New compounds for promoting hair growth and compositions containing them

Abstract

The present invention relates to novel compounds which can be used as the main component of a composition for stimulating hair growth, and also to their use in stimulating hair growth, wherein the novel compounds are 9-deoxy-31-O-demethyl-proline-FK506, 9-deoxy-36,37-dihydro-FK506, 31-O-demethyl-36,37-dihydro-FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506, 9-deoxy-FK520, 31-O-demethyl-FK520, 9-deoxy-31-O-demethyl-FK520, 9-deoxy-FK523 and 9-deoxy-31-O-demethyl-FK523.

Description

Technical Field

[0001] The present invention relates to the preparation and use of novel compounds that can be used as the main component of a composition for promoting hair growth, wherein the novel compounds are 9-deoxy-31-O-demethyl-prolyl-FK506, 9-deoxy-36,37-dihydro-FK506, 31-O-demethyl-36,37-dihydro-FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506, 9-deoxy-FK520, 31-O-demethyl-FK520, 9-deoxy-31-O-demethyl-FK520, 9-deoxy-FK523 and 9-deoxy-31-O-demethyl-FK523. More specifically, the present invention relates to a method for preparing the novel compounds and the composition for promoting hair growth, wherein the composition comprises each novel compound as an active ingredient. Background Art

[0002] Hair loss (alopecia) has become increasingly common among modern people, not only due to aging or genetics, but also due to acquired factors such as hormonal imbalances caused by environmental pollution, smoking, work stress, and changes in eating patterns. Although the growth of the population suffering from hair loss is mainly dominated by middle-aged and elderly men in their 40s and 50s, the number of young people and women suffering from hair loss has also increased in recent years.

[0003] According to the statistical analysis of the Korean Hair Restoration Therapy Society, including potential hair loss patients, the domestic population with hair loss accounts for about 10 million. Medical expenditure in 2016 was about 35.5 billion won, and the number of people with hair loss overseas and in Korea is increasing. According to the estimation of IBIS World, an industry market research company, the market size of hair loss management in the United States in 2016 was 3.6 billion US dollars, and it is expected to grow at an annual rate of 0.7% by 2021.

[0004] Meanwhile, according to a survey by the Korean Chronic Disease Management Association, seven out of ten Korean adults consider hair loss to be a disease and believe that hair loss causes direct or indirect losses in social life. And, about 23% of adults experience hair loss.

[0005] Hair loss is divided into non-scarring alopecia, which is temporary hair loss, and scarring alopecia, which is caused by permanent destruction of hair follicles or hair roots. Non-scarring alopecia is a common form of hair loss, which is divided into infectious alopecia, traumatic alopecia, inflammatory alopecia, congenital alopecia, endocrine alopecia, tumor alopecia, malnutrition alopecia, and drug-induced alopecia. Hair loss caused by abnormal hair structure, male and female pattern hair loss, and alopecia areata are also non-scarring alopecia.

[0006] With economic development and the aging of society, hair loss has been recognized as a disease rather than an uncontrollable phenomenon, and there is an urgent need to develop effective drugs to treat hair loss, which can cause psychological anxiety and stress in work and social life.

[0007] However, although more and more people are suffering from hair loss, the exact cause of hair loss has not been clarified, and there is no effective way to prevent hair loss. Due to the current situation, effective technologies for preventing hair loss and promoting hair growth have attracted more and more attention, and various preventive agents have appeared on the market.

[0008] Currently, topical minoxidil and oral Propecia are FDA-approved drugs. However, these drugs do not provide permanent therapeutic effects, but only provide the effect of delaying the progression of hair loss or maintaining the current state. In addition, topical hair growth promoters such as minoxidil can be cumbersome because they need to be used every day to maintain the effect of promoting hair growth, and the effect is not as good as oral agents. In addition, the administration of Propecia to women may increase the possibility of congenital malformations in the baby and may cause side effects such as sexual dysfunction. Therefore, there is a need to develop drugs that can provide fundamental therapeutic effects beyond the level of temporary effects. In addition, drugs that are used not only for treatment but also for prevention may be more valuable.

[0009] Although Korean Patent Publication No. 10-2005-0071491 (Invention Name: Combination of Tacrolimus (FK506) Derivatives and β2-Agonists for the Treatment of Asthma) discloses a new use of FK506 derivatives and β2-agonists for the preparation of medicaments for simultaneous, separate or sequential use to treat or prevent acute or chronic asthma, there has been no research on the use of FK506 derivatives, FK520 derivatives or FK523 derivatives to promote hair growth.

[0010] public

[0011] Technical issues

[0012] One object of the present invention is to provide nine new compounds, isomers thereof or pharmaceutically acceptable salts thereof.

[0013] Another object of the present invention is to provide a composition for promoting hair growth, which comprises at least one selected from the nine novel compounds as an active ingredient. In this regard, the composition for promoting hair growth refers to a composition for alleviating, preventing or treating hair loss.

[0014] Another object of the present invention is to provide a pharmaceutical composition for promoting hair growth, which comprises at least one of the isomers of the nine new compounds or their pharmaceutically acceptable salts as an active ingredient. In this regard, the pharmaceutical composition for promoting hair growth refers to a composition for alleviating, preventing or treating hair loss.

[0015] Another object of the present invention is to provide a bioproduction method for each of the nine new compounds.

[0016] Another object of the present invention is to provide a production strain that can be used in the bioproduction method of the nine new compounds, Streptomyces kanamycinensis kanamyceticus)ΔfkbD-fkbM (Accession No.: KCTC13581BP), kanamyceticus ΔfkbD,tcsD (Accession No.: KCTC13580BP), kanamyceticus ΔfkbM,tcsD (Accession No.: KCTC13584BP), kanamyceticus ΔfkbD-fkbM,tcsD (Accession No.: KCTC13585BP), kanamyceticus ΔfkbD,tcsB (Accession No.: KCTC13579BP), kanamyceticus ΔfkbM,tcsB (Accession No.: KCTC13583BP), kanamyceticus ΔfkbD-fkbM,tcsB (Accession No.: KCTC13582BP).

[0017] Another object of the present invention is to provide an over-the-counter (OTC) composition for promoting hair growth, comprising at least one selected from the nine new compounds, their isomers or pharmaceutically acceptable salts thereof as an active ingredient.

[0018] Another object of the present invention is to provide a cosmetic composition for promoting hair growth, comprising at least one selected from the nine novel compounds, their isomers or cosmetically acceptable salts thereof as an active ingredient.

[0019] Technical Solution

[0020] After extensive efforts, the present inventors have discovered novel compounds having hair growth promoting effects, namely, 9-deoxy-31-O-demethyl-prolyl-FK506, 9-deoxy-36,37-dihydro-FK506, 31-O-demethyl-36,37-dihydro-FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506, 9-deoxy-FK520, 31-O-demethyl-FK520, 9-deoxy-31-O-demethyl-FK520, 9-deoxy-FK523 and 9-deoxy-31-O-demethyl-FK523 (hereinafter collectively referred to as nine novel compounds), which can be used as main components of a hair growth promoting composition, developed a method for producing the same and a hair growth promoting composition using the same as an active ingredient, and confirmed that the composition can be effectively used as a hair growth promoting composition, thereby completing the present invention.

[0021] Beneficial effects

[0022] The hair growth promoting composition, which comprises at least one selected from the nine novel compounds according to the present invention as an active ingredient, can provide a hair growth promoting effect in alleviating, preventing and treating hair loss, thereby providing a basic preventive and therapeutic effect.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of HPLC analysis of 9-deoxy-31-O-demethyl-prolyl-FK506 are shown.

[0025] Figure 2 The results of NMR analysis of 9-deoxy-31-O-demethyl-prolyl-FK506 are shown ( 1 H-NMR).

[0026] Figure 3 The results of NMR analysis of 9-deoxy-31-O-demethyl-prolyl-FK506 are shown ( 13 C-NMR).

[0027] Figure 4 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-31-O-demethyl-prolyl-FK506 are shown.

[0028] Figure 5 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-31-O-demethyl-prolyl-FK506 are shown.

[0029] Figure 6The results of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-31-O-demethyl-prolyl-FK506 are shown.

[0030] Figure 7 The results of HPLC analysis of 9-deoxy-36,37-dihydro-FK506 are shown.

[0031] Figure 8 The results of NMR analysis of 9-deoxy-36,37-dihydro-FK506 are shown ( 1 H-NMR).

[0032] Fig. 9 The results of NMR analysis of 9-deoxy-36,37-dihydro-FK506 are shown ( 13 C-NMR).

[0033] Fig.10 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-36,37-dihydro-FK506 are shown.

[0034] Fig.11 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-36,37-dihydro-FK506 are shown.

[0035] Fig.12 The results of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-36,37-dihydro-FK506 are shown.

[0036] Fig.13 The results of HPLC analysis of 31-O-desmethyl-36,37-dihydro-FK506 are shown.

[0037] Fig.14 The results of the NMR analysis of 31-O-demethyl-36,37-dihydro-FK506 are shown ( 1 H-NMR).

[0038] Fig.15 The results of NMR analysis of 31-O-demethyl-36,37-dihydro-FK506 are shown ( 13 C-NMR).

[0039] Fig.16 The results of nuclear magnetic resonance analysis (1H-NMR) of 31-O-demethyl-36,37-dihydro-FK506 are shown.

[0040] Fig.17 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 31-O-desmethyl-36,37-dihydro-FK506 are shown.

[0041] Fig.18 The results of nuclear magnetic resonance analysis (HMBC-NMR) of 31-O-desmethyl-36,37-dihydro-FK506 are shown.

[0042] Fig.19 The results of HPLC analysis of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 are shown.

[0043] Fig. 20 The results of NMR analysis of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 are shown ( 1 H-NMR).

[0044] Fig.21 The results of NMR analysis of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 are shown ( 13 C-NMR).

[0045] Fig. 22 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 are shown.

[0046] Fig.23 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 are shown.

[0047] Fig.24 The results of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 are shown.

[0048] Fig.25 The results of HPLC analysis of 9-deoxy-FK520 are shown.

[0049] Fig.26 The results of NMR analysis of 9-deoxy-FK520 are shown ( 1 H-NMR).

[0050] Fig. 27 The results of NMR analysis of 9-deoxy-FK520 are shown ( 13 C-NMR).

