Novel prolyl FK506 derivatives with neurite outgrowth and synapse formation activity and uses thereof

Abstract

The present invention relates to any one novel compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, 9-deoxy-prolyl FK520 and 9-deoxy-31-O-demethyl-prolyl FK520 and use thereof.

Description

Technical Field

[0001] The present invention relates to a novel polyol selected from 9-deoxy-36,37-dihydro-prolyl FK506 (9-deoxo-36,37-dihydro-prolyl FK506), 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK A compound from the group consisting of FK506 (9-deoxo-31-O-demethyl-36,37-dihydro-prolylFK506), 9-deoxy-prolylFK520 (9-deoxo-prolylFK520) and 9-deoxy-31-O-demethyl-prolylFK520 (9-deoxo-31-O-demethyl-prolylFK520); a pharmaceutical composition for preventing or treating nervous system diseases, comprising the above-mentioned compounds, their isomers or pharmaceutically acceptable salts thereof as active ingredients; a quasi-pharmaceutical composition; a method for preventing or treating nervous system diseases using the above-mentioned pharmaceutical composition; and a method for producing the above-mentioned novel compound using a strain of Streptomyces kanamyceticus. Background Art

[0002] With the rapid aging of the world's population, the number of patients with degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, and stroke has increased rapidly. However, for these neurological diseases, it is currently impossible to implement fundamental treatments such as repairing damaged nerves and regenerating nerve cells, but treatments are being carried out for the purpose of alleviating pain or delaying deterioration. This is because there is no drug that can not only delay nerve damage, but also provide nerve regeneration and repair that can be used to treat nerve damage diseases. It is expected that the number of patients with neurodegenerative diseases will continue to increase, so the development of DMTs (Disease-modifying treatments, disease-modifying treatments) pharmaceuticals that can fundamentally inhibit the progression of the disease has become increasingly important. This is because the roles of disabled patients with disorders caused by nerve damage in society and family are greatly limited, which will cause personal losses and deterioration of quality of life. Therefore, it is urgent to develop a drug that can provide nerve regeneration, which can be used alone or in combination to treat various diseases related to nerve damage.

[0003] FK506 inhibits interleukin transcription by binding to FK506-binding protein (FKBP) 12 in human cells and then binding to calcineurin (CaN) to inhibit its activity, thereby showing immunosuppressive effects, and exhibits nerve regeneration activity through a mechanism that has not yet been precisely determined [Nat. Chemistry, Biology, November 33, 2015]. However, there are still no reports on nerve regeneration substances that have no side effects caused by immunosuppressive activity and can be used as therapeutic agents for nerve damage diseases. Summary of the invention

[0004] Technical issues

[0005] Therefore, the inventors have made great efforts to identify novel compounds 9-deoxy-36,37-dihydro-prolyl FK506 (9-deoxo-36,37-dihydro-prolyl FK506), 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 (9-deoxo-31-O-demethyl-36,37-dihydro-prolyl FK506), 9-deoxy-prolyl FK520 (9-deoxo-36,37-dihydro-prolyl FK520) that can be used as the main component of a pharmaceutical composition for preventing or treating a nervous system disease. The invention has been developed to study the neuron growth promoting effects of four novel compounds, namely, 9-deoxo-prolylFK520) and 9-deoxy-31-O-demethyl-prolylFK520 (hereinafter referred to as "four novel compounds"), develop their preparation processes, and further develop pharmaceutical compositions for preventing or treating nervous system diseases using them as active ingredients, confirm that the compositions have no side effects caused by immunosuppressive activity and can be effectively used as pharmaceutical compositions for preventing or treating nervous system diseases, thereby completing the present invention. In particular, the significance of the present invention is that, compared with the FK506 compound, the immunosuppressive activity of the four novel compounds is significantly reduced, and the effectiveness of the neuron growth promoting activity, etc. is improved.

[0006] Technical Solution

[0007] One object of the present invention is to provide any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, 9-deoxy-prolyl FK520 and 9-deoxy-31-O-demethyl-prolyl FK520.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating nervous system diseases, which comprises the above-mentioned compound, its isomer, or its pharmaceutically acceptable salt as an active ingredient.

[0009] Another object of the present invention is to provide a method for preventing or treating nervous system diseases, which comprises the step of administering the above four novel compounds to an individual other than a human.

[0010] Another object of the present invention is to provide a composition for preventing or improving nervous system diseases, which comprises the step of administering the above four novel compounds to an individual.

[0011] Another object of the present invention is to provide a method for producing the four novel compounds, which comprises the step of culturing Streptomyces kanamyceticus.

[0012] Another object of the present invention is to provide a strain for producing the four novel compounds.

[0013] Beneficial Effects

[0014] It was confirmed that the immunosuppressive activity of the pharmaceutical composition for preventing or treating nervous system diseases according to the present invention, which contains at least one selected from four novel compounds as an active ingredient, was significantly reduced, but the neurite growth and synapse formation activity effects were excellent, and in the Parkinson's disease mouse model induced by the neurotoxic substance MPTP, it was confirmed that the therapeutic effect on nervous system diseases, especially degenerative nervous system diseases, was excellent. In addition, excellent safety was confirmed in cytotoxicity and safety experiments. Therefore, the four novel compounds of the present application can be effectively used without side effects on immunosuppressive activity in the treatment of nervous system diseases, and a more fundamental therapeutic effect can be expected compared with existing drug treatments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result of HPLC analysis of 9-deoxy-36,37-dihydro-prolyl FK506.

[0016] Figure 2 This is the NMR analysis of 9-deoxy-36,37-dihydro-prolyl FK506 ( 1 H-NMR) results.

[0017] Figure 3 This is the NMR analysis of 9-deoxy-36,37-dihydro-prolyl FK506 ( 13 C-NMR) results.

[0018] Figure 4 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-36,37-dihydro-prolyl FK506 are shown in FIG.

[0019] Figure 5 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-36,37-dihydro-prolyl FK506 are shown in FIG.

[0020] Figure 6 This is the result of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-36,37-dihydro-prolyl FK506.

[0021] Figure 7 This is the result of HPLC analysis of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506.

[0022] Figure 8 This is the NMR analysis of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 ( 1 H-NMR) results.

[0023] Fig. 9 This is the NMR analysis of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 ( 13 C-NMR) results.

[0024] Fig.10 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 are shown in FIG.

[0025] Fig.11 This is the result of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506.

[0026] Fig.12 This is the result of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506.

[0027] Fig.13 This is the result of HPLC analysis of 9-deoxy-prolyl FK520.

[0028] Fig.14 is the NMR analysis of 9-deoxy-prolyl FK520 ( 1 H-NMR) results.

[0029] Fig.15is the NMR analysis of 9-deoxy-prolyl FK520 ( 13 C-NMR) results.

[0030] Fig.16 This is the result of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-prolyl FK520.

[0031] Fig.17 This is the result of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-prolyl FK520.

[0032] Fig.18 This is the result of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-prolyl FK520.

[0033] Fig.19 This is the result of HPLC analysis of 9-deoxy-31-O-demethyl-prolyl FK520.

[0034] Fig. 20 This is the NMR analysis of 9-deoxy-31-O-demethyl-prolyl FK520 ( 1 H-NMR) results.

[0035] Fig.21 This is the NMR analysis of 9-deoxy-31-O-demethyl-prolyl FK520 ( 13 C-NMR) results.

[0036] Fig. 22 This is the result of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-31-O-desmethyl-prolyl FK520.

[0037] Fig.23 This is the result of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-31-O-desmethyl-prolyl FK520.

[0038] Fig.24 This is the result of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-31-O-desmethyl-prolyl FK520.

[0039] Fig.25 It is the identification result of the degree of reduction of immunosuppressive activity of the four novel compounds of the present invention.

[0040] Fig.26 These are the identification results of the nerve cell growth promoting abilities of the four novel compounds of the present invention.

[0041] Fig. 27These are the results of identifying the therapeutic ability of the synaptic activity effects of the four novel compounds of the present invention on nervous system diseases (a: hippocampal neurons cultured by exposure to FK506 or the four novel compounds, b: determination of excitatory synaptic currents in neurons, c: frequency of excitatory synaptic currents in neurons).

[0042] Fig.28 The present invention is based on the identification results of the four novel compounds of the present invention for their ability to restore and treat dopaminergic neural pathways in Parkinson's disease animal models (A: changes in nerve fiber density, B: the number of dopaminergic nerve cells).

[0043] Fig.29 The following are the results of MTT analysis used to identify the cytotoxicity of the four novel compounds of the present invention. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below. At the same time, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the following specific description.

