Aminothiourea tyrosinase inhibitor and preparation method and application thereof

Abstract

The application relates to the technical field of fine chemicals, in particular to an aminothiourea tyrosinase inhibitor and a preparation method and application thereof. The application discloses a brand-new aminothiourea compound which can be used as a tyrosinase inhibitor, has good tyrosinase inhibition activity and a melanin generation inhibition effect, and can be used as a novel whitening agent. The aminothiourea derivative provided by the application has good water solubility, a wide application range, low toxicity to human normal cells and high safety. Moreover, the preparation method is simple, the synthesis route is short, the aminothiourea derivative is easy to mass-produce, and the price is low.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fine chemical technology, and particularly relates to an aminothiourea tyrosinase inhibitor and a preparation method and application thereof. BACKGROUND

[0002] In daily life, the human epidermis inevitably contacts with ultraviolet rays. Under the irradiation of ultraviolet rays, in order to avoid the damage of the skin by ultraviolet rays, the melanocytes in the basal layer of the epidermis produce melanin to protect the skin tissue. However, when the local melanin production of the skin is excessive or unevenly distributed, various pigmentary diseases such as chloasma, freckle, seborrheic keratosis and post-inflammatory hyperpigmentation occur, which affect the appearance. Relevant research shows that 75% of Chinese women and 60% of Japanese women hope to have fair skin. With the development of economy and the improvement of living standards, women pay more and more attention to the beauty of their own skin. Due to the influence of age, natural aging and environmental factors, excessive melanin in the epidermis makes the skin dark and brings various beauty problems. In order to pursue the quality of life and adapt to social needs, women use various brands of whitening cosmetics to achieve the purpose of whitening the skin.

[0003] Tyrosinase (EC 1.14.18.1) is a complex oxidoreductase containing binuclear copper ion active center, which is distributed in microorganisms, animals and plants. Tyrosinase is the main rate-limiting enzyme in the process of melanin formation. It first exerts its monophenolase activity to catalyze the hydroxylation of tyrosine in melanocytes to dopa. Then, tyrosinase exerts its diphenolase activity to oxidize dopa to dopaquinone. Dopaquinone generates melanin through a series of biochemical reactions. Therefore, by inhibiting tyrosinase, the production of melanin can be effectively reduced. The common tyrosinase inhibitor kojic acid is questioned by many countries due to its unstable nature, certain irritation and allergenicity. Arbutin, which is used in clinical, is not widely used due to its upper concentration limit, poor effect when used alone, instability to light, oxygen and pH, and other shortcomings.

[0004] Therefore, how to develop an efficient and safe whitening agent is still a technical problem to be solved by those skilled in the art. SUMMARY

[0005] In order to solve the above technical problems in the prior art, the present application provides an aminothiourea tyrosinase inhibitor and a preparation method and application thereof, which are as follows.

[0006] An aminothiourea tyrosinase inhibitor has the structure shown in the following general formula (I):

[0007]

[0008] Further, R in the general formula I is any one or more of hydrogen, halogen, C1-C5 alkyl, C1-C5 alkoxy, nitro, amino, hydroxyl, thiol, sulfonamide.

[0009] Exemplarily, the structural formula of the aminothiourea tyrosinase inhibitor includes:

[0010]

[0011] The specific names of the structural formula of the aminothiourea tyrosinase inhibitor are respectively: 2-(4-methoxybenzyl)hydrazine-1-thioamide, 2-(4-methylbenzyl)hydrazine-1-thioamide, 2-(2-fluorobenzyl)hydrazine-1-thioamide, 2-(4-bromobenzyl)hydrazine-1-thioamide, 2-(5-bromo-2-hydroxybenzyl)hydrazine-1-thioamide, 2-(2-chlorobenzyl)hydrazine-1-thioamide, 2-(3-fluorobenzyl)hydrazine-1-thioamide, 2-benzylhydrazine-1-thioamide, 2-(2-hydroxybenzyl)hydrazine-1-thioamide, 2-(3,5-dichloro-2-hydroxybenzyl)hydrazine-1-thioamide, 2-(2-hydroxy-5-methylbenzyl)hydrazine-1-thioamide, 2-(2-hydroxy-5-nitrobenzyl)hydrazine-1-thioamide, 2-(5-chloro-2-hydroxybenzyl)hydrazine-1-thioamide.

[0012] The aminothiourea tyrosinase inhibitor is used for preparing a medicine.

[0013] The aminothiourea tyrosinase inhibitor is used for preparing a whitening product.

