Polycyclic substituted methylene acetone compound and 5 alpha-reductase inhibition application thereof

Abstract

The invention discloses application of a compound shown in a formula I or a salt or a deuterated substance thereof in preparation of a 5 alpha-reductase inhibitor, in which ring A is a single benzene ring, a single methylenedioxy benzene ring, or a single five-membered ring containing O or S; wherein the ring A may be substituted by m Ra, and m is 0, 1, 2, 3 or 4; wherein Ra is hydroxyl, methyl, halogen, methoxyl or nitro; wherein the deuterated substance is a partial deuterated substance and / or a perdeuterated substance. In a 5AR activity inhibition experiment, the compound provided by the invention has an obvious inhibition effect: under the concentration of 250 mu M, the 5AR activity inhibition rate (the final administration concentration of 250 mu M) of the aromatic methyleneacetone active small molecule compound protected by the invention is 9-56%, and part of the compound approaches or exceeds the inhibition rate (53%) of finasteride of radix scrophulariae. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a polycyclic substituted methyleneacetone compound and a 5α-reductase inhibitory application thereof. Background Art

[0002] 5α-Reductase (5AR) is a key enzyme in the metabolism of the androgen testosterone (T). Under the condition of electrons provided by reduced coenzyme II (NADPH), it can reduce the unsaturated double bonds at positions 4 and 5 on testosterone and convert it into dihydrotestosterone (DHT). 5AR in the human body is mainly found in the sebaceous glands, liver, prostate, seminal vesicles, epididymis, hair follicles and other organs including most areas of the skin on the head. Testosterone enters the cells of the target organs and is irreversibly reduced to DHT through the catalysis of testosterone 5AR. This DHT binds to the receptor and acts as an androgen. As a more potent androgen than testosterone, DHT has 5 times the ability to bind to the androgen receptor (AR) as testosterone. It plays an important role in multiple organs and tissues such as the reproductive system, skin and hair. However, excessive levels of DHT can easily cause androgen-dependent diseases, such as androgenic alopecia, seborrheic dermatitis, acne, hirsutism, male precocious puberty, benign prostatic hyperplasia and prostate tumors.

[0003] As a common disease, hair loss is gradually becoming younger. Hair loss often brings great psychological pressure to patients, and has a great impact on personal appearance, self-confidence, mental health, social interaction and quality of life. Androgenic alopecia (AGA), also known as seborrheic alopecia, is one of the most common types of hair loss in clinical practice. Men often show a receding hairline in the front in an "M" shape, or hair loss on the top of the head in an "O" shape, or both in a "U" shape. Women often show sparse, fine and soft hair, and widening of the hair parting. The main pathological characteristics are greasy scalp, abnormal levels of dihydrotestosterone (DHT) in the blood, miniaturization of hair follicles, shortened growth phase and prolonged resting phase. Clinically, the main clinical features are the replacement of terminal hairs by villi (villi are defined as diameter <30 μm, length <30 μm; transitional hair width is between 30 and 40 μm; terminal hairs >40 μm) and a decrease in total hair density (hair / cm2). Histologically, it can also be seen that terminal hair follicles are replaced by villus follicles, with perifollicular infiltration of macrophages, increased size of sebaceous glands and thinning of the dermis. The cyclical growth of hair follicles is divided into four periods: the growth phase (anagen), in which the hair matrix keratinocytes located around the dermal papilla cells (DPCs) proliferate and the hair shaft grows; the regression phase (catagen), in which the hair follicles undergo strictly controlled cell apoptosis and extracellular matrix remodeling; the resting phase (telogen), in which the hair shaft is attached to the bulbous base of the hair follicle epithelium and can fall off due to grooming; and the active shedding phase (exogen), which is the shedding stage of the resting hair. 5AR plays an important role in the pathological process of seborrheic alopecia by converting testosterone in hair follicles into DHT. After testosterone is catalyzed and converted into more active DHT by 5AR expressed in DPCs, DHT binds to androgen receptors (AR) on hair matrix cells to form a DHT-AR complex that enters the cell nucleus and acts as a transcription factor to induce the hair follicle papilla cells to secrete a variety of cytokines, such as transforming growth factor-β (TGF-β), interleukin-1α (1L-1α) and tumor necrosis factor-α (TNF-α), which can induce the premature termination of the growth phase of hair follicles and lead to AGA. Androgenic alopecia is the result of abnormal sensitivity of scalp hair follicles to circulating androgens. Under the induction of androgen DHT, androgen receptor expression increases, thereby changing the interaction between mesenchymal and epithelial cells in hair follicles, affecting hair growth, dermal size, dermal cells, and the activity of keratinocytes and melanocytes.So far, there are only two FDA-approved drugs for the treatment of androgenic alopecia, namely the oral drug finasteride and the topical application minoxidil. Finasteride is a 5AR inhibitor that treats hair loss by reducing the DHT content in the body. Minoxidil is a vasodilator that improves the microcirculation of hair follicles to achieve the effect of treating hair loss. Other treatments include laser therapy, hair transplantation, stem cell therapy and other non-drug treatments. However, these treatments have significant side effects, such as sexual dysfunction, hirsutism, and high costs.

