TWI500594B - Crystal form a of 3(2-acetylamino-acetylamino)-propionic acid, its preparing method and application thereof - Google Patents

Description

Crystalline form A of compound 3 (2-acetamido-acetamido)-propionic acid, and a preparation method thereof application

The present invention relates to the field of skin whitening active ingredients, in particular to a compound 3 (2-acetamido-acetamido)-propionic acid crystal form A, and a preparation method thereof and application compound 3 (2-B) A whitening method for the crystalline form A of amidino-acetamido)-propionic acid in non-therapeutic uses.

Tyrosine is naturally present in epidermal cells, and the precursor of melanin is produced in the melanin production process via tyrosine → Dopa → Dopaquinone → Dopachrome → melanin. Tyrosinase is an important enzyme that converts tyrosine to dopa by the activity of its hydroxylase, which converts dopa to dopaquinone by the activity of oxidase. Therefore, as long as it is an effective component which can effectively act on epidermal melanocytes, inhibit melanogenesis, or inhibit any process of melanin biosynthesis, it can be used as an active ingredient for skin whitening.

CN 103298778 A has disclosed a compound 3 (2-acetamido-acetamido)-propionic acid for inhibiting melanin production, such as a compound of formula (I):

Example 1 of CN 103298778 A merely discloses that the filtrate is evaporated in vacuo to obtain a white powder of 3(2-acetamido-acetamido)-propionic acid, and the purity of the powder is only greater than 95 by liquid chromatography. %, but CN 103298778A does not disclose and teach the related purification method of 3(2-acetamido-acetamido)-propionic acid, and the properties such as crystal form, stability and solubility of the powder are not discussed. This has led to a significant limitation in the practical application and storage of the compound 3 (2-acetamido-acetamido)-propionic acid in the cosmetic skin care industry related to skin whitening.

In view of the above background of the invention, in order to meet the industrial requirements, one of the objects of the present invention is to provide a crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid, which has a crystal form A Good thermal stability and storage stability.

Another object of the present invention is to provide a process for preparing a crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid, which uses a low toxicity alcohol solvent, thus preparing the above The method is safe and easy to handle, and at the same time, the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid of high purity can be obtained, which is particularly advantageous for commercial mass production.

It is still another object of the present invention to provide a whitening method for non-therapeutic use, A non-therapeutic whitening method involves the use of a formulation of the addition of the compound 3 (2-acetamido-acetamido)-propionic acid Form A on the surface of the skin. Wherein the above formula 3 of the compound 3 (2-acetamido-acetamido)-propionic acid in the formula ranges from 0.1 to 10% by weight, the added compound 3 (2-B) The formulation of Form A of guanamine-acetamido)-propionic acid has a skin whitening effect. Next, the above formula of the addition of the compound 3 (2-acetamido-acetamido)-propionic acid crystal form A further comprises a vitamin C derivative selected from the group consisting of vitamin C ethyl ether. , vitamin C glycosides, vitamin C phosphates and combinations thereof. Further, the form of the formula A of the added compound 3 (2-acetamido-acetamido)-propionic acid contains a cream, an emulsion, a gel, a powder, a paste, a mask liquid, and a lotion. .

The object of the present invention and solving the technical problem thereof are to adopt the following technical solutions. Now. According to the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid provided by the present invention, the powder X-ray diffraction pattern of the above crystal form A comprises the following winding represented by 2 θ angle Peak: 10.4±0.2°, 11.0±0.2°, 12.9±0.2°, 16.0±0.2°, 16.4±0.2°, 17.6±0.2°, 19.1±0.2°, 19.5±0.2°, 20.0±0.2°, 21.2± 0.2°, 23.2±0.2°, 23.5±0.2°, 24.1±0.2°, 24.9±0.2°, 25.3±0.2°, 26.2±0.2°, 30.2±0.2°, 39.5±0.2° and 40.1±0.2°.

In one embodiment, the Fourier transform infrared spectroscopy FT-IR of the crystalline form A of the above compound 3 (2-acetamido-acetamido)-propionic acid comprises the following characteristic peaks: 3315 ± 2 cm ^-1 , 1697 ± 2cm ^-1 , 1670 ± 2cm ^-1 , 1543 ± 2cm ^-1 , 1441 ± 2cm ^-1 , 1376 ± 2cm ^-1 , 1277 ± 2cm ^-1 , 1248 ± 2cm ^-1 , 1144 ± 2cm ^-1 , 1089 ± 2cm ^{- 1} , 1071 ± 2cm ^-1 , 1044 ± 2cm ^-1 , 998 ± 2cm ^-1 , 983 ± 2cm ^-1 , 949 ± 2cm ^-1 , 888 ± 2cm ^-1 , 796 ± 2cm ^-1 , 712 ± 2cm ^-1 , And 666 ± 2cm ^-1 .

