second crossover. Two achieved double crossover mutations, ΔfkbD, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
제작한 fkbD 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbD에 p△tcsD를 도입하여 fkbD 유전자 결손 방법과 동일한 방법을 이용하여 tcsD 유전자를 결실시켰다. ΔfkbD,tcsD는 아프라마이신-민감성의 발현형질로 선택하였고, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The prepared fkbD gene was deleted by introducing pΔtcsD into ΔfkbD of Streptomyces kanamyceticus deficient, and the tcsD gene was deleted using the same method as the fkbD gene deletion method. ΔfkbD,tcsD was selected as apramycin-sensitive expression trait, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD, tcsD 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13580BP).The produced fkbD and tcsD gene-deficient strains, Streptomyces kanamyceticus ΔfkbD,tcsD, were deposited with the Korea Institute of Bioscience and Biotechnology (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession No. KCTC13580BP ).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP)의 배양을 통하여 9-deoxo-36,37-dihydro-FK506을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 9-deoxo-36,37-dihydro-FK506을 추출하였다. 9-deoxo-36,37-dihydro-FK506 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ΔfkbD,tcsD (Accession No. KCTC13580BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 9-deoxo-36,37-dihydro-FK506 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 9-deoxo-36,37-dihydro-FK506이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 9-deoxo-36,37-dihydro-FK506 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 2]로 표시되는 물질인 9-deoxo-36,37-dihydro-FK506을 얻을 수 있었다.This was lyophilized to obtain 9-deoxo-36,37-dihydro-FK506, a substance represented by [Formula 2] in powder form.
제조한 9-deoxo-36,37-dihydro-FK506의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 9-deoxo-36,37-dihydro-FK506에 대한 분석 결과는 표 4와 도 7 내지 도 12로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD로부터 9-deoxo-36,37-dihydro-FK506이 생산됨을 확인할 수 있었다.Confirmation of the prepared 9-deoxo-36,37-dihydro-FK506 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The analysis results for 9-deoxo-36,37-dihydro-FK506 are summarized in Table 4 and FIGS. 7 to 12, and the production strain Strepttomyces canaaceticus ΔfkbD,tcsD from 9-deoxo produced from these results It was confirmed that -36,37-dihydro-FK506 was produced.
9-deoxo-36,37-dihydro-FK506 (분자식 C44H73NO11, 분자량 792.06)에 대한 분석 결과를 하기의 표 4에 나타내었다.The results of the analysis for 9-deoxo-36,37-dihydro-FK506 (molecular formula C 44 H 73 NO 11 , molecular weight 792.06) are shown in Table 4 below.
Figure PCTKR2019017519-appb-T000004
Figure PCTKR2019017519-appb-T000004
Figure PCTKR2019017519-appb-I000015
Figure PCTKR2019017519-appb-I000015
Figure PCTKR2019017519-appb-I000016
Figure PCTKR2019017519-appb-I000016
Figure PCTKR2019017519-appb-I000017
Figure PCTKR2019017519-appb-I000017
1H-NMR, 13C-NMR 및 gHSQC로부터 총 탄소수 44개로써, 각각 메톡시 (δH 3.39, δC 56.56) 1개와 CH2 (δH 2.24, δH 1.70, δC 20.70) 1개가 추가 관측되었고, exomethylene의 작용기는 관측되지 않았으며, triplet coupling으로 관측되는 메틸기 (δH 0.88, δC 13.91)와 CH2 (δH 1.21, δC 20.41)가 관측되었다. 정확한 구조를 확인하기 위하여 2D-NMR을 확인하였다. gCOSY로부터 proton의 연결을 확인한 결과, H-2~H-5사이의 coupling으로부터 본 화합물은 pipecolyl 골격의 FK506인 것을 확인하였고, CH2 작용기 (δH 2.24, δH 1.70, δC 20.70) 1개가 pipecolyl 골격에 위치함을 확인하였다. gHMBC로부터 3개의 메톡시 수소 (δH 3.39, 3.36, 3.35)는 각각 C-31 (δC 77.11), C-15 (δC 77.11) 및 C-13 (δC 74.33) correlation된 것을 확인하였고, triplet coupling으로 관측되는 메틸기 (δH 0.88, δC 13.91)과 CH2 (δH 1.21, δC 20.41)가 C-21/33과 상호 correlation을 확인할 수 있었다. 따라서, tcsD가 제거된 균주로부터 만들어진 화합물로서, C-36/37 사이에 존재하던 이중결합이 환원된 형태의 9-deoxo-36,37-dihydro-FK506으로 구조 결정되었다.44 carbon atoms from 1 H-NMR, 13 C-NMR, and gHSQC, adding 1 methoxy (δ H 3.39, δ C 56.56) and 1 CH 2 (δ H 2.24, δ H 1.70, δ C 20.70), respectively No functional groups of exomethylene were observed, and methyl groups (δ H 0.88, δ C 13.91) and CH 2 (δ H 1.21, δ C 20.41) observed by triplet coupling were observed. 2D-NMR was confirmed to confirm the correct structure. As a result of confirming the proton connection from gCOSY, it was confirmed from the coupling between H-2 and H-5 that this compound was FK506 of the pipecolyl skeleton, and one CH 2 functional group (δ H 2.24, δ H 1.70, δ C 20.70) It was confirmed to be located in the pipecolyl skeleton. Three methoxy hydrogens (δ H 3.39, 3.36, 3.35) from gHMBC were correlated with C-31 (δ C 77.11), C-15 (δ C 77.11) and C-13 (δ C 74.33), respectively. The methyl group observed by triplet coupling (δ H 0.88, δ C 13.91) and CH 2 (δ H 1.21, δ C 20.41) were able to confirm cross-correlation with C-21/33. Therefore, as a compound made from the strain from which tcsD was removed, the double bond existing between C-36/37 was structurally determined to be a reduced form of 9-deoxo-36,37-dihydro-FK506.
실시예 3: 31-Example 3: 31- OO -demethyl-36,37-dihydro-FK506 제조-demethyl-36,37-dihydro-FK506 production
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbM 및 tcsD 유전자의 비활성화를 초래하여 31-O-demethyl-36,37-dihydro-FK506의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP)를 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 Streptomyces kanamyceticus ΔfkbM, a production strain of 31- O- demethyl-36,37-dihydro-FK506, resulting in inactivation of the fkbM and tcsD genes using the in-frame deletion method by) ,tcsD (Accession No. KCTC13584BP) was produced.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbM 및 tcsD 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 to produce a deletion mutant of the fkbM and tcsD genes in the Streptomyces kanamyceticus strain producing FK506, and then transferred to the conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbM 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbMLF/FkbMLR, 오른쪽-인접 절편의 프라이머 쌍 FkbMRF/FkbMRR을 설계하였고, tcsD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsDLF/TcsDLR, 오른쪽-인접 절편의 프라이머 쌍 TcsDRF/TcsDRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다. The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbM gene, the primer pair FkbMLF/FkbMLR of the left-neighbor segment and the primer pair FkbMRF/FkbMRR of the right-neighbor segment were designed, and for the deletion of the tcsD gene, the primer pair of the left-neighbor segment TcsDLF/TcsDLR, right- Primer pairs TcsDRF/TcsDRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. 31-Ｏ-메틸변환효소를 제거하기 위한 플라스미드, p△fkbM은 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbM을 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid for removing 31- O -methyltransferase, pΔfkbM, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation, thereby deleting the target gene by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant. The colonies thus secured were allowed to multiply three times without selection at 28°C to allow the second crossover. Two achieved double crossover mutations, ΔfkbM, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
제작한 fkbM 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbM에 p△tcsD를 도입하여 fkbM 유전자 결손 방법과 동일한 방법을 이용하여 tcsD 유전자를 결실시켰다. ΔfkbM,tcsD는 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The prepared fkbM gene was introduced into pΔtcsD in ΔfkbM of Streptomyces kanamyceticus deficient, and the tcsD gene was deleted using the same method as the fkbM gene deletion method. ΔfkbM,tcsD was selected as the expression trait of apramycin-sensitivity, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbM,tcsD 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13584BP).The produced fkbM and tcsD gene-deficient strains, Streptomyces kanamyceticus ΔfkbM,tcsD, were deposited with the Korea Institute of Biotechnology and Biotechnology Resource Center (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession number KCTC13584BP ).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP)의 배양을 통하여 31-O-demethyl-36,37-dihydro-FK506을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 31-O-demethyl-36,37-dihydro-FK506을 추출하였다. 31- O- demethyl-36,37-dihydro-FK506 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ΔfkbM,tcsD (Accession No. KCTC13584BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 31- O- demethyl-36,37-dihydro-FK506 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 31-O-demethyl-36,37-dihydro-FK506이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 31- O- demethyl-36,37-dihydro-FK506 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 3]으로 표시되는 물질인 31-O-demethyl-36,37-dihydro-FK506을 얻을 수 있었다.This was lyophilized to obtain 31- O- demethyl-36,37-dihydro-FK506, a substance represented by [Formula 3] in powder form.
제조한 31-O-demethyl-36,37-dihydro-FK506의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 31-O-demethyl-36,37-dihydro-FK506에 대한 분석 결과는 표 5와 도 13 내지 도 18로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD로부터 31-O-demethyl-36,37-dihydro-FK506이 생산됨을 확인할 수 있었다.Confirmation of the prepared 31- O- demethyl-36,37-dihydro-FK506 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The analysis results for 31- O- demethyl-36,37-dihydro-FK506 are summarized in Table 5 and FIGS. 13 to 18, and from the results, the production strain Streptomyces canaaceticus ΔfkbM,tcsD 31 -It was confirmed that O- demethyl-36,37-dihydro-FK506 was produced.
31-O-demethyl-36,37-dihydro-FK506 (분자식 C43H69NO12, 분자량 792.02)에 대한 분석 결과를 하기의 표 5에 나타내었다.The analysis results for 31- O- demethyl-36,37-dihydro-FK506 (molecular formula C 43 H 69 NO 12 , molecular weight 792.02) are shown in Table 5 below.
Figure PCTKR2019017519-appb-T000005
Figure PCTKR2019017519-appb-T000005
Figure PCTKR2019017519-appb-I000018
Figure PCTKR2019017519-appb-I000018
Figure PCTKR2019017519-appb-I000019
Figure PCTKR2019017519-appb-I000019
Figure PCTKR2019017519-appb-I000020
Figure PCTKR2019017519-appb-I000020
1H-NMR, 13C-NMR 및 gHSQC로부터, 총 탄소수 43개로써, ketone 탄소 (δC 196.54)가 추가 관측되었고, 2개의 메톡시가 관측되어, ΔfkbM 유전자로부터 만들어진 31-O-demethyl에서 기인한 것임을 유추할 수 있었다. gHMBC로부터 2개의 메톡시 수소 (δH 3.40, 3.30)는 각각 C-15 (δC 75.21) 및 C-13 (δC 74.03) correlation된 것을 확인되어, 본 화합물 역시 tcsD가 제거된 균주로부터 만들어진 화합물로서, C-36/37 사이에 존재하던 이중결합이 환원된 형태의 31-O-demethyl-35,37-dihydro-FK506으로 구조 결정되었다. From 1 H-NMR, 13 C-NMR and gHSQC, ketone carbon (δ C 196.54) was additionally observed with 43 carbon atoms, and 2 methoxy were observed, resulting from 31- O- demethyl made from the ΔfkbM gene. It was inferred that it was done. From gHMBC, it was confirmed that two methoxy hydrogens (δ H 3.40, 3.30) were correlated with C-15 (δ C 75.21) and C-13 (δ C 74.03), respectively, and this compound was also made from a strain in which tcsD was removed. As, the structure of the double bond existing between C-36/37 was reduced to 31- O- demethyl-35,37-dihydro-FK506.
실시예 4: 9-deoxo-31-Example 4: 9-deoxo-31- OO -demethyl-36,37-dihydro-FK506 제조-demethyl-36,37-dihydro-FK506 production
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbD-fkbM 및 tcsD 유전자의 비활성화를 초래하여 9-deoxo-31-O-demethyl-36,37-dihydro-FK506의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP)을 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 Strepttomym , a production strain of 9-deoxo-31- O- demethyl-36,37-dihydro-FK506, resulting in inactivation of the fkbD-fkbM and tcsD genes using the in-frame deletion method by Seth kanamyceticus ΔfkbD-fkbM,tcsD (Accession No. KCTC13585BP) was produced.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbD-fkbM 및 tcsD 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002, in order to prepare a deletion mutant of the fkbD-fkbM and tcsD genes in the Streptomyces kanamyceticus strain producing FK506, and then transferred to Escherichia coli ET12567/pUZ8002, It was transformed into the FK506 production strain Streptomyces kanamyceticus through conjugation.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbD-fkbM 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbD-MLF/FkbD-MLR, 오른쪽-인접 절편의 프라이머 쌍 FkbD-MRF/FkbD-MRR을 설계하였고, tcsD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsDLF/TcsDLR, 오른쪽-인접 절편의 프라이머 쌍 TcsDRF/TcsDRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다. The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbD-fkbM gene, the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed, and the left-adjacent for deletion of the tcsD gene. The primer pair TcsDLF/TcsDLR of the fragment and the primer pair TcsDRF/TcsDRR of the right-adjacent fragment were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. C9 수산화효소 (Hydroxylase)와 31-Ｏ-메틸변환효소를 함께 제거하기 위한 플라스미드, p△fkbD-fkbM는 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbD-fkbM을 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid for removing C9 hydroxylase (Hydroxylase) and 31- O -methyltransferase together, pΔfkbD-fkbM, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to target genes. It was deleted by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant. The colonies thus secured were allowed to multiply three times without selection at 28°C to allow the second crossover. The two achieved double crossover mutations, ΔfkbD-fkbM, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD-fkbM 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM에 p△tcsD를 도입하여 fkbD-fkbM 유전자 결손 방법과 동일한 방법을 이용하여 tcsD 유전자를 결실시켰다. ΔfkbD-fkbM,tcsD는 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The prepared fkbD-fkbM gene was deleted by introducing pΔtcsD into ΔfkbD-fkbM of Streptomyces kanamyceticus, and the tcsD gene was deleted using the same method as the fkbD-fkbM gene deletion method. ΔfkbD-fkbM,tcsD was selected as an apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD-fkbM 및 tcsD 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13585BP).The produced fkbD-fkbM and tcsD gene-deficient strains, Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD, were deposited on July 17, 2018 at the Korean Collection for Type Cultures (KCTC). (Accession number KCTC13585BP).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP)의 배양을 통하여 9-deoxo-31-O-demethyl-36,37-dihydro-FK506을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 9-deoxo-31-O-demethyl-36,37-dihydro-FK506을 추출하였다. 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 was prepared through cultivation of the produced strain S. pneumoniae streptomyces kanamyceticus ΔfkbD-fkbM,tcsD (Accession No. KCTC13585BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 9-deoxo-31-O-demethyl-36,37-dihydro-FK506이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 4]로 표시되는 물질인 9-deoxo-31-O-demethyl-36,37-dihydro-FK506을 얻을 수 있었다.This was lyophilized to obtain 9-deoxo-31- O- demethyl-36,37-dihydro-FK506, a substance represented by [Formula 4] in powder form.
