Diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivatives and preparation method and application thereof

Abstract

The present application provides a diaminopyrimidine oxide and low molecular cationic hyaluronic acid zinc coupled derivative and its preparation method and application. The structure of the coupled derivative is shown in Formula I: #imgabs0# wherein R1, R2, and R3 are each independently selected from hydroxypropyltrimethylammonium chloride and hydrogen, and R1, R2, and R3 are not hydrogen at the same time; R4 is pyrrolidinyl or hydrogen; m≥1, n≥0. The conjugate has good water solubility and sustained-release transdermal effect, and has excellent anti-hair loss and hair growth functions, which solves the defects of poor solubility and permeability of diaminopyrimidine oxide hair growth agents in the prior art, and strong irritation caused by high-dose and high-frequency use.

Description

Technical Field

[0001] The present application relates to the fields of medicine and cosmetics, and specifically to a diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative, and a preparation method and application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] In recent years, the incidence of hair loss has been on the rise, and the trend of younger people is becoming more and more obvious. Hair loss can occur at any age. Currently, the effective ingredients available for hair loss are extremely limited, mainly Chinese herbal ingredients and chemical hair growth agents. Although there are many Chinese herbal ingredients that have anti-hair loss effects, such as angelica, ginseng, ginkgo leaves, eucommia, etc., Chinese herbal hair growth products produced with plant extracts as raw materials have poor stability of extract ingredients, uncertain effective substances and contents, and unclear mechanisms of action, making it difficult to ensure stable efficacy. Commonly used chemical hair growth agents for the treatment of hair loss include minoxidil, finasteride, etc., but these substances have obvious side effects. Minoxidil can cause dry scalp, dandruff, scalp erythema, inflammation, irritation, etc., and can affect blood pressure. Finasteride has an inhibitory effect on male hormone activity.

[0004] The molecular structure of diaminopyrimidine oxides, such as diaminopyrimidine oxide and pyrrolidine diaminopyrimidine oxide, is similar to that of minoxidil, but they are not drugs, but raw materials approved for use in cosmetics. Among them, diaminopyrimidine oxide can inhibit the activity of lysine hydroxylase, thereby slowing down the hardening and contraction of hair follicles, and inhibit the activity of 5α-reductase, reducing the conversion of testosterone into dihydrotestosterone, while relaxing the blood vessels around the hair follicles, and promoting the transition of hair from the resting phase to the growth phase. Pyrrolidine diaminopyrimidine oxide can promote the proliferation of epidermal hair papilla cells, improve scalp microcirculation, and provide the nutrients needed for hair growth by affecting potassium ion channels. However, due to the poor water solubility of diaminopyrimidine oxides, a large amount of alcohol substances are usually added to increase the solubility when they are prepared into hair growth agents. In addition, when using such products, it is necessary to keep the hair dry to prevent the liquid from entering the face and neck through the hair, causing abnormal hair growth in these areas. During use, discomfort such as itching and dryness will also occur, which all need to be paid attention to. At the same time, due to the skin's own barrier function, this type of hair growth agent has poor transdermal absorption and requires large doses, high concentrations and repeated use.

[0005] In order to improve the shortcomings of this type of hair growth agent, many improvement measures have been disclosed, for example, β-cyclodextrin derivatives are used to encapsulate pyrrolidino diaminopyrimidine oxide to improve the solubility of pyrrolidino diaminopyrimidine oxide in water, but the particle size of the inclusion compound obtained in this way is large and cannot penetrate deeply into the hair follicle structure. For example, nano drug targeted carrier preparation technology is used to prepare a nano-composition containing diaminopyrimidine oxide and pyrrolidino diaminopyrimidine oxide to improve skin permeability and bioavailability, but the production process of this nano-preparation is cumbersome and inconvenient to use.

[0006] The purpose of the present invention is to overcome or improve at least one disadvantage of the prior art, or to provide a useful alternative. Unless the context clearly requires otherwise, throughout the specification and claims, the words "include", "comprises", etc. should be interpreted from an inclusive perspective rather than an exclusive or exhaustive perspective; that is, from the perspective of "including, but not limited to". Summary of the invention

[0007] The present invention provides a diaminopyrimidine oxide and low molecular cationic hyaluronic acid zinc coupling derivative and its preparation method and application. The derivative provided by the present invention has good water solubility and sustained-release transdermal effect, has the functions of hair solidification, hair loss prevention and hair growth, solves the defects of diaminopyrimidine oxide hair growth agents in the prior art, such as poor solubility, poor permeability, short action time, high-dose and high-frequency use, and thus strong irritation, and also has the advantages of moisturizing, good adsorption and affinity to skin and hair; at the same time, the method of the present invention is simple and easy to implement, conforms to the concept of green chemistry, and is environmentally friendly.

[0008] Specifically, the present invention provides the following technical features, and the combination of one or more of the following technical features constitutes the technical solution of the present invention.

[0009] In the first aspect of the present invention, the present invention provides a substance, which is a coupling derivative of diaminopyrimidine oxide and low molecular cationic hyaluronic acid zinc, and its structure is shown in Formula I:

[0010]

[0011] Wherein, R1, R2, R3 are each independently selected from hydroxypropyltrimethylammonium chloride and hydrogen, and R1, R2, and R3 are not hydrogen at the same time;

[0012] R4 is pyrrolidinyl or hydrogen;

[0013] m≥1, n≥0.

[0014] In some embodiments of the present invention, the cationic hyaluronic acid is a low molecular cationic hyaluronic acid, and its molecular weight is 2kDa to 100kDa, preferably 5kDa to 10kDa.