In still another embodiment, the differential scanning calorimetry (DSC) of the crystalline form A of the above compound 3 (2-acetamido-acetamido)-propionic acid is at 168 ± 2 ° C and 177 ± 2 ° C. Has a characteristic peak.

According to another object of the present invention, there is provided a method for preparing a crystalline form A of the above compound 3 (2-acetamido-acetamido)-propionic acid, which comprises the following steps: (1). Dissolving 3(2-acetamido-acetamido)-propionic acid in water and then filtering to obtain a filtrate; (2). concentrating between 20 and 100 torr at a temperature of 50 to 70 ° C The filtrate is precipitated to a solid; (3). The pressure is returned to normal pressure, then an alcohol solvent is added, and the temperature is raised to between 60 and 90 ° C to completely dissolve the solid described in the step (2) to form a solution; Cooling the solution to between 20 and 50 ° C, allowing a crystal to precipitate and stirring for at least 8 hours; and (5) filtering the precipitated crystal between 0 and 15 ° C to obtain a compound 3 after a drying procedure Form A of (2-acetamido-acetamido)-propionic acid.

The method for preparing the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid, wherein the alcohol solvent described in the step (3) is selected from the group consisting of methanol, ethanol, and n-propanol. Isopropanol and combinations thereof.

The above preparation method of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid, because the alcohol solvent used is low in toxicity, and the step is easy to handle, and is particularly advantageous for commercialization. Form A of compound 3 (2-acetamido-acetamido)-propionic acid.

The powder X-ray diffraction pattern provided by the present invention uses a Bruker D8 Advanced X-ray diffractometer, and the Cu target wavelength λ is 1.540600 angstroms, the X-ray operating condition is 40Kv, and the range of 40 mA. 2 θ angle analysis is From 5 to 60 degrees.

The data of the Fourier transform infrared spectroscopy FT-IR provided by the present invention is a model using a PerkinElmer Spectrum 100FT-IR, and the measurement range is from 650 to 4000 cm ^-1 .

The differential scanning calorimetry (DSC) provided by the present invention was a Perkinelmer analyzer type differential scanning calorimeter under the conditions of raising the temperature from 40 ° C to 270 ° C at 10 ° C per minute under nitrogen.

Stability of Form A of Compound 3 (2-acetamido-acetamido)-propionic acid.

In one embodiment, the compound 3 (2-acetamido-acetamido) of the present invention is used. - The crystal form A of propionic acid was placed in an oven at 140 ° C for 4 hours in the presence of air, and the powder X-ray diffraction pattern contained the following diffraction peak expressed as 2 θ angle: 10.4 ± 0.2 °, 11.0 ± 0.2°, 12.9±0.2°, 16.0±0.2°, 16.4±0.2°, 17.6±0.2°, 19.1±0.2°, 19.5±0.2°, 20.0±0.2°, 21.2±0.2°, 23.2±0.2°, 23.5± 0.2°, 24.1±0.2°, 24.9±0.2°, 25.3±0.2°, 26.2±0.2°, 30.2±0.2°, 39.5±0.2° and 40.1±0.2°.

According to the above examples, the compound 3 (2-acetamido-acetamido)-propyl The position and intensity of the diffraction peak of the powder X-ray diffraction pattern of the acid form A of the acid did not change, demonstrating that the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid has a high temperature. The thermal stability does not cause the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid to be destroyed during the polishing process because of the high temperature due to friction in the polishing process.

In another embodiment, the compound 3 (2-acetamido-acetamido) of the present invention is used. - The crystal form of propionic acid is stored in a normal temperature and pressure environment for one year, and its powder X-ray diffraction spectrum contains the following diffraction peaks expressed by 2 θ angle: 10.4 ± 0.2 °, 11.0 ± 0.2 °, 12.9 ± 0.2 °,16.0±0.2°, 16.4±0.2°, 17.6±0.2°, 19.1±0.2°, 19.5±0.2°, 20.0±0.2°, 21.2±0.2°, 23.2±0.2 °, 23.5 ± 0.2 °, 24.1 ± 0.2 °, 24.9 ± 0.2 °, 25.3 ± 0.2 °, 26.2 ± 0.2 °, 30.2 ± 0.2 °, 39.5 ± 0.2 ° and 40.1 ± 0.2 °.

According to the above examples, the compound 3 (2-acetamido-acetamido)-propyl The position and intensity of the diffraction peak of the powder X-ray diffraction pattern of the acid form A of the acid did not change, demonstrating the long-term storage of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid. Good stability.

In still another embodiment, according to the above compound 3 (2-acetamido-acetamido) - Preparation method of crystal form A of propionic acid The crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid prepared by the method is subjected to purity analysis by a high pressure liquid chromatography apparatus. The purity of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid is more than 99% (the UV analysis wavelength is 214 nm), and the above-mentioned crystal form A is stored in a normal temperature and normal pressure environment for one year. Purity analysis was carried out using a high pressure liquid chromatography, and the purity of the crystal form A was still more than 99% (the UV analysis wavelength was 214 nm).