제조한 9-deoxo-31-O-demethyl-36,37-dihydro-FK506의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 9-deoxo-31-O-demethyl-36,37-dihydro-FK506에 대한 분석 결과는 표 6과 도 19 내지 도 24로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD로부터 9-deoxo-31-O-demethyl-36,37-dihydro-FK506이 생산됨을 확인할 수 있었다.Confirmation of the prepared 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. Analysis results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 are summarized in Table 6 and FIGS. 19 to 24, and the production strain Streptomyces kanamyceticus ΔfkbD produced from these results It was confirmed that 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 was produced from -fkbM,tcsD.
9-deoxo-31-O-demethyl-36,37-dihydro-FK506 (분자식 C43H71NO11, 분자량 778.04)에 대한 분석 결과를 하기의 표 6에 나타내었다.The analysis results for 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 (molecular formula C 43 H 71 NO 11 , molecular weight 778.04) are shown in Table 6 below.
Figure PCTKR2019017519-appb-T000006
Figure PCTKR2019017519-appb-T000006
Figure PCTKR2019017519-appb-I000021
Figure PCTKR2019017519-appb-I000021
Figure PCTKR2019017519-appb-I000022
Figure PCTKR2019017519-appb-I000022
Figure PCTKR2019017519-appb-I000023
Figure PCTKR2019017519-appb-I000023
1H-NMR, 13C-NMR 및 gHSQC로부터, 총 탄소수 43개로 동일한 개수로 이뤄져 있으나, ΔfkbD의 영향으로 인해, 1번, 2번 물질과 같이, ketone대신에 나타나는 CH2 작용기 (δH 2.67, 2.49, δC 37.54)가 관측되었다. 2D-NMR data를 분석한 결과, 4번 화합물은 ΔfkbD-fkbM 유전자로부터 만들어진 9-deoxo-31-O-demethyl-35,37-dihydro-FK506로 구조 결정하였다. From 1 H-NMR, 13 C-NMR, and gHSQC, the total number of carbon atoms is 43, but due to the effect of ΔfkbD, CH 2 functional groups appearing in place of ketone, like substances 1 and 2 (δ H 2.67, 2.49, δ C 37.54) was observed. As a result of analyzing 2D-NMR data, compound 4 was structured with 9-deoxo-31- O- demethyl-35,37-dihydro-FK506 made from the ΔfkbD-fkbM gene.
실시예 5: 9-deoxo-FK520 제조Example 5: Preparation of 9-deoxo-FK520
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbD 및 tcsB 유전자 또는 fkbD 및 tcsD 유전자의 비활성화를 초래하여 9-deoxo-FK520의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP)을 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 Streptomyces kanamyceticus ΔfkbD,tcsB (producing strain of 9-deoxo-FK520, resulting in inactivation of fkbD and tcsB genes or fkbD and tcsD genes using the in-frame deletion method by) Accession No. KCTC13579BP) or Streptomyces kanamyceticus ΔfkbD,tcsD (Accession No. KCTC13580BP) was prepared.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbD 및 tcsB 유전자의 결손 돌연변이체 또는 fkbD 및 tcsD 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and Escherichia coli in order to produce a deletion mutant of the fkbD and tcsB genes or a deletion mutant of the fkbD and tcsD genes in the Streptomyces kanamyceticus strain producing FK506. After transfer to ET12567/pUZ8002, it was transformed into FK506 production strain Streptomyces kanamyceticus through conjugation.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbDLF/FkbDLR, 오른쪽-인접 절편의 프라이머 쌍 FkbDRF/FkbDRR을 설계하였고, tcsB 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsBLF/TcsBLR, 오른쪽-인접 절편의 프라이머 쌍 TcsBRF/TcsBRR을 설계하였다. tcsD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsDLF/TcsDLR, 오른쪽-인접 절편의 프라이머 쌍 TcsDRF/TcsDRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다. The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbD gene, the primer pair FkbDLF/FkbDLR of the left-adjacent segment and the primer pair FkbDRF/FkbDRR of the right-adjacent segment were designed, and for the deletion of the tcsB gene, the primer pair of the left-adjacent segment TcsBLF/TcsBLR, right- Primer pairs TcsBRF/TcsBRR of adjacent sections were designed. For deletion of the tcsD gene, the primer pair TcsDLF/TcsDLR of the left-adjacent segment and the primer pair TcsDRF/TcsDRR of the right-adjacent segment were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. C9 수산화효소 (Hydroxylase)를 제거하기 위한 플라스미드, p△fkbD는 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbD를 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid to remove C9 hydroxylase (Hydroxylase), pΔfkbD, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to delete the target gene by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected as a culture of a transmycin-resistant transconjugant. Colonies secured thereafter were allowed to multiply three times without selection at 28°C to allow the second crossover. Two achieved double crossover mutations, ΔfkbD, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
제작한 fkbD 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbD에 p△tcsB 또는 p△tcsD를 도입하여 fkbD 유전자 결손 방법과 동일한 방법을 이용하여 tcsB 유전자 또는 tcsD 유전자를 결실시켰다. ΔfkbD,tcsB 또는 ΔfkbD,tcsD는 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The prepared fkbD gene was introduced into pΔtcsB or pΔtcsD in ΔfkbD of Streptomyces kanamyceticus deficient, and the tcsB gene or tcsD gene was deleted using the same method as the fkbD gene deletion method. ΔfkbD, tcsB or ΔfkbD, tcsD was selected as apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD,tcsB 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였고 (수탁번호 KCTC13579BP), fkbD, tcsD 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD도 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13580BP).The produced fkbD and tcsB gene-deficient strains, Streptomyces kanamyceticus ΔfkbD,tcsB, were deposited with the Korea Institute of Biotechnology and Biotechnology Resource Center (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession No. KCTC13579BP ), fkbD and tcsD gene-deficient strains, Streptomyces kanamyceticus ΔfkbD,tcsD, were also deposited with the Korea Institute of Biotechnology and Biotechnology Resource Center (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession number KCTC13580BP ).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP)의 배양을 통하여 9-deoxo-FK520을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 9-deoxo-FK520을 추출하였다. 9-deoxo-FK520 was prepared by culturing the production strain of Streptomyces kanamyceticus ΔfkbD,tcsB (Accession No. KCTC13579BP) or Streptomyces kanamyceticus ΔfkbD,tcsD (Accession No. KCTC13580BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 9-deoxo-FK520 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 9-deoxo-FK520이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 9-deoxo-FK520 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 5]로 표시되는 물질인 9-deoxo-FK520을 얻을 수 있었다.This was lyophilized to obtain 9-deoxo-FK520, a substance represented by [Formula 5] in powder form.
제조한 9-deoxo-FK520의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 9-deoxo-FK520에 대한 분석 결과는 표 7과 도 25 내지 도 30으로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD로부터 9-deoxo-FK520이 생산됨을 확인할 수 있었다.Confirmation of the prepared 9-deoxo-FK520 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The results of the analysis for 9-deoxo-FK520 are summarized in Table 7 and FIGS. 25 to 30, and the production strains Streptomyces kanamyceticus ΔfkbD,tcsB or Streptomyces kanamyceticus ΔfkbD produced from these results, It was confirmed that 9-deoxo-FK520 was produced from tcsD.
9-deoxo-FK520 (분자식 C43H71NO11, 분자량 778.04)에 대한 분석 결과를 하기의 표 7에 나타내었다.The results of the analysis for 9-deoxo-FK520 (molecular formula C 43 H 71 NO 11 , molecular weight 778.04) are shown in Table 7 below.
Figure PCTKR2019017519-appb-T000007
Figure PCTKR2019017519-appb-T000007
Figure PCTKR2019017519-appb-I000024
Figure PCTKR2019017519-appb-I000024
Figure PCTKR2019017519-appb-I000025
Figure PCTKR2019017519-appb-I000025
Figure PCTKR2019017519-appb-I000026
Figure PCTKR2019017519-appb-I000026
1H-NMR, 13C-NMR 및 gHSQC 데이터로부터, 탄소수가 총 43개로 매우 유사한 형태로 확인되었으나, 메톡시 (δH 3.41, δC 56.81)가 추가 관측되었고, CH2 작용기가 한 개가 덜 관측되는 구조 이성질체임을 확인할 수 있었다. gCOSY로부터 proton의 연결을 확인한 결과, H-2~H-5사이의 coupling으로부터 본 화합물은 pipecolyl 골격이 확인되었고, gHMBC로부터 triplet coupling으로 관측되는 메틸기 (δH 0.88, δC 12.01)와 CH2 (δH 1.72, 1.52, δC 20.41)가 C-21 (δH 1.72, 1.52, δC 20.41) 사이에 상호 correlation이 이뤄지는 것으로부터, FK506의 propyl기가 아닌 FK520 형태를 이루는 ethyl가 C-21에 존재함을 확인하였다. 이로부터 5번 화합물은 9-deoxo-FK520으로 구조결정하였다. From 1 H-NMR, 13 C-NMR and gHSQC data, a total of 43 carbon atoms were found in a very similar form, but methoxy (δ H 3.41, δ C 56.81) was additionally observed and one CH 2 functional group was observed less. It was confirmed that it is a structural isomer. As a result of confirming the connection of the proton from gCOSY, the pipecolyl skeleton of the compound was confirmed from the coupling between H-2 and H-5, and methyl groups (δ H 0.88, δ C 12.01) and CH 2 (observed by triplet coupling from gHMBC) Since δ H 1.72, 1.52, δ C 20.41) cross-correlate between C-21 (δ H 1.72, 1.52, δ C 20.41), ethyl in the form of FK520 rather than the propyl group of FK506 exists in C-21. Was confirmed. From this, compound 5 was structured with 9-deoxo-FK520.
실시예 6: 31-Example 6: 31- OO -demethyl-FK520 제조-demethyl-FK520 manufacture
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbM 및 tcsB 유전자 또는 fkbM 및 tcsD 유전자의 비활성화를 초래하여 31-O-demethyl-FK520의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsB (수탁번호 KCTC13583BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP)을 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 Streptomyces kanamyceticus ΔfkbM, a production strain of 31- O- demethyl-FK520, resulting in inactivation of the fkbM and tcsB genes or fkbM and tcsD genes using the in-frame deletion method by) tcsB (Accession No. KCTC13583BP) or Streptomyces kanamyceticus ΔfkbM,tcsD (Accession No. KCTC13584BP) was prepared.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbM 및 tcsB 유전자의 결손 돌연변이체 또는 fkbM 및 tcsD 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and Escherichia coli in order to produce a deletion mutant of the fkbM and tcsB genes or a deletion mutant of the fkbM and tcsD genes in the Streptomyces kanamyceticus strain producing FK506. After transfer to ET12567/pUZ8002, it was transformed into FK506 production strain Streptomyces kanamyceticus through conjugation.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbM 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbMLF/FkbMLR, 오른쪽-인접 절편의 프라이머 쌍 FkbMRF/FkbMRR을 설계하였고, tcsB 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsBLF/TcsBLR, 오른쪽-인접 절편의 프라이머 쌍 TcsBRF/TcsBRR을 설계하였다. tcsD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsDLF/TcsDLR, 오른쪽-인접 절편의 프라이머 쌍 TcsDRF/TcsDRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다. The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbM gene, a primer pair FkbMLF/FkbMLR of the left-neighbor segment and a primer pair FkbMRF/FkbMRR of the right-neighbor segment were designed, and for deletion of the tcsB gene, a primer pair of the left-neighbor segment TcsBLF/TcsBLR, right- Primer pairs TcsBRF/TcsBRR of adjacent sections were designed. For deletion of the tcsD gene, the primer pair TcsDLF/TcsDLR of the left-adjacent segment and the primer pair TcsDRF/TcsDRR of the right-adjacent segment were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. 31-Ｏ-메틸변환효소를 제거하기 위한 플라스미드, p△fkbM은 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbM을 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid for removing 31- O -methyltransferase, pΔfkbM, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation, thereby deleting the target gene by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant. The colonies thus secured were allowed to multiply three times without selection at 28°C to allow the second crossover. Two achieved double crossover mutations, ΔfkbM, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