[0015] In some embodiments of the present invention, the average molecular weight of the substance represented by Formula I is less than 100 kDa, preferably 2 kDa-20 kDa.

[0016] In some embodiments of the present invention, the content of hydroxypropyltrimethylammonium chloride in the compound of formula I is 15-35%.

[0017] In some embodiments of the present invention, the zinc content in the compound of formula I is not less than 5%.

[0018] In some embodiments of the present invention, the degree of substitution of the diaminopyrimidine oxide in the compound of formula I is 0.01-0.7, preferably 0.1-0.5.

[0019] In the second aspect of the present invention, the present invention provides a method for preparing the substance represented by formula I described in the first aspect, comprising: subjecting a diaminopyrimidine oxide and a cationic hyaluronic acid to a coupling reaction; adding zinc acetate to perform salt conversion;

[0020] Wherein, the diaminopyrimidine oxide is shown in Formula II, and the cationic hyaluronic acid is shown in Formula III:

[0021]

[0022] Wherein, R1, R2, R3 are each independently selected from hydroxypropyltrimethylammonium chloride and hydrogen, and R1, R2, and R3 are not hydrogen at the same time;

[0023] R4 is pyrrolidinyl or hydrogen;

[0024] m≥1, n≥0.

[0025] In some embodiments of the present invention, the method comprises: conducting a coupling reaction of diaminopyrimidine oxide, cationic hyaluronic acid and a coupling reagent in water, ultrafiltering after the reaction, washing and then precipitating the reactant with alcohol, and adding zinc acetate for salt conversion.

[0026] In some embodiments of the present invention, after the salt conversion is completed, freeze-drying or alcohol precipitation followed by drying can be performed.

[0027] In the third aspect of the present invention, the present invention provides a composition comprising the substance represented by formula I described in the first aspect above.

[0028] In some embodiments of the present invention, the composition is a pharmaceutical composition or a cosmetic composition.

[0029] In the fourth aspect of the present invention, the present invention provides a preparation comprising the substance represented by formula I described in the first aspect above, and at least one necessary auxiliary material.

[0030] In some embodiments of the present invention, the preparation is a pharmaceutical preparation or a cosmetic preparation.

[0031] In some embodiments of the present invention, the composition or preparation is usually a topically applicable preparation. "Topical application" in the sense of the present invention means that the preparation is for local external use, that is, the preparation must be suitable for scalp and / or hair (especially hair) and the like.

[0032] In some embodiments of the present invention, the composition or preparation may further include a carrier to further improve permeability and therapeutic effect. The carrier may be in the form of an emulsion, gel, powder, solution or other suitable application. The pH value of the preparation may be adjusted by introducing an acid or alkaline substance at an appropriate position to ensure the stability and high efficiency of the composition and preparation.

[0033] In some embodiments of the present invention, the composition or preparation may also include other hair nutrients or bioactive substances, and may also contain mild cleaning ingredients. For example, natural plant ingredients such as ginger, mint, licorice, onion, etc., which can promote scalp blood circulation, regulate scalp oil secretion, and increase hair elasticity; for example, nutrients such as vitamins, minerals, amino acids, etc., which can provide necessary nutrition to nourish hair and scalp; for example, proteins such as serine, collagen, ergotamine, etc.; for example, essential oils such as rosemary, lavender, tea tree essential oil, jojoba oil, etc., to promote scalp blood circulation and reduce scalp inflammation and itching; for example, herbal extracts such as extracts of sappan wood, cypress bark, black sesame, mulberry, etc.

[0034] Specifically, the preparation of the present invention can be in a form acceptable in cosmetics, especially hair growth products, such as liquid (dew, liquid, water, oil, oil-water separation, etc.), gel (gel, glue, etc.), cream (cream, cream, honey, fat, milk, emulsion, milk, milk liquid, etc.), powder (loose powder, granules, etc.), block (block powder, large solid, etc.), mud (mud solid, etc.), spray (without propellant), aerosol (with propellant), freeze-dried (freeze-dried powder, freeze-dried tablets, etc.), wax-based (with wax as the main base material), patch, film, containing substrate (patch, film, containing substrate for use with cosmetics), etc.; specifically, examples of products in these forms include but are not limited to shampoo, conditioner, hair care essence, rinse-off hair mask, leave-in products, scalp lotion, shampoo soap, spray, etc.

[0035] In some embodiments of the present invention, the present invention provides a gel preparation, which comprises the substance of formula I described in the first aspect above and sodium acetyl alginate, wherein sodium acetyl alginate is used as a carrier. In one embodiment provided in the present application, a method for preparing the gel preparation is provided, comprising: adding the substance of formula I to a sodium acetyl alginate solution, and then adding a calcium chloride solution.

[0036] In a specific embodiment, the preparation method of the gel preparation includes: dissolving sodium acetyl alginate in deionized water to prepare a solution; adding the substance represented by formula I to the sodium acetyl alginate solution under stirring; adding calcium chloride solution, stirring and then standing to obtain the gel preparation.

[0037] The acetyl sodium alginate of the present invention can be purchased or obtained by modifying sodium alginate with acetic anhydride, wherein the substitution degree of acetyl group in the acetyl sodium alginate is 10-25%.

[0038] The composition and preparation for scalp and hair provided by the present invention can enhance the metabolism of hair follicles and improve the scalp environment, increase the growth rate and quality of hair, and reduce hair loss, thereby achieving the effect of enhancing the health and beauty of hair.