[0014] The preparation method of the aminothiourea tyrosinase inhibitor includes the following steps: adding different substituted benzaldehyde and aminothiourea into an ethanol solution, then refluxing at 80℃ for 4 hours; after monitoring the completion of the reaction, cooling the reaction solution to room temperature, adding sodium borohydride into the solution in three times, continuing to reflux at 80℃ for 6 hours, after the completion of the reaction, diluting the reaction solution with water, extracting with ethyl acetate, collecting the organic layer, and concentrating the organic layer under reduced pressure to obtain a crude product; and purifying the crude product by column chromatography with petroleum ether: ethyl acetate = 2:1 to obtain the target compound, i.e. the aminothiourea tyrosinase inhibitor.

[0015] Further, the different substituted benzaldehyde is specifically the benzaldehyde substituted by R in the general formula I.

[0016] Further, the ratio of the substituted benzaldehyde, aminothiourea, acetic acid, sodium borohydride and ethanol is 4mmol:4.4mmol:20μL:40mmol:20mL.

[0017] The specific synthesis preparation route is as follows:

[0018]

[0019] Compared with the prior art, the technical effects created by the application are embodied in:

[0020] 1. The application discloses a novel aminothiourea compound which can be used as a tyrosinase inhibitor, has good tyrosinase inhibiting activity and melanin generation inhibiting effect, and can be used as a novel whitening agent.

[0021] 2. The aminothiourea derivative provided by the application has good water solubility and a wide application range.

[0022] 3. The preparation method of the aminothiourea derivative provided by the application is simple, the synthesis route is short, the aminothiourea derivative is easy to prepare in large quantities, and the aminothiourea derivative is low in price.

[0023] 4. The aminothiourea tyrosinase inhibitor provided by the application has low toxicity to human normal cells and is high in safety. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a preparation route map of the aminothiourea tyrosinase inhibitor.

[0025] Figure 2 It is a water solubility comparison chart; wherein, (A) is an HPLC chromatographic peak chart of compound 6; (B) is an HPLC chromatographic peak chart of an intermediate before reduction of a double bond of compound 6.

[0026] Figure 3 It is a zebrafish melanin generation inhibition chart of compound 6; wherein, (A) is a front view of zebrafish at different concentrations; (B) is a side view of zebrafish at different concentrations.

[0027] Figure 4 It is a nuclear magnetic resonance hydrogen spectrum chart of compound 6.

[0028] Figure 5 It is a nuclear magnetic resonance carbon spectrum chart of compound 6.

[0029] Figure 6 It is a high-resolution mass spectrum chart of compound 6. DETAILED DESCRIPTION

[0030] The technical solutions of the application will be further limited in combination with specific implementation manners, but the scope of protection is not limited to the description.

[0031] Example 1:

[0032] Preparation of 2-(4-methoxybenzyl)hydrazine-1-thioamide (compound 1)

[0033] The structural formula of compound 1 is as follows:

[0034]

[0035] The specific preparation steps are as follows:

[0036] Step: 4-methoxybenzaldehyde and thiosemicarbazide were added into an ethanol solution, and refluxed at 80°C for 4 hours, during which acetic acid was slowly added dropwise. After monitoring the completion of the reaction, the reaction solution was cooled to room temperature, sodium borohydride was added to the solution, and refluxed at 80°C for 6 hours. After the reaction was completed, water was added to dilute the reaction solution, and the organic layer was collected after extraction with ethyl acetate, and the organic layer was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography with petroleum ether: ethyl acetate = 2: 1 to obtain the target compound 1.

[0037] Compound 1 is a light yellow solid, with a yield of 10%, and a melting point of 125-129°C; and the nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and high resolution mass spectrum data of compound 1 are as follows:

[0038] 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 7.43 (s, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.11 (s, 1H), 6.83 (d, J = 8.0 Hz, 2H), 5.15 (s, 1H), 3.69 (s, 5H).

[0039] 13 C NMR (100 MHz, DMSO-d6) δ 181.42, 159.06, 130.50, 130.31, 114.13, 55.59, 54.32.

[0040] HRMS (ESI) calcd for [M+Na] + C9H 13 N3OSNa + : 234.0672, found: 234.0671.

[0041] The preparation method of the following examples is similar to that of Example 1, and the raw material ratio used is the same as that of Example 1, except that the 4-methoxybenzaldehyde in Example 1 is replaced by other corresponding substituted benzaldehyde.