제작한 fkbM 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbM에 p△tcsB 또는 p△tcsD를 도입하여 fkbM 유전자 결손 방법과 동일한 방법을 이용하여 tcsB 유전자 또는 tcsD 유전자를 결실시켰다. ΔfkbM,tcsB 또는 ΔfkbM,tcsD는 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The prepared fkbM gene was introduced into pΔtcsB or pΔtcsD in ΔfkbM of Streptomyces kanamyceticus deficient, and the tcsB gene or tcsD gene was deleted using the same method as the fkbM gene deletion method. ΔfkbM,tcsB or ΔfkbM,tcsD was selected as apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbM,tcsB 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsB는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였고 (수탁번호 KCTC13583BP), fkbM,tcsD 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD도 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13584BP).The produced fkbM and tcsB gene-deficient strains, Streptomyces kanamyceticus ΔfkbM,tcsB, were deposited with the Korea Institute of Bioscience and Biotechnology (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession No. KCTC13583BP ), fkbM and tcsD gene-deficient strains, Streptomyces kanamyceticus ΔfkbM,tcsD, were also deposited with the Korea Institute of Biotechnology and Biotechnology Resource Center (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession No. KCTC13584BP ).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsB (수탁번호 KCTC13583BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP)의 배양을 통하여 31-O-demethyl-FK520을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 31-O-demethyl-FK520을 추출하였다. 31- O- demethyl-FK520 was prepared by culturing the above-produced production strain Streptomyces kanamyceticus ΔfkbM,tcsB (Accession No. KCTC13583BP) or Streptomyces kanamyceticus ΔfkbM,tcsD (Accession No. KCTC13584BP). . Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 31- O- demethyl-FK520 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 31-O-demethyl-FK520이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 31- O- demethyl-FK520 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 6]으로 표시되는 물질인 31-O-demethyl-FK520을 얻을 수 있었다.This was lyophilized to obtain 31- O- demethyl-FK520, a substance represented by [Formula 6] in powder form.
제조한 31-O-demethyl-FK520의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 31-O-demethyl-FK520에 대한 분석 결과는 표 8과 도 31 내지 도 36으로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsB 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD로부터 31-O-demethyl-FK520이 생산됨을 확인할 수 있었다.Confirmation of the prepared 31- O- demethyl-FK520 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The analysis results for 31- O- demethyl-FK520 are summarized in Table 8 and FIGS. 31 to 36, and the production strains Streptomyces kanamyceticus ΔfkbM,tcsB or Streptomyces kanamyceticus produced from these results It was confirmed that 31- O- demethyl-FK520 was produced from ΔfkbM,tcsD.
31-O-demethyl-FK520 (분자식 C42H67NO12, 분자량 777.99)에 대한 분석 결과를 하기의 표 8에 나타내었다.The analysis results for 31- O- demethyl-FK520 (molecular formula C 42 H 67 NO 12 , molecular weight 777.99) are shown in Table 8 below.
Figure PCTKR2019017519-appb-T000008
Figure PCTKR2019017519-appb-T000008
Figure PCTKR2019017519-appb-I000027
Figure PCTKR2019017519-appb-I000027
Figure PCTKR2019017519-appb-I000028
Figure PCTKR2019017519-appb-I000028
Figure PCTKR2019017519-appb-I000029
Figure PCTKR2019017519-appb-I000029
분자량 800.4563으로 확인된 6번 화합물은 분자량 800.4924 및 800.4927 매우 유사한 분자량을 보였으나, 1H-NMR, 13C-NMR 및 gHSQC 데이터로부터, 탄소수가 총 42개로, 메톡시기가 적은 것이 탄소 1개의 차이임을 확인할 수 있었고, ketone 탄소 (δC 196.50) 관측되었다. 2D-NMR 데이터를 종합한 결과, 31-O-demethyl-FK520으로 구조결정되었다. Compound 6 identified with a molecular weight of 800.4563 showed very similar molecular weights of 800.4924 and 800.4927, but from 1 H-NMR, 13 C-NMR and gHSQC data, the total number of carbon atoms was 42, and the less methoxy group was the difference of 1 carbon. It was confirmed, and ketone carbon (δ C 196.50) was observed. As a result of synthesizing 2D-NMR data, the structure was determined with 31- O- demethyl-FK520.
실시예 7: 9-deoxo-31-Example 7: 9-deoxo-31- OO -demethyl-FK520 제조-demethyl-FK520 manufacture
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbD-fkbM 및 tcsB 유전자 또는 fkbD-fkbM 및 tcsD 유전자의 비활성화를 초래하여 9-deoxo-31-O-demethyl-FK520의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP)를 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 Strepto, a production strain of 9-deoxo-31- O- demethyl-FK520, resulting in inactivation of the fkbD-fkbM and tcsB genes or the fkbD-fkbM and tcsD genes using the in-frame deletion method by Myces kanamyceticus ΔfkbD-fkbM,tcsB (Accession No. KCTC13582BP) or Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD (Accession No. KCTC13585BP) was prepared.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbD-fkbM 및 tcsB 유전자의 결손 또는 fkbD-fkbM 및 tcsD 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. More specifically, the cloning of the respective genes to pKC1139 vector to from Streptomyces Kana Mai Shetty kusu strain producing FK506 to produce a defect mutant of defect or fkbD-fkbM and tcsD genes fkbD-fkbM and tcsB gene After transferring to Escherichia coli ET12567/pUZ8002, it was transformed into FK506 production strain Streptomyces kanamyceticus through conjugation.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbD-fkbM 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbD-MLF/FkbD-MLR, 오른쪽-인접 절편의 프라이머 쌍 FkbD-MRF/FkbD-MRR을 설계하였다. tcsB 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsBLF/TcsBLR, 오른쪽-인접 절편의 프라이머 쌍 TcsBRF/TcsBRR을 설계하였고, tcsD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsDLF/TcsDLR, 오른쪽-인접 절편의 프라이머 쌍 TcsDRF/TcsDRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다.The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbD-fkbM gene, the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed. For deletion of the tcsB gene, the primer pair TcsBLF/TcsBLR of the left-neighbor segment and the primer pair TcsBRF/TcsBRR of the right-neighbor segment were designed, and for the deletion of the tcsD gene, the primer pair TcsDLF/TcsDLR of the left-neighbor segment, right- Primer pairs TcsDRF/TcsDRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. C9 수산화효소 (Hydroxylase)와 31-Ｏ-메틸변환효소를 함께 제거하기 위한 플라스미드, p△fkbD-fkbM는 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbD-fkbM을 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid for removing C9 hydroxylase (Hydroxylase) and 31- O -methyltransferase together, pΔfkbD-fkbM, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to target genes. It was deleted by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant. The colonies thus secured were allowed to multiply three times without selection at 28°C to allow the second crossover. The two achieved double crossover mutations, ΔfkbD-fkbM, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD-fkbM 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM에 p△tcsB 또는 p△tcsD를 도입하여 fkbD-fkbM 유전자 결손 방법과 동일한 방법을 이용하여 tcsB 유전자 또는 tcsD 유전자를 결실시켰다. ΔfkbD-fkbM,tcsB 또는 ΔfkbD-fkbM,tcsD는 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The prepared fkbD-fkbM gene was deleted by introducing pΔtcsB or p△tcsD into ΔfkbD-fkbM of Streptomyces kanamyceticus, and the tcsB gene or tcsD gene was deleted using the same method as the fkbD-fkbM gene deletion method. . ΔfkbD-fkbM,tcsB or ΔfkbD-fkbM,tcsD was selected as an apramycin-sensitive expression trait, and then confirmed by PCR, and optionally by Southern blot analysis.
제작한 fkbD-fkbM 및 tcsB 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였고 (수탁번호 KCTC13582BP), fkbD-fkbM 및 tcsD 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD도 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13585BP).The produced fkbD-fkbM and tcsB gene-deficient strains, Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB, were deposited with the Korean Collection for Type Cultures (KCTC) on July 17, 2018. (Accession No. KCTC13582BP), fkbD-fkbM and tcsD gene-deficient strains Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD were also published at the Korea Collection for Type Cultures (KCTC) on July 17, 2018. Deposited on the date (Accession No. KCTC13585BP).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP)의 배양을 통하여 9-deoxo-31-O-demethyl-FK520을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 9-deoxo-31-O-demethyl-FK520을 추출하였다. 9-deoxo-31- through the culture of the above-produced production strain Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB (Accession No. KCTC13582BP) or Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD (Accession No. KCTC13585BP) O- demethyl-FK520 was prepared. Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 9-deoxo-31- O- demethyl-FK520 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 9-deoxo-31-O-demethyl-FK520이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 9-deoxo-31- O- demethyl-FK520 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 7]로 표시되는 물질인 9-deoxo-31-O-demethyl-FK520을 얻을 수 있었다.This was lyophilized to obtain 9-deoxo-31- O- demethyl-FK520, a substance represented by [Formula 7] in powder form.
제조한 9-deoxo-31-O-demethyl-FK520의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 9-deoxo-31-O-demethyl-FK520에 대한 분석 결과는 표 9와 도 37 내지 도 42로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD로부터 9-deoxo-31-O-demethyl-FK520이 생산됨을 확인할 수 있었다.Confirmation of the prepared 9-deoxo-31- O- demethyl-FK520 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The results of the analysis for 9-deoxo-31- O- demethyl-FK520 are summarized in Table 9 and FIGS. 37 to 42, and the production strains Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB or strepto produced from these results It was confirmed that 9-deoxo-31- O- demethyl-FK520 was produced from ΔfkbD-fkbM,tcsD of Myces Kanamyceticus.
9-deoxo-31-O-demethyl-FK520 (분자식 C42H69NO11, 분자량 764.01)에 대한 분석 결과를 하기의 표 9에 나타내었다.The analysis results for 9-deoxo-31- O- demethyl-FK520 (molecular formula C 42 H 69 NO 11 , molecular weight 764.01) are shown in Table 9 below.
Figure PCTKR2019017519-appb-T000009
Figure PCTKR2019017519-appb-T000009
Figure PCTKR2019017519-appb-I000030
Figure PCTKR2019017519-appb-I000030
Figure PCTKR2019017519-appb-I000031
Figure PCTKR2019017519-appb-I000031
Figure PCTKR2019017519-appb-I000032
Figure PCTKR2019017519-appb-I000032
1H-NMR, 13C-NMR 및 gHSQC 데이터로부터, 탄소수가 총 42개로 31-O-demethyl-FK520와 유사한 결과를 보였으나, ΔfkbD-fkbM 유전자로부터 만들어진 CH2 작용기 (δH 2.67, 2.49, δC 37.54)가 관측되었다. 7번 화합물은 2D-NMR 데이터를 종합한 결과, 9-deoxo-31-O-demethyl-FK520으로 구조결정되었다. From 1 H-NMR, 13 C-NMR and gHSQC data, a total of 42 carbon atoms showed similar results to 31- O- demethyl-FK520, but a CH 2 functional group made from the ΔfkbD-fkbM gene (δ H 2.67, 2.49, δ C 37.54) was observed. As a result of synthesizing 2D-NMR data, compound 7 was structurally determined to be 9-deoxo-31- O- demethyl-FK520.
실시예 8: 9-deoxo-FK523 제조Example 8: Preparation of 9-deoxo-FK523
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbD 및 tcsB 유전자 의 비활성화를 초래하여 9-deoxo-FK523의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP)를 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 Streptomyces kanamyceticus ΔfkbD,tcsB (Accession No. KCTC13579BP), a production strain of 9-deoxo-FK523, is caused by inactivation of the fkbD and tcsB genes using the in-frame deletion method by ). It was produced.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbD 및 tcsB 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002 in order to construct a mutant of the fkbD and tcsB genes in a strain of Streptomyces kanamyceticus producing FK506, and then transferred to a conjugation ( Conjugation) was transformed into FK506 producing strain Streptomyces kanamyceticus.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbD 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbDLF/FkbDLR, 오른쪽-인접 절편의 프라이머 쌍 FkbDRF/FkbDRR을 설계하였고, tcsB 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsBLF/TcsBLR, 오른쪽-인접 절편의 프라이머 쌍 TcsBRF/TcsBRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다. The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbD gene, the primer pair FkbDLF/FkbDLR of the left-adjacent segment and the primer pair FkbDRF/FkbDRR of the right-adjacent segment were designed, and for the deletion of the tcsB gene, the primer pair of the left-adjacent segment TcsBLF/TcsBLR, right- Primer pairs TcsBRF/TcsBRR of adjacent sections were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. C9 수산화효소 (Hydroxylase)를 제거하기 위한 플라스미드, p△fkbD는 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbD를 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid to remove C9 hydroxylase (Hydroxylase), pΔfkbD, was transferred to E. coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to delete the target gene by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected as a culture of a transmycin-resistant transconjugant. Colonies secured thereafter were allowed to multiply three times without selection at 28°C to allow the second crossover. Two achieved double crossover mutations, ΔfkbD, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally by Southern blot analysis.