[0051] Fig.28 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-FK520 are shown.

[0052] Fig.29 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-FK520 are shown.

[0053] Fig.30 The results of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-FK520 are shown.

[0054] Fig.31 The results of HPLC analysis of 31-O-desmethyl-FK520 are shown.

[0055] Fig.32 The results of NMR analysis of 31-O-desmethyl-FK520 are shown ( 1 H-NMR).

[0056] Fig.33 The results of NMR analysis of 31-O-desmethyl-FK520 are shown ( 13 C-NMR).

[0057] Fig.34 The results of nuclear magnetic resonance analysis (COSY-NMR) of 31-O-desmethyl-FK520 are shown.

[0058] Fig.35 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 31-O-desmethyl-FK520 are shown.

[0059] Fig.36 The results of nuclear magnetic resonance analysis (HMBC-NMR) of 31-O-desmethyl-FK520 are shown.

[0060] Fig.37 The results of HPLC analysis of 9-deoxy-31-O-desmethyl-FK520 are shown.

[0061] Fig.38 The results of NMR analysis of 9-deoxy-31-O-demethyl-FK520 are shown ( 1 H-NMR).

[0062] Fig.39 The results of NMR analysis of 9-deoxy-31-O-demethyl-FK520 are shown ( 13 C-NMR).

[0063] Fig.40 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-31-O-desmethyl-FK520 are shown.

[0064] Fig.41 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-31-O-desmethyl-FK520 are shown.

[0065] Fig.42The results of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-31-O-desmethyl-FK520 are shown.

[0066] Fig.43 The results of HPLC analysis of 9-deoxy-FK523 are shown.

[0067] Fig.44 The results of NMR analysis of 9-deoxy-FK523 are shown ( 1 H-NMR).

[0068] Fig.45 The results of NMR analysis of 9-deoxy-FK523 are shown ( 13 C-NMR).

[0069] Fig.46 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-FK523 are shown.

[0070] Fig.47 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-FK523 are shown.

[0071] Fig.48 The results of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-FK523 are shown.

[0072] Fig.49 The results of HPLC analysis of 9-deoxy-31-O-desmethyl-FK523 are shown.

[0073] Fig.50 The results of NMR analysis of 9-deoxy-31-O-demethyl-FK523 are shown ( 1 H-NMR).

[0074] Fig.51 The results of NMR analysis of 9-deoxy-31-O-demethyl-FK523 are shown ( 13 C-NMR).

[0075] Fig.52 The results of nuclear magnetic resonance analysis (COSY-NMR) of 9-deoxy-31-O-desmethyl-FK523 are shown.

[0076] Fig.53 The results of nuclear magnetic resonance analysis (HSQC-NMR) of 9-deoxy-31-O-desmethyl-FK523 are shown.

[0077] Fig.54 The results of nuclear magnetic resonance analysis (HMBC-NMR) of 9-deoxy-31-O-desmethyl-FK523 are shown.

[0078] Fig.55 The extent of reduction in immunosuppressive activity of the nine novel compounds of the present invention is shown.

[0079] Fig.56 The novel compounds of the present invention showed hair growth promoting activity.

[0080] Fig.57 The hair growth promoting activity of the novel compounds of the present invention as determined by recognizing an increase in vibrissa length was shown.

[0081] Fig.58 The results of studies on hair growth promoting activity based on histological results of vibrissae treated with the novel compounds of the present invention are shown.

[0082] [Best Mode]

[0083] Hereinafter, the present invention will be described in detail.

[0084] At the same time, each description and embodiment disclosed herein can be applied to describe different descriptions and embodiments. That is to say, all combinations of various factors disclosed herein belong to the scope of the present invention. In addition, the scope of the present invention should not be limited by the detailed description provided below.

[0085] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention. Such equivalents are intended to be encompassed within the scope of the following claims.

[0086] To solve these problems, the present invention provides methods for preparing nine novel compounds, compositions for promoting hair growth comprising each novel compound by applying these preparation methods, and methods for alleviating, preventing and treating hair loss.

[0087] In order to achieve the above-mentioned purpose, one aspect of the present invention provides a compound, which is selected from the following nine new compounds, namely, 9-deoxy-31-O-demethyl-proline-FK506 as shown in the following formula 1, 9-deoxy-36,37-dihydro-FK506 as shown in the following formula 2, 31-O-demethyl-36,37-dihydro-FK506 as shown in the following formula 3, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 as shown in the following formula 4, 9-deoxy-FK520 as shown in the following formula 5, 31-O-demethyl-FK520 as shown in the following formula 6, 9-deoxy-31-O-demethyl-FK520 as shown in the following formula 7, 9-deoxy-FK523 as shown in the following formula 8, 9-deoxy-31-O-demethyl-FK523 as shown in the following formula 9, their isomers, or pharmaceutically acceptable salts thereof.

[0088] [Formula 1]

[0089]

[0090] [Formula 2]

[0091]

[0092] [Formula 3]

[0093]

[0094] [Formula 4]

[0095]

[0096] [Formula 5]

[0097]

[0098] [Formula 6]

[0099]

[0100] [Formula 7]

[0101]

[0102] [Formula 8]

[0103]

[0104] [Formula 9]

[0105]

[0106] In certain embodiments, the compounds of the present invention may include isomers thereof or pharmaceutically acceptable salts thereof.

[0107] Isomers refer to compounds that have the same chemical formula but different arrangements of the atoms and may include, for example, structural isomers, geometric isomers, optical isomers (enantiomers), stereoisomers, and diastereomers.

[0108] Pharmaceutically acceptable salts can refer to any organic or inorganic addition salts of the original compound, which do not cause side effects and damage the beneficial effects of the compound at concentrations effective for the patient, but are relatively nontoxic and harmless to the patient. For example, a pharmaceutically acceptable salt can be an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts can be prepared by any method commonly used in the art, for example, by dissolving the compound in an excess of aqueous solution and precipitating the salt using a water-miscible organic solvent (e.g., methanol, ethanol, acetone or acetonitrile). The same molar amount of the compound and acid or alcohol (e.g., ethylene glycol monomethyl ether) can be heated in water, and then the mixture can be dried by evaporation, or the salt precipitated by suction filtration. In this regard, the free acid can be an organic acid or an inorganic acid. The salt can be a pharmaceutically acceptable metal salt prepared using an alkali.

[0109] In another embodiment, the compounds of the present invention may be in the form of solvates or prodrugs, which are within the scope of the present invention. The solvates may preferably include hydrates and glycolates.

[0110] The composition of the present invention can be used as a single preparation or in combination with various drugs known to have preventive or therapeutic effects on hair loss, and the composition can be formulated into a single-dose dosage form or a unit dosage form in a multi-dose container using a pharmaceutically acceptable carrier or excipient.

[0111] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent used that does not cause irritation to living organisms and does not impair the biological activity or properties of the administered compound. The types of carriers available in the present invention are not particularly limited, and any type of pharmaceutically acceptable carrier commonly used in the art can be used. Non-limiting examples of the carrier may include co-surfactants such as Transcutol, polyethylene glycol, triacetin, and any mixture thereof; surfactants such as Cremophor, Tween, Myrj, Poloxamer, Pluronic, Lutrol, Imwitor, Span, and Labrafil, alone or in combination; oils such as Miglyol, Captex, and ethyl oleate, alone or in combination; and organic acids such as erythrocarboxylic acid and citric acid, alone or in combination. These carriers may be used alone or in mixture of at least two kinds.

[0112] In addition, if necessary, the composition may also include any known additives, such as antioxidants, buffers and / or antibacterial agents. The composition may be formulated into injectable dosage forms such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets in combination with diluents, dispersants, surfactants, binders, lubricants, etc.

[0113] In order to achieve the above object, another aspect of the present invention provides a method for preventing or treating hair loss, comprising administering the composition to an individual.

[0114] As used herein, the term "subject" refers to all animals that have hair loss or are at risk of hair loss.

[0115] The composition of the present invention may include a pharmaceutically effective amount of at least one selected from nine new compounds, isomers thereof, or salts thereof. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment, and the daily dose of the composition is 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, once a day to several times. However, for the purposes of the present invention, it is preferred to apply a specific therapeutically effective amount differently to a specific patient according to various factors, including the type and degree of response to be achieved, a specific composition, including whether other preparations are used according to the case, the patient's age, weight, general health, sex and diet, administration time, administration route, excretion rate of the composition, duration of treatment, and drugs used in combination or simultaneously with a specific composition, and similar factors well known in the medical field.

[0116] Although not particularly limited thereto, the composition of the present invention may be administered once or several times a day in divided doses.

[0117] The composition of the present invention can be administered alone or in combination with other therapeutic agents, or can be administered sequentially or simultaneously with existing therapeutic agents. The composition can be administered in a single dosage form or in multiple dosage forms. Taking all of the above factors into account, it is important to administer the composition in a minimum amount that can show the maximum effect without causing side effects. The amount can be easily determined by a person skilled in the art.

[0118] As used herein, the term "administering" refers to introducing the composition of the present invention into the patient's body by any appropriate method. The composition of the present invention can be administered by any oral or parenteral route as long as the composition reaches the target tissue.

[0119] The mode of administration of the composition of the present invention is not particularly limited, and any mode of administration commonly available in the art can be used. Non-limiting examples of modes of administration include oral or parenteral administration. Depending on the desired mode of administration, the composition of the present invention can be prepared into various dosage forms.

[0120] The hair growth promoting composition of the present invention can be used to reduce, prevent or treat hair loss.

[0121] In the present invention, hair loss includes both non-scarring alopecia, i.e. temporary alopecia, and scarring alopecia caused by permanent destruction of hair follicles or hair roots. Non-scarring alopecia includes infectious alopecia, traumatic alopecia, inflammatory alopecia, congenital alopecia, endocrine alopecia, tumor alopecia, malnutrition alopecia, drug-induced alopecia, alopecia caused by abnormal hair structure, male pattern baldness, female pattern baldness, and alopecia areata.

[0122] As used herein, the term "alleviation" refers to all actions to delay the progression of hair loss or alleviate the symptoms of hair loss by administering the composition of the present invention to an individual.

[0123] As used herein, the term "prevention" refers to all actions to inhibit or delay hair loss by administering the composition of the present invention to an individual.