[0004] In summary, inhibiting 5AR is one of the important means to treat androgenic alopecia. Currently, common 5AR inhibitors include finasteride and dutasteride, which have achieved significant results. However, these synthetic inhibitors often cause some side effects, such as erectile dysfunction, sexual dysfunction, male breast development, impaired muscle growth, etc., and are relatively expensive.

[0005] Therefore, it is an urgent need to find and develop new 5AR inhibitors that are green, safe, abundant in sources, efficient and inexpensive. The polycyclic substituted methyleneacetone compounds of the present invention and their application in inhibiting 5α-reductase have not been found in the prior art. Summary of the invention

[0006] Based on this, the present invention provides a use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a 5α-reductase inhibitor,

[0007]

[0008] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0009] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0010] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0011] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0012] Further, the compound of formula I is

[0013]

[0014] Further, the compound of formula II is

[0015]

[0016] wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, hydroxy, halogen, methyl, trifluoromethyl, methoxy and nitro.

[0017] Further, R1 is hydrogen, halogen or methyl, R2 is hydrogen, hydroxyl, methyl or methoxy, R3 is hydrogen, hydroxyl, halogen, trifluoromethyl, methoxy or nitro, R4 is hydrogen, hydroxyl or methoxy, and R5 is hydrogen, hydroxyl, methoxy, halogen or methyl.

[0018] Further, the compound of formula I is any one of the following compounds:

[0019]

[0020]

[0021] Furthermore, the compound of formula I is a compound having 5α-reductase inhibitory activity for reducing the production of dihydrotestosterone.

[0022] Furthermore, the compound of formula I is synthesized or derived from an extract, a crude extract and / or an enriched fraction of a natural raw material.

[0023] Furthermore, the natural raw materials include plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products of any of the above.

[0024] According to another aspect of the present invention, there is provided a use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a medicament for androgenic alopecia associated with 5α-reductase inhibition,

[0025]

[0026] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0027] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0028] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0029] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0030] According to another aspect of the present invention, there is provided a use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a skin care product, a medicine and / or an extract for oil control and / or prevention and treatment of acne:

[0031]

[0032] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0033] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0034] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0035] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0036] According to another aspect of the present invention, there is provided a 5α-reductase inhibitory composition, the 5α-reductase inhibitory composition comprising a compound of formula I or a salt or deuterated substance thereof;

[0037]

[0038] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0039] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0040] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0041] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0042] Furthermore, the compound of formula I is a compound having 5α-reductase inhibitory activity for reducing the production of dihydrotestosterone.

[0043] Furthermore, the compound of formula I is a compound having 5α-reductase inhibitory activity for reducing dihydrotestosterone production in vitro.

[0044] Furthermore, the compound of formula I is synthesized or derived from an extract, a crude extract and / or an enriched fraction of a natural raw material.

[0045] Furthermore, the natural raw materials include plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products of any of the above.

[0046] According to another aspect of the present invention, there is provided a use of the above-mentioned 5α-reductase inhibitory composition in the preparation of a 5α-reductase inhibitor, wherein the 5α-reductase inhibitory composition is a skin composition, a pharmaceutical composition, a health product, a functional food, a food additive and / or a hair care product.

[0047] According to another aspect of the present invention, there is provided a use of the above 5α-reductase inhibitory composition in the preparation of a medicament for androgenic alopecia associated with 5α-reductase inhibition.

[0048] According to another aspect of the present invention, there is provided a use of the above-mentioned 5α-reductase inhibitory composition in the preparation of skin care products, medicines and / or extracts for oil control and / or prevention and treatment of acne.

[0049] Furthermore, the skin composition further comprises one or more skin care substances for preventing and / or improving androgen-related skin abnormalities, such as fructose acid, salicylic acid, azelaic acid and derivatives thereof, enzymes and colloidal sulfur.

[0050] Furthermore, the skin composition is in the form of an emulsion, an essence, a toner, a face cream or a face mask.

[0051] Furthermore, the skin composition further comprises one or more auxiliary materials selected from the group consisting of a thickener, a pH regulator, a preservative, an antibacterial agent and a stabilizer.

[0052] Furthermore, the thickener is selected from one or more of the following: carbomer, sodium polyacrylate, xanthan gum and hydroxyethyl cellulose.

[0053] Further, the pH adjuster is selected from one or more of the following: triethanolamine, aminomethyl propanol, citric acid and lactic acid.

[0054] Further, the preservative is selected from one or more of the following: methylparaben, propylparaben, butylparaben, p-hydroxyacetophenone and phenoxyethanol.