제작한 fkbD 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbD에 p△tcsB를 도입하여 fkbD 유전자 결손 방법과 동일한 방법을 이용하여 tcsB 유전자를 결실시켰다. ΔfkbD,tcsB는 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. By introducing a p △ tcsB the produced fkbD gene defect Streptomyces Kana Mai Shetty kusu ΔfkbD, using the same method as fkbD gene-deficient deletion method was the tcsB gene. ΔfkbD,tcsB was selected as the expression trait of apramycin-sensitivity, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD,tcsB 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13579BP).The produced fkbD and tcsB gene-deficient strains, Streptomyces kanamyceticus ΔfkbD,tcsB, were deposited with the Korea Institute of Bioscience and Biotechnology (Korean Collection for Type Cultures, KCTC) on July 17, 2018 (Accession No. KCTC13579BP ).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP)의 배양을 통하여 9-deoxo-FK523을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 9-deoxo-FK523을 추출하였다. 9-deoxo-FK523 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ΔfkbD,tcsB (Accession No. KCTC13579BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 9-deoxo-FK523 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 9-deoxo-FK523이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. 9-deoxo-FK523 was identified in aliquot 3. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 8]로 표시되는 물질인 9-deoxo-FK523을 얻을 수 있었다.This was lyophilized to obtain 9-deoxo-FK523, a substance represented by [Formula 8] in powder form.
제조한 9-deoxo-FK523의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 9-deoxo-FK523에 대한 분석 결과는 표 10과 도 43 내지 도 48로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB로부터 9-deoxo-FK523이 생산됨을 확인할 수 있었다.Confirmation of the prepared 9-deoxo-FK523 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The results of the analysis for 9-deoxo-FK523 are summarized in Table 10 and FIGS. 43 to 48, and it was confirmed that 9-deoxo-FK523 was produced from the production strain Streptomyces canaaceticus ΔfkbD,tcsB produced from these results. Could.
9-deoxo-FK523 (분자식 C42H69NO11, 분자량 764.01)에 대한 분석 결과를 하기의 표 10에 나타내었다.The results of the analysis for 9-deoxo-FK523 (molecular formula C 42 H 69 NO 11 , molecular weight 764.01) are shown in Table 10 below.
Figure PCTKR2019017519-appb-T000010
Figure PCTKR2019017519-appb-T000010
Figure PCTKR2019017519-appb-I000033
Figure PCTKR2019017519-appb-I000033
Figure PCTKR2019017519-appb-I000034
Figure PCTKR2019017519-appb-I000034
Figure PCTKR2019017519-appb-I000035
Figure PCTKR2019017519-appb-I000035
9-deoxo-31-O-demethyl-FK520 분자량과 동일한 분자량을 갖고 있으며, 1H-NMR, 13C-NMR 및 gHSQC 데이터로부터, 탄소수가 총 42개로 매우 유사한 형태로 확인되었으나, 9-deoxo-31-O-demethyl-FK520과 달리 메톡시 (δH 3.41, δC 56.81)가 추가 관측되었고, CH2 작용기가 한 개가 덜 관측되는 구조 이성질체임을 확인할 수 있었다. gCOSY로부터 proton의 연결을 확인한 결과, H-2~H-5사이의 coupling으로부터 본 화합물은 pipecolyl 골격이 확인되었고, gHMBC로부터 doublet coupling으로 관측되는 메틸기 (δH 1.18, δC 17.13)가 C-21 (δH 3.21, δC 53.45) 사이에 상호 correlation이 이뤄지는 것으로부터, FK523 형태를 이루는 methyl가 C-21에 존재함을 확인하였다. 이로부터 9-deoxo-FK523으로 구조결정하였다.9-deoxo-31- O- demethyl-FK520 has the same molecular weight as the molecular weight, and from 1 H-NMR, 13 C-NMR and gHSQC data, a total of 42 carbon atoms was confirmed in a very similar form, but 9-deoxo-31 -Unlike O- demethyl-FK520, methoxy (δ H 3.41, δ C 56.81) was additionally observed, and it was confirmed that one of the CH 2 functional groups was a structural isomer with less observation. As a result of confirming the connection of the proton from gCOSY, the pipecolyl skeleton was confirmed in this compound from the coupling between H-2 and H-5, and the methyl group (δ H 1.18, δ C 17.13) observed by doublet coupling from gHMBC was C-21 From the correlation between (δ H 3.21 and δ C 53.45), it was confirmed that methyl forming the FK523 form was present in C-21. From this, the structure was determined with 9-deoxo-FK523.
실시예 9: 9-deoxo-31-Example 9: 9-deoxo-31- OO -demethyl-FK523 제조-demethyl-FK523 manufacture
FK506을 생산하는 균주인 스트렙토마이세스 카나마이세티쿠스에 대하여 Ban, Y. H. 외 (J. Nat. Prod. 2013, 76, 1091-1098)에 기재된 방법에 따라 이중교차 상동 재조합 (Double cross-over homologous recombination)에 의한 인-프레임 (In-frame) 결실 방법을 사용하여 fkbD-fkbM 및 tcsB 유전자의 비활성화를 초래하여 9-deoxo-31-O-demethyl-FK523의 생산균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP)를 제작하였다.Double cross-over homologous recombination according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098) for Streptomyces kanamyceticus, a strain producing FK506 ), resulting in inactivation of the fkbD-fkbM and tcsB genes using the in-frame deletion method by Streptomyces kanamyceticus ΔfkbD, a production strain of 9-deoxo-31- O- demethyl-FK523 -fkbM,tcsB (Accession No. KCTC13582BP) was produced.
구체적으로 설명하면, FK506을 생산하는 스트렙토마이세스 카나마이세티쿠스 균주에서 fkbD-fkbM 및 tcsB 유전자의 결손 돌연변이체를 제작하기 위하여 각각의 유전자를 pKC1139 벡터에 클로닝하여 Escherichia coli ET12567/pUZ8002로 옮긴 후, 접합 (Conjugation)을 통해 FK506 생산균주 스트렙토마이세스 카나마이세티쿠스로 형질전환하였다. Specifically, each gene was cloned into a pKC1139 vector and transferred to Escherichia coli ET12567/pUZ8002, in order to produce a deletion mutant of the fkbD-fkbM and tcsB genes in the Streptomyces kanamyceticus strain producing FK506, and then transferred to Escherichia coli ET12567/pUZ8002, It was transformed into the FK506 production strain Streptomyces kanamyceticus through conjugation.
균주제작 방법은 보다 구체적으로 인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작, 유전자 결실 균주 제작으로 설명할 수 있다. The method for producing a strain can be described in more detail by production of an in-frame gene deletion plasmid (Plasmids) and production of a gene deletion strain.
인-프레임 (In-frame) 유전자 결실 플라스미드 (Plasmids)의 제작은 대장균-방선균 셔틀 (E. coli-Streptomyces shuttle) 벡터 pKC1139를 인-프레임 (In-frame) 유전자 결실을 위해서 사용하였다. 플라스미드 (Plasmid) 제작은 스트렙토마이세스 카나마이세티쿠스로부터 유래된 포스미드 (Fosmid) DNA로부터 삭제를 위한 표적 유전자의 왼쪽- 및 오른쪽-인접 절편 (Left- and right-flanking fragments)의 PCR 증폭에 의해 실시하였다. fkbD-fkbM 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 FkbD-MLF/FkbD-MLR, 오른쪽-인접 절편의 프라이머 쌍 FkbD-MRF/FkbD-MRR을 설계하였다. tcsB 유전자의 결실을 위해서는 왼쪽-인접 절편의 프라이머 쌍 TcsBLF/TcsBLR, 오른쪽-인접 절편의 프라이머 쌍 TcsBRF/TcsBRR을 설계하였다. PCR 절편 모두는 분리하여 HindIII-XbaI 또는 XbaI-EcoRI으로 절단한 후에 pKC1139 벡터에 클로닝 하였다. 본 실시예에서 사용된 균주, 플라스미드 및 프라이머에 대한 정보는 표 1과 표 2에 제시하였다.The production of the in-frame gene deletion plasmid ( E. coli - Streptomyces shuttle) The vector pKC1139 was used for in-frame gene deletion. Plasmid production was accomplished by PCR amplification of left- and right-flanking fragments of target genes for deletion from Fosmid DNA derived from Streptomyces kanamyceticus. It was carried out. For deletion of the fkbD-fkbM gene, the primer pair FkbD-MLF/FkbD-MLR of the left-adjacent segment and the primer pair FkbD-MRF/FkbD-MRR of the right-adjacent segment were designed. For deletion of the tcsB gene, a primer pair TcsBLF/TcsBLR of the left-adjacent segment and a primer pair TcsBRF/TcsBRR of the right-adjacent segment were designed. All of the PCR fragments were separated, cut with HindIII-XbaI or XbaI-EcoRI, and then cloned into the pKC1139 vector. Information about the strains, plasmids and primers used in this Example is presented in Table 1 and Table 2.
유전자 결실 균주 제작을 위해 사용한 플라스미드는 표 1에 요약하였다. C9 수산화효소 (Hydroxylase)와 31-Ｏ-메틸변환효소를 함께 제거하기 위한 플라스미드, p△fkbD-fkbM은 대장균 ET12567/pUZ8002에 옮긴 후 접합에 의해 스트렙토마이세스 카나마이세티쿠스 내로 도입하여 표적 유전자를 상동 재조합으로 결실시켰다. 결실 플라스미드와 스트렙토마이세스 카나마이세티쿠스 염색체 사이에서 단일 교차가 일어난 균주는 아프라마이신 (Apramycin)이 있는 37℃ (pSG5-기본으로 하는 복제단위 (Replicon)를 위한 비-증식 허용온도)에서 아프라마이신-저항성이 있는 피전달접합균주 (Transconjugant)의 배양으로 선택하였다. 이후 확보된 콜로니는 28℃에서 선택 없이 3회 증식을 수행하여 두 번째 교차를 허용하였다. 2개의 달성된 이중 교차 돌연변이, 즉 ΔfkbD-fkbM을 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The plasmid used to construct the gene deletion strain is summarized in Table 1. The plasmid for removing C9 hydroxylase (Hydroxylase) and 31- O -methyltransferase together, pΔfkbD-fkbM, is transferred to Escherichia coli ET12567/pUZ8002 and introduced into Streptomyces kanamyceticus by conjugation to target genes. It was deleted by homologous recombination. Strains with a single cross between the deletion plasmid and the Streptomyces kanamyceticus chromosome were subdivided at 37°C with apramycin (non-proliferable temperature for pSG5-based replication unit (Replicon)). It was selected by cultivation of a pramycin-resistant transconjugant. The colonies thus secured were allowed to multiply three times without selection at 28°C to allow the second crossover. The two achieved double crossover mutations, ΔfkbD-fkbM, were selected as apramycin-sensitive expression traits, then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD-fkbM 유전자가 결손된 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM에 p△tcsB를 도입하여 fkbD-fkbM 유전자 결손 방법과 동일한 방법을 이용하여 tcsB 유전자를 결실시켰다. ΔfkbD-fkbM,tcsB는 아프라마이신-민감성의 발현형질로 선택한 다음, 그 후에 PCR로 확인하였고, 선택적으로 서던 블롯 분석으로 확인하였다. The prepared fkbD-fkbM gene was introduced into pΔtcsB into ΔfkbD-fkbM of Streptomyces kanamyceticus deficient, and the tcsB gene was deleted using the same method as the fkbD-fkbM gene deletion method. ΔfkbD-fkbM,tcsB was selected as an apramycin-sensitive expression trait, and then confirmed by PCR, and optionally confirmed by Southern blot analysis.
제작한 fkbD-fkbM 및 tcsB 유전자 결손 균주인 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB는 한국생명공학연구원 생물자원센터 (Korean Collection for Type Cultures, KCTC)에 2018년 7월 17일자로 기탁하였다 (수탁번호 KCTC13582BP).The produced fkbD-fkbM and tcsB gene-deficient strains, Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB, were deposited with the Korean Collection for Type Cultures (KCTC) on July 17, 2018. (Accession number KCTC13582BP).
상기 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP)의 배양을 통하여 9-deoxo-31-O-demethyl-FK523을 제조하였다. 구체적으로 설명하면 다음과 같다. 250 ml의 베플 삼각 플라스크 (Baffled flask)에 50 ml의 R2YE 배지 (수크로오즈 103 g/L, 글루코오즈 10 g/L, 황산칼륨 0.25 g/L, 염화마그네슘 6수화물 10.12 g/L, 카사미노 엑시드 0.1 g/L, 효모 추출물 (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, 인산칼륨 (0.5%) 10 ml/L, 염화칼슘 2수화물 (3.68%) 80 ml/L, L-프롤린 (20%) 15 ml/L, 미량 원소 용액 2 ml/L, 수산화나트륨 (1 N) 5 ml/L)를 첨가하고, 여기에 생산균주를 접종한 다음에 회전식 진탕 배양기에서 28℃, 180 rpm 조건에서 이틀 동안 전배양을 실시하였다. 그 다음에 1 L의 R2YE 배지가 첨가되어 있는 3 L 삼각 플라스크 (Erlenmeyer flask)에 이틀 동안 전배양한 배양액 10 ml를 접종하였다. 접종 후에 28℃, 180 rpm 조건에서 6일 동안 배양을 실시하였다. 6일간 배양 후에 1차 추출공정을 통해 생산된 9-deoxo-31-O-demethyl-FK523을 추출하였다. 9-deoxo-31- O- demethyl-FK523 was prepared through cultivation of the produced strain S. pneumoniae kanamyceticus ΔfkbD-fkbM,tcsB (Accession No. KCTC13582BP). Specifically, it is as follows. 50 ml of R2YE medium (sucrose 103 g/L, glucose 10 g/L, potassium sulfate 0.25 g/L, magnesium chloride hexahydrate 10.12 g/L, cassamino in a 250 ml Baffled flask) Acid 0.1 g/L, yeast extract (10%) 50 ml/L, TES buffer (5.73%, pH 7.2) 100 ml/L, potassium phosphate (0.5%) 10 ml/L, calcium chloride dihydrate (3.68%) 80 ml/L, L-proline (20%) 15 ml/L, trace element solution 2 ml/L, sodium hydroxide (1 N) 5 ml/L) is added, and the production strain is inoculated, followed by rotary shaking Pre-incubation was performed for 2 days at 28° C. and 180 rpm in the incubator. Then, a 3 L Erlenmeyer flask to which 1 L of R2YE medium was added was inoculated with 10 ml of pre-cultured culture for two days. After inoculation, culture was performed for 6 days at 28°C and 180 rpm. After incubation for 6 days, 9-deoxo-31- O- demethyl-FK523 produced through the primary extraction process was extracted.