[0124] As used herein, the term "treatment" refers to all actions that alleviate or beneficially change the symptoms of hair loss by administering the composition of the present invention to an individual suspected of having hair loss. In one embodiment of the present invention, the immunosuppressive effects of the nine new compounds were confirmed by in vitro T cell activity assays. The results confirmed that their immunosuppressive effects were reduced compared to FK506.

[0125] Furthermore, in another embodiment of the present invention, the effects of nine novel compounds on promoting the growth of antennae were confirmed.

[0126] In another embodiment of the present invention, the effects of nine new compounds in promoting the growth of antennae were confirmed, and cell growth markers were confirmed based on histological results, thereby confirming that the antennae continued to grow.

[0127] These results suggest that these nine new compounds have hair growth-promoting effects without side effects caused by immunosuppressive activity and can therefore be effectively used to alleviate, prevent or treat hair loss.

[0128] In order to achieve the above-mentioned object, another aspect of the present invention provides nine new compounds, 9-deoxy-31-O-demethyl-propenyl-FK506, 9-deoxy-36,37-dihydro-FK506, 31-O demethyl-36,37-dihydro-FK506, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506, 9-deoxy-FK520, 31-O demethyl-FK520, 9-deoxy-31-O demethyl-FK520, 9-deoxy-FK523 and a method for biomanufacturing 9-deoxy-31-O-demethyl-FK523.

[0129] In the bioproduction process, a culture temperature adopted in a general process of culturing microorganisms belonging to the genus Streptomyces is used. The culture temperature suitable for the embodiment of the present invention may preferably be in the range of 23°C to 30°C, more preferably in the range of 25°C to 28°C.

[0130] Furthermore, during the production process, the pH of the culture process is maintained in the range of 6.5 to 9, preferably in the range of 7 to 8.

[0131] At the same time, it is also important to maintain a high dissolved oxygen level during the manufacturing process. When the dissolved oxygen level at the beginning of the culture is assumed to be 100%, the dissolved oxygen level should be maintained at 30% or higher until the end of the culture. For this purpose, stirring can be performed at a rate of 800 rpm to 1500 rpm.

[0132] The nine new compounds produced by the cultured microbial cells during the manufacturing process can be extracted by the first extraction process, the second extraction process and the third extraction process. In the present invention, the first extraction process is carried out by an organic solvent extraction method, wherein the solvent used can be ethyl acetate, methanol or acetone, preferably ethyl acetate or methanol. The second extraction process can be carried out using silica gel chromatography, and methanol or dichloromethane can be used as a solvent. In addition, the third extraction process can be carried out using chromatography, wherein the solvent used can be acetonitrile, ammonium acetate buffer, acetic acid or formic acid, preferably acetonitrile. The application of these methods promotes the recovery of the nine new compounds and improves the yield.

[0133] In order to achieve the above-mentioned object, another aspect of the present invention provides a production strain that can be used to produce nine new compounds, namely, Streptomyces kanamycinensis. kanamyceticus)ΔfkbD-fkbM (Accession No.: KCTC13581BP), kanamyceticus ΔfkbD,tcsD (Accession No.: KCTC13580BP), kanamyceticus ΔfkbM,tcsD (Accession No.: KCTC13584BP), kanamyceticus ΔfkbD-fkbM,tcsD (Accession No.: KCTC13585BP), kanamyceticus ΔfkbD,tcsB (Accession No.: KCTC13579BP), kanamyceticus ΔfkbM,tcsB (Accession No.: KCTC13583BP), kanamyceticus ΔfkbD-fkbM,tcsB (Accession No.: KCTC13582BP).

[0134] Another object of the present invention is to provide an over-the-counter (OTC) composition for promoting hair growth, comprising at least one selected from the nine new compounds, their isomers or pharmaceutically acceptable salts thereof as an active ingredient.

[0135] When a composition including at least one selected from the nine novel compounds is added to an OTC composition for the purpose of promoting hair growth, the compound may be added as it is, or may be used together with other OTC components, and may be appropriately used according to any method commonly used in the art. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.

[0136] As used herein, the term "OTC composition" means fibers, rubber products, or similar products used to diagnose, treat, alleviate, cure, or prevent disease in humans or animals; non-device, non-machine, or similar products that have no significant effect or direct action on the human body; and preparations used for sterilization, insecticide, and similar purposes to prevent infectious diseases. OTC products do not include products used to diagnose, treat, alleviate, cure, or prevent disease in humans or animals, excluding appliances, machines, and equipment; or products used to exert a pharmacological effect on the structure or function of humans or animals, except appliances, machines, or equipment. OTC products may include external formulations and personal hygiene products.

[0137] Although not particularly limited thereto, the skin external agent may be specifically prepared in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel. The personal hygiene product may be, but is not particularly limited to, specifically, soap, cosmetics, wet wipes, tissues, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, base makeup, foundation, blush, mascara, eye shadow, sunscreen lotion, hair care products, air freshener gel or washing gel.

[0138] In addition, the OTC composition of the present invention can be a disinfectant cleaner, shower foam, ointment, wet wipes, coating agent, etc., but is not limited thereto, and the preparation method, dosage, use method, components, etc. of the OTC composition can be appropriately selected from conventional techniques known in the art.

[0139] In addition to the above components, the OTC composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent. The pharmaceutically acceptable carrier, excipient or diluent is not limited as long as it does not adversely affect the effect of the present invention, and may include fillers, extenders, binders, humectants, disintegrants, surfactants, lubricants, sweeteners, aromatics and preservatives.

[0140] In one embodiment of the present invention, nine novel compounds were confirmed to have the effect of promoting hair growth and can be used as OTC compositions for preventing hair loss and promoting hair growth.

[0141] When the preparation is a powder, the carrier may be lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate, polyamide powder or any mixture thereof.

[0142] When the formulation of the present invention is a solution or emulsion, the carrier can be a solvent, solubilizer or emulsifier, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, especially cottonseed oil, peanut oil, corn seed oil, olive oil, castor oil, sesame oil, glycerol, fatty esters, polyethylene glycol or fatty acid esters of sorbitol.

[0143] When the formulation is a suspension, the carrier may be a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar or astragalus.

[0144] Another object of the present invention is to provide a cosmetic composition for promoting hair growth, comprising at least one selected from the nine novel compounds, their isomers or cosmetically acceptable salts thereof as an active ingredient.

[0145] As used herein, "cosmetic composition" can be prepared in any commercially manufactured dosage form, such as solutions, emulsions, suspensions, muds, creams, lotions, gels, powders, sprays, surfactant-containing cleansers, oils, soaps, liquid cleansers, bath bombs, foundations, base makeup, essences, nourishing lotions, foams, packaging, softened water, sunscreens, sun oils, etc.

[0146] When the preparation of the present invention is a solution or emulsion, the carrier can be a solvent, solubilizer or emulsifier, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol, fatty esters, polyethylene glycol or fatty acid esters of sorbitol.

[0147] When the formulation is a suspension, the carrier may be a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminum hydroxide, bentonite, agar or astragalus.

[0148] When the preparation of the present invention is a paste, cream or gel, the carrier may be animal oil, vegetable oil, wax, paraffin, starch, astragalus, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc or zinc oxide.

[0149] When the preparation of the present invention is a powder or spray, the carrier may be lactose, talc, silicon dioxide, aluminum hydroxide, calcium silicate or polyamide powder. In particular, in the case of a spray, the preparation may further include a propellant such as chlorofluorocarbons, propane / butane or dimethyl ether.

[0150] When the preparation of the present invention is a detergent containing a surfactant, the carrier may be a fatty alcohol sulfate, a fatty alcohol ether sulfate, a sulfosuccinic acid monoester, an isothiocyanate, an imidazoline derivative, methyl taurine, a sarcosinate, a fatty acid amide ether sulfate, an alkylamide betaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a lanolin derivative or an ethoxylated glyceryl fatty acid ester.

[0151] In one embodiment of the present invention, the hair growth promoting effect of nine novel compounds was confirmed, and the use of compositions comprising the compounds as cosmetic compositions for preventing hair loss and promoting hair growth was confirmed.

[0152] [Best Mode]

[0153] In one embodiment of the present invention, the hair growth promoting effect of nine novel compounds was confirmed, and the use of compositions including the compounds as cosmetic compositions for preventing hair loss and promoting hair growth was confirmed.

[0154] Example 1: 9-deoxy-31- O Preparation of -desmethyl-prolyl-FK506

[0155] Streptomyces kanamyceticus ΔfkbD-fkbM (accession number: KCTC13581BP) is a 9-deoxy-31-O-demethyl-prolyl-FK506-producing strain, which is constructed by inactivating the fkbD-fkbM gene of Streptomyces kanamyceticus (FK506-producing strain) and causing in-frame deletion by double crossover homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0156] Specifically, to construct a mutant of the FK506-kanamycin-producing Streptomyces strain lacking fkbD and fkbM genes, each gene was cloned into pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506-kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0157] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0158] In the construction of the in-frame gene deletion plasmid, the Escherichia coli-Streptomyces shuttle vector pKC1139 is used for the in-frame gene deletion. The construction of the plasmid is carried out by carrying out PCR to the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbD-fkbM gene, a pair of primers FkbD-MLF / FkbD-MLR of the left fragment and a pair of primers FkbD-MRF / FkbD-MRR of the right fragment are designed. All PCR fragments are separated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned on the pKC1139 carrier. The information about the bacterial strain, plasmid and primer used in the present embodiment is shown in Table 1 and Table 2 below.

[0159] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbD-fkbM that simultaneously removes C9 hydroxylase and 31-O-methyltransferase was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains that undergo a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome are selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies are propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbD-fkbM, is selected as an apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0160] The constructed fkbD-fkbM gene deletion strain, Streptomyces kanamyceticus ΔfkbD-fkbM, was deposited in the Korea Collection of Type Cultures (KCTC) at the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (accession number: KCTC13581BP).

[0161] Table 1

[0162] Information on strains and plasmids used

[0163]

[0164]

[0165] Table 2

[0166] Information on primers used

[0167]

[0168]

[0169] 9-Deoxy-31-O-demethyl-prolyl-FK506 was prepared by culturing the constructed production strain Streptomyces kanamycinii ΔfkbD-fkbM (Accession No.: KCTC13581BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / LMgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / LTES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / L CaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured for two days in a rotary shaker incubator at 28°C and 180rpm. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28° C. and 180 rpm for 6 days. After 6 days of culture, 9-deoxy-31-O-demethyl-prolyl-FK506 was extracted using the first extraction process.