[0039] In the fifth aspect of the present invention, the present invention provides the use of the substance represented by formula I described in the first aspect above, or the composition described in the third aspect above, or the preparation described in the fourth aspect above in the preparation of a medicament (such as a hair growth agent) or cosmetic (such as a hair growth product) for treating hair and / or scalp conditions. Wherein, the hair is head hair or body hair, especially refers to head hair.

[0040] In some embodiments of the invention, treating hair and / or scalp conditions is preventing and / or ameliorating hair loss and / or stimulating hair growth.

[0041] In the present invention, "preventing and / or improving hair loss" refers to reducing, reversing, slowing down or preventing hair loss symptoms including but not limited to hair loss, thinning hair, uneven hair thickness, etc. The above symptoms may be hereditary hair loss, alopecia areata, seborrheic alopecia, hair breakage and loss caused by Trichomonas infection, seasonal hair loss, stress hair loss and malnutrition hair loss, etc.

[0042] In the present invention, "stimulating hair growth" refers to promoting hair growth, regrowth or increasing the amount of hair, such as stimulating hair follicles to regenerate hair, promoting the thickening of naturally thin or sparse hair (increasing hair volume), optimizing the hair growth cycle so that the hair has a longer growth period, promoting hair growth in bald or hair loss areas, and accelerating the rate of new hair growth.

[0043] In some embodiments of the present invention, the cosmetics are mainly hair growth products, and the dosage forms of the hair growth products include liquids (dew, liquid, water, oil, oil-water separation, etc.), gels (gel, glue, etc.), creams and lotions (cream, cream, honey, fat, milk, emulsion, milk, milk liquid, etc.), powders (loose powder, granules, etc.), blocks (block powder, large solid blocks, etc.), mud (mud solids, etc.), sprays (without propellants), aerosols (with propellants), freeze-dried (freeze-dried powder, freeze-dried tablets, etc.), wax-based (with wax as the main base material), patches, films, containing substrates (patches, films, containing substrates used in conjunction with cosmetics), etc.; specifically, examples of products in these forms include but are not limited to shampoos, conditioners, hair care essences, rinse-off hair masks, wash-free products, scalp lotions, shampoo soaps, sprays, etc.

[0044] In the sixth aspect of the present invention, the present invention provides a method for treating hair and / or scalp conditions, which comprises topically applying the substance represented by formula I described in the first aspect above, or a composition or preparation comprising the substance represented by formula I, in an effective amount to the scalp and / or hair to prevent and / or improve hair loss and / or stimulate hair growth.

[0045] In the present invention, "preventing and / or improving hair loss" refers to reducing, reversing, slowing down or preventing hair loss symptoms including but not limited to hair loss, thinning hair, uneven hair thickness, etc. The above symptoms may be hereditary hair loss, alopecia areata, seborrheic alopecia, hair breakage and loss caused by Trichomonas infection, seasonal hair loss, stress hair loss and nutritional alopecia, etc.

[0046] In the present invention, "stimulating hair growth" refers to promoting hair growth, regrowth and / or increasing the amount of hair, such as stimulating hair follicles to regenerate hair, promoting the thickening of naturally thin or sparse hair (increasing hair volume), optimizing the hair growth cycle so that the hair has a longer growth period, promoting hair growth in bald or hair loss areas, and accelerating the rate of new hair growth.

[0047] The effective amount of the present invention refers to the amount of a substance, composition or formulation that can significantly induce a positive effect on the scalp and / or hair during treatment (e.g., during the use period). The positive effect is such as a beneficial effect in appearance and / or feel, such as, in one embodiment, the growth of new hair, the thickening of thinning hair, the reduction of hair loss, etc. In some cases, an effective amount can be demonstrated using ex vivo and / or in vitro methods.

[0048] The topical application described in the present invention refers to contacting the component or preparation to be applied with the scalp and / or hair, and its methods include but are not limited to spreading, dripping, spraying, massaging and the like.

[0049] In an embodiment of the present invention, application of the substance of formula I of the present invention or a composition or preparation comprising the substance can achieve at least one of the following effects to achieve the effects of hair strengthening, hair loss prevention and / or hair growth: preventing hair follicle blockage, nourishing hair follicles, and strengthening hair roots to hair tips; promoting keratin fiber production composition and increasing hair concentration; extending the hair growth cycle and accelerating hair growth; making fine hair grow healthily and strengthening hair; softening the collagen fiber network and preventing hair loss; and acting on the deep structure of the hair root to induce new hair and accelerate the growth of new hair.

[0050] Compared with the prior art, the advantages of the present invention include:

[0051] In the technical scheme of the present invention, the diaminopyrimidine oxide and the low molecular cationic hyaluronic acid zinc coupled derivative have good water solubility and sustained-release transdermal effect, and have excellent hair solidification, hair loss prevention and / or hair growth functions, which solves the defects of the diaminopyrimidine oxide in the prior art, such as poor permeability, short action time, large dose and high frequency use, and thus strong irritation, and at the same time has the advantages of moisturizing, good adsorption and affinity to skin and hair, etc.

[0052] The derivative of the present invention has a unique transdermal absorption mechanism. Under the action of the amidohydrolase in the body fluid, the amide bond is opened, so that the diaminopyrimidine oxide and hyaluronic acid zinc are stably transferred to the dermis of the skin. A water-containing hyaluronic acid film is formed on the surface, and together with the endogenous hyaluronic acid, a "sandwich" structure is formed on both sides of the stratum corneum. The water content of the stratum corneum is rapidly increased to form a semipermeable membrane, and the diaminopyrimidine oxide and ionic zinc are easily passed through by the osmotic pressure principle, so that the diaminopyrimidine oxide is slowly released, which has the effect of preventing hair loss and promoting hair growth. In addition to completely maintaining the function of the diaminopyrimidine oxide, the product also has the effect of low molecular cationic hyaluronic acid zinc. At the same time, the presence of zinc has a synergistic effect of preventing hair loss and promoting hair growth with the diaminopyrimidine oxide.