1차 추출공정은 다음과 같이 실시하였다. 먼저, 배양액에 동량의 메탄올을 첨가하여 30분 동안 혼합해 준 후 원심분리하여 균체를 제거하였고, 균체를 제거한 추출액에 대해서는 회전 증발기 (Rotary evaporator)를 이용한 농축을 실시해 주었다. 그 다음에 농축한 추출액을 물에 용해시키고, 2배 용량의 에틸 아세테이트 (Ethyl acetate)를 첨가 후 잘 혼합해 준 다음 층 분리가 될 때까지 방치하였다. 층이 분리된 다음에 위층의 유기용매층을 회수하고 이를 회전 증발기를 이용하여 농축시키고 농축 후의 무게를 측정하였다. 1차 추출공정을 실시하여 얻어진 추출액을 실리카겔이 충진된 칼럼에 통과시켜 주었다. 이때 실리카겔의 양은 1차 추출공정의 추출액 무게의 15배를 사용하였으며, 이동상은 5가지 비율의 메탄올과 메틸렌 클로라이드 (분액 1. 0:100, 분액 2. 1:100, 분액 3. 1:10, 분액 4. 1:1, 분액 5. 100:0)를 사용하였다. 분액 3에서 9-deoxo-31-O-demethyl-FK523이 확인되었다. 이렇게 얻어진 분액 3은 회전 증발기를 이용하여 농축하고 HPLC를 이용하여 최종적으로 정제하였다.The first extraction process was performed as follows. First, the same amount of methanol was added to the culture medium, mixed for 30 minutes, and then centrifuged to remove the cells. Concentration using a rotary evaporator was performed on the extracts from which the cells were removed. Then, the concentrated extract was dissolved in water, and twice the volume of ethyl acetate was added, mixed well, and then allowed to stand until layer separation. After the layers were separated, the organic solvent layer on the upper layer was collected, concentrated using a rotary evaporator, and weighed after concentration. The extract obtained by performing the first extraction process was passed through a column filled with silica gel. At this time, the amount of silica gel used was 15 times the weight of the extract in the first extraction process, and the mobile phase was composed of 5 ratios of methanol and methylene chloride (fraction 1. 0:100, partition 2. 1:100, partition 3. 1:10, Fraction 4. 1:1 and fraction 5. 100:0) were used. In fraction 3, 9-deoxo-31- O- demethyl-FK523 was identified. The obtained fraction 3 was concentrated using a rotary evaporator and finally purified using HPLC.
이를 동결건조시켜 분말형태의 [화학식 9]로 표시되는 물질인 9-deoxo-31-O-demethyl-FK523을 얻을 수 있었다.This was lyophilized to obtain 9-deoxo-31- O- demethyl-FK523, a substance represented by [Formula 9] in powder form.
제조한 9-deoxo-31-O-demethyl-FK523의 확인은 다음과 같이 실시하였다. 구체적으로, 고성능액체크로마토그래피 분석 (High performance liquid chromatography analysis), 질량 분석 (Mass spectrometry), 핵자기공명 분석 (Nuclear magnetic resonance analysis)을 실시하였다. 9-deoxo-31-O-demethyl-FK523에 대한 분석 결과는 표 11과 도 49 내지 도 54로 정리되며, 이러한 결과들로부터 제작한 생산균주 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB로부터 9-deoxo-31-O-demethyl-FK523이 생산됨을 확인할 수 있었다.Confirmation of the prepared 9-deoxo-31- O- demethyl-FK523 was performed as follows. Specifically, high performance liquid chromatography analysis (Mass spectrometry), nuclear magnetic resonance analysis (Nuclear magnetic resonance analysis) was performed. The analysis results for 9-deoxo-31- O- demethyl-FK523 are summarized in Table 11 and FIGS. 49 to 54, and the production strains Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB produced from these results are 9 It was confirmed that -deoxo-31- O -demethyl-FK523 was produced.
9-deoxo-31-O-demethyl-FK523 (분자식 C41H67NO11, 분자량 752.01)에 대한 분석 결과를 하기의 표 11에 나타내었다.The results of the analysis for 9-deoxo-31- O- demethyl-FK523 (molecular formula C 41 H 67 NO 11 , molecular weight 752.01) are shown in Table 11 below.
Figure PCTKR2019017519-appb-T000011
Figure PCTKR2019017519-appb-T000011
Figure PCTKR2019017519-appb-I000036
Figure PCTKR2019017519-appb-I000036
Figure PCTKR2019017519-appb-I000037
Figure PCTKR2019017519-appb-I000037
Figure PCTKR2019017519-appb-I000038
Figure PCTKR2019017519-appb-I000038
1H-NMR, 13C-NMR 및 gHSQC 데이터로부터, 탄소수가 총 41개로 9-deoxo-FK523와 달리, 메톡시 1개가 부족함을 알 수 있었다. 이와 함께, ΔfkbD-fkbM 유전자로부터 만들어진 CH2 작용기 (δH 2.70, 2.51, δC 36.25)가 관측되었다. 2D-NMR 데이터를 종합한 결과, 9-deoxo-31-O-demethyl-FK523으로 구조결정되었다. From the 1 H-NMR, 13 C-NMR and gHSQC data, it was found that, unlike 9-deoxo-FK523 with 41 carbon atoms in total, one methoxy was lacking. Along with this, a CH 2 functional group (δ H 2.70, 2.51, δ C 36.25) made from the ΔfkbD-fkbM gene was observed. As a result of synthesizing 2D-NMR data, the structure was determined to be 9-deoxo-31- O- demethyl-FK523.
실시예 10: 9종 신규 화합물의 면역억제활성 조사Example 10: Investigation of immunosuppressive activity of 9 new compounds
9종 신규 화합물의 면역억제활성 감소 정도를 통상의 인비트로 (In vitro) T-세포 활성 분석법 (J. Immunol. 143: 718-726, 1989)을 이용하여 조사하였다. CD4+ T 세포가 분열되는 것은 면역반응이 일어나고 있음을 보여주는 지표로 CD4+ T 세포를 Cell TraceTM Violet (CTV)으로 염색하여 세포가 면역반응에 따른 분열을 하여 T 세포가 증식하는 경우에 각 세포의 CTV 보유량이 감소되는 현상이 나타나므로 이를 지표로 이용하여 면역억제활성 정도를 조사하였다.The degree of immunosuppressive activity reduction of 9 novel compounds was investigated using a conventional in vitro T-cell activity assay (J. Immunol. 143: 718-726, 1989). Cleavage of CD4+ T cells is an indicator that an immune response is taking place. CD4+ T cells are stained with Cell Trace TM Violet (CTV), and when cells divide according to the immune response, CTV of each cell proliferates. As a phenomenon in which the retention amount decreases, the degree of immunosuppressive activity was investigated using this as an indicator.
6~8주령의 B6J 실험용 쥐의 지라 (Spleen)로부터 단세포 (Single cell)를 박리하고 MagniSort® Mouse CD4 T cell Enrichment Kit (eBioscience)를 사용하여 CD4+ T 세포를 분리하였다. CD4+ T 세포를 Cell TraceTM Violet (CTV) Cell Proliferation Kit (Molecular Probes)로 염색하고 FK506 또는 9종 신규 화합물을 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng/ml, 1000 ng/ml 농도가 되게 첨가한 다음에 72시간 동안 배양하였다. T 세포의 활성화를 위해 Dynabeads® Mouse T-Activator CD3/CD28 (Gibco)를 사용하였다. 대조군으로는 활성화되지 않은 T 세포를 사용하였다. 배양 후에 유세포 분석기 (Flow cytometry)로 CTV 강도 (Intensity)를 분석하였다. Single cells were detached from spleens of 6-8 weeks old B6J experimental mice and CD4+ T cells were separated using MagniSort ® Mouse CD4 T cell Enrichment Kit (eBioscience). CD4+ T cells were stained with Cell Trace TM Violet (CTV) Cell Proliferation Kit (Molecular Probes) and FK506 or 9 new compounds were 0.01 ng/ml, 0.1 ng/ml, 1 ng/ml, 10 ng/ml, 100 ng /ml, added to a concentration of 1000 ng/ml, and then incubated for 72 hours. Dynabeads ® Mouse T-Activator CD3/CD28 (Gibco) was used for T cell activation. As a control, inactivated T cells were used. After cultivation, CTV intensity was analyzed by flow cytometry.
하기 표 12와 도 55는 유세포 분석기를 이용하여 CTV 강도를 측정한 것으로 T 세포 증식 정도를 보여주어, FK506 및 9종 신규 화합물의 면역억제활성 정도를 보여준다. 하기 표 12와 도 55에 나타난 바와 같이 본원발명에서 제시하고 있는 모든 신규화합물들은 FK506보다 감소된 면역억제활성을 나타내었다.Table 12 and FIG. 55 show the degree of T cell proliferation as measured by CTV intensity using a flow cytometer, and show the degree of immunosuppressive activity of FK506 and nine new compounds. As shown in Table 12 and FIG. 55, all the new compounds proposed in the present invention showed reduced immunosuppressive activity than FK506.
Figure PCTKR2019017519-appb-T000012
Figure PCTKR2019017519-appb-T000012
이러한 결과로부터, 본 발명에 따른 9종 신규 화합물들이 FK506에 비교하여 면역억제활성이 크게 감소되었음을 확인할 수 있었으며, 9종의 신규 화합물이 최소 100배 이상의 IC50 (ng/ml) 농도를 보임에 따라 면역억제활성이 현저하게 감소됨을 확인할 수 있었다. 이로부터 9종 신규 화합물 중 선택된 하나 이상을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물이 면역억제활성에 기인한 부작용에 대한 염려 없이 사용할 수 있다고 판단하였다.From these results, it was confirmed that 9 new compounds according to the present invention significantly reduced immunosuppressive activity compared to FK506, and 9 new compounds showed an IC 50 (ng/ml) concentration of at least 100 times or more. It was confirmed that the immunosuppressive activity was significantly reduced. From this, it was determined that a pharmaceutical composition for the prevention or treatment of a nervous system disease comprising at least one selected from 9 new compounds as an active ingredient can be used without concern for side effects due to immunosuppressive activity.
실시예 11: 9종 신규 화합물들의 신경세포 성장촉진 활성 조사 (Example 11: Investigation of nerve cell growth promoting activity of 9 new compounds ( In vitroIn vitro ))
9종 신규 화합물의 신경세포 성장 촉진능을 랫드 PC12 세포 (Pheochromocytoma cell)를 이용하여 Mo, S. J. 외에 의해 보고된 방법 (J. Am. Chem. Soc. 133: 976-985, 2011)에 따라 조사하였다. 구체적으로, 상기 PC12 세포에 신경돌기 증식을 유도하는 신경 성장 인자 (NGF; KOMA Biotech; 10 ng/ml)를 96시간 동안 처리하였는데, 이 처리 시에 FK506 또는 9종 신규 화합물 중 하나를 함께 처리하였고 (처리 농도: 1 ng/ml, 10 ng/ml), 대조군은 아무 처리도 해 주지 않았다. 신경돌기의 길이는 인화된 사진을 이용하여 기존에 보고된 방법 (J. Pharmacol. Exp. Ther. 302: 1278-1285, 2002)에 따라 측정하였다.The ability to promote neuronal growth of 9 new compounds was investigated according to the method reported by Mo, SJ et al. (J. Am. Chem. Soc. 133: 976-985, 2011) using rat PC12 cells (Pheochromocytoma cells). . Specifically, the PC12 cells were treated with neural growth factor (NGF; KOMA Biotech; 10 ng/ml) for inducing neurite outgrowth for 96 hours, during which treatment either FK506 or one of 9 new compounds was treated together. (Treatment concentration: 1 ng/ml, 10 ng/ml), the control group did not give any treatment. The length of the neurites was measured according to a previously reported method (J. Pharmacol. Exp. Ther. 302: 1278-1285, 2002) using a printed photograph.
그 결과를 도 56에 제시하였다. 도 56에서 알 수 있듯이 본 발명의 신규 화합물 9종은 우수한 신경 축삭 생성 (Neurite outgrowth) 효과를 나타내었다. 즉, 본 발명의 신규 화합물 9종은 우수한 신경세포 성장 촉진능을 갖고 있음을 확인할 수 있었다. 특히, 처리 농도 1 ng/ml 경우에는 9-deoxo-FK520 및 9-deoxo-FK523의 경우 FK506에 비하여 우수한 신경 축삭 생성효과를 보였으며, 처리 농도 10 ng/ml 경우에는 9-deoxo-FK520, 9-deoxo-FK523 및 9-deoxo-31-O-demethyl-FK523이 FK506에 비하여 우수한 신경 축삭 생성효과를 보임을 확인할 수 있었다. 따라서, 본 발명에 따른 9종 신규 화합물들은 신경계 질환의 예방 또는 치료 목적으로 사용할 수 있다고 결론지을 수 있었다.The results are presented in Figure 56. As can be seen in FIG. 56, 9 new compounds of the present invention exhibited an excellent neurite outgrowth effect. That is, it was confirmed that the nine new compounds of the present invention have excellent nerve cell growth promoting ability. Particularly, 9-deoxo-FK520 and 9-deoxo-FK523 exhibited superior nerve axon production effect when treated concentration of 1 ng/ml, and 9-deoxo-FK520, 9 when treated concentration of 10 ng/ml. It was confirmed that -deoxo-FK523 and 9-deoxo-31- O- demethyl-FK523 showed superior nerve axon production effects compared to FK506. Therefore, it was concluded that the nine new compounds according to the present invention can be used for the purpose of preventing or treating neurological diseases.
실시예 12: 신규 화합물들의 신경세포 성장촉진 활성 조사 (Example 12: Investigation of the neuronal growth promoting activity of new compounds ( In vivoIn vivo ))
신규 화합물의 신경세포 성장 촉진능을 통상의 면역조직화학기법을 이용하여 E. Petroske 외에 의해 보고된 방법 (Neuroscience. 106: 590-601, 2001)에 따라 조사하였다. 구체적으로, 9~10주령의 C57BL/6J 마우스에 신경독성 화합물인 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)를 처리하여 (처리 농도: 25 mg/kg), 운동 신경을 관장하는 흑색질-선조체 도파민성 신경경로(nigrostriatal pathway)를 퇴행시켰다. MPTP 처리를 전후로 하여, 캐뉼라(cannula) 주입 시스템을 이용하여, 깨어 있는 실험동물의 뇌실에 FK506 또는 2종의 신규 화합물을 처리하였고 (처리 농도: 1 mg/kg), 대조군에는 saline을 처리하였다. In vivo 신경세포 성장 촉진능을 평가하기 위해 기존의 보고된 방법에 따라 (Int. J. Mol. Med. 1656: 870-878, 2014), 흑색질-선조체 신경경로의 시작부인 흑색질 치밀부(pars compacta)에서는 도파민성 신경 세포체의 개수를 계측하였고, 신경경로의 말단부인 선조체에서는 흑색질로부터 뻗어 있는 신경 섬유의 밀도 변화를 정량 하였다. The ability to promote neuronal growth of new compounds was investigated according to the method reported by E. Petroske et al. (Neuroscience. 106: 590-601, 2001) using conventional immunohistochemical techniques. Specifically, 9-10 weeks old C57BL/6J mice treated with a neurotoxic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (treatment concentration: 25 mg/kg), The black matter- striatum dopaminergic nigrostriatal pathway, which governs the motor nerves, was regressed. Before and after MPTP treatment, a cannula injection system was used to treat FK506 or two new compounds in the ventricles of awake experimental animals (treatment concentration: 1 mg/kg), and saline was treated in the control group. In order to evaluate the ability to promote nerve cell growth in vivo, according to the previously reported method (Int. J. Mol. Med. 1656: 870-878, 2014), the black matter-pars compacta, the beginning of the black matter-progenitor neuropathy In ), the number of dopaminergic neuronal cell bodies was measured, and the density change of nerve fibers extending from the black matter was quantified in the striatum, the distal end of the neuropath.
그 결과를 도 57에 제시하였다. 도 57에서 알 수 있듯이, 본 발명의 신규 화합물 9종 중 9-deoxo-31-O-demethyl-FK520과 9-deoxo-31-O-demethyl-FK523의 혼합물 및 9-deoxo-31-O-demethyl-FK520의 경우, 선조체로 뻗은 도파민성 신경섬유의 밀도변화가 saline과 FK506 대비 더 높아 우수함을 확인하였다. 즉, 본 발명의 신규 화합물 9종 화합물의 경우 우수한 신경세포 성장 촉진능을 갖고 있음을 확인할 수 있었으며, 신경계 질환의 예방 또는 치료 목적으로 사용할 수 있다고 결론지을 수 있었다.The results are presented in Figure 57. As can be seen in FIG. 57, 9-deoxo-31- O of the 9 kinds of novel compounds of the present invention -demethyl-FK520 and 9-deoxo-31- O -demethyl- FK523 mixtures of 9-deoxo-31- O -demethyl of -In the case of FK520, it was confirmed that the density change of the dopaminergic nerve fibers extending to the striatum was higher than that of saline and FK506, and was excellent. That is, it was confirmed that the novel compound 9 of the present invention has excellent neuron growth promoting ability, and it can be concluded that it can be used for the purpose of preventing or treating neurological diseases.
실시예 13: 신규 화합물들의 신경계 질환 치료 효과 확인 ΙExample 13: Confirmation of the treatment effect of new compounds on nervous system diseases Ι
신규 화합물의 신경계 질환 치료 효과 확인은, 상기의 신경독성 화합물인 MPTP를 처리하여 신경을 손상시킨 마우스에 신규 화합물을 각각 투여한 다음, 로타로드 테스트 및 오픈필드 테스트를 수행하여 조사하였다. 구체적으로는 9~10주령의 C57BL/6J 마우스에 신경독성 화합물인 MPTP를 처리하여 (처리 농도: 25 mg/kg), 운동 신경을 관장하는 흑색질-선조체 도파민성 신경경로를 퇴행시켰다. MPTP 처리를 전후로 하여, 캐뉼라 주입 시스템을 이용하여, 실험동물의 뇌실에 FK506 또는 9종 신규 화합물을 처리하였고 (처리 농도: 1 mg/kg), 대조군에는 saline을 3일간 처리하였다. Confirmation of the therapeutic effect of the new compound on the nervous system disease was investigated by administering a new compound to a mouse that has damaged nerves by treating the neurotoxic compound MPTP, and then performing a rotarod test and an open field test. Specifically, 9 to 10 weeks of age, C57BL/6J mice treated with MPTP, a neurotoxic compound (treatment concentration: 25 mg/kg), regressed the black matter-progenitor dopamine neuropath responsible for motor neurons. Before and after MPTP treatment, FK506 or 9 new compounds were treated in the ventricles of experimental animals using a cannula injection system (treatment concentration: 1 mg/kg), and saline was treated for 3 days in the control group.