[0045] One aspect of the present invention for achieving the above object is to provide any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506 represented by Chemical Formula 1, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 represented by Chemical Formula 2, 9-deoxy-prolyl FK520 represented by Chemical Formula 3, and 9-deoxy-31-O-demethyl-prolyl FK520 represented by Chemical Formula 4:

[0046] [Chemical formula 1]

[0047]

[0048] [Chemical formula 2]

[0049]

[0050] [Chemical formula 3]

[0051]

[0052] [Chemical formula 4]

[0053]

[0054] Another aspect of the present invention for achieving the above object is to provide a pharmaceutical composition for preventing or treating a nervous system disease, comprising any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506 represented by Chemical Formula 1, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 represented by Chemical Formula 2, 9-deoxy-prolyl FK520 represented by Chemical Formula 3, and 9-deoxy-31-O-demethyl-prolyl FK520 represented by Chemical Formula 4, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:

[0055] [Chemical formula 1]

[0056]

[0057] [Chemical formula 2]

[0058]

[0059] [Chemical formula 3]

[0060]

[0061] [Chemical formula 4]

[0062]

[0063] As used herein, the term "FK506, tacrolimus or fujimycin" is a substance with immunosuppressive activity originally isolated from various strains of Streptomyces tsukubaensis, and is a 23-membered ring macrolide polyketide compound. It is known that its immunosuppressive effect is stronger than that of cyclosporine, and when used for organ transplantation (especially liver), it suppresses rejection reactions. The above-mentioned FK506 can be synthesized by a PKS / NRPS (polyketide synthase / nonribosomal peptide synthetase) complex system, but the FK506 derivative of the present invention can be a novel compound as follows: that is, produced by a novel strain prepared by deleting the biosynthetic gene of Streptomyces sp.

[0064] As used herein, the term "FK520 or ascomycin" also refers to an immunosuppressant, a compound having a macrolide structure formed by 23 carbon atoms, and is an ethyl analog of FK506 at the C21 position.

[0065] As a specific example of the present invention, the compound of the present invention may include its isomers or pharmaceutically acceptable salts.

[0066] As used herein, the term "isomer" refers to the relationship between compounds that have the same chemical formula but are not actually identical, and may include, for example, conformational isomers, geometric isomers, optical isomers (enantiomers), stereoisomers, and diastereomers.

[0067] As used herein, the term "pharmaceutically acceptable salt" refers to any organic or inorganic addition salts having a concentration of effective effect that is relatively nontoxic and harmless to the patient, and the side effects caused by the salt do not reduce the beneficial effects of the parent compound. For example, the above-mentioned salt can 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 of an aqueous acid solution and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared as follows: heating an equimolar amount of the compound and an acid or alcohol (for example, ethylene glycol monomethyl ether) in water, then evaporating the above mixture and drying, or filtering the precipitated salt. At this time, organic acids and inorganic acids can be used as free acids. The above-mentioned salt can be a pharmaceutically acceptable metal salt prepared using a base.

[0068] As another specific example, the compound of the present invention may be in the form of a solvate or a prodrug, which is included in the scope of the present invention. Preferably, the solvate may include a hydrate and an ethanolate.

[0069] The pharmaceutical composition of the present invention can be used as a single preparation, or can be used as a combined preparation by further comprising a drug known to have a therapeutic effect on nervous system diseases. The pharmaceutical composition can be formulated using a pharmaceutically acceptable carrier or excipient, so that it can be prepared in a unit dose form or by being loaded into a multi-dose container.

[0070] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the injected compound. The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the above-mentioned carriers may include co-surfactants exemplified by transcutol, polyethylene glycol, triacetin, and mixtures thereof; surfactants exemplified by Cremophor, Tween, Myrj, Poloxamer, Pluronic, Lutrol, Imwitor, Span, Labrafil, etc., which may be used alone or as a mixture; oils exemplified by caprylic capric triglyceride, medium chain glyceryl acid paper (Captex), and ethyl oleate, etc., which may be used alone or as a mixture; and organic acids exemplified by erythrocortic acid and citric acid, etc., which may be used alone or as a mixture. They may be used alone or in combination of two or more.

[0071] In addition, other conventional additives such as antioxidants, buffers and / or antibacterial agents may be added as needed, and diluents, dispersants, surfactants, binders, lubricants, etc. may be added to formulate injectable preparations (e.g., aqueous solutions, suspensions, emulsions), pills, capsules, granules or tablets, etc.

[0072] As used herein, the term "neurological disease" refers to a general term for diseases of the nervous system, and in a specific example, may include diseases caused by nerve damage. Nerve damage diseases may include neurodegenerative diseases, peripheral nerve injury, traumatic brain injury, and cerebral infarction caused by cerebrovascular disorders, but are not limited thereto.

[0073] As an example, the above-mentioned neurodegenerative diseases refer to diseases in which degenerative changes occur in nerve cells of the central nervous system and cause various symptoms, such as dementia, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system strophy, olivopontocerebellaratrophy (OPCA), Shy-Drager syndrome; striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, corticobasal ganlionic degeneration, diffuse Lewy body disease (DLS), and so on. disease, Parkinson-ALS-dementia complex of Guam, or Pick's disease.

[0074] As another example, the above-mentioned neurological damage diseases may include epilepsy, stroke, cerebral infarction, ischemic brain disease, spinal cord injury disease, peripheral nervous system disease, behavioral disorder, developmental disorder, mental retardation, Down syndrome or schizophrenia, but are not limited thereto.

[0075] As another example, the nerve damage disease may be a condition caused by nerve cell damage or cell death, etc.

[0076] As used herein, the term "prevention" refers to the act of administering the above-mentioned pharmaceutical composition of the present invention to an individual with symptoms or conditions suspected or likely to develop a nervous system disease to reduce the possibility of developing a nervous system disease or alleviate the symptoms or conditions.

[0077] As used herein, the term "treatment" refers to all actions to improve or benefit the symptoms of a neurological disease by administering the pharmaceutical composition of the present invention to an individual suspected of having a neurological disease.

[0078] The pharmaceutical composition comprising the above four novel compounds of the present invention is characterized in that the immunosuppressive activity is reduced.

[0079] In one embodiment of the present invention, the immunosuppressive activity of the four novel compounds was confirmed by in vitro T cell activity assay, and significantly lower immunosuppressive activity was confirmed compared to FK506 (Table 7).

[0080] In another specific example of the present invention, the activity of the four novel compounds in promoting the growth of nerve cells based on neurite outgrowth was confirmed ( Fig.26 ).

[0081] In another specific example of the present invention, by confirming the functional synaptic activity of the four novel compounds, the frequency of the increased excitatory synaptic current compared to FK506 was confirmed, thereby confirming the therapeutic effects of the four novel compounds on nervous system diseases ( Fig. 27 ).

[0082] In another specific example of the present invention, in order to evaluate the in vivo neuroregenerative activity of the above four novel compounds, a disease animal model of Parkinson's disease mouse model induced by the neurotoxic compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to confirm the degree of recovery of nerve cells and nerve fibers degenerated by MPTP, thereby confirming the therapeutic effects of the above four novel compounds on neurodegenerative diseases ( Fig.28 ).

[0083] In addition, in another specific example of the present invention, the safety of the above four novel compounds can be determined by 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis for cytotoxicity identification ( Fig.29 ), the Ames test for liver genotoxicity assessment, and the human eag-related gene (hERG) analysis for potential effects on cardiac repolarization.

[0084] The above results indicate that the above four novel compounds are substances without immunosuppressive side effects and have proven therapeutic efficacy and safety for neurological diseases, and can be used to prevent or treat neurological diseases.

[0085] As another aspect to achieve the above object, the present invention provides a method for preventing or treating a nervous system disease, comprising the step of administering the above pharmaceutical composition to an individual.

[0086] As used herein, the terms "neurological disease", "prevention" and "treatment" are the same as above.

[0087] As used herein, the term "subject" may refer to all animals that have or may develop a neurological disease.

[0088] The pharmaceutical composition of the present invention may include a pharmaceutically effective amount of one or more selected from four novel compounds, its isomers or salts thereof. As used herein, the term "pharmaceutically effective dose" refers to a sufficient amount for treating diseases with a reasonable benefit / risk ratio applicable to medical treatment, and generally, 0.001 mg / kg-1000 mg / kg, preferably 0.05 mg / kg-200 mg / kg, more preferably 0.1 mg / kg-100 mg / kg, can be administered once a day to several times. However, according to the purpose of the present invention, the specific therapeutic effective dose for a specific patient is preferably different according to the type and degree of the reaction to be achieved, including whether to use a specific composition of other preparations according to the situation, the patient's age, weight, general health, sex and diet, administration time, route of administration and secretion rate of the composition, treatment duration, drugs used in combination or simultaneously with a specific composition, and similar factors well known in the medical field.

[0089] Although the administration frequency of the pharmaceutical composition of the present invention is not particularly limited thereto, it may be administered once a day or several times in divided doses.

[0090] The dosage of the pharmaceutical composition of the present invention may be 0.001 mg / kg-1000 mg / kg, specifically 0.05 mg / kg-200 mg / kg, 0.1 mg / kg-100 mg / kg, 0.1 mg / kg-20 mg / kg, but is not limited thereto.

[0091] The pharmaceutical composition of the present invention can be administered alone or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents. In addition, the above-mentioned pharmaceutical composition can be administered in a single dose or multiple doses. Taking all the above factors into consideration, it is important to administer the drug in an amount that minimizes the induction of side effects and obtains the maximum effect with the minimum amount, which can be easily determined by those skilled in the art.

[0092] As used herein, the term "administering" refers to introducing the pharmaceutical composition of the present invention into the patient's body by any appropriate method. The administration route of the composition of the present invention can be any oral or parenteral route as long as it can reach the target tissue.

[0093] The administration method of the pharmaceutical composition of the present invention is not particularly limited, and any administration method commonly used in the art can be used. Non-limiting examples of administration methods include oral or parenteral administration. The pharmaceutical composition of the present invention can be prepared into various dosage forms according to the target administration method.