[0053] For the derivatization reaction of hyaluronic acid, the traditional salt transfer and organic solvents are avoided, and water is directly used as the solvent for coupling. For the zinc salt transfer operation, ion exchange resin and other operations are avoided, which is in line with the concept of green chemistry and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The drawings constituting part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. The implementation scheme of the present application is described in detail below in conjunction with the drawings, wherein:

[0055] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the compound of formula I1 prepared in Preparation Example 1 of the present invention is shown.

[0056] Figure 2 The hydrogen nuclear magnetic resonance spectrum of the compound of formula I2 prepared in Preparation Example 2 of the present invention is shown.

[0057] Figure 3 The graph showing the comparison results of the water solubility test of Experimental Example 1 of the present invention is shown.

[0058] Figure 4 A comparison chart of the cumulative permeation amounts of the test samples in different experimental groups of Experimental Example 2 of the present invention is shown.

[0059] Figure 5 A comparison chart of the retention amounts of the test samples in different experimental groups of Experimental Example 2 of the present invention is shown.

[0060] Figure 6 A comparison chart of the efficacy of the test samples in different experimental groups of Experimental Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0061] The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0062] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0063] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in this application are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the content described herein can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0064] The present invention provides a derivative of a diaminopyrimidine oxide and a low molecular cationic hyaluronic acid zinc coupling, the structure of which is shown in Formula I1 and I2:

[0065]

[0066] R1, R2, R3 are each independently selected from hydroxypropyltrimethylammonium chloride and hydrogen, and R1, R2, and R3 are not hydrogen at the same time; m≥1, n≥0. The average molecular weight of the substance represented by formula I is less than 100 kDa, preferably 2 kDa-20 kDa; the content of hydroxypropyltrimethylammonium chloride in the compound of formula I is 15-35%, and the zinc content is not less than 5%.

[0067] The compound of the present invention can be prepared by the following reaction route:

[0068]

[0069] R1, R2, R3 are each independently selected from hydroxypropyltrimethylammonium chloride and hydrogen, and R1, R2, and R3 are not hydrogen at the same time; R4 is pyrrolidinyl or hydrogen; m≥1, n≥0. The average molecular weight of the substance represented by formula I is less than 100 kDa, preferably 2 kDa-20 kDa; the content of hydroxypropyltrimethylammonium chloride in the compound of formula I is 15-35%, and the zinc content is not less than 5%.

[0070] Specifically, the preparation and properties of the conjugates of the present invention will be illustrated by the following examples. It should be understood that the examples are provided for illustration only and are difficult to be exhaustive. Those skilled in the art can easily prepare derivatives having the structure of the present invention or other derivatives similar to the structure of the present invention according to the present invention, and verify the structure by NMR spectra.

[0071] The low molecular cationic sodium hyaluronate described in the present invention is obtained by modifying low molecular hyaluronic acid with glycidyl trimethyl ammonium chloride. For example, the low molecular cationic sodium hyaluronate described in the following embodiment of the present invention is prepared by the following method: adding low molecular sodium hyaluronate to 4 times the volume of deionized water, stirring evenly, adding 2.5 equivalents of glycidyl trimethyl ammonium chloride and 0.1 equivalents of tetrabutyl ammonium hydroxide, adding 3.0 equivalents of 3 mol / L sodium hydroxide solution dropwise, stirring at 20-30°C for 4-5h, after the reaction is completed, pouring the reaction solution into 95 ethanol for precipitation, decanting the supernatant, solid-phase precipitating with anhydrous ethanol, washing the precipitate with 95 ethanol 2-3 times, and drying the precipitate to obtain low molecular cationic sodium hyaluronate.

[0072] Preparation Example, Preparation of Compounds of Formula I

[0073] Preparation Example 1. Preparation of Compound of Formula I1

[0074] Add 100g of low molecular weight cationic sodium hyaluronate with a molecular weight of 5kDa to 10kDa (cationic substitution degree of 18.6%) to 2000g of purified water and stir until dissolved. Add 100g of O-benzotriazole-tetramethyluronium hexafluorophosphate, adjust the pH to 6-7 with sodium hydroxide, add 3g of compound II (R4 is H) in batches, and stir at 20-30℃ for 16 hours; add 50g of O-benzotriazole-tetramethyluronium hexafluorophosphate and 3g of compound II (R4 is H), adjust the pH to 6-7 with sodium hydroxide, and stir at 20-30℃ for 24 hours; add 50g of O-benzotriazole-tetramethyluronium hexafluorophosphate and 3g of compound II (R4 is H), adjust the pH to 6-7 with sodium hydroxide, and stir at 20-30℃ for 16 hours. After the reaction is completed, add to 2000g of purified water. The product was ultrafiltered through a nanofiltration membrane (molecular weight cutoff 1000Da), washed, concentrated and precipitated with alcohol to obtain a white solid.

[0075] The above solid was added to 1L of 95% ethanol containing 6% zinc acetate, stirred for 16 hours, allowed to stand, the supernatant was poured off, the above steps were repeated 6 times, dehydrated with anhydrous ethanol 2 times, filtered and dried to obtain 110g of the compound of formula I1, with a molecular weight of 8kDa, a degree of substitution of diaminopyrimidine oxide of 0.27, a hydroxypropyltrimethylammonium chloride content of 17.1%, and a zinc content of 6.0%. The nuclear magnetic resonance hydrogen spectrum of the compound of formula I1 (also referred to as diaminopyrimidine oxide and low molecular cationic hyaluronic acid zinc coupling derivative in the present invention) is shown in Figure 1 .