A non-therapeutic whitening method according to the present invention, the non-treatment A whitening method for use comprises the use of a formulation of the addition of the compound 3 (2-acetamido-acetamido)-propionic acid Form A on the surface of the skin.

In one embodiment, the non-therapeutic whitening method, wherein the compound Formulation weight percent of 3(2-acetamido-acetamido)-propionic acid Form A in this formulation The formulation of the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid is between 0.1 and 10% by weight, and has a skin whitening effect.

In a preferred embodiment, when the compound 3 (2-acetamido-acetamido)-propyl When the added weight percentage of the acid form A in the formulation is 2% by weight, the in vitro test (Ex-vivo) shows that the use of the formula can reduce melanin production by 30% after 9 days (existing melanin level -30%); The skin spot desalination test (In-vivo) showed that the average spot desaturation of the formulation after 14 days reached 51% (age spot reduction: 51%).

In still another embodiment, wherein the above-mentioned additive compound 3 (2-acetamido-ethyl hydrazine) The formulation of the crystalline form A of the amino)-propionic acid further comprises a derivative of vitamin C selected from the group consisting of vitamin C ethyl ether, vitamin C glycoside, vitamin C phosphate, and combinations thereof.

In another embodiment, wherein the compound 3 (2-acetamido-ethyl hydrazine) is added as described above The form of the formulation of the crystalline form A of the amino)-propionic acid comprises a cream, an emulsion, a gel, a powder, a paste, a masking liquid, and a lotion.

The present invention has significant advantages and advantageous effects as compared with the prior art. By using The examples illustrate that the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid provided by the invention and the preparation method and application thereof have the following advantages and beneficial effects: The crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid provided by the invention has good thermal stability and storage stability.

Next, the present invention provides a method for preparing a crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid, wherein the solvent used in the preparation method is a low toxicity alcohol solvent. Therefore, it is advantageous to commercialize the crystal form A of the compound 3(2-acetamido-acetamido)-propionic acid, and the compound 3 can be analyzed by a high pressure liquid chromatography to have a purity greater than 99% ( Form A of 2-acetamido-acetamido)-propionic acid.

Meanwhile, the present invention provides a non-therapeutic whitening method which can be achieved on the surface of the skin by using a formula A of the addition of the compound 3 (2-acetamido-acetamido)-propionic acid. Skin whitening effect.

In summary, the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid provided by the present invention has good thermal stability and storage stability, and is provided by the present invention. The preparation method of the crystal form A is safe and the steps are easy to operate, which is favorable for commercial production, and can obtain the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid with high purity and reproducibility. It can effectively solve the shortcomings in the current technology and the problems faced by the industry. Meanwhile, the present invention provides a non-therapeutic whitening method by using an additive compound 3 (2-acetamide). Form A of acetyl-acetamido)-propionic acid is formulated on the surface of the skin to achieve skin whitening.

The above description is only an overview of the technical solution of the present invention, in order to be able to understand more clearly. The above and other objects, features, and advantages of the present invention will become more apparent from the aspects of the invention. as follows.

The above and other technical contents, features and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments. In order to thoroughly understand the present invention, detailed steps and compositions thereof will be set forth in the following description. Obviously, the practice of the invention is not limited to the specific details that are apparent to those skilled in the art. On the other hand, well-known components or steps are not described in detail to avoid unnecessarily limiting the invention. The preferred embodiments of the present invention are described in detail below, but the present invention may be widely practiced in other embodiments, and the scope of the present invention is not limited by the scope of the following patents. .

According to an embodiment of the present invention, the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid for inhibiting melanin production, wherein the above-mentioned crystal form A is powder X-ray winding The spectroscopy, as shown in Figure 1, contains the following diffraction peaks expressed as 2 θ angles: 10.4 ± 0.2 °,11.0±0.2°,12.9±0.2°,16.0±0.2°, 16.4±0.2°, 17.6±0.2°, 19.1±0.2°, 19.5±0.2°, 20.0±0.2°, 21.2±0.2°, 23.2±0.2 °, 23.5 ± 0.2 °, 24.1 ± 0.2 °, 24.9 ± 0.2 °, 25.3 ± 0.2 °, 26.2 ± 0.2 °, 30.2 ± 0.2 °, 39.5 ± 0.2 ° and 40.1 ± 0.2 °.

According to an embodiment of the present invention, the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid for inhibiting melanin production, wherein the above-mentioned crystal form A has a Fourier transform infrared spectrum As shown in Fig. 2, the FT-IR includes the following characteristic peaks: 3315 ± 2 cm ^-1 , 1697 ± 2 cm ^-1 , 1670 ± 2 cm ^-1 , 1543 ± 2 cm ^-1 , 1441 ± 2 cm ^-1 , 1376 ± 2 cm ^{- 1} , 1277 ± 2cm ^-1 , 1248 ± 2cm ^-1 , 1144 ± 2cm ^-1 , 1089 ± 2cm ^-1 , 1071 ± 2cm ^-1 , 1044 ± 2cm ^-1 , 998 ± 2cm ^-1 , 983 ± 2cm ^-1 , 949 ± 2 cm ^-1 , 888 ± 2 cm ^-1 , 796 ± 2 cm ^-1 , 712 ± 2 cm ^-1 , and 666 ± 2 cm ^-1 .