[0055] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0056] Furthermore, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant.

[0057] Furthermore, the dosage form of the pharmaceutical composition is a patch, emulsion, tablet, pill, capsule, powder, film, lozenge, granule or oral solution.

[0058] Furthermore, the pharmaceutical composition further comprises one or more drugs and / or extracts for preventing and / or treating androgen-dependent diseases.

[0059] Furthermore, the androgen-dependent disease is androgenic alopecia.

[0060] Furthermore, the pharmaceutical composition further comprises one or more skin care products, drugs and / or extracts for oil control and / or prevention and treatment of acne.

[0061] Furthermore, the hair care product is shampoo.

[0062] Beneficial effects of the present invention:

[0063] In the 5AR activity inhibition experiment, the compounds of the present invention have obvious inhibitory effects: at a concentration of 250 μM, the 5AR activity inhibition rate of the aromatic methyleneacetone active small molecule compounds protected by the present invention (final administration concentration 250 μM) is between 9% and 56%, and some compounds approach or exceed the inhibition rate of finasteride (53%). BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0065] Figure 1 This is a schematic diagram of the H NMR spectrum of ZK-1 (Osmundacetone).

[0066] Figure 2 The results of the inhibitory activity (inhibition rate %) of aromatic methylene acetone compounds 5AR are shown in Figure 1. ***P<0.001vs control (only adding the same amount of the same solvent as the sample to be tested to the 5AR activity test system), FNR: Finasteride, 20μM. DETAILED DESCRIPTION

[0067] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0068] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0069] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0070] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0071] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0072] As described in the background technology section, it is an urgent need to find and develop new 5AR inhibitors that are green, safe, abundant in sources, efficient and inexpensive, and the prior art has not found a polycyclic substituted methyleneacetone compound of the present invention and its 5α-reductase inhibitory application. In order to solve the above problems, the present invention provides a compound of the following formula I or its salt or deuterated product for use in the preparation of a 5α-reductase inhibitor,

[0073]

[0074] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0075] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0076] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0077] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0078] The 5AR activity inhibiting molecule of the present invention is a polycyclic substituted methyleneacetone type active small molecule compound, part of which is derived from natural plants and has good safety.

[0079] The polycyclic substituted methyleneacetone active small molecule compound of the present invention has a relatively simple structure, and is relatively easy to chemically synthesize or extract and separate. In particular, chemical synthesis can obtain a target product with a high yield by reacting cheap raw materials, and the production cost is relatively low, which is suitable for large-scale production. Industrial synthesis of dehydrozingerone (i.e., compound ZK-2) is often prepared by condensing with acetone in a sodium hydroxide solution using vanillin, a bulk raw material, and the yield of the product is as high as 85%-91%. Protocatechuic aldehyde (3,4-dihydroxybenzaldehyde) reacts with acetone under alkaline conditions to react with aldol condensation to synthesize the target product osmundacetone (i.e., compound ZK-1), and the yield can be as high as 89%.

[0080] The polycyclic substituted methyleneacetone active small molecule compound of the present invention has relatively good stability and is suitable for being widely used in various fields such as medical drugs, foods, food additives, health products, skin care products, cosmetics, etc.

[0081] In a preferred embodiment, the compound of formula I is

[0082]

[0083] In a preferred embodiment, the compound of formula II is

[0084]

[0085] wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, hydroxy, halogen, methyl, trifluoromethyl, methoxy and nitro.

[0086] In a preferred embodiment, R1 is hydrogen, halogen or methyl, R2 is hydrogen, hydroxyl, methyl or methoxy, R3 is hydrogen, hydroxyl, halogen, trifluoromethyl, methoxy or nitro, R4 is hydrogen, hydroxyl or methoxy, and R5 is hydrogen, hydroxyl, methoxy, halogen or methyl.

[0087] In a preferred embodiment, the compound of formula I is any one of the following compounds:

[0088]

[0089]

[0090] In a preferred embodiment, the compound of formula I is a compound having 5α-reductase inhibitory activity for reducing the production of dihydrotestosterone.

[0091] In a preferred embodiment, the compound of formula I is synthesized or is derived from an extract, a crude extract and / or an enriched fraction of a natural raw material.

[0092] In a preferred embodiment, the natural raw material includes plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products of any of the above.

[0093] According to another aspect of the present invention, there is provided a use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a medicament for androgenic alopecia associated with 5α-reductase inhibition,

[0094]

[0095] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0096] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0097] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0098] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0099] According to another aspect of the present invention, there is provided a use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a skin care product, a medicine and / or an extract for oil control and / or prevention and treatment of acne:

[0100]

[0101] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0102] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0103] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0104] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0105] According to another aspect of the present invention, there is provided a 5α-reductase inhibitory composition, the 5α-reductase inhibitory composition comprising a compound of formula I or a salt or deuterated substance thereof;

[0106]

[0107] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0108] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0109] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0110] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0111] In a preferred embodiment, the compound of formula I is a compound having 5α-reductase inhibitory activity for reducing the production of dihydrotestosterone.