[0094] In one embodiment of the present invention, the four novel compounds have significantly reduced immunosuppressive activity, and are excellent in neurite growth and synapse formation activity effects, degenerative nervous system disease therapeutic effects, and safety.

[0095] These results suggest that the four novel compounds can be used to treat persistent neurological diseases by significantly reducing the side effects caused by immunosuppressive activity.

[0096] Another aspect of the present invention for achieving the above object is to provide a method for producing the above four novel compounds, which comprises a step of culturing Streptomyces kanamyceticus, as a bioproduction method for the above four novel compounds.

[0097] Among the four novel compounds, 9-deoxy-36,37-dihydro-prolyl FK506 or 9-deoxy-prolyl FK520 can be prepared by culturing kanamycin Streptomyces ΔfkbD, tcsD, fkbL (Accession No.: KCTC14171BP).

[0098] In addition, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 or 9-deoxy-31-O-demethyl-prolyl FK520 can be prepared by culturing kanamycin Streptomyces ΔfkbD-fkbM, tcsD, fkbL (Accession No.: KCTC14170BP).

[0099] In the present application, the term "culture" refers to the cultivation of microorganisms under appropriately adjusted environmental conditions. The culture process of the present application can be carried out according to appropriate culture media and culture conditions known in the art. Those skilled in the art can easily adjust and use this culture process according to the selected strain. Specifically, the above-mentioned culture can be batch, continuous and fed-batch, but is not limited thereto.

[0100] In the preparation methods of the above four novel compounds, the culture temperature generally used in the culture process of Streptomyces is used. The suitable culture temperature for implementing the present invention is preferably 23°C to 30°C, more preferably 25°C to 28°C.

[0101] Furthermore, in the above preparation method, the pH during the culture process is maintained between 6.5 and 9, preferably between 7 and 8.

[0102] On the one hand, in the above preparation method, it is important to maintain a high dissolved oxygen level in the culture medium. When the dissolved oxygen level at the beginning of the culture is 100%, the dissolved oxygen level should be maintained above 30% until the end of the culture. For this purpose, stirring can usually be performed at 800 rpm to 1500 rpm.

[0103] The extraction of the four novel compounds produced by the cultured cell body in the above-mentioned preparation method can be achieved by implementing the first extraction process, the second extraction process and the third extraction process. In the present invention, as the first extraction process, an organic solvent extraction method is used, and the solvent that can be used at this time is ethyl acetate (Ethyl acetate), methanol (Methanol) or acetone (Acetone), etc., preferably ethyl acetate or methanol. As the second extraction process, silica gel chromatography (Silica gel chromatography) can be used, and the solvent that can be used at this time is preferably methanol and methylene chloride (Methylene chloride). In addition, chromatography can be used as the third extraction process, and the solvent that can be used at this time is acetonitrile (Acetonitrile), ammonium acetate buffer (Ammonium acetate buffer), acetic acid (Acetic acid) or formic acid (Formic acid), etc., preferably acetonitrile. The application of these methods is conducive to the recovery of the four novel compounds and improves the yield.

[0104] As another aspect to achieve the above-mentioned purpose, the present invention provides a production strain for preparing four new compounds, and the above-mentioned production strains are kanamycin Streptomyces ΔfkbD-fkbM, tcsD, fkbL (preservation number: KCTC14170BP) and kanamycin Streptomyces ΔfkbD, tcsD, fkbL (preservation number: KCTC14171BP).

[0105] The above-mentioned production strain may be a recombinant strain, and the recombination may be achieved by genetic modification, such as transformation.

[0106] As another aspect to achieve the above-mentioned object, the present invention provides a quasi-pharmaceutical composition for preventing or ameliorating nervous system diseases, comprising any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, 9-deoxy-prolyl FK520, 9-deoxy-31-O-demethyl-prolyl FK520, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0107] The above four compounds, isomers, pharmaceutically acceptable salts, nervous system diseases and prevention are the same as above.

[0108] The term "quasi-drug" as used in the present invention refers to articles used for diagnosing, curing, alleviating, treating or preventing human or animal diseases, excluding instruments, machines or devices; and articles used for producing pharmacological effects on the structure and function of humans or animals, excluding instruments, machines or devices.

[0109] In the present invention, the quasi-drug composition may have the effect of preventing or improving nervous system diseases, but is not limited thereto.

[0110] In addition to the above ingredients, the quasi-medical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent as required. The above pharmaceutically acceptable carrier, excipient or diluent is not limited, as long as it does not damage the effect of the present invention, for example, it may include a filler, a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, a lubricant, a sweetener, a fragrance, a preservative, etc.

[0111] The above-mentioned "pharmaceutically acceptable carrier" refers to a carrier, excipient or diluent that does not cause irritation to the organism and does not damage the biological activity and properties of the injected compound. Specifically, it can be a non-naturally occurring carrier. The type of carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the above-mentioned carriers can include physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, glucose solution, maltodextrin solution, glycerol and ethanol, etc. They can be used alone or in combination of two or more.

[0112] The above-mentioned composition comprising a pharmaceutically acceptable carrier can be various dosage forms for oral or non-oral administration, preferably oral dosage forms, but not limited thereto. When formulation is performed, a commonly used filler, extender, adhesive, wetting agent, disintegrant, surfactant and other diluents or excipients are used to prepare. Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and this solid preparation is prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. to the above-mentioned compound. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, internal liquids, emulsions, syrups, etc., and in addition to water and liquid paraffin as commonly used simple diluents, a variety of excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can be included. Preparations for non-oral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As non-aqueous solvents and suspending solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, synthetic fatty acid esters (Witepsol), polyethylene glycol (Macrogol), Tween 61, cocoa butter, laurel fat, glycerin gelatin, etc. can be used.

[0113] The quasi-drug composition of the present invention may include disinfectant cleaners, shower foams, ointments, wet wipes, coating agents, etc., but is not limited thereto. The preparation method, dosage, method of use, components, etc. of the quasi-drug may be appropriately selected from conventional techniques known in the art.

[0114] In another aspect of the present invention, a food composition for preventing or improving nervous system diseases is provided, comprising any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, 9-deoxy-prolyl FK520 and 9-deoxy-31-O-demethyl-prolyl FK520, an isomer thereof, or a food-acceptable salt thereof as an active ingredient.

[0115] The above four compounds, isomers, food-acceptable salts, nervous system diseases and prevention are the same as those described above.

[0116] The food composition of the present invention can be taken daily, and unlike general medicines, it uses natural products as raw materials, has the advantages of no side effects even when taking medicines for a long time, and therefore can be usefully used to prevent or improve nervous system diseases.

[0117] As used herein, the term "improvement" refers to all actions that alleviate or reduce parameters associated with a neurological disease (eg, severity of symptoms) by ingesting a food composition.

[0118] As used herein, the term "food" includes meat, sausage, bread, chocolate, sugar, snacks, biscuits, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, health drinks, alcoholic beverages, vitamin complexes, health functional foods, health foods, etc., and may include all foods in a general sense.

[0119] The health functional food and food for special health use (FoSHU) are the same terms, which refer to foods with high medical and therapeutic effects. After processing, in addition to providing nutrition, they can also effectively play a biological regulation function.

[0120] Here, "functionality" refers to the effect of nutrients regulating the structure and function of the human body, or obtaining useful effects in health care such as physiological effects. The above-mentioned health food refers to food that has a positive health or health-promoting effect compared with general food, and health supplement food refers to food that is intended to supplement health. Depending on the situation, the terms health functional food, health food and health supplement food can be used interchangeably.

[0121] Specifically, the above-mentioned health functional foods refer to foods prepared by adding the above-mentioned four new compounds to food materials such as beverages, teas, spices, chewing gum, biscuits, etc., or by encapsulation, powderization, suspension, etc., which bring specific effects on health when ingested. Unlike general medicines, since food is used as raw material, it has the advantage of no side effects even if taken for a long time.

[0122] The food of the present invention can be prepared by methods commonly used in the art, and can be prepared by adding raw materials and ingredients commonly used in the art.

[0123] Furthermore, the above-mentioned food composition can be prepared in various dosage forms without limitation as long as it is recognized as a food.

[0124] In addition, the above-mentioned food composition may further include a food-acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the art can be used.

[0125] In addition, the above-mentioned food composition may also include additional ingredients commonly used in food compositions to improve smell, taste, and vision. For example, the above-mentioned composition may include vitamin A, vitamin C, vitamin D, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, biotin, folic acid, pantothenic acid, etc. In addition, the above-mentioned composition may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc.; and amino acids such as lysine, tryptophan, cysteine, and valine.

[0126] In addition, the above-mentioned food composition can also include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG) (monosodium glutamate, etc.), sweeteners (sweetness, sodium cyclamate, saccharin, sodium, etc.), spices (vanillin, lactone, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), fortifiers, emulsifiers, thickeners (foaming agents), film-forming agents, gum bases, foam suppressants, solvents, improvers, etc. The above-mentioned additives can be selected according to the type of food and used in appropriate amounts.

[0127] In another aspect of the present invention, there is provided a use of a composition for preventing, improving or treating a nervous system disease, the composition comprising any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, 9-deoxy-prolyl FK520, 9-deoxy-31-O-demethyl-prolyl FK520, an isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. Specific embodiments

[0129] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are only used to illustrate the present invention, and the scope of the present invention is not limited thereto.