According to an embodiment of the present invention, the combination for inhibiting melanin production Form 3 of 2-(2-acetamido-acetamido)-propionic acid, wherein the differential scanning calorimetry (DSC) of the above Form A is shown in Figure 3, which is at 168 ± Characteristic peaks at 2 ° C and 177 ± 2 ° C.

Compound 3 (2-acetamido-acetamido)-propionic acid provided according to the present invention The preparation method of the crystal form A, the preparation method of the above crystal form A comprises the following steps: (1) Dissolving 3(2-acetamido-acetamido)-propionic acid in water, and then filtering to obtain a filtrate; (2). at a temperature of 50 to 70 ° C, a pressure of 20 to The filtrate is concentrated between 100 torr to a solid precipitate; (3). The pressure is returned to normal pressure, then an alcohol solvent is added, and the temperature is raised to between 60 and 90 ° C to completely dissolve the solid described in the step (2) to form a Solution; (4) cooling the solution to between 20 and 50 ° C, allowing a crystal to precipitate and stirring for at least 8 hours; and (5). filtering the precipitated crystal between 0 and 15 ° C for a drying procedure Form A of compound 3 (2-acetamido-acetamido)-propionic acid is obtained.

In the above preparation method, the alcohol solvent described in the step (3) is selected from the group consisting of methanol. Ethanol, n-propanol, isopropanol and combinations thereof. Preferably, the above alcohol solvent is selected from the group consisting of ethanol and isopropanol.

Compound 3 (2-acetamido-acetamido)-propionic acid provided according to the present invention Preparation of Form A The crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid prepared by the method of purity analysis using a high pressure liquid chromatography apparatus, the compound 3 (2- The purity of Form A of acetamino-acetamido)-propionic acid is greater than 99% (UV analysis wavelength is 214 nm).

In one embodiment, the present invention provides the compound 3 (2-acetamido-acetamido) Form A of propionic acid is prepared by using a 95% aqueous solution of ethanol as the alcohol solvent, and the purity of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid prepared is prepared. It was 99.4% (UV analysis wavelength was 214 nm).

In another embodiment, the present invention provides Compound 3 (2-acetamido-acetamide) The crystal form A of propionate is prepared by using isopropanol as the alcohol solvent, and the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid is prepared. The purity was 99.9% (UV analysis wavelength was 214 nm).

In order to enhance the compound 3 (2-acetamido-acetamido) provided by the present invention - The yield of the preparation method of the form A of propionic acid, the saturation solubility of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid in different solvents is necessary to be further studied and explored. .

The method for measuring the saturation solubility of the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid in different solvents is as follows: First, the compound 3 (2-acetamido-acetamido) is prepared. - a supersaturated solution of the form A of propionic acid, followed by a filtration procedure, and the obtained filtrate is quantitatively analyzed by a high pressure liquid chromatography to obtain a compound 3 (2-acetamido-acetamido)-propyl The saturation solubility of acid Form A in various solvents at room temperature (24 to 26 ° C).

In one embodiment, the above-mentioned compound A of the compound 3 (2-acetamido-acetamido)-propionic acid for inhibiting melanin production is a mixed solvent of ethanol and water (95% by weight of ethanol). The saturated solubility in the solution was 18.4 mg/g (24 to 26 ° C).

In another embodiment, the above-mentioned saturated form of the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid for inhibiting melanin production in methanol is 26.3 mg/g (24~ 26 ° C).

In still another embodiment, the above-mentioned crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid for inhibiting melanin production has a saturated solubility in water of 177 mg/g (24 to 26 ° C). ).

According to the present invention, a non-therapeutic whitening method comprising the use of a compound 3 (2-acetamido-acetamido)-propionic acid Type A is formulated on the surface of the skin.

In one embodiment, the above compound 3 (2-acetamido-acetamido)-propyl The crystal form A of the acid is added in the formula in a range of 0.1 to 10% by weight, and the above-mentioned addition of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid The formula has a skin whitening effect.

In a preferred embodiment, the above compound 3 (2-acetamido-acetamido) - When the added weight percentage of propionic acid Form A in this formulation is 2% by weight, the in vitro test (ex-vivo) shows that using this formula can reduce melanin production by 30% after 9 days (existing melanin level-30%) The skin spotting test (in-vivo) showed that the spots were faded by 51% after 14 days (age spot reduction: 51%).