[0112] In a preferred embodiment, the compound of formula I is a compound having 5α-reductase inhibitory activity for reducing dihydrotestosterone production in vitro.

[0113] In a preferred embodiment, the compound of formula I is synthesized or is derived from an extract, a crude extract and / or an enriched fraction of a natural raw material.

[0114] In a preferred embodiment, the natural raw material includes plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products of any of the above.

[0115] According to another aspect of the present invention, there is provided a use of the above-mentioned 5α-reductase inhibitory composition in the preparation of a 5α-reductase inhibitor, wherein the 5α-reductase inhibitory composition is a skin composition, a pharmaceutical composition, a health product, a functional food, a food additive and / or a hair care product.

[0116] According to another aspect of the present invention, there is provided a use of the above 5α-reductase inhibitory composition in the preparation of a medicament for androgenic alopecia associated with 5α-reductase inhibition.

[0117] According to another aspect of the present invention, there is provided a use of the above-mentioned 5α-reductase inhibitory composition in the preparation of skin care products, medicines and / or extracts for oil control and / or prevention and treatment of acne.

[0118] In a preferred embodiment, the skin composition further comprises one or more skin care substances for preventing and / or improving androgen-related skin abnormalities, such as fruit acid, salicylic acid, azelaic acid and derivatives thereof, enzymes and colloidal sulfur.

[0119] In a preferred embodiment, the skin composition is in the form of an emulsion, an essence, a toner, a cream or a mask.

[0120] In a preferred embodiment, the skin composition further comprises one or more auxiliary materials selected from the group consisting of a thickener, a pH adjuster, a preservative, an antibacterial agent and a stabilizer.

[0121] In a preferred embodiment, the thickener is selected from one or more of the following: carbomer, sodium polyacrylate, xanthan gum and hydroxyethyl cellulose.

[0122] In a preferred embodiment, the pH adjuster is selected from one or more of the following: triethanolamine, aminomethyl propanol, citric acid and lactic acid.

[0123] In a preferred embodiment, the preservative is selected from one or more of the following: methylparaben, propylparaben, butylparaben, p-hydroxyacetophenone and phenoxyethanol.

[0124] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0125] In a preferred embodiment, the auxiliary material is selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, glidants, lubricants, dispersants, plasticizers, opacifiers and antioxidants.

[0126] In a preferred embodiment, the pharmaceutical composition is in the form of a patch, emulsion, tablet, pill, capsule, powder, film, lozenge, granule or oral solution.

[0127] In a preferred embodiment, the pharmaceutical composition further comprises one or more drugs and / or extracts for preventing and / or treating androgen-dependent diseases.

[0128] In a preferred embodiment, the androgen-dependent disease is androgenic alopecia.

[0129] In a preferred embodiment, the pharmaceutical composition further comprises one or more skin care products, drugs and / or extracts for oil control and / or prevention and treatment of acne.

[0130] In a preferred embodiment, the hair care product is shampoo.

[0131] In a preferred embodiment, the use is for inhibiting 5α-reductase in vitro.

[0132] In a preferred embodiment, the pharmaceutical concentration of the compound of the following formula I is 250 μM.

[0133] According to another aspect of the present invention, there is provided a use of a compound of the following formula I or a salt or deuterated form thereof for inhibiting 5α-reductase in vitro:

[0134]

[0135] Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S;

[0136] wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4;

[0137] Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro;

[0138] The deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

[0139] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0140] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0141] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0142] Example 1 5AR Inhibitory Activity Test Experimental Method

[0143] 1. Experimental Principle

[0144] 5AR (5α-reductase) is a membrane protease that depends on reduced coenzyme II (NADPH). It is an important androgen metabolic enzyme in the skin and can irreversibly convert testosterone (T) into dihydrotestosterone (DHT).

[0145] An in vitro evaluation system for screening 5AR inhibitors was established. 5α-reductase prepared from SD rat liver (the liver is rich in 5AR) was used, and finasteride (FNR) was used as a positive control. The change in T content in the system before and after the reaction was determined by high performance liquid chromatography, and the inhibition rate of 5AR activity was calculated.