[0170] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract to remove the microbial cells is concentrated by a rotary evaporator. After the concentrated extract is redissolved in water, ethyl acetate is added in an amount twice the volume of water and shaken for extraction. The mixture is placed until the aqueous layer and the organic layer are separated. After the layer separation, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phases. In sample 3, 9-deoxy-31-O-demethyl-prolyl-FK506 is confirmed. The above-obtained sample 3 is concentrated using a rotary evaporator and finally purified by HPLC.

[0171] The concentrate was lyophilized to obtain 9-deoxy-31-O-demethyl-prolyl-FK506 represented by Formula 1 in the form of powder.

[0172] The prepared 9-deoxy-31-O-demethyl-prolyl-FK506 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 9-deoxy-31-O-demethyl-prolyl-FK506 are shown in Tables 3 and Figures 1 to 6 As shown, and based on the results, it was confirmed that 9-deoxy-31-O-demethyl-prolyl-FK506 was produced by the constructed production strain Streptomyces kanamyciniensis ΔfkbD-fkbM.

[0173] 9-Deoxy-31-O-demethyl-prolyl-FK506 (molecular formula: C 41 H 57 NO 11 The analysis results of the 200 μg / ml (molecular weight: 749.4714) are shown in Table 3 below.

[0174] Table 3

[0175]

[0176]

[0177]

[0178] based on 1 H-NMR and 13 C-NMR results show that a ketone carbon (δ C 214.00), two carbonyl carbons (δ C 171.99 and 170.09), an exomethylene backbone (δ C 116.84 and 135.80) and two olefin backbones (δ C 141.22 and 122.04; and δ C 132.64 and 129.79) were confirmed as characteristic functional groups, and a doubly oxidized quaternary carbon (δ C 98.73), 7 oxidized methylene carbons (δ C 78.28, 77.39, 75.28, 75.81, 74.73, 71.14 and 69.23), 2 methoxy carbons (δ C 57.98 and 56.48), and 5 methyl carbons (δ C19.10, 17.20, 15.90, 14.41 and 10.13). The obtained compound was identified as a FK506 derivative consisting of 42 carbon atoms. In order to accurately identify the structure of the compound, 2D-NMR confirmation was performed. As a result of proton coupling identification using gCOSY, based on the coupling of H-2 and H-4, it was confirmed that the compound was not FK506 having a piperidine skeleton but a compound including a prolyl skeleton without a CH2 functional group. According to gHMBC data, based on H-9 (δ H 2.56,2.63) and C-8(δ C 171.99) and C-10 (δ C 98.73), confirming that C-9 of the compound is not a ketone but a skeleton reduced to CH2. In addition, since two methoxy functional groups are connected to C-13 and C-15, it is confirmed that there is no methoxy group on C-31. In conclusion, the compound was confirmed to be 9-deoxy-31-O-demethyl-prolyl-FK506.

[0179] Example 2: Preparation of 9-deoxy-36,37-dihydro-FK506

[0180] Streptomyces kanamyceticus ΔfkbD,tcsD (Accession No.: KCTC13580BP) is a 9-deoxy-36,37-dihydro-FK506-producing strain, which was constructed by inactivating the fkbD and tcsD genes of Streptomyces kanamyceticus (FK506-producing strain) and causing in-frame deletion by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0181] Specifically, to construct a mutant of the FK506-kanamycin-producing Streptomyces strain lacking the fkbD and tcsD genes, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506-kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0182] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0183] In the construction of the in-frame gene deletion plasmid, the Escherichia coli-Streptomyces shuttle vector pKC1139 is used for the in-frame gene deletion. The construction of the plasmid is carried out by carrying out PCR to the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbD gene, the primers FkbDLF / FkbDLR of a pair of left fragments and the primers FkbDRF / FkbDRR of a pair of right fragments are designed. In order to delete the tcsD gene, the primers TcsDLF / TcsDLR of a pair of left fragments and the primers TcsDRF / TcsDRR of a pair of right fragments are designed. All PCR fragments are separated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned on the pKC1139 carrier. The information of the bacterial strain, plasmid and primer used in the present embodiment is presented in Table 1 and Table 2.

[0184] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbD that removes C9 hydroxylase was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains that undergo a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome are selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies are propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbD, is selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0185] The tcsD gene was deleted by introducing pΔtcsD into the constructed fkbD gene-deficient Streptomyces ΔfkbD using the same method as the fkbD gene deletion. ΔfkbD,tcsD was selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0186] The constructed fkbD and tcsD gene deletion strain, Streptomyces kanamyceticus ΔfkbD,tcsD, was deposited in the Korea Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (accession number: KCTC13580BP).

[0187] 9-Deoxy-36,37-dihydro-FK506 was prepared by culturing a mutant strain of Streptomyces kanamycinii ΔfkbD,tcsD (Accession No.: KCTC13580BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / L MgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / L TES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / L CaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured in a rotary shaker incubator at 28°C and 180rpm for two days. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28° C. and 180 rpm for 6 days. After 6 days of culture, 9-deoxy-36,37-dihydro-FK506 was extracted using the first extraction process.

[0188] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the supernatant is concentrated using a rotary evaporator. After the concentrated extract is dissolved in water, two volumes of ethyl acetate are added and mixed. The mixture is left until the layers are separated. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phases. In sample 3, 9-deoxy-36,37-dihydro-FK506 is confirmed. The above-obtained sample 3 is concentrated using a rotary evaporator and finally purified by HPLC.

[0189] The concentrate was lyophilized to obtain 9-deoxy-36,37-dihydro-FK506 represented by Formula 2 in the form of a powder.

[0190] The prepared 9-deoxy-36,37-dihydro-FK506 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 9-deoxy-36,37-dihydro-FK506 are shown in Tables 4 and Figures 7 to 12As shown, and based on the results, it was confirmed that 9-deoxy-36,37-dihydro-FK506 was produced by the constructed production strain Streptomyces kanamycinii ΔfkbD,tcsD.

[0191] 9-Deoxy-36,37-dihydro-FK506 (molecular formula: C 44 H 73 NO 11 The analysis results of the above-mentioned compounds are shown in Table 4 below.

[0192] Table 4

[0193]

[0194]

[0195]

[0196] according to 1 H-NMR, 13 C-NMR and gHSQC results showed that the obtained compound was composed of 44 carbon atoms, and a methoxy group (δ H 3.39 and δ C 56.56) and a CH2 group (δ H 2.24, δ H 1.70 and δ C 20.70), no exomethylene functional groups were observed, and the methyl group (δ H 0.88 and δ C 13.91) and CH2 group (δ H 1.21 and δ C 20.41) was observed. In order to accurately identify the structure, 2D-NMR was performed. The results of proton coupling identified by gCOSY confirmed that the compound was FK506 containing a piperidine backbone, and based on the coupling from H-2 to H-5, there was a CH2 functional group (δ H 2.24, δ H 1.70 and δ C 20.70). Based on gHMBC, three methoxy hydrogens (δ H 3.39, 3.36 and 3.35) respectively with C-31 (δ C 77.11), C-15(δ C 77.11) and C-13 (δ C 74.33) and the methyl group (δ H 0.88 and δ C 13.91) and CH2 groups (δH 1.21 and δ C 20.41) is related to C-21 / 33. Therefore, as a compound produced by the tcsD gene deletion strain, 9-deoxy-36,37-dihydro-FK506 was determined to have a structural form in which the double bond between C-36 / 37 was reduced.

[0197] Example 3: Preparation of 31-O-demethyl-36,37-dihydro-FK506

[0198] Streptomyces kanamyceticus ΔfkbM,tcsD (accession number: KCTC13584BP) is a 31-O-demethyl-36,37-dihydro-FK506-producing strain, which is constructed by inactivating the fkbM and tcsD genes of Streptomyces kanamyceticus (FK506-producing strain) and causing in-frame deletion by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0199] Specifically, to construct a mutant of the FK506-kanamycin-producing Streptomyces strain lacking the fkbM and tcsD genes, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506-kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0200] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0201] In the construction of the in-frame gene deletion plasmid, the Escherichia coli-Streptomyces shuttle vector pKC1139 is used for the in-frame gene deletion. The construction of the plasmid is carried out by carrying out PCR to the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbM gene, a pair of primers FkbMLF / FkbMLR of the left fragment and a pair of primers FkbMRF / FkbMRR of the right fragment are designed. In order to delete the tcsD gene, a pair of primers TcsDLF / TcsDLR of the left fragment and a pair of primers TcsDRF / TcsDRR of the right fragment are designed. All PCR fragments are separated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned on the pKC1139 carrier. The information of the bacterial strain, plasmid and primer used in the present embodiment is shown in Table 1 and Table 2.

[0202] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbM with 31-O-methyltransferase removed was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains with a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome were selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies were propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbM, was selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0203] The tcsD gene was deleted by introducing pΔtcsD into the constructed fkbM gene-deficient Streptomyces ΔfkbM using the same method as the fkbM gene deletion. ΔfkbM,tcsD was selected as an apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0204] The constructed fkbM and tcsD gene deletion strain, Streptomyces kanamyceticus ΔfkbM, tcsD, was deposited in the Korea Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (accession number: KCTC13584BP).

[0205] 31-O-demethyl-36,37-dihydro-FK506 was prepared by culturing the constructed production strain Streptomyces kanamycinii ΔfkbM,tcsD (Accession No.: KCTC13584BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / LMgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / LTES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / L CaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured for two days in a rotary shaker incubator at 28°C and 180rpm. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28° C. and 180 rpm for 6 days. After 6 days of culture, 31-O-desmethyl-36,37-dihydro-FK506 was extracted using the first extraction process.

[0206] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract to remove the microbial cells is concentrated by a rotary evaporator. After the concentrated extract is dissolved in water, ethyl acetate twice the volume of the extract is added and mixed, and the mixture is placed until the layers are separated. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phases. In sample 3, 31-O-demethyl-36,37-dihydro-FK506 is confirmed. The above-obtained sample 3 is concentrated using a rotary evaporator and finally purified by HPLC.