[0076] Preparation Example 2, Preparation of Compound of Formula I2

[0077] Add 100g of low molecular weight cationic sodium hyaluronate with a molecular weight of 5kDa to 10kDa (cationic substitution degree of 18.6%) to 2000g of purified water and stir until dissolved. Add 100g of O-benzotriazole-tetramethyluronium hexafluorophosphate, adjust the pH to 6-7 with sodium hydroxide, add 4.5g of compound II (R4 is pyrrolidinyl) in batches and stir at 20-30℃ for 16 hours; add 50g of O-benzotriazole-tetramethyluronium hexafluorophosphate and 4.5g of compound II (R4 is pyrrolidinyl), adjust the pH to 6-7 with sodium hydroxide, and stir at 20-30℃ for 24 hours; add 50g of O-benzotriazole-tetramethyluronium hexafluorophosphate and compound II (R4 is pyrrolidinyl), adjust the pH to 6-7 with sodium hydroxide, and stir at 20-30℃ for 16 hours. After the reaction is completed, add to 2000g of purified water. The product was ultrafiltered through a nanofiltration membrane (molecular weight cutoff 1000Da), washed, concentrated and precipitated with alcohol to obtain a white solid.

[0078] Add 1L of 95% ethanol containing 6% zinc acetate, stir for 16 hours, let stand, pour off the supernatant, repeat the above steps 6 times, concentrate under reduced pressure to remove ethanol, add the solid to 1L deionized water, filter at 0.22μm, and freeze-dry to obtain 108g of compound of formula I2, with a molecular weight of 9kDa, a degree of substitution of pyrrolidinyl diaminopyrimidine oxide of 0.30, a hydroxypropyltrimethylammonium chloride content of 17.4%, and a zinc content of 6.6%. The nuclear magnetic resonance hydrogen spectrum of the compound of formula I2 (also referred to as pyrrolidinyl diaminopyrimidine oxide and low molecular cationic hyaluronic acid zinc coupling derivative in the present invention) is shown in Figure 2 .

[0079] Experimental Example 1: Water Solubility Test Comparison

[0080] Compound I1 and compound I2 prepared in Preparation Example 1 and Preparation Example 2 were dissolved in purified water at a concentration of 0.1 g / mL. 0.1 g / mL of diaminopyrimidine oxide (i.e., Formula II, R4 is hydrogen) and pyrrolidine diaminopyrimidine oxide (i.e., Formula II, R4 is pyrrolidine) were used as controls. The results are shown in FIG. Figure 3 According to the water solubility test results, it can be confirmed that diaminopyrimidine oxide and pyrrolidine diaminopyrimidine oxide are almost insoluble in water at the same concentration and are therefore in an opaque state, while the two compounds of the present invention are completely soluble in water.

[0081] Experimental Example 2

[0082] This experimental example tests the test samples in 1) in vitro cytotoxicity test and irritation test, 2) skin permeation test and 3) hair growth activity test.

[0083] 1. Experimental groups and test samples:

[0084] Experimental group 1: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, low molecular cationic hyaluronic acid zinc was added to the sodium acetyl alginate solution in an amount of 0.08mol / L; a 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain a low molecular cationic hyaluronic acid zinc gel as the test sample of experimental group 1.

[0085] Experimental Group 2: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; zinc acetate was added to the sodium acetyl alginate solution under mechanical stirring at an addition amount of 0.08mol / L; calcium chloride solution with a concentration of 0.2wt% was added, stirred for 20min, and then allowed to stand for 24h to obtain zinc acetate gel as the test sample of Experimental Group 2.

[0086] Experimental Group 3: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, diaminopyrimidine oxide was added to the sodium acetyl alginate solution in an amount of 0.08mol / L; a 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain diaminopyrimidine oxide gel as the test sample of Experimental Group 3.

[0087] Experimental Group 4: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, diaminopyrimidine oxide and low molecular cationic zinc hyaluronate were added to the sodium acetyl alginate solution, and the added amount was 0.08mol / L; 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain the test samples of Experimental Group 4.

[0088] Experimental Group 5: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, diaminopyrimidine oxide and zinc acetate were added to the sodium acetyl alginate solution, and the added amount was 0.08mol / L; 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain the test samples of Experimental Group 5.

[0089] Experimental Group 6: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, zinc acetate and low molecular cationic zinc hyaluronate were added to the sodium acetyl alginate solution, and the added amount was 0.08mol / L; 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain the test samples of Experimental Group 6.

[0090] Experimental Group 7: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, diaminopyrimidine oxide, zinc acetate and low molecular cationic zinc hyaluronate were added to the sodium acetyl alginate solution, and the added amount was 0.08mol / L; 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain the test samples of Experimental Group 7.

[0091] Experimental Group 8: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2 wt%; under mechanical stirring conditions, the compound of formula I1 prepared in Preparation Example 1 was added to the sodium acetyl alginate solution in an amount of 0.08 mol / L; a calcium chloride solution with a concentration of 0.2 wt% was added, stirred for 20 min, and then allowed to stand for 24 h to obtain the test sample of Experimental Group 8.

[0092] Experimental Group 9: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, pyrrolidino diaminopyrimidine oxide was added to the sodium acetyl alginate solution in an amount of 0.08 mol / L; a 0.2wt% calcium chloride solution was added, stirred for 20 minutes, and then allowed to stand for 24 hours to obtain diaminopyrimidine oxide gel as the test sample of Experimental Group 9.