MPTP로 유도한 파킨슨 질환 마우스 모델로 퇴행성 신경계 질환을 특정하여, FK506 또는 신규 화합물 처리에 따른 하지의 보행 및 운동 기능 변화에 대한 기본 평가를 실행하였다. 로타로드 검사의 경우, 10 rpm의 속도로 회전하는 막대기 위에 실험 동물을 올려 놓고 낙하하기까지 걸린 시간을 측정하여, 하지의 보행 및 운동능력의 변화를 확인하였다. 오픈필드 테스트는, 벽으로 둘러싸인 비어 있는 공간 안에서 10분 동안 실험 동물의 총 이동거리를 측정함으로써, 기본적인 운동기능의 변화를 비교 분석하였다. Degenerative neurological diseases were identified using the MPTP-induced Parkinson's disease mouse model, and a basic evaluation was performed on changes in gait and motor function in the lower extremities following treatment with FK506 or new compounds. In the case of the rotarod test, the experimental animal was placed on a stick rotating at a speed of 10 rpm, and the time taken to drop was measured to confirm changes in walking and athletic ability of the lower extremities. In the open field test, the change in basic motor function was compared and analyzed by measuring the total travel distance of the experimental animal for 10 minutes in an empty space surrounded by walls.
로타로드 테스트 및 오픈필드 테스트 결과가 도 58에 제시되어 있다. 상기 결과는 본 발명의 신규 화합물 9종 중 9-deoxo-31-O-demethyl-FK520과 9-deoxo-31-O-demethyl-FK523의 혼합물을 처리한 그룹이, 생리식염수를 처리한 그룹 대비하여 유의미한 하지 보행 및 운동 능력의 향상을 보여줌을 가리킨다. 한편 오픈 필드 검사 결과, 9-deoxo-31-O-demethyl-FK520과 9-deoxo-31-O-demethyl-FK523의 혼합물을 처리한 그룹이, 생리식염수를 처리한 그룹 대비하여 높은 운동성을 보이는 것으로 확인되었다. 이 결과로부터, 본 발명의 신규 화합물 9종 중 선택되는 어느 하나 이상을 유효 성분으로 포함하는 신경계 질환 예방 또는 치료용 약학적 조성물이 퇴행성 신경질환 치료에 효과가 있음을 확인할 수 있었다. The results of the rotarod test and open field test are presented in FIG. 58. The results are compared to the group treated with a mixture of 9-deoxo-31- O- demethyl-FK520 and 9-deoxo-31- O -demethyl-FK523 among 9 new compounds of the present invention, compared to the group treated with physiological saline. Indicates a significant improvement in gait and motor skills. Meanwhile, as a result of the open field test, the group treated with the mixture of 9-deoxo-31- O- demethyl-FK520 and 9-deoxo-31- O- demethyl-FK523 showed higher mobility compared to the group treated with physiological saline. Was confirmed. From these results, it was confirmed that a pharmaceutical composition for preventing or treating neurological diseases comprising at least one selected from 9 new compounds of the present invention as an active ingredient is effective in treating degenerative neurological diseases.
실시예 14: 신규 화합물들의 기능성 시냅스 형성 활성 조사Example 14: Functional synapse formation activity investigation of new compounds
신규 화합물의 기능성 시냅스 형성 활성을, 초대 배양한 해마 신경 세포를 이용하여 D. Park. 외에 의해 보고된 방법 (Sci. Rep. 7: 7260, 2017)에 따라 조사하였다. 구체적으로 FK506 또는 신규 화합물에 노출시켜 (처리 농도: 1 ng/ml) 초대 배양한 10일-14일차 해마 신경 세포를 대상으로 패치-고정 레코딩을 시행하여, 흥분성 시냅스 전류의 빈도와 진폭을 측정하였다. 흥분성 시냅스 전류의 빈도는 시냅스 수의 변화를 확인할 수 있는 지표로 사용되고, 진폭은 시냅스 가소성을 알 수 있는 지표로서, 신규 화합물에 의한 시냅스의 질적 변화를 파악할 수 있다. The functional synapse-forming activity of the novel compound was analyzed using D. Park. In addition, it was investigated according to the method reported by (Sci. Rep. 7: 7260, 2017). Specifically, exposure to FK506 or a new compound (processing concentration: 1 ng/ml) was performed for patch-fixed recording on hippocampal neurons from the first to 10th to 14th days of primary culture to measure the frequency and amplitude of excitatory synaptic currents. . The frequency of the excitatory synaptic current is used as an index for confirming a change in the number of synapses, and the amplitude is an index for synaptic plasticity, and it is possible to grasp the qualitative change of synapses by a new compound.
측정한 흥분성 시냅스 전류의 빈도를 나타낸 결과를 도 59에 제시하였다. 도 56에서 알 수 있듯이 본 발명의 신규 화합물 9종 중 9-deoxo-31-O-demethyl-FK520과 9-deoxo-31-O-demethyl-FK520의 혼합물을 처리한 그룹에서 흥분성 시냅스 전류의 빈도가 유의미하게 증가했음을 확인하였다. 이는 상기 실시예 12와 13에서 신규 화합물을 처리한 그룹이 보인 in vivo 신경세포 성장촉진 활성 향상과 퇴행성 신경질환 치료 효과가, 기능성 시냅스 형성의 증가에서 기인할 수 있음을 시사한다. The results showing the frequency of the measured excitatory synaptic current are presented in FIG. 59. As can be seen from FIG. 56, the frequency of excitatory synaptic currents in the group treated with a mixture of 9-deoxo-31- O- demethyl-FK520 and 9-deoxo-31- O -demethyl-FK520 among 9 new compounds of the present invention It was confirmed that it increased significantly. This suggests that the in vivo neuron growth promoting activity and the treatment effect of degenerative neurological diseases shown by the groups treated with the novel compounds in Examples 12 and 13 may be attributable to an increase in functional synapse formation.
도 60은 흥분성 시냅스 전류의 진폭을 나타낸 그래프로서, 생리식염수와 FK506을 처리한 그룹이 유사한 진폭을 보인 것에 반해서, 신규 화합물 9종 중 9-deoxo-31-O-demethyl-FK520과 9-deoxo-31-O-demethyl-FK520의 혼합물을 처리한 그룹에서 평균 진폭이 증가하는 것을 관찰하였다. 이는 상기 신규화합물 9종 중 선택되는 어느 하나 이상을 유효 성분으로 포함하는 신경계 질환 예방 또는 치료용 약학적 조성물이 단순히 신경 세포 성장을 촉진하는 이상의, 시냅스 가소성을 높이는 작용에 관여함을 시사한다.Figure 60 is a graph showing the amplitude of the excitatory synaptic current, whereas the group treated with physiological saline and FK506 showed similar amplitude, 9-deoxo-31- O- demethyl-FK520 and 9-deoxo- out of 9 new compounds The average amplitude was observed to increase in the group treated with the mixture of 31- O- demethyl-FK520. This suggests that a pharmaceutical composition for preventing or treating a nervous system disease comprising at least one selected from the nine new compounds as an active ingredient is involved in the action of enhancing synaptic plasticity, more than simply promoting growth of nerve cells.
실시예 15: 9종 신규 화합물의 신경계 질환 치료 효과 확인 ⅡExample 15: Confirmation of treatment effects of 9 new compounds on nervous system diseases Ⅱ
9종 신규 화합물의 신경계 질환 치료 효과 확인은 신경을 손상시킨 랫드에 9종 신규 화합물 각각을 투여한 다음 BBB 테스트 및 그리드 워크 테스트를 수행하여 조사하였다. 구체적으로는 체중 220 g 정도의 2개월령 암컷 랫드 (Sprague-Dawley)에 대하여 신경손상 수술 (Experimental Neurology, 176: 143, 2002)을 실시하였다. 신경손상 수술은 25 mg/ml의 케타민 (Ketamine)과 1.3 mg/ml의 럼푼 (Rompun) 혼합물 2 mg/kg을 투여하여 완전히 마취시킨 랫드에 L2 복부척추후궁절제술 (L2 Ventral Laminectomy)을 실시하는 방식으로 수행하였다. 자세하게는 랫드의 제2요추를 열고 마이크로겸자 (Microrongeur)를 이용하여 정형추궁판 (Arcus Vertebra)의 좌외측 부위에 작은 구멍 (1 mm2)을 낸 다음 이 구멍에 블레이드 홀더 (Blade Holder)의 칼날을 삽입하고 경질막 (Dura Mater)을 지나 정형추궁판의 우외측 부위까지 칼집을 내어 척수의 복부 부위에 외상을 입혔다. 이후, 척수신경손상 부위의 등쪽근육조직 (Dorsal Musculature)을 봉합하고 피부를 수술용 클립으로 결찰시킴으로써 신경손상 수술을 실시하였다. 신경손상 수술을 마친 후 랫드를 따듯한 톱밥위에 두어 체온을 유지시키고 자율 방광 조절 (Autonomic Bladder Control)이 완전히 회복될 때까지 약 7일간 매일 3 내지 4회씩 복부 아래쪽을 마사지하여 방광의 내용물을 배출시켰다. 수술 시와 수술 이후의 감염예방을 위해 체중 100 g당 5 mg의 항생물질 세팔렉신 (Cefalexin)을 매일 근육 내 주사하였다. Confirmation of the treatment effect of the 9 new compounds on the nervous system disease was investigated by administering each of the 9 new compounds to the rats with nerve damage and then performing a BBB test and a grid walk test. Specifically, 2 months old female rats (Sprague-Dawley) weighing about 220 g were subjected to neurological surgery (Experimental Neurology, 176: 143, 2002). Neuroinjury surgery is performed by administering L2 Ventral Laminectomy to completely anesthetized rats by administering 2 mg/kg of a mixture of 25 mg/ml ketamine and 1.3 mg/ml rumpun. Was carried out. In detail, open the second lumbar vertebra of the rat and use a microrongeur to make a small hole (1 mm 2 ) in the left and outer part of the orthotic arch plate (Arcus Vertebra), and then cut the blade of the blade holder into the hole. And trauma to the abdominal region of the spinal cord by inserting the sheath through the dura mater and cutting the sheath to the right outer region of the orthopedic bladder. Subsequently, nerve injury surgery was performed by suturing the Dorsal Musculature of the spinal nerve injury site and ligating the skin with a surgical clip. After the nerve injury surgery, the rats were placed on a warm sawdust to maintain body temperature, and the contents of the bladder were discharged by massaging the lower abdomen 3 to 4 times daily for about 7 days until autonomic bladder control was fully restored. To prevent infection during and after surgery, 5 mg of antibiotic cephalexin per 100 g of body weight was injected intramuscularly daily.
신경계 질환 치료 효과 확인을 위한 BBB 테스트와 그리드 워크 테스트는 신경손상 수술 14일 전부터 실시하였고, 수술 3일전에 행동 및 운동기능에 대한 기본 평가를 실시하여 9종 신규 화합물의 투여 후 결과와 비교 분석하였다. BBB 테스트는 랫드를 표면이 거친 투명 플렉시글래스 박스에 넣고 4-5분간 관찰하여 관절운동, 체중 지탱, 앞다리-뒷다리 공조, 꼬리 위치 등과 같은 기준에 따라 점수를 매겨 0점은 뒷다리의 운동능이 없는 것으로 규정하고 최대 21점은 정상적인 보행능을 갖는 것으로 규정하였다. 그리드 워크 테스트는 랫드가 1 m 길이의 금속 평행봉을 걷게 하여 규칙적인 발걸음 능력을 평가하고, 비스듬한 봉을 내려갈 때의 뒷다리에 의한 조절능을 발걸음의 실수 횟수로 계수하여 10회의 실수는 랫드가 규칙적으로 걷지 못할 뿐만 아니라 다리를 자발적으로 조절할 수 없음을 나타내고, 0 내지 1회의 실수는 손상이 없고 정상적인 랫드의 운동능을 나타내는 것으로 규정하였다.The BBB test and grid walk test for confirming the effect of treating neurological diseases were performed 14 days before the nerve injury surgery, and a basic evaluation of behavior and motor function was performed 3 days before the operation, and the results were compared and analyzed after the administration of 9 new compounds. . In the BBB test, the rats were placed in a transparent plexiglass box with a rough surface, and observed for 4-5 minutes to score according to criteria such as joint exercise, weight bearing, forelimb-hindlimb coordination, and tail position. The maximum 21 points were defined as having normal walking ability. In the grid walk test, the rat walked a 1 m long metal parallel rod to evaluate the regular step ability, and the regulating ability of the hind leg when descending an oblique rod was counted as the number of steps, so the rat made 10 mistakes regularly. In addition to not being able to walk, it indicates that the legs cannot be controlled spontaneously, and mistakes from 0 to 1 are defined as being intact and indicating normal motor performance.
신경손상 수술 이후 3일째에 BBB 테스트와 그리드 워크 테스트에서 유사한 점수를 보인 랫드 100마리를 임의 선정하여 이를 총 10개 그룹으로 나누고 서로 다른 처치를 38일간 실시하였다 (G1: 부형제 매일 경구 투여, G2: 5 mg/kg 용량으로 9-deoxo-31-O-demethyl-prolyl-FK506 매일 경구 투여, G3: 5 mg/kg 용량으로 9-deoxo-36,37-dihydro-FK506 매일 경구 투여, G4: 5 mg/kg 용량으로 31-O-demethyl-36,37-dihydro-FK506 매일 경구 투여, G5: 5 mg/kg 용량으로 9-deoxo-31-O-demethyl-36,37-dihydro-FK506 매일 경구 투여, G6: 5 mg/kg 용량으로 9-deoxo-FK520 매일 경구 투여, G7: 5 mg/kg 용량으로 31-O-demethyl-FK520 매일 경구 투여, G8: 5 mg/kg 용량으로 9-deoxo-31-O-demethyl-FK520 매일 경구 투여, G9: 5 mg/kg 용량으로 9-deoxo-FK523 매일 경구 투여, G10: 5 mg/kg 용량으로 9-deoxo-31-O-demethyl-FK523 매일 경구 투여).On the third day after the nerve injury surgery, 100 rats with similar scores in the BBB test and the grid walk test were randomly selected, divided into 10 groups, and treated with different treatments for 38 days (G1: daily oral administration of excipients, G2: 9-deoxo-31- O- demethyl-prolyl-FK506 daily oral dose at 5 mg/kg dose, G3: 9-deoxo-36,37-dihydro-FK506 daily oral dose at 5 mg/kg dose, G4: 5 mg 31- O- demethyl-36,37-dihydro-FK506 daily oral dose at /kg dose, G5: 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 daily oral dose at 5 mg/kg dose, G6: 9-deoxo-FK520 daily oral dose at 5 mg/kg dose, G7: 31- O- demethyl-FK520 daily oral dose at 5 mg/kg dose, G8: 9-deoxo-31- at 5 mg/kg dose O -demethyl-FK520 daily oral administration, G9: 5 mg/kg dose 9-deoxo-FK523 daily oral dose, G10: 5 mg/kg dose 9-deoxo-31- O -demethyl-FK523 daily oral dose).
신경손상 수술 후 3일째부터 1주일 간격으로 BBB 테스트 및 그리드 워크 테스트를 수행한 결과가 도 57 및 도 58에 제시되어 있다. 상기 결과는 부형제 투여군에 비하여 9종의 신규화합물 투여군의 점수가 뛰어났으며, 이 결과로부터 9종 신규 화합물 중 선택된 하나 이상을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물이 신경계 질환 치료에 효과가 있음을 확인할 수 있었다.The results of performing the BBB test and the grid walk test at intervals of one week from the third day after the nerve injury surgery are presented in FIGS. 57 and 58. The result was superior to that of the excipient-administered group, and the score of the nine new compound-administered groups was superior, and from this result, the pharmaceutical composition for preventing or treating a neurological disease comprising at least one selected from the nine new compounds as an active ingredient was a nervous system disease. It was confirmed that the treatment was effective.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the above detailed description and equivalent concepts thereof.
Figure PCTKR2019017519-appb-I000039
Figure PCTKR2019017519-appb-I000039
Figure PCTKR2019017519-appb-I000040
Figure PCTKR2019017519-appb-I000040
Figure PCTKR2019017519-appb-I000041
Figure PCTKR2019017519-appb-I000041
Figure PCTKR2019017519-appb-I000042
Figure PCTKR2019017519-appb-I000042
Figure PCTKR2019017519-appb-I000043
Figure PCTKR2019017519-appb-I000043
Figure PCTKR2019017519-appb-I000044
Figure PCTKR2019017519-appb-I000044
Figure PCTKR2019017519-appb-I000045
Figure PCTKR2019017519-appb-I000045