[0042] Example 2:

[0043] Preparation of 2-(4-methylbenzyl)hydrazine-1-thioamide (compound 2)

[0044] Compared with Example 1, the difference is that only 4-methoxybenzaldehyde is replaced by 4-methylbenzaldehyde.

[0045] The structural formula of compound 2 is shown as follows:

[0046]

[0047] Compound 2 is a white solid, the yield is 10%, the melting point is 152-157°C; and the data of the nuclear magnetic hydrogen spectrum, the nuclear magnetic carbon spectrum and the high resolution mass spectrum of compound 2 are shown as follows:

[0048] 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 7.42 (s, 1H), 7.21 (d, J = 6.8 Hz, 2H), 7.08 (d, J = 7.2 Hz, 3H), 5.20 (t, J = 5.2 Hz, 1H), 3.75 (d, J = 5.2 Hz, 2H), 2.24 (s, 3H);

[0049] 13 C NMR (100 MHz, DMSO-d6) δ 181.43, 136.68, 135.37, 129.24, 129.18, 54.62, 21.22;

[0050] HRMS (ESI) calcd for [M-H] - C9H 12 N3S - : 196.0903, found: 196.0916.

[0051] Example 3:

[0052] Preparation of 2-(2-fluorobenzyl)hydrazine-1-carbothioamide (compound 3)

[0053] The difference between Example 1 and Example 3 is that only 4-methoxybenzaldehyde is replaced by 2-fluorobenzaldehyde.

[0054] The structural formula of compound 3 is shown as follows:

[0055]

[0056] Compound 3 is a light yellow solid, the yield is 17%, the melting point is 100-104°C; and the data of the nuclear magnetic hydrogen spectrum, the nuclear magnetic carbon spectrum and the high resolution mass spectrum of compound 3 are shown as follows:

[0057] 1 H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 7.46-7.42 (m, 2H), 7.31-7.25 (m, 1H), 7.13-7.08 (m, 3H), 5.31 (t, J = 5.2 Hz, 1H), 3.85 (d, J = 5.2 Hz, 2H).

[0058] 13 C NMR (100 MHz, DMSO-d6) δ 181.58, 162.45, 160.02 (d, 1C, J = 243 Hz, 1 J CF ), 131.72, 129.80, 129.73 (d, 1C, J = 7 Hz, 3 J CF ), 125.26, 125.11 (d, 1C, J = 15 Hz, 2 J CF ), 124.70, 115.67, 115.45 (d, 1C, J = 22 Hz, 2 J CF ), 48.04;

[0059] HRMS (ESI) calcd for [M-H] - C8H9FN3S - : 200.0652, found: 200.0659.

[0060] Example 4:

[0061] Preparation of 2-(4-bromobenzyl)hydrazine-1-thioamide (Compound 4)

[0062] The difference between Example 1 and this example is that only 4-methoxybenzaldehyde is replaced by 4-bromobenzaldehyde.

[0063] The structural formula of Compound 4 is shown below:

[0064]

[0065] Compound 4 is a white solid with a yield of 12% and a melting point of 163-167 °C. The data of the hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum and high resolution mass spectrum of Compound 4 are shown below:

[0066] 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 7.46 (d, J = 8.4 Hz, 3H), 7.31 (d, J = 8.4 Hz, 2H), 7.14 (s, 1H), 5.32 (t, J = 5.0 Hz, 1H), 3.77 (d, J = 4.9 Hz, 2H);

[0067] 13 C NMR (100 MHz, DMSO-d6) δ 181.46, 138.03, 131.49, 120.74, 54.08;

[0068] HRMS (ESI) calcd for [M-H] - C8H9BrN3S - :261.9832, found:261.9840.

[0069] Example 5:

[0070] Preparation of 2-(5-bromo-2-hydroxybenzyl)hydrazine-1-thioamide (Compound 5)

[0071] The difference between Example 1 and this example is that only 4-methoxybenzaldehyde is replaced by 5-bromo-2-hydroxybenzaldehyde.

[0072] The structural formula of Compound 5 is shown below:

[0073]

[0074] wherein Compound 5 is a light yellow solid with a yield of 11%; melting point: 180-184°C; and the data of the nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and high resolution mass spectrum of Compound 5 are shown below:

[0075] 1 H NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H), 8.61 (s, 1H), 7.52 (s, 1H), 7.35 (d, J = 2.6 Hz, 1H), 7.27 (s, 1H), 7.20 (dd, J = 8.4, 2.4 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 5.11 (s, 1H), 3.75 (d, J = 5.6 Hz, 2H);

[0076] 13 C NMR (100 MHz, DMSO-d6) δ 181.17, 155.34, 132.78, 131.16, 127.15, 117.48, 110.44, 49.36;

[0077] HRMS (ESI) calcd for [M-H] - C8H9BrN3OS - :277.9781, found:277.9790. Example 6:

[0078] Preparation of 2-(2-chlorobenzyl)hydrazine-1-thioamide (Compound 6)

[0079] The difference between Example 1 and this example is that only 4-methoxybenzaldehyde is replaced by 2-chlorobenzaldehyde.