[0130] Example 1: Preparation of 9-deoxy-36,37-dihydro-prolyl FK506

[0131] For Streptomyces kanamyceticus, which produces FK506, according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098), double cross-over homologous recombination was used to cause in-frame gene deletion, resulting in inactivation of fkbD, tcsD and fkbL genes, and Streptomyces kanamyceticus ΔfkbD, tcsD, fkbL (Accession No.: KCTC14171BP) as a production strain of 9-deoxy-36,37-dihydro-prolyl FK506 was prepared.

[0132] Specifically, in order to prepare deletion mutants of fkbD, tcsD and fkbL genes in the FK506 kanamycin-producing Streptomyces strain, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then transformed into the kanamycin-producing Streptomyces as the FK506-producing strain by conjugation.

[0133] The strain preparation method can be more specifically described as the preparation of in-frame gene deletion plasmids and the preparation of gene deletion strains.

[0134] In the preparation of in-frame gene deletion plasmid, E. coli-Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. The preparation of plasmid was carried out by PCR amplification of left- and right-flanking fragments of the target gene for deletion in Fosmid DNA derived from Streptomyces kanamycin. For the deletion of fkbD gene, primer pair FkbDLF / FkbDLR for the left flank fragment and primer pair FkbDRF / FkbDRR for the right flank fragment were designed. For the deletion of tcsD gene, primer pair TcsDLF / TcsDLR for the left flank fragment and primer pair TcsDRF / TcsDRR for the right flank fragment were designed. For the deletion of fkbL gene, primer pair FkbLLF / FkbLLR for the left flank fragment and primer pair FkbLRF / FkbLRR for the right flank fragment were designed. All PCR fragments were isolated, cut with HindIII-XbaI or XbaI-EcoRI, and cloned into pKC1139 vector. Information about strains, plasmids and primers used in this example are listed in Tables 1 and 2 below.

[0135] The plasmids used to prepare gene-deficient strains are summarized in Table 1. After the plasmid pΔfkbD for knocking out C9 hydroxylase (HydroxylaseHydroxylase) was transferred to Escherichia coli ET12567 / pUZ8002, it was introduced into kanamycin streptomyces by conjugation, and the target gene was deleted by homologous recombination. The strains that produced a single crossover between the deletion plasmid and the kanamycin streptomyces chromosome were selected by culturing the transconjugant strain with apramycin resistance at 37°C (non-proliferation permissive temperature for pSG5-based replicon) in the presence of apramycin. Afterwards, the obtained colonies were propagated three times at 28°C without selection, and a second crossover was allowed. Two double exchange mutations (i.e., ΔfkbD) were selected by apramycin-sensitive phenotype, and then confirmed by PCR, and optionally confirmed by Southern blot hybridization.

[0136] By introducing the plasmid pΔtcsD for modifying the C21 side chain into the prepared fkbD gene-deficient kanamycin streptomyces ΔfkbD, the tcsD gene was deleted using the same method as the fkbD gene deletion method. ΔfkbD and tcsD were selected by apramycin-sensitive phenotype and identified by PCR, optionally by Southern blot hybridization. By introducing the plasmid pΔfkbL for forming the C1 prolyl ring into the additionally prepared fkbD and tcsD gene-deficient kanamycin streptomyces ΔfkbD and tcsD, the fkbL gene was deleted using the same method as the fkbD and tcsD gene deletion method. ΔfkbD, tcsD, and fkbL were selected by apramycin-sensitive phenotype and then confirmed by PCR.

[0137] The prepared fkbD, tcsD, and fkbL gene-deficient strains of Streptomyces kanamycinus ΔfkbD, tcsD, and fkbL were deposited in the Korean Collection for Type Cultures (KCTC) at the Korea Institute of Bioscience on April 14, 2020 (accession number: KCTC14171BP).

[0138] Table 1

[0139] Information on strains and plasmids used

[0140]

[0141]

[0142] Table 2

[0143] Information on primers used

[0144] Primers Sequence 5' to 3' (restriction sites are underlined) Restriction enzymes FWf <![CDATA[TATA AAGCTT CGGAGCCCCGGTGGACCT]]> HindIII FkB <![CDATA[TTAA TCTAGA CGTCGCCTCGTCGTCGCT]]> Xgf FWf <![CDATA[GTAA TCTAGA GTCGGCTACTGCCTCTAC]]> Xgf FqV <![CDATA[GAAT GAATTC CGACGAACAGCGGTTCCT]]> EcoRI FkbD-MLF <![CDATA[TATA AAGCTT CGGAGCCCCGGTGGACCT]]> HindIII FkbD-MLR <![CDATA[TTAA TCTAGA CGTCGCCTCGTCGTCGCT]]> Xgf FkbD-MRF <![CDATA[TATA TCTAGA GACACCGAAGGCGCGCTC]]> Xgf FkbD-MRR <![CDATA[TTAA GAATTC GAACACCGAGGCCGTCCA]]> EcoRI TxDV <![CDATA[GCT AAGCTT CTCAGGCGTCTGCGGATGC]]> HindIII TcsDLR <![CDATA[ATC GGATCC TTCGCTCACCGGGGCTGCC]]> B H TxDRF <![CDATA[AGC AGATCT GGCATGTTCTGGTCAGTCC]]> BgI TcD <![CDATA[GTC GAATTC CATGCCACGAACGGGTCGA]]> EcoRI FWf <![CDATA[AAT AAGCTT CCACGAGCCCGGT]]> HindIII FqV <![CDATA[AAA TCTAGA CACATCGCGTTCGAC]]> Xgf FWf <![CDATA[AAT TCTAGA CACGGAGAGGATCTG]]> Xgf FqV <![CDATA[AAA GAATTC CCACCACCCCCG]]> EcoRI

[0145] 9-Deoxy-36,37-dihydro-prolyl FK506 is prepared by culturing the above-prepared production strains Streptomyces kanamyciniensis ΔfkbD, tcsD, fkbL (Accession No.: KCTC14171BP), which are specifically described as follows. 50 ml of R2YE medium (103 g / L sucrose, 10 g / L glucose, 0.25 g / L K2SO4, 10.12 g / L MgCl2·6H2O, 0.1 g / L casamino acids, 50 ml / L yeast extract (10%), 100 ml / L TES buffer (5.73%, pH 7.2), 10 ml / L potassium phosphate (0.5%), 80 ml / L CaCl2·2H2O (3.68%), 15 ml / L L-proline (20%), 2 ml / L trace element solution and 5 ml / L NaOH (1N)) was added to a 250 ml baffled flask, inoculated with the production strain, and pre-cultured in a rotary shaker incubator at 28°C and 180 rpm for two days. Subsequently, 10 ml of the culture solution pre-cultured for two days was inoculated into a 3-liter conical flask containing 1 liter of R2YE medium. After inoculation, the culture was cultured for 6 days at 28°C and 180 rpm. After 6 days of culture, the produced 9-deoxy-36,37-dihydro-prolyl FK506 was extracted by the first extraction process.

[0146] The first extraction process was performed as follows. First, an equal amount of methanol was added to the culture medium, and after mixing for 30 minutes, the cells were removed by centrifugation, and the extract from which the cells were removed was concentrated by a rotary evaporator. Then, the concentrated extract was dissolved in water, ethyl acetate was added in an amount twice the amount used, and the mixture was fully mixed and allowed to stand until the layers separated. After the layers were separated, the upper organic solvent layer was recovered, concentrated using a rotary evaporator, and the weight after concentration was measured. The extract obtained by the first extraction process was passed through a silica gel-filled column. In this case, the amount of silica gel was 15 times the weight of the extract obtained by the first extraction process, and five ratios of methanol and dichloromethane were used as the mobile phase (fractionation 1.0:100, fractionation 2.1:100, fractionation 3.1:10, fractionation 4.1:1, fractionation 5.100:0). 9-deoxy-36,37-dihydro-prolyl FK506 was confirmed in fraction 3. The fraction 3 thus obtained was concentrated using a rotary evaporator, and finally purified using HPLC.

[0147] This was freeze-dried to obtain a substance represented by Chemical Formula 1, ie, 9-deoxy-36,37-dihydro-prolyl FK506, in a powdered form.

[0148] The prepared 9-deoxy-36,37-dihydro-prolyl FK506 was confirmed as follows. Specifically, high performance liquid chromatography analysis, mass spectrometry analysis, and nuclear magnetic resonance analysis were performed. The analysis results of 9-deoxy-36,37-dihydro-prolyl FK506 are shown in Tables 3 and Figures 1 to 6 As shown, based on the results, it can be confirmed that 9-deoxy-36,37-dihydro-prolyl FK506 is produced from the prepared production strains Streptomyces kanamycins ΔfkbD, tcsD, and fkbL.

[0149] 9-Deoxy-36,37-dihydro-prolyl FK506 (molecular formula: C 43 H 71 NO 11 , molecular weight: 777.50) are shown in Table 3 below.

[0150] Table 3

[0151]

[0152]

[0153]

[0154] according to 1 H, 13 C-NMR confirmed a keto carbon (δ C 214.8), two carbonyl carbons (δ C 171.6 and 169.7) and two olefin skeletons (δ C 140.6 and 122.6; and δ C 132.3 and 129.4), dioxygenated quaternary carbon (δ C 98.4), seven oxidized methylene carbons (δ C 84.2, 77.8, 77.4, 74.4, 73.6, 70.8, 69.1), three methoxy carbons (δ C 57.7, 56.6, 56.2), six methyl carbons were observed (δ C 18.9, 16.9, 15.4, 14.0, 9.8). And the FK506 derivative was observed to be composed of 43 carbon atoms.