[0146] 2. Main materials, reagents and instruments

[0147] Testosterone (T), NADPH, Beijing Solaibao Technology Co., Ltd.; Tris, Sigma-Aldrich, Co.; 5α-reductase extract (5α-reductase prepared from male SD rat liver extract), Wuhan Hongren Biopharmaceutical Co., Ltd.; Finasteride (>98%), Hunan Yuxin Pharmaceutical Co., Ltd.; NADPH Beijing Bailingwei Technology Development Co., Ltd.; deep-well plate (1.0mL 96 round-hole U-shaped bottom), sealing film, 1mL syringe (with needle), 75% ethanol (AR grade), methanol (AR, Adamas), acetonitrile (AR, Adamas), hydrochloric acid (AR grade), Shanghai Titan Technology Co., Ltd.; phosphoric acid (HPLC grade), Thermo Fisher Scientific (China) Co., Ltd.; PTFE syringe filter (0.22μm, 1.3mm), CNWAthena C18-WP chromatographic column, Shanghai Anpu Experimental Technology Co., Ltd.; HPLC bottle (1.5mL, including cap mat), Kexi Biological (Ningbo) Co., Ltd.; ultrapure water, homemade in the laboratory (Think Lab brand Labonova Ultra Pro ultrapure water system). The aromatic methyleneacetone compound ZK-1 to be tested was homemade (identified as osmundacetone by hydrogen spectrum and mass spectrometry, purity>95%, hydrogen spectrum as Figure 1 ZK-2, ZK-4, ZK-17, ZK-19, ZK-20, ZK-21, ZK-22, ZK-23, and ZK-25 were purchased from Anhui Zesheng Technology Co., Ltd. (Annaiji brand) with a purity of >95%. ZK-3, ZK-5, ZK-6, ZK-7, ZK-8, ZK-9, ZK-10, ZK-11, ZK-12, ZK-13, ZK-14, ZK-15, ZK-16, ZK-24, and ZK-26 were purchased from Nanchang Chennuo Biotechnology Co., Ltd. with a purity of >95%.

[0148]

[0149]

[0150] Microplate constant temperature oscillator, Hangzhou Aosheng Instrument Co., Ltd.; XSR205DU / A analytical balance (one hundred thousandth), Mettler-Toledo Instrument Co., Ltd.; single-channel micropipette 100-1000μL, Research plus / 8-channel micropipette 30-300μL, Research plus / 8-channel micropipette 0.5-10μL, EppendorfAG; LT53 low-speed centrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; Vanquish F ultra-high performance liquid chromatograph (VF-P10-A pump, vacuum degassing pump, column oven, autosampler, DAD-3000 detector, Chromeleon liquid chromatography data workstation), Thermo Fisher Scientific Inc.

[0151] 3. Experimental methods

[0152] 3.1. Sample solution preparation

[0153] 1) Tris-HCl Buffer: Dissolve 1 packet of Tris powder in 800 mL of ultrapure water, adjust the pH to 7.0 with hydrochloric acid, and make up to 1000 mL;

[0154] 2) T solution: Weigh an appropriate amount and dissolve it in 75% ethanol to 5mM. Add 5μL to 1000μl system, the final concentration is 25μM;

[0155] 3) NADPH solution: Weigh an appropriate amount and dissolve it in prepared Tris-HCl buffer solution to 10mM. Take 25μL to 1000μL system, the final concentration is 250μM;

[0156] 4) Finasteride: Weigh an appropriate amount and dissolve it in DMSO to 667 μM. Take 30 μL to 1000 μL system, the final concentration is 20 μM;

[0157] 5) Sample to be tested: Weigh an appropriate amount of the sample to be tested and dissolve it in DMSO to make a 100mM solution, then take an appropriate amount and dilute it 12 times with 75% ethanol as the sample solution to be tested. Take 30μL to 1000μL system, the final concentration is 250μM;

[0158] 6) Negative control: Take an appropriate amount of DMSO and add 75% ethanol to dilute it 12 times as the negative control solution.

[0159] 3.2 Reaction system and sample testing

[0160] [1] According to the volumes in Table 1, add Tris-HCl, rat liver 5AR extract, T, test sample / finasteride / 75% ethanol and NADPH in a 1.0 mL-96 round well U-shaped deep well plate in sequence to a final volume of 1000 μL. Mix thoroughly with a 300 μL micropipette.

[0161] [2] Immediately take two 300μL portions of the mixture and transfer them to two new 1.0mL-96 round-hole U-shaped deep-well plates. Immediately add 600μL of methanol to one of the plates to terminate the reaction. Mix thoroughly and centrifuge at 2000rpm / min for 5min, marking it as "0min". Seal the other plate with a sealing film and place it on a microplate constant temperature oscillator, set to 200rpm / min, incubate at 37℃ for 45min, marking it as "45min".

[0162] [3] After centrifugation of the 96-well plate at 0 min, filter with a 0.22 μm needle filter and take 0.5 mL of the filtrate into an HPLC injection bottle to prepare the 0 min HPLC test sample;

[0163] [4] After 45 min, take out another 96-well plate from the microplate thermostat and perform the same operations as steps 2 and 3 to prepare HPLC test samples for 45 min;

[0164] [5] The T content of each sample at 0 min and 45 min was detected by high performance liquid chromatography.