In one embodiment, the above-mentioned addition compound 3 (2-acetamido-acetamido) The formulation of Form A of propionic acid further comprises a derivative of vitamin C selected from the group consisting of vitamin C ethyl ether, vitamin C glycoside, vitamin C phosphate, and combinations thereof.

In another embodiment, the above-mentioned addition compound 3 (2-acetamido-acetamido) The form of the formulation of the form A of propionic acid comprises a cream, an emulsion, a gel, a powder, a paste, a mask liquid, and a lotion.

Example 1. Preparation of 3(2-acetamido-acetamido)-propionic acid

Acetylglycine (Ac-Gly-OH; 3.8 g) and triethylamine (7 ml;) were dissolved in 160 ml of tetrahydrofuran (THF), cooled to -10 ° C, and then added with chloroformic acid. Butyl ester (isobutylchloroformate; 5.5 g). The mixed solution was stirred at -10 ° C for 1 hour. Further, β-alanine benzyl p-toluenesulfonate (H-β-Ala-OBzl.PTSA; 11.5 g) and triethylamine (3 ml;) were dissolved in 160 ml of tetrahydrofuran (THF). Thereafter, it was added to the above mixed acid anhydride solution. The reaction mixture was stirred at room temperature overnight. The salt was filtered and dried under vacuum to remove THF. The residue was purified by column chromatography, using ethyl acetate and heptane as eluent to afford about 5.9 g of intermediate (Ac-Gly-beta-Ala-OBzl).

The intermediate was dissolved in 200 ml of tetrahydrofuran, a palladium carbon catalyst (10 wt% Pd/C) was added, and the mixture was stirred overnight under a hydrogen atmosphere. Then, the catalyst was removed by filtration, and the filtrate was evaporated in vacuo to give about 2.5 g of white powder (yield 41.0%, purity > 95%). The obtained compound was analyzed by a hydrogen-nuclear magnetic resonance spectrometer to obtain an H ¹ -NMR spectrum (1.84, s , 3H; 2.35-2.38, t , 2H; 3.27-3.31, m , 2H; 3.61, d , 2H; 7.85-7.87). , t , 1H; 8.03-8.05, t , 1H; 12.22, s , 1H), identified as 3(2-acetamido-acetamido)-propionic acid, the above white powder was re-isolated with isopropanol Crystallization, heating and dissolving the white powder, cooling to crystal precipitation, holding the crystal at room temperature for 2 hours, filtering the precipitated crystals, and subjecting the precipitated crystals to a vacuum drying procedure to obtain 3 (2-acetamido-ethylamine) - White powder crystal of propionic acid (1.67 g), and the purity of the white powder crystal was analyzed by a high pressure liquid chromatography to have a purity of 98.4% (UV analysis wavelength was 214 nm).

3(2-Ethylamino-acetamido)-propionic acid prepared in the above Example 1 The powder X-ray diffraction pattern of the white powder crystals contains the following diffraction peaks expressed as 2 θ angles: 9.3 ± 0.2°, 15.1 ± 0.2°, 16.5 ± 0.2°, 21.3 ± 0.2°, 22.4 ± 0.2°, 23.2 ± 0.2°, 24.6±0.2°, 25.2±0.2°, 26.1±0.2°, 28.6±0.2°, 30.4±0.2°, 31.0±0.2°, 32.1±0.2°, 38.4±0.2°, 40.0±0.2° and 41.6± 0.2°. Its powder X-ray diffraction pattern is shown in Figure 5.

The 3(2-acetamido-acetamido)-propionic acid prepared in Example 1 was prepared. The white powder crystals were placed in an oven at 140 ° C for 4 hours in the presence of air, and then subjected to powder X-ray diffraction analysis, and the powder X-ray diffraction pattern was as shown in Fig. 6. Further analysis and comparison of Fig. 5 and Fig. 6 clearly show the change of the intensity of the diffraction peak and the position of the 2θ angle in the X-ray diffraction pattern of the powder, which clearly indicates the 3(2-acetamidine) prepared in the first example. The crystalline form of the white powder crystal of amino-acetamido)-propionic acid is not thermally stable.

Example 2 Preparation of crystal form A of 3(2-acetamido-acetamido)-propionic acid

According to the procedure described in Example 1, a white powder (20 g) of 3(2-acetamido-acetamido)-propionic acid was first prepared, and the white powder was dissolved in water (100 g), and the insoluble matter was filtered to obtain clarification. The filtrate was concentrated at 65 ° C with a reduced pressure concentrator until the solid was precipitated. After returning to normal pressure, isopropanol (200 g) and water (20 g) were added, and the temperature was raised to 70 ° C to dissolve the precipitated solid to form a solution, followed by cooling. However, the solution was allowed to precipitate at 45 ° C, and stirred under temperature for 12 hours, and then cooled to 5 ° C, and the precipitated crystals were filtered; the precipitated crystals were vacuum dried to obtain 3 (2-acetamido-acetamido group). The white crystalline solid of propionic acid (16.7 g) was analyzed by a high-pressure liquid chromatography to a purity of 99.9% (UV analysis wavelength: 214 nm).