Claims (12)

  1. 하기의 화학식 1로 표시되는 9-deoxo-31-O-demethyl-prolyl-FK506 (9-데옥소-31-O-디메틸-프롤릴-FK506), 하기의 화학식 2로 표시되는 9-deoxo-36,37-dihydro-FK506 (9-데옥소-36,37-디히드로-FK506), 하기의 화학식 3으로 표시되는 31-O-demethyl-36,37-dihydro-FK506 (31-O-디메틸-36,37-디히드로-FK506), 하기의 화학식 4로 표시되는 9-deoxo-31-O-demethyl-36,37-dihydro-FK506 (9-데옥소-31-O-디메틸-36,37-디히드로-FK506), 하기의 화학식 5로 표시되는 9-deoxo-FK520 (9-데옥소-FK520), 하기의 화학식 6으로 표시되는 31-O-demethyl-FK520 (31-O-디메틸-FK520), 하기의 화학식 7로 표시되는 9-deoxo-31-O-demethyl-FK520 (9-데옥소-31-O-디메틸-FK520), 하기의 화학식 8로 표시되는 9-deoxo-FK523 (9-데옥소-FK523), 및 하기의 화학식 9로 표시되는 9-deoxo-31-O-demethyl-FK523 (9-데옥소-31-O-디메틸-FK523)로 이루어진 군으로부터 선택되는 어느 하나의 화합물, 이의 이성질체, 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물:9-deoxo-31- O- demethyl-prolyl-FK506 represented by Formula 1 below (9-deoxo-31- O -dimethyl-prolyl-FK506), 9-deoxo-36 represented by Formula 2 below ,37-dihydro-FK506 (9-deoxo-36,37-dihydro-FK506), 31- O- demethyl-36,37-dihydro-FK506 (31- O -dimethyl-36) represented by Chemical Formula 3 below , 37-dihydro--FK506), formula 4 9-deoxo-31- O -demethyl -36,37-dihydro-FK506 (9- Deoxo -31- represented by O in the to-dimethyl-di -36,37- Hydro-FK506), 9-deoxo-FK520 (9-deoxo-FK520) represented by Formula 5 below, 31- O- demethyl-FK520 (31- O -dimethyl-FK520) represented by Formula 6 below, 9-deoxo-31- O- demethyl-FK520 (9-deoxo-31- O -dimethyl-FK520) represented by Formula 7 below, 9-deoxo-FK523 (9-deoxo) represented by Formula 8 below -FK523), and any one compound selected from the group consisting of 9-deoxo-31- O- demethyl-FK523 (9-deoxo-31- O -dimethyl-FK523) represented by Formula 9 below, isomers thereof , Or a pharmaceutical composition for the prevention or treatment of a nervous system disease comprising a pharmaceutically acceptable salt thereof as an active ingredient:
    [화학식 1][Formula 1]
    Figure PCTKR2019017519-appb-I000046
    Figure PCTKR2019017519-appb-I000046
    [화학식 2][Formula 2]
    Figure PCTKR2019017519-appb-I000047
    Figure PCTKR2019017519-appb-I000047
    [화학식 3][Formula 3]
    Figure PCTKR2019017519-appb-I000048
    Figure PCTKR2019017519-appb-I000048
    [화학식 4][Formula 4]
    Figure PCTKR2019017519-appb-I000049
    Figure PCTKR2019017519-appb-I000049
    [화학식 5][Formula 5]
    Figure PCTKR2019017519-appb-I000050
    Figure PCTKR2019017519-appb-I000050
    [화학식 6][Formula 6]
    Figure PCTKR2019017519-appb-I000051
    Figure PCTKR2019017519-appb-I000051
    [화학식 7][Formula 7]
    Figure PCTKR2019017519-appb-I000052
    Figure PCTKR2019017519-appb-I000052
    [화학식 8][Formula 8]
    Figure PCTKR2019017519-appb-I000053
    Figure PCTKR2019017519-appb-I000053
    [화학식 9][Formula 9]
    Figure PCTKR2019017519-appb-I000054
    .
    Figure PCTKR2019017519-appb-I000054
    .
  2. 제1항에 있어서, 상기 9-deoxo-31-O-demethyl-prolyl-FK506이 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM (수탁번호 KCTC13581BP)을 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method according to claim 1, wherein the 9-deoxo-31- O- demethyl-prolyl-FK506 is produced through culture using Streptomyces kanamyceticus ΔfkbD-fkbM (Accession No. KCTC13581BP) as a production strain. Pharmaceutical composition for the prevention or treatment of diseases of the nervous system.
  3. 제1항에 있어서, 상기 9-deoxo-36,37-dihydro-FK506이 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the 9-deoxo-36,37-dihydro-FK506 is a neurological disease characterized by being produced by culturing Streptomyces kanamyceticus ΔfkbD,tcsD (Accession No. KCTC13580BP) as a production strain. Pharmaceutical composition for the prevention or treatment of.
  4. 제1항에 있어서, 상기 31-O-demethyl-36,37-dihydro-FK506이 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the 31- O- demethyl-36,37-dihydro-FK506 is characterized by being produced through culture using Streptomyces kanamyceticus ΔfkbM,tcsD (Accession No. KCTC13584BP) as a production strain. Pharmaceutical composition for the prevention or treatment of diseases of the nervous system.
  5. 제1항에 있어서, 상기 9-deoxo-31-O-demethyl-36,37-dihydro-FK506이 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the 9-deoxo-31- O- demethyl-36,37-dihydro-FK506 is cultured using Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD (Accession No. KCTC13585BP) as a production strain. Pharmaceutical composition for the prevention or treatment of diseases of the nervous system, characterized in that produced.
  6. 제1항에 있어서, 상기 9-deoxo-FK520이 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsD (수탁번호 KCTC13580BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The culture according to claim 1, wherein the 9-deoxo-FK520 is produced by producing a strain of Streptomyces kanamyceticus ΔfkbD,tcsB (Accession No. KCTC13579BP) or Streptomyces kanamyceticus ΔfkbD,tcsD (Accession No. KCTC13580BP). Pharmaceutical composition for the prevention or treatment of diseases of the nervous system, characterized in that produced through.
  7. 제1항에 있어서, 상기 31-O-demethyl-FK520이 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsB (수탁번호 KCTC13583BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbM,tcsD (수탁번호 KCTC13584BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method according to claim 1, wherein the 31- O- demethyl-FK520 produces Streptomyces kanamyceticus ΔfkbM,tcsB (Accession No. KCTC13583BP) or Streptomyces kanamyceticus ΔfkbM,tcsD (Accession No. KCTC13584BP) as a production strain. Pharmaceutical composition for the prevention or treatment of diseases of the nervous system, characterized by being produced through culture.
  8. 제1항에 있어서, 상기 9-deoxo-31-O-demethyl-FK520이 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP) 또는 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsD (수탁번호 KCTC13585BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the 9-deoxo-31- O- demethyl-FK520 is Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB (Accession No. KCTC13582BP) or Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD ( Pharmaceutical composition for the prevention or treatment of diseases of the nervous system, characterized in that it is produced through culture using accession number KCTC13585BP) as a production strain.
  9. 제1항에 있어서, 상기 9-deoxo-FK523이 스트렙토마이세스 카나마이세티쿠스 ΔfkbD,tcsB (수탁번호 KCTC13579BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the 9-deoxo-FK523 is produced by cultivation using Streptomyces kanamyceticus ΔfkbD,tcsB (Accession No. KCTC13579BP) as a production strain. Ever composition.
  10. 제1항에 있어서, 상기 9-deoxo-31-O-demethyl-FK523이 스트렙토마이세스 카나마이세티쿠스 ΔfkbD-fkbM,tcsB (수탁번호 KCTC13582BP)를 생산균주로 이용한 배양을 통해 제조되는 것을 특징으로 하는 신경계 질환의 예방 또는 치료용 약학적 조성물.The method according to claim 1, wherein the 9-deoxo-31- O- demethyl-FK523 is produced by culturing Streptomyces kanamyceticus ΔfkbD-fkbM,tcsB (Accession No. KCTC13582BP) as a production strain. Pharmaceutical composition for the prevention or treatment of diseases of the nervous system.
  11. 하기의 화학식 1로 표시되는 9-deoxo-31-O-demethyl-prolyl-FK506, 하기의 화학식 2로 표시되는 9-deoxo-36,37-dihydro-FK506, 하기의 화학식 3으로 표시되는 31-O-demethyl-36,37-dihydro-FK506, 하기의 화학식 4로 표시되는 9-deoxo-31-O-demethyl-36,37-dihydro-FK506, 하기의 화학식 5로 표시되는 9-deoxo-FK520, 하기의 화학식 6으로 표시되는 31-O-demethyl-FK520, 하기의 화학식 7로 표시되는 9-deoxo-31-O-demethyl-FK520, 하기의 화학식 8로 표시되는 9-deoxo-FK523, 및 하기의 화학식 9로 표시되는 9-deoxo-31-O-demethyl-FK523로 이루어진 군으로부터 선택되는 어느 하나의 화합물, 이의 이성질체, 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 포함하는 신경계 질환의 예방 또는 치료용 약학적 조성물을 인간을 제외한 개체에게 투여하는 단계를 포함하는 것을 특징으로 하는, 신경계 질환의 예방 또는 치료 방법:9-deoxo-31- O- demethyl-prolyl-FK506 represented by Formula 1 below, 9-deoxo-36,37-dihydro-FK506 represented by Formula 2 below, 31- O represented by Formula 3 below -demethyl-36,37-dihydro-FK506, 9-deoxo-31- O represented by Formula 4 below, -demethyl-36,37-dihydro-FK506, 9-deoxo-FK520 represented by Formula 5 below, 31- O- demethyl-FK520 represented by Formula 6, 9-deoxo-31- O- demethyl-FK520 represented by Formula 7 below, 9-deoxo-FK523 represented by Formula 8 below, and the following Formula For the prevention or treatment of a nervous system disease comprising any compound selected from the group consisting of 9-deoxo-31- O- demethyl-FK523 represented by 9, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient A method of preventing or treating a nervous system disease, comprising administering a pharmaceutical composition to an individual other than a human:
    [화학식 1][Formula 1]
    Figure PCTKR2019017519-appb-I000055
    Figure PCTKR2019017519-appb-I000055
    [화학식 2][Formula 2]
    Figure PCTKR2019017519-appb-I000056
    Figure PCTKR2019017519-appb-I000056
    [화학식 3][Formula 3]
    Figure PCTKR2019017519-appb-I000057
    Figure PCTKR2019017519-appb-I000057
    [화학식 4][Formula 4]
    Figure PCTKR2019017519-appb-I000058
    Figure PCTKR2019017519-appb-I000058
    [화학식 5][Formula 5]
    Figure PCTKR2019017519-appb-I000059
    Figure PCTKR2019017519-appb-I000059
    [화학식 6][Formula 6]
    Figure PCTKR2019017519-appb-I000060
    Figure PCTKR2019017519-appb-I000060
    [화학식 7][Formula 7]
    Figure PCTKR2019017519-appb-I000061
    Figure PCTKR2019017519-appb-I000061
    [화학식 8][Formula 8]
    Figure PCTKR2019017519-appb-I000062
    Figure PCTKR2019017519-appb-I000062
    [화학식 9][Formula 9]
    Figure PCTKR2019017519-appb-I000063
    .
    Figure PCTKR2019017519-appb-I000063
    .
  12. 제11항에 있어서, 상기 신경계 질환은 퇴행성 신경질환 (Neurodegenerative disease), 말초신경장애 (Peripheral nerve injury), 외상성뇌손상 (Traumatic brain injury), 뇌졸중 (Cerebral infarction)인 것을 특징으로 하는, 신경계 질환의 예방 또는 치료 방법.The method of claim 11, wherein the neurological disease is a neurodegenerative disease (Neurodegenerative disease), peripheral nerve disorder (Peripheral nerve injury), Traumatic brain injury (Traumatic brain injury), Stroke (Cerebral infarction), Prevention or treatment methods.
PCT/KR2019/017519 2018-12-11 2019-12-11 Novel compound and pharmaceutical composition comprising same for treating neurological disorders WO2020122609A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20180158865 2018-12-11
KR10-2018-0158865 2018-12-11
KR10-2019-0086647 2019-07-17
KR1020190086647A KR102134782B1 (en) 2018-12-11 2019-07-17 A novel compound and a pharmaceutical composition for treating neurological disorder comprising the same