[0165] Table 1 Reaction system solution composition

[0166] composition Positive Control Samples to be tested Negative control Tris-HCl 840μL 840μL 840μL 5AR extract 100μL 100μL 100μL T(final 25μM) 5μL 5μL 5μL Sample to be tested (final concentration 250 μM) - 30μL - Finasteride (final 20 μM) 30μL - - Negative control solution - - 30μL NADPH (final 250 μM) 25μL 25μL 25μL Total volume 1000μL 1000μL 1000μL

[0167] Note: Final 25μM, Final 250μM, Final 20μM means that after the reagent is added to the reaction system, the final concentration of the reagent participating in the biochemical reaction in the reaction system is 25μM, 250μM, and 20μM; "-" means no addition

[0168] 3.3 T content detection method

[0169] Chromatographic column: CNW Athena C18-WP, 2.1×50mm, 5μm; column temperature: 30℃; flow rate: 0.6mL / min; detection wavelength: 243nm; injection volume: 10μL; mobile phase: 5%-95% acetonitrile / 0.1% formic acid water gradient elution for 20min; T content was determined by external standard one-point method.

[0170] 3.4 Data Analysis

[0171] The calculation formula of T conversion rate and 5AR activity inhibition rate is as follows:

[0172] 5α-reductase activity inhibition rate % = (reaction system without inhibitor T 45 Conversion amount-reaction system with inhibitor T 45 Conversion amount) / reaction system without inhibitor T 45 Conversion amount × 100%

[0173] Where:

[0174] The T0 concentration measurement value is the T concentration measurement value (μM) measured after the reaction of each sample was terminated at 0 min;

[0175] T 45 The concentration measurement value is the concentration measurement value (μM) of T measured after the reaction of each sample was terminated at 45 min;

[0176] T 45 The conversion amount was the concentration value of T measured at 45 minus the concentration value of T measured after the reaction was terminated at 0 min (μM) for each sample.

[0177] The significance T-test T-test is calculated using the T.TEST function of Microsoft Office365 excel, and the specific parameter selection is T.TEST(data-1, data-2, tails=2, type=2), that is, T.TEST(array-1, array-2, tails=2, type=2).

[0178] 4. 5AR inhibition rate test results:

[0179] The experimental results are as follows Figure 1 As shown in Table 2. In the 5AR activity inhibition experiment, the inhibitory effect is obvious: at a concentration of 250 μM, the 5AR activity inhibition rate of the aromatic methylene acetone active small molecule compound protected by the present invention (final concentration of administration 250 μM) is between 9% and 56%, and some compounds are close to or exceed the inhibition rate of Yangshen finasteride (53%). Specifically, compounds ZK-1 to ZK-14, ZK-16 to ZK-21, and ZK-23 to ZK-26 all show obvious 5AR inhibitory activity at 250 μM, and the same type of aromatic methylene acetone compounds ZK-15 and ZK-22 have no 5AR inhibitory activity.

[0180] Table 2 AR inhibitory activity (inhibition rate %) and significance T test of compound 5 (compared with negative control)

[0181]

[0182]

[0183] Example 2 Compound anti-hair loss effect test:

[0184] The MTT method was used to determine the activity of human dermal papilla cells (HDPCs, purchased from Boxi Biotechnology (Suzhou) Co., Ltd.) after dihydrotestosterone stimulation:

[0185] Digest the cells in logarithmic growth phase, count them with a hemocytometer, inoculate 1×104 cells per well in a 96-well cell culture plate, and discard the supernatant after 24 hours of culture. Blank control group: a group with only culture medium added, no test compound, and no dihydrotestosterone (DHT) added; negative control group: DHT was added to the cell culture medium as a negative control group, so that the DHT concentration was 180 μM, and no test compound was added to this group; experimental group: DHT was added to the cell culture medium so that the final DHT concentration was 180 μM, and then the test sample solution (dissolved in DMSO) was added to the cell culture medium of each well as shown in Table 1, so that the final concentration of the test sample in the cell culture medium was 250 μM, and after 24 hours of culture, the supernatant was discarded and washed with PBS, and 100 μL of freshly prepared MTT solution of 0.5 mg / mL (dissolved in DMEM culture medium) was added to each well. After incubation for 4 hours, the supernatant was discarded, 100 μL DMSO was added to each well to dissolve the crystals, and the cells were shaken at 37°C for 5 minutes. The absorbance was read at 490 nm on an ELISA reader. No cells were added, and only the test solvent was added as the solvent group. The cell proliferation calculation formula was:

[0186] Cell proliferation rate (%) = (A1-A0) / (A2-A0) × 100%

[0187] Wherein: A0 represents the absorbance value of the solvent group; A1 represents the absorbance value of each sample group (including the negative control group and each experimental group); A2 represents the absorbance value of the blank control group.