Example 3 Preparation of crystal form A of 3(2-acetamido-acetamido)-propionic acid

According to the procedure described in Example 1, first, 3 (2-acetamido-acetamido) was prepared. a white powder of propionic acid (20 g), the white powder was dissolved in water (100 g), and the insoluble material was filtered to obtain a clear filtrate, and the filtrate was concentrated to a solid at 65 ° C using a vacuum concentrator, and returned to normal pressure. After adding isopropanol (200 g), a solid solution was dissolved by refluxing to form a solution, and then the solution was cooled to 45 ° C to precipitate crystals, and stirred under temperature for 10 hours, cooled to 10 ° C, and the precipitated crystals were filtered; The precipitated crystals were dried under vacuum to give a white crystalline solid (17.5 g) of 3 (2-ethylamino-ethylamino)-propionic acid, and the purity of the white crystalline solid was analyzed by high-pressure liquid chromatography. Its purity was 99.2% (UV analysis wavelength was 214 nm).

Example 4 Preparation of crystal form A of 3(2-acetamido-acetamido)-propionic acid

According to the procedure described in Example 1, first, 3 (2-acetamido-acetamido) was prepared. a white powder of propionic acid (3 g), the white powder was dissolved in water (100 g), and the insoluble matter was filtered to obtain a clear filtrate, and the filtrate was concentrated to a solid at 65 ° C using a vacuum concentrator, and returned to normal pressure. After adding ethanol (14.25 g) and water (0.75 g), the solid was precipitated by heating to 75 ° C to form a solution, and then the solution was cooled to 35 ° C to precipitate crystals, and stirred at a temperature for 12 hours, and then cooled to 10 ° C. filter The precipitated crystals were subjected to vacuum drying to obtain a white crystalline solid (2.4 g) of 3(2-acetamido-acetamido)-propionic acid, which was analyzed by high-pressure liquid chromatography. The purity of the crystalline solid was 99.4% (UV analysis wavelength was 214 nm).

Example 2, 3 and 4 give 3(2-acetamido-acetamido)-propionic acid The powder X-ray diffraction pattern of the white crystalline solid contains the following diffraction peaks expressed as 2 θ angles: 10.4 ± 0.2 °, 11.0 ± 0.2 °, 12.9 ± 0.2 °, 16.0 ± 0.2 °, 16.4 ± 0.2 °, 17.6 ± 0.2 °, 19.1±0.2°, 19.5±0.2°, 20.0±0.2°, 21.2±0.2°, 23.2±0.2°, 23.5±0.2°, 24.1±0.2°, 24.9±0.2°, 25.3±0.2°, 26.2±0.2 °, 30.2 ± 0.2 °, 39.5 ± 0.2 ° and 40.1 ± 0.2 °.

The Fourier-converted infrared spectroscopy FT-IR of the white crystalline solid of 3 (2-acetamido-acetamido)-propionic acid obtained in Examples 2, 3 and 4 contains the following characteristic peaks: 3315 ± 2 cm ^-1 , 1697 ±2cm ^-1 , 1670 ± 2cm ^-1 , 1543 ± 2cm ^-1 , 1441 ± 2cm ^-1 , 1376 ± 2cm ^-1 , 1277 ± 2cm ^-1 , 1248 ± 2cm ^-1 , 1144 ± 2cm ^-1 , 1089 ± 2cm ^-1 , 1071 ± 2cm ^-1 , 1044 ± 2cm ^-1 , 998 ± 2cm ^-1 , 983 ± 2cm ^-1 , 949 ± 2cm ^-1 , 888 ± 2cm ^-1 , 796 ± 2cm ^-1 , 712 ± 2cm ^-1 , and 666 ± 2cm ^-1 .

According to the above example two, three and four obtained 3 (2-acetamido-acetamide) The powder X-ray diffraction pattern of the white crystalline solid of propionate and the FT-IR data of Fourier transform infrared spectroscopy confirmed that the white crystalline solid of 3(2-acetamido-acetamido)-propionic acid was Chemical Form 3 of 2-(2-acetamido-acetamido)-propionic acid.