[0093] Experimental group 10: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, pyrrolidino diamino pyrimidine oxide and low molecular cationic zinc hyaluronate were added to the sodium acetyl alginate solution, and the added amount was 0.08mol / L; 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain the test samples of experimental group 10.

[0094] Experimental Group 11: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, pyrrolidino diaminopyrimidine oxide and zinc acetate were added to the sodium acetyl alginate solution, and the added amount was 0.08mol / L; 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain the test samples of Experimental Group 11.

[0095] Experimental Group 12: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, pyrrolidino diaminopyrimidine oxide, zinc acetate and low molecular cationic zinc hyaluronate were added to the sodium acetyl alginate solution, and the added amount was 0.08mol / L; 0.2wt% calcium chloride solution was added, stirred for 20min, and then allowed to stand for 24h to obtain the test samples of Experimental Group 12.

[0096] Experimental Group 13: Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, the compound of formula I2 prepared in Preparation Example 2 was added to the sodium acetyl alginate solution in an amount of 0.08mol / L; a calcium chloride solution with a concentration of 0.2wt% was added, stirred for 20min, and then allowed to stand for 24h to obtain the test sample of Experimental Group 13.

[0097] Experimental group 14 (blank group): Sodium acetyl alginate was dissolved in deionized water to prepare a solution with a concentration of 2wt%; under mechanical stirring conditions, a calcium chloride solution with a concentration of 0.2wt% was added, stirred for 20min, and then allowed to stand for 24h to obtain a gel as the blank group 14 test sample.

[0098] The acetyl sodium alginate used in the present invention is obtained by modifying sodium alginate with acetic anhydride. The preparation method of the acetyl sodium alginate in this embodiment comprises: placing sodium alginate (mannuronic acid: guluronic acid = 1.40-1.70) in an acetic acid / acetic anhydride / triethylamine (1:1:0.1) solution, the mass volume ratio of the two being 1:10, stirring and reacting at 80°C for 10 hours. After the reaction is completed, pouring the reaction solution into ethanol for precipitation, decanting the supernatant, washing the precipitate with 95% ethanol for 2 to 3 times, drying the precipitate, adding water to dissolve the dried solid, filtering, and then freeze-drying the filtrate into powder to obtain acetyl sodium alginate with an acetyl substitution degree of 12%.

[0099] The preparation method of low molecular cationic hyaluronic acid zinc described in this embodiment includes: adding 100g of low molecular cationic sodium hyaluronate with a molecular weight of 5kDa to 10kDa (cationic substitution degree of 18.6%) to 1L of 95 ethanol containing 6% zinc acetate, stirring for 16 hours, standing, pouring off the supernatant, repeating the above steps 6 times, dehydrating with anhydrous ethanol 2 times, filtering and drying to obtain 96g of low molecular cationic hyaluronic acid zinc, with a molecular weight of 10kDa, a hydroxypropyltrimethylammonium chloride content of 17.6%, and a zinc content of 6.7%.

[0100] 2. In vitro cytotoxicity and irritation experiments

[0101] According to GB T16886.5-2017 / ISO10993-5:2009 "Biological Evaluation of Medical Devices Part 5: In vitro Cytotoxicity Test", in vitro cytotoxicity tests were conducted on experimental groups 8 and 13 to verify biocompatibility. The MTT cytotoxicity test evaluation method was used, L929 cells were selected as the cell line, 10% DMSO solution was used as the positive control group, 3% fetal calf serum culture medium was used as the negative control group, and the test group was added with 3% fetal calf serum culture medium extract of the test sample, and cultured at 37°C for 24 hours.

[0102] The specific experimental steps are as follows: L929 cells cultured for 48 h were prepared into 1×10 5 Cell suspension of 1×10 5 The cell suspension of 100 μL / well was inoculated into a 96-well cell culture plate (100 μL / well) and cultured in a 37°C, 5% CO2 incubator for 24 hours to form a semi-confluent monolayer; after 24 hours, the original culture medium was discarded, and then 100 μL of sample extract, positive control solution, and negative control solution were added, 5 wells per group, and cultured in a 37°C, 5% CO2 incubator for 24 hours. 50 μL of MTT solution was added to each well, and then continued to incubate in a 37°C, 5% CO2 incubator for 2 hours;

[0103] The MTT solution was discarded, and 100 μL of DMSO solution was added to each well. The plate was shaken and placed on a microtiter plate photometer to measure the absorbance at 570 nm. The cell proliferation rate (RGR, %) was calculated and the cytotoxicity was evaluated according to the standards in the table below.

[0104] Table 1 In vitro cytotoxicity grading table

[0105] level 0 1 2 3 4 5 Relative proliferation rate / % ≥100 75-99 50-74 25-49 1-24 0

[0106] The experimental results are shown in Table 2. The in vitro cytotoxicity of the compound of formula I1 and the compound of formula I2 prepared by the present invention are both level 1, indicating that both have good biocompatibility and will not produce toxicity or cause toxic residues.

[0107] Table 2 In vitro cytotoxicity test results

[0108] sample Toxicity level Experimental Group 8 1 Experimental Group 13 1 Positive control group 5

[0109] Fifteen healthy rabbits, weighing about 2 kg, were randomly divided into three groups, with 5 rabbits in each group. The hair on both sides of the rabbits' back skin was removed 24 hours before the experiment. The skin in the removed area was tested for injury 24 hours after the hair removal. Rabbits with injured skin were not suitable for skin irritation experiments. The samples of experimental group 8, experimental group 13 and experimental group 14 (blank group) were applied three times a day for 7 consecutive days, and the experimental results were observed. The experimental results are shown in Table 3.