[0155] In order to determine the exact structure, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) was confirmed. As a result of confirming the proton connection by gCOSY, it was confirmed that the compound includes a prolyl skeleton without a CH2 functional group, rather than FK506 having a piperidine skeleton, based on the gHMBC data. H 2.55, 2.62) and C-8 (δ C 171.6), C-10(δ C 98.4), confirming that the compound is a skeleton reduced to CH2 at C-9 rather than a ketone. Based on the fact that no exomethylene was observed between C36-C37 of the basic skeleton of FK506, and H37, which was observed as a triplet by gCOSY 2D-NMR, showed a coupling correlation with H36a / b and between H36a / b and H35a / b, respectively, it was confirmed that the skeleton of C36-C37 was dehydrogenated. In addition, it was confirmed that the three methoxy functional groups were structures with C-13, C-15 and C-31. In summary, it was confirmed that the compound was 9-deoxy-36,37-dihydro-prolyl FK506.

[0156] Example 2: Preparation of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506

[0157] For the kanamycin Streptomyces that produces FK506, according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098), double crossover homologous recombination was used to cause in-frame gene deletion, resulting in inactivation of fkbD-fkbM, tcsD and fkbL genes, and the kanamycin Streptomyces ΔfkbD-fkbM, tcsD, fkbL (preservation number: KCTC14170BP) that is a production strain of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 was prepared.

[0158] Specifically, in order to prepare deletion mutants of fkbD-fkbM, tcsD and fkbL genes in the FK506-producing Streptomyces kanamycin strain, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then transformed into the Streptomyces kanamycin as the FK506-producing strain by conjugation.

[0159] The strain preparation method can be more specifically described as the preparation of an in-frame gene deletion plasmid and the preparation of a gene deletion strain.

[0160] In the preparation of in-frame gene deletion plasmid, E. coli-Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. The preparation of plasmid was implemented by PCR amplification of the left and right flanking fragments of the target gene for deletion in the Fosmid DNA derived from Streptomyces kanamycin. For the deletion of fkbD-fkbM genes, the primer pair FkbD-MLF / FkbD-MLR for the left flank fragment and the primer pair FkbD-MRF / FkbD-MRR for the right flank fragment were designed. For the deletion of tcsD gene, the primer pair TcsDLF / TcsDLR for the left flank fragment and the primer pair TcsDRF / TcsDRR for the right flank fragment were designed. For the deletion of fkbL gene, the primer pair FkbLLF / FkbLLR for the left flank fragment and the primer pair FkbLRF / FkbLRR for the right flank fragment were designed. All PCR fragments were isolated, cut with HindIII-XbaI or XbaI-EcoRI, and cloned into pKC1139 vector. Information on strains, plasmids and primers used in this example is listed in Tables 1 and 2 above.

[0161] The plasmids used to prepare gene-deficient strains are summarized in Table 1. After the plasmid pΔfkbD-fkbm for simultaneously knocking out C9 hydroxylase and 31-O-methyltransferase was transferred to Escherichia coli ET12567 / pUZ8002, it was introduced into Streptomyces kanamycinis by conjugation, and the target gene was deleted by homologous recombination. Strains that produced a single crossover between the deletion plasmid and the chromosome of Streptomyces kanamycinis were selected by culturing a transconjugant strain with apramycin resistance at 37°C (a non-proliferation permissive temperature for pSG5-based replicons) in the presence of apramycin. Afterwards, the obtained colonies were propagated three times at 28°C without selection, and a second crossover was allowed.

[0162] Two double crossover mutations (ie, ΔfkbD-fkbM) were selected by the apramycin-sensitive phenotype and subsequently confirmed by PCR and optionally by Southern blot hybridization.

[0163] By introducing the plasmid pΔtcsD for modifying the C21 side chain into the prepared fkbD-fkbM gene-deleted kanamycin Streptomyces ΔfkbD-fkbM, the tcsD gene was deleted using the same method as the fkbD-fkbM gene deletion method. ΔfkbD-fkbM and tcsD were selected by the apramycin sensitivity phenotype and identified by PCR, optionally by Southern blot hybridization. By introducing the plasmid pΔfkbL for forming the C1 prolyl ring into the additionally prepared fkbD-fkbM and tcsD gene-deleted kanamycin Streptomyces ΔfkbD-fkbM and tcsD, the fkbL gene was deleted using the same method as the fkbD-fkbM and tcsD gene deletion method. ΔfkbD-fkbM, tcsD, and fkbL were selected by the apramycin sensitivity phenotype and then identified by PCR.

[0164] The prepared fkbD-fkbM, tcsD, fkbL gene-deficient strains of Streptomyces kanamycinus ΔfkbD-fkbM, tcsD, fkbL were deposited at the Bioresource Center (KCTC) of the Korea Institute of Bioscience on April 14, 2020 (Deposit No.: KCTC14170BP).

[0165] 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 was prepared by culturing the above-prepared production strain Streptomyces kanamycins ΔfkbD-fkbM, tcsD, fkbL (Accession No.: KCTC14170BP). The specific description is as follows. 50 ml of R2YE medium

[0166] (103 g / L sucrose, 10 g / L glucose, 0.25 g / L K2SO4, 10.12 g / L MgCl2·6H2O, 0.1 g / L casamino acids, 50 ml / L yeast extract (10%), 100 ml / L TES buffer (5.73%, pH 7.2), 10 ml / L potassium phosphate (0.5%), 80 ml / L CaCl2·2H2O (3.68%), 15 ml / L L-proline (20%), 2 ml / L trace element solution and 5 ml / L NaOH (1N)) were added to a 250 ml baffled flask, inoculated with the production strain, and pre-cultured in a rotary shaker incubator at 28°C and 180 rpm for two days. Subsequently, 10 ml of the culture solution pre-cultured for two days was inoculated into a 3-liter conical flask containing 1 liter of R2YE medium. After inoculation, the culture was cultured for 6 days at 28°C and 180 rpm. After 6 days of culture, the produced 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 was extracted by the first extraction process.

[0167] The first extraction process was performed as follows. First, an equal amount of methanol was added to the culture medium, and after mixing for 30 minutes, the cells were removed by centrifugation, and the extract from which the cells were removed was concentrated by a rotary evaporator. Then, the concentrated extract was dissolved in water, ethyl acetate was added in an amount twice that used, and the mixture was fully mixed and allowed to stand until the layers separated. After the layers were separated, the upper organic solvent layer was recovered, concentrated using a rotary evaporator, and the weight after concentration was measured. The extract obtained by the first extraction process was passed through a silica gel-filled column. In this case, the amount of silica gel was 15 times the weight of the extract obtained by the first extraction process, and five ratios of methanol and dichloromethane were used as mobile phases (fractionation 1, 0:100, fractionation 2, 1:100, fractionation 3, 1:10, fractionation 4, 1:1, fractionation 5, 100:0). 9-deoxy-31-O-demethyl-prolyl FK506 was confirmed in fractionation 3. The fractionation 3 thus obtained was concentrated using a rotary evaporator, and finally purified using HPLC.

[0168] The concentrate was freeze-dried to obtain a substance represented by Chemical Formula 2, ie, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, in a powdered form.

[0169] The prepared 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 was confirmed as follows. Specifically, high performance liquid chromatography analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were performed. The analysis results of 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 are shown in Tables 4 and Figures 7 to 12 As shown, based on the results, it can be confirmed that 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 is produced from the prepared production strain Streptomyces kanamycinus ΔfkbD-fkbM, tcsD, and fkbL.

[0170] 9-Deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 (molecular formula: C 42 H 73 NO 11 , molecular weight: 763.49) are shown in Table 4 below.

[0171] Table 4

[0172]

[0173]

[0174] according to 1 H, 13 C-NMR confirmed a keto carbon (δC 216.3), two carbonyl carbons (δ C 171.7 and 169.8) and two olefin skeletons (δ C 140.8 and 122.6; δ C 132.4 and 129.4), and double quaternary carbon oxides (δ C 98.4), seven oxidized methylene carbons (δ C 77.9, 77.3, 75.5, 75.0, 74.4, 70.8, 69.1), two methoxy carbons (δ C 58.7, 57.7), five methyl carbons were observed (δ C 18.9, 16.9, 15.4, 14.0, 9.8). It was observed that the FK506 derivative consisted of 42 carbon atoms. In order to determine the exact structure, 2D-NMR was confirmed. As a result of confirming the proton connection by gCOSY, it was confirmed that the compound had a prolyl skeleton based on the coupling between H-2 to H-4. According to the gHMBC data, based on H-9 (δ H 2.54, 2.63) and C-8 (δ C 171.7) and C-10 (δ C 98.4), confirming that the compound is a skeleton in which C-9 is reduced to CH2 rather than a ketone. In addition, since two methoxy functional groups are bound to C-13 and C-15, it was confirmed that there is no methoxy group at C-31.

[0175] In summary, the compound was confirmed to be 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506.