Whitening efficacy test: Melaninogenesis inhibition test: Injecting mouse melanoma metastasis cells (Mus musculus skin melanoma; B16-F10) into a 96-well plate, so that each well contains 5000 Cells (5000 cells/well) were cultured in 5% CO ₂ and 10% fetal bovine serum (Fetal Bovine Serum Dulbecco's modified Eagle's medium; FBS DMEM) at 37 °C. The background control group (negative control; negative control group) contains 1 μM melatonin (α-MSH, α-melanocyte stimulating hormone), the blank control group does not contain α-MSH, the test sample solution first 5 wt% of compound 3 (2 Form A of acetaminos-acetamido)-propionic acid is added to phenol red free DMEM and is prepared to contain 0.1 mg/ml tyrosine and 1 μM melatonin. 5wt% FBS medium (5wt% FBS DMEM), filtered by 0.22μm screening program (because 0.1mg/ml tyrosine will cause supersaturation of the culture solution, so centrifugal filtration is required), prepared in the culture solution The sample is tested to obtain a test sample solution. After the cells have completely confluence, test sample solutions or reference samples (blank control group and background control group) at concentrations of 625 ppm, 1250 ppm, 2500 ppm, and 5000 ppm are added to each well, and after treatment for 3 days, 100 μL of 1N is added. Sodium hydroxide (NaOH), after shaking for 10 minutes, observe the color of the test sample solution. As shown in Fig. 7, when the concentration of the test sample solution was 2500 ppm and 5000 ppm, there was a remarkable effect of suppressing melanin production.

Melanin inhibition test (Ex-vivo)

In this test, artificially cultured epidermal tissue is prepared by coculturing human epidermal cells and melanocytes in a cell culture dish, wherein compound 3 (2-acetamidoamine- The formulation of the form A of acetaminos-propionic acid was 2% by weight for the melanin inhibition test, and the results showed that the use of the formulation reduced the melanin production by 30% after 9 days (existing melanin level -30%). From the cross-sectional view of the epidermal tissue, it is clearly observed that the deposition of melanin is significantly less, as shown in Fig. 8.

Fade skin spots Test (In-vivo age spot reducing study )

The test was conducted on 10 healthy volunteers aged 25 to 50 years, and the compound 3 (2-acetamido-acetamido)-propionic acid was continuously used on local spots on the volunteer's face. The amount of Form A added was 2% by weight of the formulation for 14 days, and the degree of speckle desalination was analyzed by a skin analyzer to an average of 51% of the degree of speckle desalination, and the human test effect is shown in FIG.

Hereinafter, the use of the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid according to the present invention will be described using a formulation example, but the present invention is not limited to the formulation examples.

Formulation 1: A lotion of Compound 3 (2-acetamido-acetamido)-propionic acid Form A and Vitamin C ethyl ether was added.

Lotion

In a hybrid manner, existing conventional methods can be used, such as first in A The components are uniformly mixed, and the components in B are separately added. After stirring, the components of C which have been previously mixed are added to the mixture of A and B, and stirred uniformly.

Formulation 2, adding the emulsion of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid

Lotion (lotion)

In a hybrid manner, existing conventional methods can be used, such as A and B first. The components in the mixture were respectively heated to 80 ° C, stirred evenly, and then the mixture of the components of B was added to the mixture of the components of A, stirred for 5 minutes, then cooled, neutralized by adding sodium hydroxide, and the temperature was lowered to 45 ° C. Add the ingredients in C one by one and mix well.

Formulation III, adding compound 3 (2-acetamido-acetamido)-propionic acid crystal Type A mask fluid

Mask liquid

First, the components of A are glycerin polymethacrylate, propylene glycol and The PVM/MA copolymer and the PVM/MA decadiene cross-linked polymer were added to pure water, and after standing for 20 to 30 minutes, they were uniformly shaken until completely dissolved. Then, the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid is added to the component which is finally added to C, and uniformly mixed to prepare a mask liquid.

The above description is only a preferred embodiment of the present invention and is not intended to be an invention. The present invention has been disclosed in the above preferred embodiments, but is not intended to limit the present invention, and any person skilled in the art can leave without departing from the scope of the present invention. In the following, when the method and the technical content disclosed above are used to make some modifications or modifications to the equivalent embodiment, the present embodiment does not depart from the technical solution of the present invention, and the above embodiments are made according to the technical essence of the present invention. Any simple modifications, equivalent changes and modifications are still within the scope of the technical solutions of the present invention.

Figure 1 is a powder X-ray diffraction pattern of the crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid of the present invention; and Fig. 2 is a compound 3 (2-B) of the present invention. Fourier transform infrared spectroscopy FT-IR spectrum of crystalline form A of guanamine-acetamido)-propionic acid; Fig. 3 is a compound of the invention 3 (2-acetamido-acetamido)-propionic acid Differential Scanning Thermal Analysis Pattern (DSC) of Form A; Figure 4 is a plot of Compound 3 (2-acetamido-acetamido)-propionic acid Form A of the present invention placed in an oven at 140 ° C Powder X-ray diffraction pattern after heating for 4 hours in the presence of air; Figure 5 is a white powder crystal of compound 3 (2-acetamido-acetamido)-propionic acid prepared in Example 1. Powder X-ray diffraction pattern; Figure 6 is a white powder crystal of compound 3 (2-acetamido-acetamido)-propionic acid prepared in Example 1, placed in an oven at 140 ° C in the presence of air Powder X-ray diffraction pattern after heating for 4 hours; Figure 7 is a graph of inhibition of melanin production of Form A of Compound 3 (2-acetamido-acetamido)-propionic acid of the present invention (In vitro) Effect diagram; Figure 8 is the combination of the present invention Form 3 of 2-(2-acetamido-acetamido)-propionic acid was added in an amount of 2% by weight of the formulation after 9 days of the melanin inhibition test (Ex-vivo) on the cultured epidermal tissue. Cross-sectional view of epidermal tissue; and Figure 9 is a formula of the compound 3 (2-acetamido-acetamido)-propionic acid of the present invention added in an amount of 2 wt% for skin on human skin Effect map after 14 days of spot intensification test (In vivo).