[0188] MTT test results: The absorbance value of the cell viability determination of the blank control group was recorded as 1, the average cell viability of the negative control group (180 μM DHT) was 0.5, and the average cell viability of each experimental group to which each sample to be tested (ZK1-ZK14, ZK16-ZK21, ZK23-ZK26) was added was between 0.7 and 0.9.

[0189] Compared with the blank control group, the negative control group (180 μM dihydrotestosterone) significantly induced apoptosis of dermal papilla cells and inhibited the proliferation of dermal papilla cells. When DHT and the test samples (ZK1-ZK14, ZK16-ZK21, ZK23-ZK26) were added to the experimental group at the same time, the apoptosis of dermal papilla cells by DHT was significantly alleviated, and the cell viability was improved (the average cell viability range was between 0.7-0.9, while the average cell viability of the negative control group (180 μM DHT) was 0.5), that is, each test compound reduced the apoptotic effect of DHT on dermal papilla cells, and the results were statistically significant by T test. The above results indicate that this class of compounds protected by the present invention (ZK1-ZK14, ZK16-ZK21, ZK23-ZK26) can significantly enhance the viability of dermal papilla cells damaged by DHT, and have the effect of preventing hair loss and hair growth. This result is consistent with the 5AR inhibitory activity results of this class of compounds. All samples tested showed no obvious cytotoxicity in the experiment.

[0190] Example 3 Experiment on the inhibition of sebaceous gland oil secretion by compounds

[0191] 3.1 Experimental Principle

[0192] The human sebaceous gland cell line SZ95 was selected for the construction of an in vitro skin oil secretion and acne model. The oil secretion of the cells simulated the sebum secretion of human skin. Nile red dye was used to perform fluorescent staining on neutral lipids. The oil content of sebaceous gland cells was quantitatively detected by the fluorescent quantitative method, thereby evaluating the oil control and acne-removing effects of the test samples.

[0193] 3.2 Experimental Materials:

[0194] Cell line: human sebaceous gland cells (SZ95); culture medium: sodium pyruvate-free DMEM medium containing 10% FBS; maintenance medium: sodium pyruvate-free DMEM medium containing 1% FBS; culture conditions: cultured at 37°C, 5% CO2, and saturated humidity; Nile red, FDA (fluorescein diacetate), linoleic acid, Titan Technology.

[0195] 3.3 Sample preparation:

[0196] 1) Test sample: Weigh an appropriate amount of sample, add DMSO to prepare a stock solution, and then dilute it into a working solution using DMEM (without sodium pyruvate) medium containing 1% FBS.

[0197] 2) Positive control: Isotretinoin

[0198] Weigh isotretinoin powder, add DMSO to prepare a stock solution, and then dilute it into a working solution using DMEM (without sodium pyruvate) medium containing 1% FBS.

[0199] 3) Negative control: DMEM containing 1% FBS (without sodium pyruvate)

[0200] Pipette 5 mL of FBS, add 45 mL of DMEM (without sodium pyruvate) to prepare DMEM (without sodium pyruvate) containing 10% FBS, filter and set aside, then pipette 5 mL of DMEM (without sodium pyruvate) containing 10% FBS, add 45 mL of DMEM (without sodium pyruvate) to prepare DMEM (without sodium pyruvate) containing 1% FBS, filter and set aside.

[0201] 3.4 Test steps:

[0202] Neutral lipid fluorescence quantification experiment

[0203] 1) The cell suspension was inoculated into a 96-well cell culture plate and cultured for 18-24 hours until the cells reached 80% confluence.

[0204] 2) Linoleic acid, linoleic acid + each sample to be tested (ZK1-ZK4, ZK16-ZK21, ZK23-ZK26) and positive control (linoleic acid + isotretinoin) were added respectively, and the supernatant was discarded after incubation for 48 hours. After washing twice with PBS, Nile red dye was added to the experimental group and FDA was added to the control group. The cells were incubated for 5 minutes, and the released fluorescence was detected on a multifunctional microplate reader. Excitation wavelengths of 485nm and 494nm and absorption wavelengths of 565nm and 523nm were used to detect the fluorescence intensity of Nile red and FDA, respectively.

[0205] 3) The results were determined by the ratio of Nile red and FDA (OD ratio): intracellular neutral lipid percentage (%) = experimental group OD ratio / control group OD ratio × 100%. Statistical analysis was performed using T test. P < 0.05 was considered statistically significant.

[0206] 3.5 Experimental Results

[0207] Compared with the negative control group, the content of neutral lipids in the model control group was significantly increased (P<0.05); compared with the model control group, the content of neutral lipids in the positive control group was significantly decreased (P<0.05), indicating that the model was successfully established. Compared with the model control group (only linoleic acid), the content of neutral lipids in the test sample group (linoleic acid and ZK1-ZK14, ZK16-ZK21, ZK23-ZK26) decreased by 8%-40%, and the percentage of neutral lipids (%) in the test sample group and the model control group was statistically different (P<0.05). This indicates that it has the effect of significantly inhibiting the secretion of oil by sebaceous gland cells, thereby having the effect of controlling oil and removing acne.