[0110] Table 3 Skin irritation test results

[0111]

[0112] Note: "+" indicates that the rabbits have skin redness, swelling, inflammation and congestion; "++" indicates that the rabbits have skin redness, swelling, inflammation and congestion, and there is an increasing trend; "-" indicates that the rabbits have no skin redness, swelling, inflammation and congestion.

[0113] From the experimental results in Table 3, it can be seen that the samples of Experimental Group 8 and Experimental Group 13 have no irritation to the skin.

[0114] 3. Transdermal Experiment

[0115] The vertical Franz diffusion cell method was used to conduct the in vitro mouse skin permeation experiment. The abdominal skin of SD male rats was fixed between the receiving chamber and the supply chamber, and the experimental group 3, experimental group 4, experimental group 5, experimental group 7, experimental group 8, experimental group 9, experimental group 10, experimental group 11, experimental group 12, and experimental group 13 were placed in the supply chamber, and physiological saline containing 2% sodium dodecyl sulfate and 20% ethanol by mass fraction was used as the receiving liquid and stirred and diffused at 300 speed at 37°C. Samples were taken at 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 hours, and analyzed by UV-visible spectrophotometry to calculate the cumulative permeation of diaminopyrimidine oxide, compound of formula I1, pyrrolidine diaminopyrimidine oxide and compound of formula I2 per unit area of ​​skin at different times. After the 24-hour in vitro permeation test, the mouse skin with effective permeation area was cut into pieces, homogenized after adding an appropriate amount of receiving solution, and the homogenate was centrifuged at 8,000 r / min for 30 minutes. The supernatant was taken, filtered through a 0.22 μm filter membrane, and analyzed by UV-visible spectrophotometry to calculate the retention of diaminopyrimidine oxide, compound of formula I1, pyrrolidine diaminopyrimidine oxide and compound of formula I2 per unit area of ​​skin. The experimental data are shown in Figure 4 and Figure 5 .

[0116] Figure 4 It is the cumulative permeation amount of different groups in the transdermal experiment; Figure 5 is the retention amount of different groups in the transdermal experiment. Figure 4 and Figure 5 It can be seen that the compounds I1 and I2 of the present invention have good skin permeability and skin retention ability. Figure 4 The cumulative permeation per unit area of ​​the I1 compound of the present invention is about 2.5 times that of diaminopyrimidine oxide, and the transdermal effect is significantly improved. The addition of zinc acetate and low molecular cationic zinc hyaluronate can increase the transdermal permeability of diaminopyrimidine oxide, but the physical mixture thereof is not as good as the compound of formula I1 of the present application; the cumulative permeation per unit area of ​​the I2 compound of the present invention is about 3.5 times that of pyrrolidino diaminopyrimidine oxide, and the transdermal effect is significantly improved. The addition of zinc acetate and low molecular cationic zinc hyaluronate can increase the transdermal permeability of pyrrolidino diaminopyrimidine oxide, but the physical mixture thereof is not as good as the compound of formula I2 of the present application. Figure 5 After the I1 compound of the present invention penetrated the skin for 24 hours, its skin retention per unit area reached 11.5 μg·cm -2 However, the skin retention of diaminopyrimidine oxide permeates for 24 hours per unit area is only 3.6 μg cm -2 The I2 compound of the present invention permeated the skin for 24 hours, and its skin retention per unit area reached 10.8 μg·cm -2 However, the skin retention per unit area of ​​pyrrolidinodiaminopyrimidine oxide was only 2.8 μg cm after 24 h of percutaneous transdermal administration. -2Diaminopyrimidine oxides are difficult to penetrate the stratum corneum of the skin, and cannot act on the deep structure of hair follicles, so their bioavailability is low. The diaminopyrimidine oxides and low-molecular cationic hyaluronic acid zinc coupled derivatives of the present invention can penetrate deeply into the hair follicles, directly reach the target, and stay in the hair follicles for a long time, with sustained and controlled release, which significantly improves their bioavailability and enhances the anti-hair loss and hair growth effects.

[0117] 4. Hair growth activity test

[0118] A total of 280 patients aged 25-50 years with grade III-V hair loss according to Hamilton-Norwood scale were selected, including 200 males and 80 females, and were randomly divided into 14 groups.

[0119] Inclusion criteria: no hair loss treatment within six months, willing to accept follow-up and actively cooperate with treatment, no hair perming or dyeing during treatment. Exclusion criteria: patients with systemic diseases accompanied by heart, lung, liver or kidney damage, patients with endocrine or mental diseases, patients with scalp damage or other skin diseases.

[0120] Treatment method: Encapsulate the sample gels of experimental groups 1-14 into a container with a metering pump head (the pump head can pump out 1 mL each time). Use the samples of experimental groups 1-14 twice a day, morning and evening, pump out 1 mL of the drug each time for smear treatment. Keep the scalp and hair dry before use. After use, massage the scalp with your hands for 3-5 minutes.

[0121] Treatment cycle: 4 weeks as one course of treatment. After 4 courses of treatment, observe the treatment effect.

[0122] Treatment effect judgment criteria: 1) Significant effect: stop hair loss, hair grows back in hairless areas; 2) Effective: hair loss is significantly improved, black hair grows back in some hairless areas; 3) Ineffective: hair loss is not improved or alleviated, and new hair does not grow in hairless areas. The test results are shown in Table 4. The significant effect is calculated. The results are as follows Figure 6 As shown.