[0207] The concentrate was lyophilized to obtain 31-O-demethyl-36,37-dihydro-FK506 represented by Formula 3 in the form of a powder.

[0208] The prepared 31-O-demethyl-36,37-dihydro-FK506 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 31-O-demethyl-36,37-dihydro-FK506 are shown in Tables 5 and Figures 13 to 18 As shown, and based on the results, it was confirmed that 31-O-demethyl-36,37-dihydro-FK506 was produced by the constructed production strain Streptomyces kanamycinii ΔfkbM,tcsD.

[0209] 31-O-Demethyl-36,37-dihydro-FK506 (molecular formula: C 43 H 69 NO 12 The analysis results of the above-mentioned compounds are shown in Table 5 below.

[0210] Table 5

[0211]

[0212]

[0213]

[0214]

[0215] according to 1 H-NMR, 13 C-NMR and gHSQC results showed that the obtained compound was composed of 43 carbon atoms. C 196.54), and two methoxy groups were observed, suggesting that the compound was derived from 31-O-demethylation produced by the ΔfkbM gene. Based on gHMBC, two methoxy hydrogens (δ H 3.40 and 3.30) respectively with C-15 (δ C 75.21) and C-13 (δ C As a compound produced by the tcsD gene deletion strain, 31-O-demethyl-35,37-dihydro-FK506 was determined to have a structure in which the double bond existing between C-36 / 37 was reduced.

[0216] Example 4: Preparation of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506

[0217] Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD (Accession No.: KCTC13585BP) is a 9-deoxy-31-O-demethyl-36,37-dihydro-FK506-producing strain, which was constructed by inactivating the fkbD-fkbM and tcsD genes of Streptomyces kanamyceticus (FK506-producing strain) and causing in-frame deletion by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0218] Specifically, to construct a mutant of the FK506-kanamycin-producing Streptomyces strain lacking fkbD-fkbM and tcsD genes, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506-kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0219] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0220] In the construction of the in-frame gene deletion plasmid, the Escherichia coli-Streptomyces shuttle vector pKC1139 is used for the in-frame gene deletion. The construction of the plasmid is carried out by carrying out PCR to the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbD-fkbM gene, a pair of primers FkbD-MLF / FkbD-MLR of the left fragment and a pair of primers FkbD-MRF / FkbD-MRR of the right fragment are designed. In order to delete the tcsD gene, a pair of primers TcsDLF / TcsDLR of the left fragment and a pair of primers TcsDRF / TcsDRR of the right fragment are designed. All PCR fragments are separated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned on the pKC1139 carrier. The information of the bacterial strain, plasmid and primer used in the present embodiment is shown in Table 1 and Table 2.

[0221] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbD-fkbM that simultaneously removes C9 hydroxylase and 31-O-methyltransferase was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains that undergo a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome are selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies are propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbD-fkbM, is selected as an apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0222] The tcsD gene was deleted by introducing pΔtcsD into the constructed fkbD-fkbM gene-deleted Streptomyces ΔfkbD-fkbM using the same method as the deletion of the fkbD-fkbM gene. ΔfkbD-fkbM, tcsD was selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0223] The constructed fkbD-fkbM and tcsD gene deletion strain, Streptomyces kanamyceticus ΔfkbD-fkbM,tcsD, was deposited in the Korea Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (accession number: KCTC13585BP).

[0224] 9-Deoxy-31-O-demethyl-36,37-dihydro-FK506 was prepared by culturing the constructed production strain Streptomyces kanamycinii ΔfkbD-fkbM,tcsD (Accession No.: KCTC13585BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / L MgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / L TES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / L CaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured in a rotary shaker incubator at 28°C and 180rpm for two days. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28° C. and 180 rpm for 6 days. After 6 days of culture, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 was extracted using the first extraction process.

[0225] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract from which the microbial cells are removed is concentrated by a rotary evaporator. After the concentrated extract is dissolved in water, ethyl acetate twice the volume of the extract is added and mixed, and the mixture is placed until stratification. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phases. In sample 3, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 is confirmed. The above-obtained sample 3 is concentrated using a rotary evaporator and finally purified by HPLC.

[0226] The concentrate was lyophilized to obtain 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 represented by Formula 4 in the form of a powder.

[0227] The prepared 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 are shown in Tables 6 and Figures 19 to 24 As shown, and based on the results, it was confirmed that 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 was produced by the constructed production strain Streptomyces kanamycinii ΔfkbD-fkbM,tcsD.

[0228] 9-Deoxy-31-O-demethyl-36,37-dihydro-FK506 (molecular formula: C 43 H 71 NO 11 The analysis results of the above-mentioned compounds are shown in Table 6 below.

[0229] Table 6

[0230]

[0231]

[0232]

[0233] according to 1 H-NMR, 13C-NMR and gHSQC results showed that although the obtained compounds consisted of 43 carbon atoms, in substances 1 and 2, CH2 functional groups (δ H 2.67, 2.49 and δ C Based on the analysis results of 2D-NMR data, the structure of compound No. 4 was determined to be 9-deoxy-31-O-demethyl-35,37-dihydro-FK506 produced by the ΔfkbD-fkbM gene.

[0234] Example 5: Preparation of 9-deoxy-FK520

[0235] Streptomyces kanamyceticus ΔfkbD, tcsB (accession number: KCTC13579BP) or Streptomyces kanamyceticus ΔfkbD, tcsD (accession number: KCTC13580BP) is a 9-deoxy-FK520-producing strain, which is constructed by inactivating the fkbD and tcsB genes or the fkbD and tcsD genes of Streptomyces kanamyceticus (FK506-producing strain), and causing in-frame deletion by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0236] Specifically, to construct mutants of the FK506 kanamycin-producing Streptomyces strain lacking fkbD and tcsB genes or fkbD and tcsD genes, each gene was cloned into pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506 kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0237] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0238] In the construction of the in-frame gene deletion plasmid, the E. coli-Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. The construction of the plasmid was performed by PCR on the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbD gene, a pair of primers FkbDLF / FkbDLR for the left fragment and a pair of primers FkbDRF / FkbDRR for the right fragment were designed. In order to delete the tcsB gene, a pair of primers TcsBLF / TcsBLR for the left fragment and a pair of primers TcsBRF / TcsBRR for the right fragment were designed. In order to delete the tcsD gene, a pair of primers TcsDLF / TcsDLR for the left fragment and a pair of primers TcsDRF / TcsDRR for the right fragment were designed. All PCR fragments were isolated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned into pKC1139 vector. The information of strains, plasmids and primers used in this example are shown in Tables 1 and 2.

[0239] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbD that removes C9 hydroxylase was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains that undergo a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome are selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies are propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbD, is selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0240] The tcsB or tcsD gene was deleted by introducing pΔtcsB or pΔtcsD into the constructed fkbD gene-deficient Streptomyces ΔfkbD using the same method as the deletion of the fkbD gene. ΔfkbD, tcsB or ΔfkbD, tcsD was selected as the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0241] The constructed fkbD and tcsB gene deletion strain, Streptomyces kanamyceticus ΔfkbD,tcsB, was deposited in the Korea Collection of Type Culture (KCTC) at the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (Accession No.: KCTC13579BP), and the constructed fkbD and tcsD gene deletion strain, Streptomyces kanamyceticus ΔfkbD,tcsD, was also deposited in the Korea Collection of Type Culture (KCTC) at the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (Accession No.: KCTC13580BP).

[0242] 9-deoxy-FK520 is prepared by culturing the constructed production strain Streptomyces kanamyciniensis ΔfkbD, tcsB (Accession No.: KCTC13579BP) or Streptomyces kanamyciniensis ΔfkbD, tcsD (Accession No.: KCTC13580BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / LMgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / L TES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / L CaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured for two days in a rotary shaker incubator at 28°C and 180rpm. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28°C and 180 rpm for 6 days. After 6 days of culture, 9-deoxy-FK520 was extracted using the first extraction process.

[0243] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract to remove the microbial cells is concentrated by a rotary evaporator. After the concentrated extract is dissolved in water, ethyl acetate twice the volume of the extract is added and mixed, and the mixture is placed until stratification. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phase. In sample 3, 9-deoxy-FK520 is confirmed. The above-obtained sample 3 is concentrated using a rotary evaporator and finally purified by HPLC.

[0244] The concentrate was lyophilized to obtain 9-deoxy-FK520 represented by Formula 5 in the form of a powder.

[0245] The prepared 9-deoxy-FK520 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 9-deoxy-FK520 are shown in Tables 7 and Figures 25 to 30 As shown, and based on the results, it was confirmed that 9-deoxy-FK520 was produced by the constructed production strain S. kanamycinensis ΔfkbD, tcsB or S. kanamycinensis ΔfkbD, tcsD.

[0246] 9-Deoxy-FK520 (Molecular formula: C 43 H 71 NO 11 The analysis results of the above-mentioned compounds are shown in Table 7 below.

[0247] Table 7

[0248]

[0249]

[0250]

[0251] based on 1 H-NMR, 13 C-NMR and gHSQC data showed that the compound composed of 43 carbon atoms was similar to 9-deoxy-31-O-demethyl-36,37-dihydro-FK506, but due to the additional observation of methoxy groups (δ H 3.41 and δ C56.81), and less CH2 functional groups were observed, so the compound was confirmed as a structural isomer. The results of proton coupling identified by gCOSY confirmed that the compound has a piperidine backbone based on coupling from H-2 to H-5. Based on the observation of triple coupled methyl groups from gHMBC, and the presence of C-21 (δ H 1.72 and 1.52, and δ C 20.41) between the CH2 groups (δ H 1.72 and 1.52, and δ C 20.41), confirming that the ethyl group at C-21 constitutes FK520, not the propyl group of FK506. On this basis, the structure of compound 5 was determined to be 9-deoxy-FK520.

[0252] Example 6: Preparation of 31-O-demethyl-FK520

[0253] Streptomyces kanamyceticus ΔfkbM, tcsB (accession number: KCTC13583BP) or Streptomyces kanamyceticus ΔfkbM, tcsD (accession number: KCTC13584BP) is a 31-O-demethyl-FK520-producing strain, which is constructed by inactivating the fkbM and tcsB genes or the fkbM and tcsD genes of Streptomyces kanamyceticus (FK506-producing strain) and causing in-frame deletion by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0254] Specifically, to construct mutants of the FK506-producing Streptomyces kanamycin strain lacking fkbM and tcsB genes or fkbM and tcsD genes, each gene was cloned into pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506-producing Streptomyces kanamycin strain was transformed with the vector by conjugation.