Publications (1)

Publication Number Publication Date
WO2020122609A1 true WO2020122609A1 (en) 2020-06-18

Family

ID=71077368

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2019/017519 WO2020122609A1 (en) 2018-12-11 2019-12-11 Novel compound and pharmaceutical composition comprising same for treating neurological disorders

Country Status (1)

Country Link
WO (1) WO2020122609A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5604234A (en) * 1991-09-05 1997-02-18 Abbott Laboratories Substituted thiol macrolactam immunomodulators
KR20050071491A (en) * 2002-11-08 2005-07-07 후지사와 야꾸힝 고교 가부시키가이샤 Use of tacrolimus (fk506) derivatives combined with beta2-agonists for the treatment of asthma
KR20060125916A (en) * 2004-03-01 2006-12-06 펩팀문, 인코포레이티드 Methods and compositions for treatment of autoimmune diseases
KR20120019360A (en) * 2010-08-24 2012-03-06 이화여자대학교 산학협력단 Novel tacrolimus derivative, neuroprotective composition comprising the same, immunosuppressive composition comprising the same, method of manufacturing the same and strain for manufacturing the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5604234A (en) * 1991-09-05 1997-02-18 Abbott Laboratories Substituted thiol macrolactam immunomodulators
KR20050071491A (en) * 2002-11-08 2005-07-07 후지사와 야꾸힝 고교 가부시키가이샤 Use of tacrolimus (fk506) derivatives combined with beta2-agonists for the treatment of asthma
KR20060125916A (en) * 2004-03-01 2006-12-06 펩팀문, 인코포레이티드 Methods and compositions for treatment of autoimmune diseases
KR20120019360A (en) * 2010-08-24 2012-03-06 이화여자대학교 산학협력단 Novel tacrolimus derivative, neuroprotective composition comprising the same, immunosuppressive composition comprising the same, method of manufacturing the same and strain for manufacturing the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GOULET, M. T. ET AL.: "ALKYL ETHER DERIVATIVES OF THE FK-506 RELATED , IMMUNOSUPPRESSIVE MACROLIDE L-683 , 742 (C3 1 -O-DESMETHYL ASCOMYCIN", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 4, no. 7, 1994, pages 927 - 930, XP026646494, DOI: 10.1016/S0960-894X(01)80265-1 *

Similar Documents

Publication Publication Date Title
WO2018056764A1 (en) Insulin analog having reduced binding force to insulin receptor, and use thereof
WO2011142514A1 (en) Composition containing pias3 as an active ingredient for preventing or treating cancer or immune disease
WO2013048164A1 (en) NOVEL COMPOUNDS AS HIF-1αINHIBITORS AND MANUFACTURING PROCESS THEREOF
WO2011159137A2 (en) Novel thiourea or urea derivative, preparation method thereof, and pharmaceutical composition for preventing or treating aids, containing same as active ingredient
WO2022035115A1 (en) Composition for prevention and treatment of skeletal muscle-related diseases containing alnus japonica extract or compound isolated therefrom and use thereof
WO2020251181A1 (en) Composition for promoting production of stem cell-derived exosomes and increasing stemness
WO2020106119A1 (en) Pharmaceutical composition comprising histone deacetylase 6 inhibitors
WO2016018117A1 (en) Fk506 derivative maintaining nerve regeneration activity without immunosuppressive activity, and use thereof
WO2020122609A1 (en) Novel compound and pharmaceutical composition comprising same for treating neurological disorders
WO2021066573A1 (en) Novel compound and use thereof in treating autoimmune diseases
WO2021194228A1 (en) Pharmaceutical composition for prevention or treatment of cancer
WO2022197100A1 (en) Pharmaceutical composition for enhancing anticancer effect of anticancer drug
WO2023080765A1 (en) Novel oxadiazole derivative and use thereof
WO2019231261A1 (en) Novel biphenyl derivative compound and use thereof
WO2014182078A1 (en) Composition for regulating immunological activity in intestines and use thereof
WO2021086038A1 (en) Novel compound and pharmaceutical composition for prevention or treatment of cancer comprising same
WO2021256894A1 (en) Novel prolylfk506 derivatives having neurite growth and synapse formation activities and uses thereof
WO2020122607A1 (en) Novel compound and pharmaceutical composition for treating fungal infection comprising same
WO2022203219A1 (en) Pharmaceutical composition for prevention or treatment of cancer
WO2022211518A1 (en) Novel compound and use thereof in treating psoriasis, asthma, or systemic lupus erythematosus
WO2020122611A1 (en) Novel compound and composition comprising same for stimulating hair growth
WO2023249228A1 (en) Novel compound and use thereof for treatment of autoimmune diseases
WO2023043261A1 (en) Pharmaceutical composition for enhancing anticancer effect of anticancer drug
WO2010064817A2 (en) Novel coumarin-based compounds, preparation method thereof, and multidrug resistance inhibitory pharmaceutical compositions containing same as active ingredients
WO2023043269A1 (en) Pharmaceutical composition for modulating immune response

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19895289

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19895289

Country of ref document: EP

Kind code of ref document: A1