[0208] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a 5α-reductase inhibitor, characterized in that: Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S; wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4; Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro; Wherein, the deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

2. The use according to claim 1, characterized in that The compound of formula I is Preferably, the compound of formula II is wherein R1, R2, R3, R4 and R5 are each independently selected from the group consisting of hydrogen, hydroxyl, halogen, methyl, trifluoromethyl, methoxy and nitro; Preferably, R1 is hydrogen, halogen or methyl, R2 is hydrogen, hydroxy, methyl or methoxy, R3 is hydrogen, hydroxy, halogen, methoxy, trifluoromethyl or nitro, R4 is hydrogen, hydroxy or methoxy, and R5 is hydrogen, methoxy, hydroxy, halogen or methyl; Preferably, the compound of formula I is any one of the following compounds: Preferably, the compound of formula I is a compound having 5α-reductase inhibitory activity that reduces dihydrotestosterone production; Preferably, the compound of formula I is synthesized or derived from an extract, crude extract and / or enriched fraction of a natural raw material; Preferably, the natural raw materials include plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products of any of the above.

3. Use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a medicament for androgenic alopecia associated with 5α-reductase inhibition, characterized in that: Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S; wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4; Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro; Wherein, the deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

4. Use of a compound of the following formula I or a salt or deuterated product thereof in the preparation of a skin care product, a medicine and / or an extract for oil control and / or prevention and treatment of acne, characterized in that: Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S; wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4; Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro; Wherein, the deuterated substance is a partially deuterated substance and / or a fully deuterated substance.

5. A 5α-reductase inhibitory composition, characterized in that: The 5α-reductase inhibitory composition comprises a compound of formula I or a salt or deuterated substance thereof; Wherein, ring A is a single benzene ring, a single methylenedioxybenzene ring, or a single five-membered ring containing O or S; wherein the ring A may be substituted by m Ra, where m is 0, 1, 2, 3 or 4; Wherein, Ra is hydroxy, methyl, halogen, trifluoromethyl, methoxy or nitro; Wherein, the deuterated substance is a partially deuterated substance and / or a fully deuterated substance; Preferably, the compound of formula I is a compound having 5α-reductase inhibitory activity that reduces dihydrotestosterone production; Preferably, the compound of formula I is synthesized or derived from an extract, crude extract and / or enriched fraction of a natural raw material; More preferably, the natural raw materials include plants, animals, insects, microorganisms, marine organisms and / or fungi, or metabolites or fermentation products of any of the above.

6. Use of the 5α-reductase inhibitor composition according to claim 5 in the preparation of a 5α-reductase inhibitor, characterized in that: The 5α-reductase inhibitory composition is a skin composition, a pharmaceutical composition, a health product, a functional food, a food additive and / or a hair care product.

7. Use of the 5α-reductase inhibitory composition according to claim 5 in the preparation of a medicament for androgenic alopecia associated with 5α-reductase inhibition.

8. Use of the 5α-reductase inhibitory composition according to claim 5 in the preparation of skin care products, medicines and / or extracts for oil control and / or prevention and treatment of acne.

9. The use according to claim 5, characterized in that The skin composition further comprises one or more skin care substances for preventing and / or improving androgen-related skin abnormalities, such as fruit acid, salicylic acid, azelaic acid and derivatives thereof, enzymes and colloidal sulfur; Preferably, the skin composition is in the form of a lotion, essence, toner, cream or mask; More preferably, the skin composition further comprises one or more excipients selected from the group consisting of a thickener, a pH adjuster, a preservative, an antibacterial agent, and a stabilizer; More preferably, the thickener is selected from one or more of the following: carbomer, sodium polyacrylate, xanthan gum and hydroxyethyl cellulose; More preferably, the pH adjuster is selected from one or more of the following: triethanolamine, aminomethyl propanol, citric acid and lactic acid; More preferably, the preservative is selected from one or more of the following: methylparaben, propylparaben, butylparaben, p-hydroxyacetophenone and phenoxyethanol; Also preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the auxiliary material is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, glidant, lubricant, dispersant, plasticizer, opacifier and antioxidant; Also preferably, the dosage form of the pharmaceutical composition is a patch, emulsion, tablet, pill, capsule, powder, film, lozenge, granule or oral solution.

10. The use according to claim 5, characterized in that The pharmaceutical composition further comprises one or more drugs and / or extracts for preventing and / or treating androgen-dependent diseases; Preferably, the androgen-dependent disease is androgenic alopecia; More preferably, the pharmaceutical composition further comprises one or more skin care products, drugs and / or extracts for oil control and / or prevention and treatment of acne; Also preferably, the hair care product is shampoo.

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