[0255] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0256] In the construction of the in-frame gene deletion plasmid, the E. coli-Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. The construction of the plasmid was performed by PCR on the left and right fragments of the target gene deleted from Fosmid DNA from Streptomyces kanamyceticus. In order to delete the fkbM gene, a pair of primers FkbMLF / FkbMLR for the left fragment and a pair of primers FkbMRF / FkbMRR for the right fragment were designed. In order to delete the tcsB gene, a pair of primers TcsBLF / TcsBLR for the left fragment and a pair of primers TcsBRF / TcsBRR for the right fragment were designed. In order to delete the tcsD gene, a pair of primers TcsDLF / TcsDLR for the left fragment and a pair of primers TcsDRF / TcsDRR for the right fragment were designed. All PCR fragments were isolated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned into pKC1139 vector. The information of strains, plasmids and primers used in this example are shown in Tables 1 and 2.

[0257] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbM with 31-O-methyltransferase removed was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains with a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome were selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies were propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbM, was selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0258] The tcsB or tcsD gene was deleted by introducing pΔtcsB or pΔtcsD into the constructed fkbM gene-deficient Streptomyces ΔfkbM using the same method as the deletion of the fkbM gene. ΔfkbM, tcsB or ΔfkbM, tcsD was selected as the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0259] The constructed fkbM and tcsB gene deletion strain, Streptomyces kanamyceticus ΔfkbM,tcsB, was deposited in the Korea Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (Accession No.: KCTC13583BP), and the constructed fkbM and tcsD gene deletion strain, Streptomyces kanamyceticus ΔfkbM,tcsD, was also deposited in the Korea Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (Accession No.: KCTC13584BP).

[0260] 31-O-desmethyl-FK520 is prepared by culturing the constructed production strain Streptomyces kanamycinii ΔfkbM,tcsB (Accession No.: KCTC13583BP) or Streptomyces kanamycinii ΔfkbM,tcsD (Accession No.: KCTC13584BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / LMgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / L TES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / L CaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured for two days in a rotary shaker incubator at 28°C and 180rpm. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28°C and 180 rpm for 6 days. After 6 days of culture, 31-O-desmethyl-FK520 was extracted using the first extraction process.

[0261] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract that removes the microbial cells is concentrated with a rotary evaporator. After the concentrated extract is dissolved in water, ethyl acetate twice the volume of the extract is added and mixed, and the mixture is placed until stratification. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phases. In sample 3, 31-O-demethyl-FK520 is confirmed. The above-obtained sample 3 is concentrated with a rotary evaporator and finally purified with HPLC.

[0262] The concentrate was lyophilized to obtain 31-O-desmethyl-FK520 represented by Formula 6 in the form of a powder.

[0263] The prepared 31-O-demethyl-FK520 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 31-O-demethyl-FK520 are shown in Tables 8 and Figures 31 to 36 As shown, and based on the results, it was confirmed that 31-O-desmethyl-FK520 was produced by the constructed production strain Streptomyces kanamycinii ΔfkbM,tcsB or Streptomyces kanamycinii ΔfkbM,tcsD.

[0264] 31-O-Demethyl-FK520 (Molecular formula: C 42 H 67 NO 12 The analysis results of the 200 μg / ml (molecular weight: 400 μg / ml) are shown in Table 8 below.

[0265] Table 8

[0266]

[0267]

[0268]

[0269] Although the molecular weight of compound 6 is 800.4563, which is very similar to the molecular weights of 800.4924 and 800.4927, based on 1 H-NMR, 13 C-NMR and gHSQC data confirmed that the compound, which consists of a total of 42 carbon atoms, has one carbon atom difference due to one less methoxy group, and a keto carbon (δC 196.50). According to 2D-NMR data, the structure of the compound was determined to be 31-O-demethyl-FK520.

[0270] Example 7: Preparation of 9-deoxy-31-O-demethyl-FK520

[0271] Streptomyces kanamyceticus ΔfkbD-fkbM, tcsB (accession number: KCTC13582BP) and Streptomyces kanamyceticus ΔfkbD-fkbM, tcsD (accession number: KCTC13585BP) are 9-deoxy-31-O-demethyl-FK520 producing strains, which are constructed by inactivating the fkbD-fkbM and tcsB genes or fkbD-fkbM and tcsD genes of Streptomyces kanamyceticus (FK506 producing strain), and causing in-frame deletions by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0272] Specifically, to construct mutants of the FK506 kanamycin-producing Streptomyces strain lacking the fkbD-fkbM and tcsB genes or the fkbD-fkbM and tcsD genes, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506 kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0273] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0274] In the construction of the in-frame gene deletion plasmid, the E. coli-Streptomyces shuttle vector pKC1139 was used for in-frame gene deletion. The construction of the plasmid was carried out by PCR of the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbD-fkbM genes, a pair of primers FkbD-MLF / FkbD-MLR for the left fragment and a pair of primers FkbD-MRF / FkbD-MRR for the right fragment were designed. In order to delete the tcsB gene, a pair of primers TcsBLF / TcsBLR for the left fragment and a pair of primers TcsBRF / TcsBRR for the right fragment were designed. In order to delete the tcsD gene, a pair of primers TcsDLF / TcsDLR for the left fragment and a pair of primers TcsDRF / TcsDRR for the right fragment were designed. All PCR fragments were isolated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned into pKC1139 vector. The information of strains, plasmids and primers used in this example are shown in Tables 1 and 2.

[0275] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbD-fkbM that simultaneously removes C9 hydroxylase and 31-O-methyltransferase was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains that undergo a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome are selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies are propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbD-fkbM, is selected as an apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0276] The tcsB or tcsD gene was deleted by introducing pΔtcsB or pΔtcsD into the constructed fkbD-fkbM gene-deficient Streptomyces ΔfkbD-fkbM using the same method as the deletion of the fkbD-fkbM gene. ΔfkbD-fkbM,tcsB or ΔfkbD-fkbM,tcsD was selected as the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0277] The constructed fkbD-fkbM and tcsB gene deletion strain, Streptomyces kanamyceticus ΔfkbD-fkbM, tcsB, was deposited in the Korea Collection of Type Culture (KCTC) at the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (Accession No.: KCTC13582BP), and the constructed fkbD-fkbM and tcsD gene deletion strain, Streptomyces kanamyceticus ΔfkbD-fkbM, tcsD, was also deposited in the Korea Collection of Type Culture (KCTC) at the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (Accession No.: KCTC13585BP).

[0278] 9-deoxy-31-O-demethyl-FK520 was prepared by culturing the constructed production strain Streptomyces kanamycins ΔfkbD-fkbM, tcsB (Accession No.: KCTC13582BP) or Streptomyces kanamycins ΔfkbD-fkbM, tcsD (Accession No.: KCTC13585BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / LMgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / LTES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / L CaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured for two days in a rotary shaker incubator at 28°C and 180rpm. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28°C and 180 rpm for 6 days. After 6 days of culture, 9-deoxy-31-O-desmethyl-FK520 was extracted using the first extraction process.

[0279] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract that removes the microbial cells is concentrated by a rotary evaporator. After the concentrated extract is dissolved in water, ethyl acetate twice the volume of the extract is added and mixed, and the mixture is placed until stratification. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phases. In sample 3, 9-deoxy-31-O-demethyl-FK520 is confirmed. The above-obtained sample 3 is concentrated using a rotary evaporator and finally purified by HPLC.

[0280] The concentrate was lyophilized to obtain 9-deoxy-31-O-desmethyl-FK520 represented by Formula 7 in the form of a powder.

[0281] The prepared 9-deoxy-31-O-demethyl-FK520 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 9-deoxy-31-O-demethyl-FK520 are shown in Tables 9 and Figures 31 to 42 As shown, and based on the results, it was confirmed that 9-deoxy-31-O-desmethyl-FK520 was produced by the constructed production strain Streptomyces kanamycinii ΔfkbD-fkbM, tcsB or Streptomyces kanamycinii ΔfkbD-fkbM, tcsD.

[0282] 9-Deoxy-31-O-demethyl-FK520 (molecular formula: C 42 H 69 NO 11 The analysis results of the molecular weight: 764.01) are shown in Table 9 below.

[0283] Table 9

[0284]

[0285]

[0286]

[0287]

[0288] based on 1 H-NMR, 13C-NMR and gHSQC data showed that although the obtained compound consisted of 42 carbon atoms, showing similar results to 31-O-desmethyl-FK520, the CH2 functional group (δ H 2.67 and 2.49 and δ C 37.54). According to 2D-NMR data, the structure of compound 7 was determined to be 9-deoxy-31-O-demethyl-FK520.

[0289] Example 8: Preparation of 9-deoxy-FK523

[0290] Streptomyces kanamyceticus ΔfkbD, tcsB (accession number: KCTC13579BP) is a 9-deoxy-FK523-producing strain, which was constructed by inactivating the fkbD and tcsB genes of Streptomyces kanamyceticus (FK506-producing strain) and causing in-frame deletion by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0291] Specifically, to construct a mutant of the FK506-kanamycin-producing Streptomyces strain lacking the fkbD and tcsB genes, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506-kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0292] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0293] In the construction of the in-frame gene deletion plasmid, the Escherichia coli-Streptomyces shuttle vector pKC1139 is used for the in-frame gene deletion. The construction of the plasmid is carried out by carrying out PCR to the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbD gene, the primers FkbDLF / FkbDLR of a pair of left fragments and the primers FkbDRF / FkbDRR of a pair of right fragments are designed. In order to delete the tcsB gene, the primers TcsBLF / TcsBLR of a pair of left fragments and the primers TcsBRF / TcsBRR of a pair of right fragments are designed. All PCR fragments are separated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned on the pKC1139 carrier. The information of the bacterial strain, plasmid and primer used in the present embodiment is presented in Table 1 and Table 2.