[0123] Table 4 Comparison of treatment effects

[0124]

[0125]

[0126] From the data results in the treatment effect comparison table in Table 4, it can be seen that experimental groups 1, 2, 6 and 14 are ineffective, indicating that low molecular cationic zinc hyaluronate and zinc acetate have no effect on hair solidification and hair loss prevention, and cannot promote hair growth. Experimental groups 4, 5, and 7 are slightly better than experimental group 3 in treating hair loss, indicating that low molecular cationic zinc hyaluronate and zinc acetate can slightly enhance the effect of diaminopyrimidine oxide in treating hair loss. The therapeutic effect of experimental group 8 is significantly higher than that of the diaminopyrimidine oxide gel of experimental group 3 alone, and is also significantly higher than that of the diaminopyrimidine oxide mixed gels of experimental groups 4, 5, and 7, respectively, with low molecular cationic zinc hyaluronate and zinc acetate. Experimental groups 10, 11, and 12 are slightly better than experimental group 9 in treating hair loss, indicating that low molecular cationic zinc hyaluronate and zinc acetate can slightly enhance the effect of pyrrolidino diaminopyrimidine oxide in treating hair loss. The therapeutic effect of experimental group 13 is significantly higher than that of the pyrrolidino diaminopyrimidine oxide gel of experimental group 9 alone, and is also significantly higher than that of the pyrrolidino diaminopyrimidine oxide mixed gels of experimental groups 10, 11, and 12, respectively, with low molecular cationic zinc hyaluronate and zinc acetate. It is fully proved that the significant improvement in the therapeutic effect of the compound of formula I1 and the compound of formula I2 prepared by the present invention is not caused by the simple combination of diaminopyrimidine oxides with low molecular cationic zinc hyaluronate and zinc acetate, but is due to the fundamentally endowed with better properties through chemical modification.

[0127] In summary, the invention solves the defects of diaminopyrimidine oxide hair growth agents in the prior art, such as poor solubility, poor permeability, short action time, and strong irritation caused by large-dose, high-frequency and high-concentration use. It has good water solubility and sustained-release transdermal effect, and has excellent hair solidification, anti-hair loss and hair growth functions. It also has the advantages of moisturizing properties and good adsorption and affinity to skin and hair. It also solves the technical problems in functional cosmetics that hyaluronic acid ingredients and diaminopyrimidine oxides need to be added separately and have a short action time.

[0128] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Although the present application is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative, the structure of which is shown in Formula I: in, R1, R2, R3 are each independently selected from hydroxypropyltrimethylammonium chloride and hydrogen, and R1, R2, and R3 are not hydrogen at the same time; R4 is pyrrolidinyl or hydrogen; m≥1, n≥0; The average molecular weight of the substance of formula I is less than 100 kDa.

2. The diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative according to claim 1, characterized in that: The average molecular weight of the substance of formula I is between 2 kDa and 20 kDa.

3. The diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative according to claim 1, characterized in that: Hydroxypropyltrimethylammonium chloride in the compound of formula I The content is 15-35%.

4. The diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative according to claim 1, characterized in that: The zinc content in the compound of formula I is not less than 5%.

5. The diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative according to claim 1, characterized in that: The degree of substitution of the diaminopyrimidine oxide in the compound of formula I is 0.01-0.

7.

6. The diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative according to claim 1, characterized in that: The degree of substitution of the diaminopyrimidine oxide in the compound of formula I is 0.1-0.

5.

7. A method for preparing the diaminopyrimidine oxide and cationic hyaluronic acid zinc coupling derivative according to claim 1, comprising: The diaminopyrimidine oxide and cationic hyaluronic acid are subjected to coupling reaction; zinc acetate is added to carry out salt conversion; Wherein, the diaminopyrimidine oxide is shown in Formula II, and the cationic hyaluronic acid is shown in Formula III: Wherein, R1, R2, R3 are each independently selected from hydroxypropyltrimethylammonium chloride and hydrogen, but not both hydrogen; R4 is pyrrolidinyl or hydrogen; m≥1, n≥0; The average molecular weight of the substance of formula I is less than 100 kDa.

8. The method according to claim 7, characterized in that The method comprises: performing a coupling reaction on diaminopyrimidine oxide, cationic hyaluronic acid and a coupling reagent in water, ultrafiltration after the reaction is completed, washing and then precipitating with alcohol to obtain a reactant, and adding zinc acetate to perform salt conversion.

9. The method according to claim 7, characterized in that: After the salt conversion is completed, freeze-drying or alcohol precipitation followed by drying is performed. 10 . A composition comprising the diaminopyrimidine oxide according to claim 1 and a cationic hyaluronic acid zinc coupled derivative.

11. The composition according to claim 10, characterized in that The composition is a pharmaceutical composition or a cosmetic composition.

12. A preparation comprising the diaminopyrimidine oxide and cationic hyaluronic acid zinc coupled derivative according to any one of claims 1 to 6, and at least one necessary auxiliary material.

13. The preparation according to claim 12, characterized in that The preparation is a pharmaceutical preparation or a cosmetic preparation.

14. The preparation according to claim 12, characterized in that The preparation is an external preparation.

15. Use of the diaminopyrimidine oxide and cationic hyaluronic acid zinc coupled derivative according to any one of claims 1 to 6, or the composition according to claim 10 or 11, or the preparation according to any one of claims 12 to 14 in the preparation of a medicament or cosmetic for treating hair and / or scalp conditions.

16. The use according to claim 15, characterized in that Treatment of hair and / or scalp conditions is to prevent and / or improve hair loss and / or stimulate hair growth.

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