[0176] Example 3: Preparation of 9-deoxy-prolyl FK520

[0177] For the kanamycin Streptomyces used for producing FK506, according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098), double crossover homologous recombination was used to cause in-frame gene deletion, resulting in inactivation of fkbD, tcsD and fkbL genes, and kanamycin Streptomyces ΔfkbD, tcsD, fkbL (Accession No.: KCTC14171BP) was prepared as a production strain of 9-deoxy-prolyl FK520.

[0178] Specifically, in order to prepare deletion mutants of fkbD, tcsD and fkbL genes in the FK506-producing Streptomyces kanamycin strain, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then transformed into the FK506-producing Streptomyces kanamycin strain by conjugation.

[0179] The strain preparation method can be more specifically described as the preparation of an in-frame gene deletion plasmid and the preparation of a gene deletion strain.

[0180] In the preparation of in-frame gene deletion plasmid, E. coli-Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. The preparation of the plasmid was implemented by PCR amplification of the left and right flanking fragments of the target gene for deletion in the Fosmid DNA derived from Streptomyces kanamycin. For the deletion of the fkbD gene, the primer pair FkbDLF / FkbDLR for the left flank fragment and the primer pair FkbDRF / FkbDRR for the right flank fragment were designed. For the deletion of the tcsD gene, the primer pair TcsDLF / TcsDLR for the left flank fragment and the primer pair TcsDRF / TcsDRR for the right flank fragment were designed. For the deletion of the fkbL gene, the primer pair FkbLLF / FkbLLR for the left flank fragment and the primer pair FkbLRF / FkbLRR for the right flank fragment were designed. All PCR fragments were isolated, cut with HindIII-XbaI or XbaI-EcoRI, and cloned into pKC1139 vector. Information about strains, plasmids and primers used in this example is listed in Tables 1 and 2.

[0181] The plasmids used to prepare gene-deficient strains are summarized in Table 1. After the plasmid pΔfkbD for knocking out C9 hydroxylase was transferred to Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation, the target gene was deleted by homologous recombination. The strains that produced a single crossover between the deletion plasmid and the kanamycin streptomyces chromosome were selected by culturing the transconjugant strain with apramycin resistance at 37°C (non-proliferation permissive temperature for pSG5-based replicon) in the presence of apramycin. Afterwards, the obtained colonies were propagated three times at 28°C without selection, and a second crossover was allowed. Two double exchange mutations (i.e., ΔfkbD) were selected by apramycin-sensitive phenotype, followed by confirmation by PCR and optionally by Southern blot hybridization.

[0182] By introducing the plasmid pΔtcsD for modifying the C21 side chain into the prepared fkbD gene-deficient kanamycin streptomyces ΔfkbD, the tcsD gene was deleted using the same method as the fkbD gene deletion method. ΔfkbD and tcsD were selected by the apramycin sensitivity phenotype and identified by PCR, optionally by Southern blot hybridization. By introducing the plasmid pΔfkbL for forming the C1 prolyl ring into the additionally prepared fkbD and tcsD gene-deficient kanamycin streptomyces ΔfkbD and tcsD, the fkbL gene was deleted using the same method as the fkbD and tcsD gene deletion method. ΔfkbD, tcsD, and fkbL were selected by the apramycin sensitivity phenotype, and then identified by PCR, optionally by Southern blot hybridization.

[0183] The prepared fkbD, tcsD, and fkbL gene-deficient strains of Streptomyces kanamycinus ΔfkbD, tcsD, and fkbL were deposited at the Bioresource Center (KCTC) of the Korea Institute of Bioscience on April 14, 2020 (accession number: KCTC14171BP).

[0184] 9-Deoxy-prolyl FK520 is prepared by culturing the above-prepared production strain Streptomyces kanamyciniensis ΔfkbD, tcsD, fkbL (Accession No.: KCTC14171BP). Specific descriptions are as follows. 50 ml of R2YE medium (103 g / L sucrose, 10 g / L glucose, 0.25 g / L K2SO4, 10.12 g / L MgCl2·6H2O, 0.1 g / L casamino acids, 50 ml / L yeast extract (10%), 100 ml / L TES buffer (5.73%, pH 7.2), 10 ml / L potassium phosphate (0.5%), 80 ml / L CaCl2·2H2O (3.68%), 15 ml / L L-proline (20%), 2 ml / L trace element solution and 5 ml / L NaOH (1N)) was added to a 250 ml baffled flask, inoculated with the production strain, and pre-cultured in a rotary shaker incubator at 28°C and 180 rpm for two days. Subsequently, 10 ml of the culture solution pre-cultured for two days was inoculated into a 3-liter conical flask containing 1 liter of R2YE medium. After inoculation, the culture was cultured for 6 days at 28°C and 180 rpm. After 6 days of culture, the produced 9-deoxy-prolyl FK520 was extracted by the first extraction process.

[0185] The first extraction process is performed as follows. First, an equal amount of methanol is added to the culture medium, and after mixing for 30 minutes, the cells are removed by centrifugation, and the extract from which the cells are removed is concentrated by a rotary evaporator. Then, the concentrated extract is dissolved in water, ethyl acetate is added in an amount twice that used, and the mixture is fully mixed and allowed to stand until the layers are separated. After the layers are separated, the upper organic solvent layer is recovered, concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a silica gel-filled column. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and five ratios of methanol and dichloromethane are used as mobile phases (fractionation 1, 0:100, fractionation 2, 1:100, fractionation 3, 1:10, fractionation 4, 1:1, fractionation 5, 100:0). 9-deoxy-prolyl FK520 was confirmed in fractionation 3. The fractionation 3 thus obtained was concentrated using a rotary evaporator, and finally purified using HPLC.

[0186] The concentrate was freeze-dried to obtain a substance represented by Chemical Formula 3, ie, 9-deoxy-prolyl FK520, in a powdered form.

[0187] The prepared 9-deoxy-prolyl FK520 was confirmed as follows. Specifically, high performance liquid chromatography analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were performed. The analysis results of 9-deoxy-prolyl FK520 are shown in Tables 5 and Figures 13 to 18 As shown, based on the results, it was confirmed that 9-deoxy-prolyl FK520 was produced from the prepared production strains Streptomyces kanamycinus ΔfkbD, tcsD, and fkbL.

[0188] 9-Deoxy-prolyl FK520 (molecular formula: C 42 H 69 NO 11 , molecular weight: 763.49) are shown in Table 5 below.

[0189] Table 5

[0190]

[0191]

[0192]

[0193] according to 1 H, 13 C-NMR confirmed a keto carbon (δ C 215.0), two carbonyl carbons (δ C 171.8, 169.9) and two olefin skeletons (δ C 141.1 and 122.6;

[0194] δ C 132.5 and 129.7), and double quaternary carbon (δ C 98.6), seven oxidized methylene carbons (δ C 84.2, 78.0, 77.4, 74.4, 73.8, 71.1, 69.4), three methoxy carbons (δ C 57.9, 57.9, 56.4), five methyl carbons were observed (δ C 19.1, 17.1, 15.7, 14.4, 10.0). It was observed that the FK506 derivative consisted of 42 carbon atoms. In order to confirm the exact structure of the compound, 2D-NMR was confirmed. As a result of confirming the proton connection by gCOSY, it was confirmed that the compound had a prolyl skeleton based on the coupling between H-2 to H-4. According to the gHMBC data, based on H-9 (δ H 2.57 and 2.62) and C-8 (δ C 171.8) and C-10 (δ C 98.6), confirming that the compound is a skeleton reduced to CH2 rather than a ketone at C-9. In addition, it was confirmed that it is a structure with three methoxy functional groups present at C-13, C-15 and C-31. In addition, by gCOSY coupling correlation (gCOSY coupling correlation) and gHMBC long range correlation (gHMBC long range correlation), it was confirmed that C-35 and C-36 are bound to C-21 through ethyl. In summary, it was confirmed that the compound is 9-deoxy-prolyl FK520.

[0195] Example 4: Preparation of 9-deoxy-31-O-demethyl-prolyl FK520

[0196] For the kanamycin Streptomyces used for producing FK506, according to the method described in Ban, YH et al. (J. Nat. Prod. 2013, 76, 1091-1098), double exchange homologous recombination was used to cause the deletion of in-frame genes, resulting in the inactivation of fkbD-fkbM, tcsD and fkbL genes, and the kanamycin Streptomyces ΔfkbD, tcsD, fkbL (preservation number: KCTC14170BP) used as the production strain of 9-deoxy-31-O-demethyl-prolyl FK520 was prepared.

[0197] Specifically, in order to prepare deletion mutants of fkbD-fkbM, tcsD and fkbL genes in the FK506-producing Streptomyces kanamycin strain, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then transformed into the Streptomyces kanamycin as the FK506-producing strain by conjugation.

[0198] The strain preparation method can be more specifically described as the preparation of an in-frame gene deletion plasmid and the preparation of a gene deletion strain.

[0199] In the preparation of in-frame gene deletion plasmid, E. coli-Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. The preparation of plasmid was implemented by PCR amplification of the left and right flanking fragments of the target gene for deletion in the Fosmid DNA derived from Streptomyces kanamycin. For the deletion of fkbD-fkbM genes, the primer pair FkbD-MLF / FkbD-MLR for the left flank fragment and the primer pair FkbD-MRF / FkbD-MRR for the right flank fragment were designed. For the deletion of tcsD gene, the primer pair TcsDLF / TcsDLR for the left flank fragment and the primer pair TcsDRF / TcsDRR for the right flank fragment were designed. For the deletion of fkbL gene, the primer pair FkbLLF / FkbLLR for the left flank fragment and the primer pair FkbLRF / FkbLRR for the right flank fragment were designed. All PCR fragments were isolated, cut with HindIII-XbaI or XbaI-EcoRI, and cloned into pKC1139 vector. Information about strains, plasmids and primers used in this example is listed in Tables 1 and 2.