[0294] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbD that removes C9 hydroxylase was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains that undergo a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome are selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies are propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbD, is selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0295] The tcsB gene was deleted using the same method as the deletion of the fkbD gene by introducing pΔtcsB into the constructed fkbD gene-deficient Streptomyces ΔfkbD. ΔfkbD,tcsB was selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0296] The constructed fkbD and tcsB gene deletion strain, Streptomyces kanamyceticus ΔfkbD, tcsB, was deposited in the Korea Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (accession number: KCTC13579BP).

[0297] 9-deoxy-FK523 was prepared by culturing a mutant strain of Streptomyces kanamycinii ΔfkbD, tcsB (Accession No.: KCTC13579BP). This will be described in more detail. 50 mL of R2YE medium (including 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 triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured in a rotary shaker incubator at 28°C and 180 rpm for two days. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28°C and 180 rpm for 6 days. After 6 days of culture, 9-deoxy-FK523 was extracted using the first extraction process.

[0298] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract to remove the microbial cells is concentrated with a rotary evaporator. After the concentrated extract is dissolved in water, ethyl acetate twice the volume of the extract is added and mixed, and the mixture is placed until stratification. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phase. In sample 3, 9-deoxy-FK523 is confirmed. The above-obtained sample 3 is concentrated with a rotary evaporator and finally purified with HPLC.

[0299] The concentrate was lyophilized to obtain 9-deoxy-FK523 represented by Formula 8 in the form of a powder.

[0300] The prepared 9-deoxy-FK523 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 9-deoxy-FK523 are shown in Tables 10 and Figures 43 to 48 As shown, and based on the results, it was confirmed that 9-deoxy-FK523 was produced by the constructed production strain S. kanamyciniensis ΔfkbD,tcsB.

[0301] 9-Deoxy-FK523 (Molecular formula: C 42 H 69 NO 11 The analysis results of the above-mentioned compounds are shown in Table 10 below.

[0302] Table 10

[0303]

[0304]

[0305] The molecular weight of the obtained compound is the same as that of 9-deoxy-31-O-desmethyl-FK520. 1 H-NMR, 13 C-NMR and gHSQC data show that the compound consisting of a total of 42 carbon atoms has a very similar structure, but due to the additional observation of a methoxy group (δ H 3.41 and δ C56.81), and less CH2 functional groups were observed, so the compound was confirmed as a structural isomer, different from 9-deoxy-31-O-demethyl-FK520. The results of proton coupling were identified by gCOSY, confirming that the compound has a piperidine backbone based on H-2 to H-5 coupling. Based on gHMBC, the methyl group (δ H 1.18 and δ C 17.13) and C-21(δ H 3.21 and δ C 53.45), thus confirming the presence of a methyl group constituting FK523 at C-21. On this basis, the structure of the compound was determined to be 9-deoxy-FK523.

[0306] Example 9: Preparation of 9-deoxy-31-O-demethyl-FK523

[0307] Streptomyces kanamyceticus ΔfkbD-fkbM, tcsB (accession number: KCTC13582BP) is a 9-deoxy-31-O-demethyl-FK523-producing strain, which was constructed by inactivating the fkbD-fkbM and tcsB genes of Streptomyces kanamyceticus (FK506-producing strain) and causing in-frame deletion by double hybrid homologous recombination according to the method introduced by Ban, YH et al., (J. Nat. Prod. 2013, 76, 1091-1098).

[0308] Specifically, to construct a mutant of the FK506-kanamycin-producing Streptomyces strain lacking fkbD-fkbM and tcsB genes, each gene was cloned into the pKC1139 vector and transferred into Escherichia coli ET12567 / pUZ8002, and then the FK506-kanamycin-producing Streptomyces strain was transformed with the vector by conjugation.

[0309] The method for constructing the strain can be more specifically explained as constructing an in-frame gene deletion plasmid and constructing a gene deletion strain.

[0310] In the construction of the in-frame gene deletion plasmid, the Escherichia coli-Streptomyces shuttle vector pKC1139 is used for the in-frame gene deletion. The construction of the plasmid is carried out by carrying out PCR to the left and right fragments of the target gene deleted from the Fosmid DNA of Streptomyces kanamyceticus. In order to delete the fkbD-fkbM gene, the primers FkbD-MLF / FkbD-MLR of a pair of left fragments and the primers FkbD-MRF / FkbD-MRR of a pair of right fragments are designed. In order to delete the tcsB gene, the primers TcsBLF / TcsBLR of a pair of left fragments and the primers TcsBRF / TcsBRR of a pair of right fragments are designed. All PCR fragments are separated, digested with HindIII-XbaI or XbaI-EcoRI, and cloned on the pKC1139 carrier. The information of the bacterial strain, plasmid and primer used in the present embodiment is shown in Table 1 and Table 2.

[0311] The plasmids used to construct gene deletion strains are shown in Table 1. A plasmid pΔfkbD-fkbM that simultaneously removes C9 hydroxylase and 31-O-methyltransferase was transformed into Escherichia coli ET12567 / pUZ8002 and introduced into kanamycin streptomyces by conjugation to delete the target gene by homologous recombination. Strains that undergo a single hybridization between the deletion plasmid and the kanamycin streptomyces chromosome are selected by culturing apramycin-resistant transfers at 37°C (a non-permissive temperature for replication of pSG5-based replicons) in the presence of apramycin. Then, the obtained colonies are propagated three times at 28°C without selection to allow a second hybridization. The obtained double hybrid mutant, ΔfkbD-fkbM, is selected as an apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0312] The tcsB gene was deleted by introducing pΔtcsB into the constructed fkbD-fkbM gene-deficient Streptomyces ΔfkbD-fkbM using the same method as the deletion of the fkbD-fkbM gene. ΔfkbD-fkbM,tcsB was selected for the apramycin-sensitive phenotype and identified by PCR and optionally by Southern blotting.

[0313] The constructed fkbD-fkbM and tcsB gene deletion strain, Streptomyces kanamyceticus ΔfkbD-fkbM, tcsB, was deposited in the Korea Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology on July 17, 2018 (accession number: KCTC13582BP).

[0314] 9-Deoxy-31-O-demethyl-FK523 was prepared by culturing the constructed production strain Streptomyces kanamycinii ΔfkbD-fkbM (Accession No.: KCTC13582BP). This will be described in more detail. 50mL R2YE medium (comprising 103g / L sucrose, 10g / L glucose, 0.25g / L K2SO4, 10.12g / L MgCl2·6H2O, 0.1g / L casamino acids, 50mL / L yeast extract (10%)), 100mL / L TES buffer (5.73%, pH 7.2), 10mL / L potassium phosphate (0.5%), 80mL / LCaCl2·2H2O (3.68%), 15mL / L L-proline (20%), 2mL / L trace element solution and 5mL / L NaOH (1N)) was added to a 250mL triangular shaking flask and inoculated with the production strain. Then, the strain was pre-cultured for two days in a rotary shaker incubator at 28°C and 180rpm. Subsequently, 10 ml of the pre-cultured culture was inoculated into 1 liter of R2YE medium in a 3-liter conical flask. After inoculation, the strain was cultured at 28°C and 180 rpm for 6 days. After 6 days of culture, 9-deoxy-31-O-demethyl-FK523 was extracted using the first extraction process.

[0315] The first extraction process is carried out as follows. First, an equal volume of methanol is added to the culture solution and mixed for 30 minutes, the microbial cells are removed by centrifugation, and then the extract that removes the microbial cells is concentrated with a rotary evaporator. After the concentrated extract is dissolved in water, ethyl acetate twice the volume of the extract is added and mixed, and the mixture is placed until stratification. After the layers are separated, the upper layer (i.e., the organic solvent layer) is recovered and concentrated using a rotary evaporator, and the weight after concentration is measured. The extract obtained by the first extraction process is passed through a column filled with silica gel. In this case, the amount of silica gel is 15 times the weight of the extract obtained by the first extraction process, and methanol and dichloromethane are mixed in five ratios (sample 1.0:100, sample 2.1:100, sample 3.1:10, sample 4.1:1, and sample 5.100:0) as mobile phase. In sample 3, 9-deoxy-31-O-demethyl-FK523 is confirmed. The above-obtained sample 3 is concentrated with a rotary evaporator and finally purified with HPLC.

[0316] The concentrate was lyophilized to obtain 9-deoxy-31-O-desmethyl-FK523 represented by Formula 9 in the form of a powder.

[0317] The prepared 9-deoxy-31-O-demethyl-FK523 was identified as follows. HPLC analysis, mass spectrometry analysis and nuclear magnetic resonance analysis were specifically performed. The analysis results of 9-deoxy-31-O-demethyl-FK523 are shown in Tables 11 and Figures 49 to 54As shown, and based on the results, it was confirmed that 9-deoxy-31-O-desmethyl-FK523 was produced by the constructed production strain Streptomyces kanamycinii ΔfkbD-fkbM,tcsB.

[0318] 9-Deoxy-31-O-demethyl-FK523 (molecular formula: C 41 H 67 NO 11 The analysis results of the above-mentioned compounds are shown in Table 11.

[0319] Table 11

[0320]

[0321]

[0322]

[0323] based on 1 H-NMR, 13 C-NMR and gHSQC data showed that the obtained compound was composed of 41 carbon atoms, which was different from 9-deoxy-FK523 in that it had one less methoxy group. The CH2 functional group (δ H 2.70 and 2.51 and δ C 36.25). According to 2D-NMR data, the structure of the compound was determined to be 9-deoxy-31-O-demethyl-FK523.

[0324] Example 10: Identification of immunosuppressive activity of nine new compounds

[0325] The extent to which the immunosuppressive activity of these nine novel compounds was reduced was determined by a known in vitro T cell activity assay (J. Immunol. 143:718-726, 1989).

[0326] CD4+T cell division is an indicator of immune response. TM When CD4+ T cells stained with Violet (CTV) proliferate due to immune response, the amount of CTV per cell decreases, and this is used as an indicator to determine the degree of immunosuppressive activity.

[0327] 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 Cell Trace TMViolet (CTV) cell proliferation kit (Molecular Probes) staining, and added FK506 or 9 new compounds at concentrations of 0.01ng / ml, 0.1ng / ml, 1ng / ml, 10ng / ml, 100ng / ml, 1000ng / ml, respectively, and then incubated for 72 hours. To activate T cells, use Mouse T activator CD3 / CD28 (Gibco). Inactivated T cells were used as a control. CTV intensity was analyzed by flow cytometry after incubation.