Claims (12)

Form A of compound 3 (2-acetamido-acetamido)-propionic acid, powder X of crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid The light diffraction pattern contains the following diffraction peaks expressed as 2 θ angles: 10.4 ± 0.2 °, 11.0 ± 0.2 °, 12.9 ± 0.2 °, 16.0 ± 0.2 °, 16.4 ± 0.2 °, 17.6 ± 0.2 °, 19.1 ± 0.2 ° , 19.5±0.2°, 20.0±0.2°, 21.2±0.2°, 23.2±0.2°, 23.5±0.2°, 24.1±0.2°, 24.9±0.2°, 25.3±0.2°, 26.2±0.2°, 30.2±0.2° , 39.5 ± 0.2 ° and 40.1 ± 0.2 °. A crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid as described in claim 1, wherein the powder X-ray diffraction pattern of the above-mentioned crystalline form A is as shown in FIG. Shown. The crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid according to claim 1, wherein the FT-IR of the above-mentioned crystal form A includes the following Characteristic peaks: 3315±2cm ^-1 , 1697±2cm ^-1 , 1670±2cm ^-1 , 1543±2cm ^-1 , 1441±2cm ^-1 , 1376±2cm ^-1 , 1277±2cm ^-1 , 1248±2cm ^-1 , 1144±2cm ^-1 , 1089±2cm ^-1 , 1071±2cm ^-1 , 1044±2cm ^-1 , 998±2cm ^-1 , 983±2cm ^-1 , 949±2cm ^-1 , 888±2cm ^-1 ,796 ±2 cm ^-1 , 712 ± 2 cm ^-1 , and 666 ± 2 cm ^-1 . The crystal form A of the compound 3 (2-acetamido-acetamido)-propionic acid according to claim 1, wherein the Fourier transform infrared spectrum of the above crystal form A is as shown in Fig. 2 Show. Form A of the compound 3 (2-acetamido-acetamido)-propionic acid as described in claim 1, wherein the differential scanning calorimetry (DSC) of the above crystalline form A is 168±2°C and 177±2°C. It has characteristic peaks. A process for preparing a crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid according to any one of claims 1 to 5, which is a form A The preparation method comprises the following steps: (1) dissolving 3(2-acetamido-acetamido)-propionic acid in water, and then filtering to obtain a filtrate; (2) at a temperature between 50 and 70 ° C. , the pressure is concentrated between 20 and 100 torr to precipitate a solid; (3). The pressure is returned to normal pressure, then an alcohol solvent is added, and the temperature is raised to between 60 and 90 ° C to make the solid described in the step (2). Completely dissolved to form a solution; (4) cooling the solution to between 20 and 50 ° C to precipitate a crystal and stirring for at least 8 hours; and (5). filtering the precipitated crystal between 0 and 15 ° C, Form A of compound 3 (2-acetamido-acetamido)-propionic acid was obtained after a drying procedure. The method for preparing the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid according to claim 6, wherein the alcohol solvent is selected from the group consisting of methanol, ethanol, and positive Propanol, isopropanol and combinations thereof. The method for preparing the crystalline form A of the compound 3 (2-acetamido-acetamido)-propionic acid according to claim 6, wherein the compound 3 obtained after the drying process is described above (2-B) The purity of Form A of the amide-acetamido)-propionic acid is greater than 99%. A non-therapeutic whitening method comprising the use of a compound 3 (2-acetamido-acetamido) as described in any one of claims 1 to 5 - The formulation of Form A of propionic acid is on the surface of the skin. The non-therapeutic whitening method according to any one of claims 1 to 5, wherein the compound 3 (2-acetamido-ethylammonium) according to any one of claims 1 to 5 The addition weight percent of Form A of propionic acid in the formulation ranges from 0.1 to 10% by weight. The non-therapeutic whitening method according to claim 9, wherein the compound 3 (2-acetamido-acetamide) according to any one of claims 1 to 5 is added. The formulation of Form A of propionic acid further comprises a derivative of vitamin C selected from the group consisting of vitamin C ethyl ether, vitamin C glycoside, vitamin C phosphate, and combinations thereof. The non-therapeutic whitening method according to claim 9, wherein the compound 3 (2-acetamido-acetamide) according to any one of claims 1 to 5 is added. The form of the formulation of Form A of propionic acid comprises a cream, an emulsion, a gel, a powder, a paste, a masking liquid, and a lotion.