[0200] The plasmids used to prepare gene deletion strains are summarized in Table 1. The plasmid pΔfkbD-fkbM for knocking out C9 hydroxylase and 31-O-methyltransferase simultaneously was transferred to Escherichia coli ET12567 / pUZ8002 and introduced into Streptomyces kanamycinis by conjugation, and the target gene was deleted by homologous recombination. The strains that produced a single crossover between the deletion plasmid and the chromosome of Streptomyces kanamycinis were selected by culturing the transconjugant strain with apramycin resistance at 37°C (non-proliferation permissive temperature for pSG5-based replicon) in the presence of apramycin. Afterwards, the obtained colonies were propagated three times at 28°C without selection, and a second crossover was allowed. Two double exchange mutations (i.e., ΔfkbD-fkbM) were selected by apramycin-sensitive phenotype, followed by confirmation by PCR and optionally by Southern blot hybridization.

[0201] By introducing the plasmid pΔtcsD for modifying the C21 side chain into the prepared fkbD-fkbM gene-deleted kanamycin Streptomyces ΔfkbD-fkbM, the tcsD gene was deleted using the same method as the fkbD-fkbM gene deletion method. ΔfkbD-fkbM and tcsD were selected by the apramycin sensitivity phenotype and identified by PCR, optionally by Southern blot hybridization. By introducing the plasmid pΔfkbL for forming the C1 prolyl ring into the additionally prepared fkbD-fkbM and tcsD gene-deleted kanamycin Streptomyces ΔfkbD-fkbM and tcsD, the fkbL gene was deleted using the same method as the fkbD-fkbM and tcsD gene deletion method. ΔfkbD-fkbM, tcsD, and fkbL were selected by the apramycin sensitivity phenotype and then confirmed by PCR.

[0202] The prepared fkbD-fkbM, tcsD, and fkbL gene-deficient strains of Streptomyces kanamycinus ΔfkbD, tcsD, and fkbL were deposited at the Bioresource Center (KCTC) of the Korea Institute of Bioscience on April 14, 2020 (accession number: KCTC14170BP).

[0203] 9-Deoxy-31-O-demethyl-prolyl FK520 is prepared by culturing the above-prepared production strain Streptomyces kanamycinus ΔfkbD-fkbM, tcsD, fkbL (Accession No.: KCTC14170BP). Specific descriptions are as follows. 50 ml of R2YE medium (103 g / L sucrose, 10 g / L glucose, 0.25 g / L K2SO4, 10.12 g / L MgCl2·6H2O, 0.1 g / L casamino acids, 50 ml / L yeast extract (10%), 100 ml / L TES buffer (5.73%, pH 7.2), 10 ml / L potassium phosphate (0.5%), 80 ml / L CaCl2·2H2O (3.68%), 15 ml / L L-proline (20%), 2 ml / L trace element solution and 5 ml / L NaOH (1N)) was added to a 250 ml baffled flask, inoculated with the production strain, and pre-cultured in a rotary shaker incubator at 28°C and 180 rpm for two days. Subsequently, 10 ml of the culture solution pre-cultured for two days was inoculated into a 3-liter conical flask containing 1 liter of R2YE medium. After inoculation, the culture was cultured for 6 days at 28°C and 180 rpm. After 6 days of culture, the produced 9-deoxy-31-O-demethyl-prolyl FK520 was extracted by the first extraction process.

[0204] The first extraction process is performed as follows. First, an equal amount of methanol is added to the culture medium, and after mixing for 30 minutes, the cells are removed by centrifugation, and the extract from which the cells are removed is concentrated by a rotary evaporator. Then, the concentrated extract is dissolved in water, ethyl acetate is added in an amount twice that used, and the mixture is fully mixed and allowed to stand until the layers are separated. After the layers are separated, the upper organic solvent layer is recovered, concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a silica gel-filled column. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and five ratios of methanol and dichloromethane are used as mobile phases (fractionation 1, 0:100, fractionation 2, 1:100, fractionation 3, 1:10, fractionation 4, 1:1, fractionation 5, 100:0). 9-deoxy-31-O-demethyl-prolyl FK520 was confirmed in fractionation 3. The fractionation 3 thus obtained was concentrated using a rotary evaporator, and finally purified using HPLC.

[0205] The concentrate was freeze-dried to obtain a substance represented by Chemical Formula 4, ie, 9-deoxy-31-O-desmethyl-prolyl FK520, in a powdery form.

[0206] The prepared 9-deoxy-31-O-demethyl-prolyl FK520 was confirmed as follows. Specifically, high performance liquid chromatography analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were performed. The analysis results of 9-deoxy-31-O-demethyl-prolyl FK520 are shown in Tables 6 and Figures 19 to 24 As shown, based on the results, it was confirmed that 9-deoxy-31-O-demethyl-prolyl FK520 was produced from the prepared production strain Streptomyces kanamycinus ΔfkbD-fkbM, tcsD, and fkbL.

[0207] 9-Deoxy-31-O-demethyl-prolyl FK520 (molecular formula: C 41 H 67 NO 11 , molecular weight: 749.97) are shown in Table 6 below.

[0208] Table 6

[0209]

[0210]

[0211]

[0212] according to 1 H, 13 C-NMR confirmed a keto carbon (δ C 214.7), two carbonyl carbons (δC 171.6 and 169.8) and two olefin skeletons (δ C 140.8 and 122.4; δ C 132.4 and 129.4), and double quaternary carbon oxides (δ C 98.4), seven oxidized methylene carbons (δ C 77.9, 77.3, 75.5, 75.0, 74.4, 70.9, 69.1), two methoxy carbons (δ C 57.7, 56.2), five methyl carbons were observed (δ C 18.9, 16.9, 15.4, 11.7, 9.8). And it was observed that the FK506 derivative consisted of 41 carbon atoms, and the number of carbon atoms thereof was reduced. In order to confirm the structure of the accurate compound, 2D-NMR was confirmed. As a result of confirming the proton connection by gCOSY, it was confirmed that the compound had a prolyl skeleton based on the coupling between H-2 to H-4, and it was confirmed that the compound had an FK520 structure based on the coupling correlation of H21-H35-36. According to the gHMBC data, based on H-9 (δ H 2.56, 2.62) and C-8 (δ C 171.6) and C-10 (δ C 98.4), confirming that the compound is a skeleton reduced to CH2 at C-9 rather than a ketone. In addition, since two methoxy functional groups are bound to C-13 and C-15, it is confirmed that there is no methoxy group at C-31. In summary, it is confirmed that the compound is 9-deoxy-31-O-demethyl-prolyl FK520.

[0213] Example 5: Study on the immunosuppressive activity of four novel compounds

[0214] The conventional in vitro T cell activity assay (J.Immunol.143:718-726,1989) was used to study the degree of reduction in immunosuppressive activity of four novel compounds. CD4+T cell division is an indicator of immune response. When CD4+T cells are stained with Cell TraceTM Violet (CTV), the amount of CTV in each cell decreases as the cells divide and T cells proliferate in response to the immune response, so this indicator was used to study the degree of immunosuppressive activity.

[0215] Single cells were isolated from the spleen of 6- to 8-week-old B6J experimental mice. CD4+ T cells were isolated using the Mouse CD4 T Cell Enrichment Kit (eBioscience). CD4+ T cells were isolated using CellTraceTM Violet (CTV) cell proliferation kit (Molecular Probes) was used for staining, and FK506 or the four novel compounds were added to make the concentrations of 0.01ng / ml, 0.1ng / ml, 1ng / ml, 10ng / ml, 100ng / ml, and 1000ng / ml, and then cultured for 72 hours. Mouse T-activator CD3 / CD28 (Gibco). Unactivated T cells were used as a control. After culture, CTV intensity was analyzed using flow cytometry.

[0216] Table 7 below and Fig.25 The T cell proliferation degree of CTV intensity measured by flow cytometer is shown, and the immunosuppressive activity degree of FK506 and four novel compounds is shown. Fig.25 As shown, all novel compounds provided by the present invention exhibited significantly reduced immunosuppressive activity compared to FK506. In particular, among the FK506 derivatives reported so far with maintained or improved neuroregenerative activity, 9-deoxy-31-O-desmethyl-prolyl FK520 exhibited the lowest immunosuppressive activity.

[0217] Table 7

[0218]

[0219]

[0220] From the above results, it can be confirmed that the immunosuppressive activity of the four novel compounds of the present invention is significantly reduced compared with FK506, because the four novel compounds show 1.14×10 5 Times more than IC 50 (ng / ml) concentration, it can be confirmed that the immunosuppressive activity is significantly reduced. It can be judged that the pharmaceutical composition for preventing or treating nervous system diseases containing at least one of the four novel compounds as an active ingredient can be used without worrying about side effects caused by immunosuppressive activity.