[0328] Table 12 below and Fig.55 The extent of T cell proliferation based on CTV intensity measured using flow cytometry is shown, indicating the extent of immunosuppressive activity of FK506 and nine new compounds. Fig.55 As shown, all novel compounds provided by the present invention showed reduced immunosuppressive activity compared to FK506.

[0329] Table 12

[0330]

[0331]

[0332] Based on the above results, it was confirmed that the nine novel compounds of the present invention had significantly lower immunosuppressive activity than FK506, and thus it was determined that the hair growth promoting composition comprising at least one compound selected from the nine novel compounds as an active ingredient can be used without considering the side effects caused by the immunosuppressive activity.

[0333] Example 11: Identification of hair growth promoting activity of nine new compounds

[0334] The hair growth promoting activity of nine novel compounds of the present invention relative to the parent drug (FK506) was evaluated using mouse whisker tissue. The mouth and mouth tissue (whisker pad) around the mouth with dense hair were peeled off from each 6 to 8 week old experimental black mouse (C57BL / 6 mouse) and treated with 70% ethanol for a short time for a few seconds. The treated mouth tissue was prepared in the following state, soaked in phosphate buffered saline (PBS) supplemented with 1% penicillin / streptomycin (P / S) on ice. The surrounding subcutaneous fat tissue and connective tissue were removed using tweezers and a blade, and the prepared mouth tissue was observed using a dissecting microscope to expose the whisker tissue. Then, the whiskers associated with the hair follicles were separated from the mouth tissue using tweezers and a blade, and the whiskers were added to PBS containing 1% P / S. Then, the well plate was filled with a vibrissa culture medium (serum-free Dulbecco's modified Eagle's medium (DMEM) containing 1% P / S and 12.5 μg / ml gentamicin), and each of the separated vibrissae was added to each well. Furthermore, they were incubated in an incubator at 37°C and 5% CO2, while the culture medium was replaced with fresh culture medium every 2 days. In order to evaluate the effects of these 9 new compounds on the growth of vibrissae, these 9 new compounds were mixed with the culture medium, and then evaluated by measuring the length, area, volume, etc. of the vibrissae relative to the reference value.

[0335] The results confirmed that these 9 new compounds can provide the same level of hair growth promoting activity as the parent drug FK506. The results of some new compounds are shown as representative examples. Fig.56 These examples are for illustrative purposes and are not intended to highlight specific compounds. In addition, the growth in antenna length measured by the novel compounds is shown in Fig.57 In. Fig.57 As shown, it was confirmed that both the new compound and FK506 can increase the length of the antennae.

[0336] In addition, histological results of vibrissae cultured under treatment with some of the novel compounds showed Fig.58 It was confirmed that the growth marker (Ki67) of the cells was overexpressed due to the novel compound 9-deoxy-31-O-desmethyl-FK520 compared to the untreated group, and thus the growth of the tentacles treated with the novel compound was confirmed to be continuously maintained. Based on this result, it was confirmed that the hair growth promoting composition including at least one of the nine novel compounds of the present invention as an active ingredient has the desired hair growth promoting effect, thereby determining that the composition can be effectively used for preventing and treating hair loss.

[0337] The above description of the present invention is provided for the purpose of illustration, and those skilled in the art will understand that various changes and modifications can be made without changing the technical concept and essential features of the present invention. Therefore, it is obvious that the above-mentioned embodiments are illustrative in all aspects and do not limit the present invention. The various embodiments disclosed herein are not intended to be limiting, and their true scope and spirit are indicated by the following claims. The present invention is limited only by the terms of the attached claims and the full scope of equivalents given by these claims.

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

Claims

1. A pharmaceutical composition for promoting hair growth, or alleviating, preventing or treating hair loss, comprising a compound selected from the group consisting of: 9-deoxy-31-O-demethyl-prolyl-FK506 (9-deoxo-31-O-demethyl-prolyl-FK506) as shown in the following formula 1, 9-deoxy-36,37-dihydro-FK506 (9-deoxo-36,37-dihydro-FK506) as shown in the following formula 2, 31-O-demethyl-36,37-dihydro-FK506 (31-O-demethyl-36,37-dihydro-FK506) as shown in the following formula 3, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 (31-O-demethyl-36,37-dihydro-FK506) as shown in the following formula 4 -dihydro-FK506), 9-deoxy-FK520 (9-deoxo-FK520) shown in the following formula 5, 31-O-demethyl-FK520 (31-O-demethyl-FK520) shown in the following formula 6, 9-deoxy-31-O-demethyl-FK520 (9-deoxo-31-O-demethyl-FK520) shown in the following formula 7, 9-deoxy-FK523 (9-deoxo-FK523) shown in the following formula 8, 9-deoxy-31-O-demethyl-FK523 (9-deoxo-31-O-demethyl-FK523) shown in the following formula 9, their isomers, or pharmaceutically acceptable salts thereof as active ingredients, wherein the compounds have reduced immunosuppressive activity compared to FK506: [Formula 1] [Formula 2] [Formula 3] [Formula 4] [Formula 5] [Formula 6] [Formula 7] [Formula 8] [Formula 9] 2. The pharmaceutical composition according to claim 1, characterized in that The 9-deoxy-31-O-demethyl-prolyl-FK506 was produced by culturing Streptomyces kanamyciniensis ΔfkbD-fkbM with accession number KCTC13581BP as a production strain.

3. The pharmaceutical composition according to claim 1, characterized in that The 9-deoxy-36,37-dihydro-FK506 was produced by culturing the kanamycin Streptomyces ΔfkbD,tcsD with accession number KCTC13580BP as a production strain.

4. The pharmaceutical composition according to claim 1, characterized in that The 31-O-demethyl-36,37-dihydro-FK506 was produced by culturing Streptomyces kanamycinii ΔfkbM,tcsD with accession number KCTC13584BP as a production strain.

5. The pharmaceutical composition according to claim 1, characterized in that The 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 was produced by culturing the kanamycin Streptomyces ΔfkbD-fkbM, tcsD with accession number KCTC13585BP as a production strain.

6. The pharmaceutical composition according to claim 1, characterized in that The 9-deoxy-FK520 is produced by culturing the kanamycin Streptomyces ΔfkbD, tcsB with the accession number KCTC13579BP or the kanamycin Streptomyces ΔfkbD, tcsD with the accession number KCTC13580BP as a production strain.

7. The pharmaceutical composition according to claim 1, characterized in that The 31-O-demethyl-FK520 is produced by culturing the kanamycin Streptomyces ΔfkbM, tcsB with the accession number KCTC13583BP or the kanamycin Streptomyces ΔfkbM, tcsD with the accession number KCTC13584BP as a production strain.

8. The pharmaceutical composition according to claim 1, characterized in that The 9-deoxy-31-O-demethyl-FK520 is produced by culturing the kanamycin Streptomyces ΔfkbD-fkbM, tcsB with the accession number KCTC13582BP or the kanamycin Streptomyces ΔfkbD-fkbM, tcsD with the accession number KCTC13585BP as a production strain.

9. The pharmaceutical composition according to claim 1, characterized in that The 9-deoxy-FK523 was produced by culturing Streptomyces kanamyciniensis ΔfkbD, tcsB with accession number KCTC13579BP as a production strain.

10. The pharmaceutical composition according to claim 1, characterized in that The 9-deoxy-31-O-demethyl-FK523 was produced by culturing the kanamycin Streptomyces ΔfkbD-fkbM, tcsB with accession number KCTC13582BP as a production strain.

11. The pharmaceutical composition according to claim 1, characterized in that The hair loss includes scarring alopecia; or at least one non-scarring alopecia selected from the group consisting of infectious alopecia, traumatic alopecia, inflammatory alopecia, congenital alopecia, endocrine alopecia, tumor alopecia, malnutrition alopecia, drug-induced alopecia, alopecia caused by abnormal hair structure, male pattern alopecia, female pattern alopecia, and alopecia areata.

12. A compound selected from the group consisting of 9-deoxy-31-O-demethyl-prolyl-FK506 as shown in the following formula 1, 9-deoxy-36,37-dihydro-FK506 as shown in the following formula 2, 9-deoxy-FK523 as shown in the following formula 8, 9-deoxy-31-O-demethyl-FK523 as shown in the following formula 9, isomers thereof, or pharmaceutically acceptable salts thereof: [Formula 1] [Formula 2] [Formula 8] [Formula 9] 13. An over-the-counter composition for promoting hair growth, comprising a compound selected from the group consisting of: 9-deoxy-31-O-demethyl-prolyl-FK506 as shown in the following formula 1, 9-deoxy-36,37-dihydro-FK506 as shown in the following formula 2, 31-O-demethyl-36,37-dihydro-FK506 as shown in the following formula 3, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 as shown in the following formula 4, 5, 31-O-demethyl-FK520 as shown in the following formula 6, 9-deoxy-31-O-demethyl-FK520 as shown in the following formula 7, 9-deoxy-FK523 as shown in the following formula 8, 9-deoxy-31-O-demethyl-FK523 as shown in the following formula 9, isomers thereof, or pharmaceutically acceptable salts thereof as active ingredients, wherein the compound has reduced immunosuppressive activity compared to FK506: [Formula 1] [Formula 2] [Formula 3] [Formula 4] [Formula 5] [Formula 6] [Formula 7] [Formula 8] [Formula 9] 14. A cosmetic composition for promoting hair growth, comprising a compound selected from the group consisting of: 9-deoxy-31-O-demethyl-prolyl-FK506 as shown in the following formula 1, 9-deoxy-36,37-dihydro-FK506 as shown in the following formula 2, 31-O-demethyl-36,37-dihydro-FK506 as shown in the following formula 3, 9-deoxy-31-O-demethyl-36,37-dihydro-FK506 as shown in the following formula 4, 5, 31-O-demethyl-FK520 as shown in the following formula 6, 9-deoxy-31-O-demethyl-FK520 as shown in the following formula 7, 9-deoxy-FK523 as shown in the following formula 8, 9-deoxy-31-O-demethyl-FK523 as shown in the following formula 9, isomers thereof, or pharmaceutically acceptable salts thereof as active ingredients, wherein the compound has reduced immunosuppressive activity compared to FK506: [Formula 1] [Formula 2] [Formula 3] [Formula 4] [Formula 5] [Formula 6] [Formula 7] [Formula 8] [Formula 9]

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