[0221] Example 6: Analysis of the neuronal cell growth promoting activity of four novel compounds

[0222] According to the method reported by Jing Sun et al. (J. Neuroinflam. 15: 180, 2018), the ability of four novel compounds to promote neuronal cell growth was studied using primary cultured mouse hippocampal neurons. Specifically, primary cultured hippocampal neurons were treated with FK506 or one of the four novel compounds (treatment concentration: 1 ng / ml), and the control group was not treated. According to the previously reported method (J. Pharmacol. Exp. Ther. 302: 1278-1285, 2002), the length of neurites was measured using printed photographs.

[0223] The results are shown in the figure Fig.26 .from Fig.26 It can be seen that the four novel compounds of the present invention exhibit excellent neurite growth effects.

[0224] In other words, it can be confirmed that the four novel compounds of the present invention have excellent ability to promote the proliferation of nerve cells. In particular, it can be confirmed that 9-deoxy-prolyl FK520 shows an excellent effect on the generation of nerve axons compared with FK506. Therefore, it can be concluded that the four novel compounds of the present invention can be used to prevent or treat nervous system diseases.

[0225] Example 7: Confirmation of the therapeutic effects of four novel compounds on nervous system diseases

[0226] According to the method reported by D. Park et al. (Sci. Rep. 7: 7260, 2017), the functional synaptic activity of four novel compounds was studied using primary cultured hippocampal neurons. Specifically, the frequency of excitatory synaptic currents was measured by performing patch clamp recordings on hippocampal neurons on days 10-14 of primary culture exposed to FK506 or four novel compounds (treatment concentration: 1 ng / ml). The frequency of excitatory synaptic currents is used as an indicator that can confirm changes in the number of synapses.

[0227] The results of the measured excitatory synaptic current frequency are shown in the figure Fig. 27 .

[0228] The results showed that the spontaneous excitatory postsynaptic current (sEPSC) of 9-deoxy-36,37-dihydro-prolyl FK506 and 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 increased to 3.29±0.22 (p<0.001, n=7) and 2.63±0.27 (p<0.05, n=6), respectively, which were equivalent to the increased values ​​compared with FK506 (2.61±0.18 (p<0.005, n=10)).

[0229] In other words, it was confirmed that among the four novel compounds of the present invention, the frequency of excitatory synaptic currents in the treatment groups of 9-deoxy-36,37-dihydro-prolyl FK506 and 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 was significantly increased. This suggests that the improvement in the neuronal growth promoting activity shown in the treatment group of the novel compound in Example 6 is also related to the increase in the formation of functional synapses.

[0230] Example 8: Confirmation of the therapeutic effects of four novel compounds on degenerative nervous system diseases

[0231] The effects of the synthetic toxin MPTP of four novel compounds on the density of dopaminergic nerve fibers were studied. Specifically, physiological saline, FK506, and four novel compounds (treatment concentration: 5 mg / ml) were administered for 3 days using a cannula injection system that can inject drugs into specific brain areas of awake animals. On the second day, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as a neurotoxic compound was injected intraperitoneally to establish an animal model of Parkinson's disease and induce the degeneration of the nigrostriatal dopaminergic pathway associated with motor ability. On the fourth day, immunohistochemistry (IHC) staining was performed using a dopaminergic nerve cell-specific marker (i.e., TH antibody) to measure changes in the number of dopaminergic nerve cells and nerve fiber density.

[0232] The results of the measurement of the number of dopaminergic neurons and changes in nerve fiber density are shown in the figure Fig.28 .

[0233] From the results, it was confirmed that the number of dopaminergic nerve cells was restored and the density of nerve fibers was restored.

[0234] In other words, the four novel compounds of the present invention, 9-deoxy-36,37-dihydro-prolyl FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, 9-deoxy-prolyl FK520 and 9-deoxy-31-O-demethyl-prolyl FK520, showed better recovery of nerve fiber density than FK506, and it was confirmed that they had almost no immunosuppressive ability.

[0235] The above results indicate that the four novel compounds can be used for the treatment of nervous system diseases, and in particular, can be used as targeted drugs for degenerative nervous system diseases.

[0236] Example 9: Confirmation of the safety of four new compounds

[0237] The safety of the four novel compounds was performed by MTT assay for cytotoxicity study, Ames test for liver genotoxicity evaluation, hERG assay for evaluating potential effects on cardiac repolarization. As a method, the study was conducted according to a commonly used method.

[0238] Specifically, in the MTT assay, a drug concentration 80 to 800 times higher (100 nM to 1000 nM) than the cytotoxicity test concentration (1 ng / ml = 1.24 nM) was set as the test concentration, and the drug toxicity of normal cells (HEK293T cells) derived from mammals was evaluated. In the Ames test, the treatment concentrations of the four novel compounds were 4 mg, 16 mg, and 64 mg per plate, respectively, and the reverse mutation induction in strains requiring histidine (Samonella Typhimurium TA98, Salmonella typhimurium TA98) and strains requiring tryptophan (Escherichia Coli WP2 uvrA, Escherichia coli WP2 uvrA) was used as an index to measure the four novel compounds. In the hERG assay, two or more cells were treated in such a way that the treatment concentrations of the four novel compounds were 0 mM, 2 mM, 8 mM, and 32 mM, respectively. Normal tyrode solution was perfused into one CHO hERG cell. After confirming that the hERG channel current was constant for 3-4 sweeps, the excipient control group and the four novel compound-treated groups were perfused for about 1-2 minutes or more until the recorded hERG channel current was constant. The relative current (pA or nA) and the suppression rate (%) were measured.

[0239] The MTT analysis results are shown in the figure. Fig.29 .from Fig.29 It can be seen that almost no cytotoxicity was observed in the four novel compounds of the present invention.

[0240] In other words, no cytotoxicity was observed in the treatment groups of the four novel compounds of the present invention, namely, 9-deoxy-36,37-dihydro-prolyl FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506, 9-deoxy-prolyl FK520 and 9-deoxy-31-O-demethyl-prolyl FK520, and it can be confirmed that the four novel compounds of the present invention are safe substances that do not cause the risk of inducing reverse mutation of the test strain used in the Ames test and cardiac repolarization in the hERG analysis.

[0241] According to the above description, those skilled in the art to which the present invention belongs will be able to understand that, without changing the technical ideas or essential features of the present invention, it can be implemented in other specific forms. In this regard, the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be understood to include all changes or modifications derived from the meaning, scope and equivalent concepts of the claims, rather than being limited to the above detailed description.

[0242]

[0243]

Claims

1. A compound, which is any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506 represented by Chemical Formula 1, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 represented by Chemical Formula 2, 9-deoxy-prolyl FK520 represented by Chemical Formula 3, and 9-deoxy-31-O-demethyl-prolyl FK520 represented by Chemical Formula 4: [Chemical formula 1] [Chemical formula 2] [Chemical formula 3] [Chemical formula 4] or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition for preventing or treating a nervous system disease, comprising any one compound selected from the group consisting of 9-deoxy-36,37-dihydro-prolyl FK506 represented by Chemical Formula 1, 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506 represented by Chemical Formula 2, 9-deoxy-prolyl FK520 represented by Chemical Formula 3, and 9-deoxy-31-O-demethyl-prolyl FK520 represented by Chemical Formula 4, or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical formula 1] [Chemical formula 2] [Chemical formula 3] [Chemical formula 4] 3. The pharmaceutical composition for preventing or treating nervous system diseases according to claim 2, characterized in that: The nervous system disease is any one or more selected from the group consisting of nerve injury disease, neurodegenerative disease, peripheral nerve injury, traumatic brain injury, and stroke.

4. The pharmaceutical composition for preventing or treating nervous system diseases according to claim 3, characterized in that: The neurological injury disease is any one or more selected from the following group: epilepsy, stroke, cerebral infarction, ischemic brain disease, spinal cord injury disease, peripheral nervous system disease, behavioral disorder, developmental disorder, mental retardation, Down syndrome and schizophrenia.

5. The pharmaceutical composition for preventing or treating nervous system diseases according to claim 3, characterized in that: The neurodegenerative disease is any one or more selected from the following group: dementia, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, corticobasal ganglionic degeneration, diffuse Lewy body disease, Guam Parkinson's-ALS-dementia complex and Pick's disease.

6. The pharmaceutical composition for preventing or treating nervous system diseases according to claim 2, characterized in that: The pharmaceutical composition has reduced immunosuppressive activity.

7. The pharmaceutical composition for preventing or treating nervous system diseases according to claim 2, characterized in that: The pharmaceutical composition exhibits neurite outgrowth and synapse formation activities.

8. A composition comprising the compound or a salt thereof according to claim 1, and an excipient.

9. A method for preparing the compound according to claim 1, comprising: (i) culturing Streptomyces kanamycinii ΔfkbD, tcsD, and fkbL with the accession number KCTC14171BP to prepare 9-deoxy-36,37-dihydro-prolyl FK506; (ii) culturing Streptomyces kanamycinii ΔfkbD, tcsD, and fkbL with the accession number KCTC14171BP to prepare 9-deoxy-prolyl FK520; (iii) culturing Streptomyces kanamycinus ΔfkbD-fkbM, tcsD, fkbL with the accession number KCTC14170BP to produce 9-deoxy-31-O-demethyl-36,37-dihydro-prolyl FK506; or (iv) Cultivating Streptomyces kanamycinus ΔfkbD-fkbM, tcsD, and fkbL with the accession number KCTC14170BP to prepare 9-deoxy-31-O-demethyl-prolyl FK520.

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