[0080] The structural formula of Compound 6 is shown below:

[0081]

[0082] Compound 6 was a light yellow solid with a yield of 13%; melting point: 120-125 °C; and the data of the nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and high resolution mass spectrum of Compound 6 were as follows:

[0083] 1 H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 7.48-7.46 (m, 2H), 7.39-7.36 (m, 1H), 7.28-7.25 (m, 2H), 7.10 (s, 1H), 5.35 (s, 1H), 3.92 (d, J = 4.4 Hz, 2H);

[0084] 13 C NMR (100 MHz, DMSO-d6) δ 181.21, 135.64, 133.56, 131.40, 129.66, 129.52, 127.59, 52.02;

[0085] HRMS (ESI) calcd for [M-H] - C8H9ClN3S - : 216.0357, found: 216.0369.

[0086] Example 7:

[0087] Preparation of 2-(3-fluorobenzyl)hydrazine-1-carbothioamide (Compound 7)

[0088] The difference between Example 1 and Example 7 was that only 4-methoxybenzaldehyde was replaced by 3-fluorobenzaldehyde.

[0089] The structural formula of Compound 7 was as follows:

[0090]

[0091] Compound 7 was a white solid with a yield of 12%; melting point: 107-112 °C; and the data of the nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and high resolution mass spectrum of Compound 7 were as follows:

[0092] 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 7.33-6.99 (m, 6H), 5.35 (t, J = 5.1 Hz, 1H), 3.83 (d, J = 4.8 Hz, 2H);

[0093] 13C NMR (100 MHz, DMSO-d6) δ 181.05, 163.91, 161.49 (d, 1C, J = 242 Hz, 1 J CF ), 141.63, 141.56 (d, 1C, J = 7 Hz, 3 J CF ), 130.47, 130.39 (d, 1C, J = 8 Hz, 3 J CF ), 125.22, 125.19 (d, 1C, J = 3 Hz, 4 J CF ), 115.89, 115.68 (d, 1C, J = 21 Hz, 2 J CF ), 114.47, 114.26 (d, 1C, J = 21 Hz, 2 J CF ), 54.08;

[0094] HRMS (ESI) calcd for [M-H] - C8H9FN3S - : 200.0652, found: 200.0662.

[0095] Example 8:

[0096] Preparation of 2-benzylhydrazine-1-thioamide (Compound 8)

[0097] The difference between Example 1 and this example is that only 4-methoxybenzaldehyde is replaced by benzaldehyde.

[0098] The structural formula of Compound 8 is shown below:

[0099]

[0100] Compound 8 is a light yellow solid with a yield of 16%, a melting point of 145-150 °C, and the data of its 1H NMR, 13C NMR and high resolution mass spectrum are shown below:

[0101] 1 H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 1H), 7.44 (s, 1H), 7.36 - 7.19 (m, 5H), 7.14 (s, 1H), 5.26 (t, J = 5.4 Hz, 1H), 3.81 (d, J = 5.2 Hz, 2H).

[0102] 13C NMR (100 MHz, DMSO-d6) δ 181.51, 156.12, 130.72, 128.81, 124.29, 119.34, 115.65, 50.32;

[0103] HRMS (ESI) calcd for [M-H] - C8H 10 N3S - : 182.0746, found: 182.0755.

[0104] Example 9:

[0105] Preparation of 2-(2-hydroxybenzyl)hydrazine-1-thioamide (Compound 9)

[0106] The difference between Example 1 and this example is that only 4-methoxybenzaldehyde is replaced by 2-hydroxybenzaldehyde.

[0107] The structural formula of Compound 9 is shown below:

[0108]

[0109] Compound 9 is a white solid with a yield of 32%, a melting point of 118-122°C, and the data of its1H NMR,13C NMR and high resolution mass spectrum are shown below:

[0110] 1 H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 8.56 (s, 1H), 7.50 (s, 1H), 7.26 (s, 1H), 7.17-7.00 (m, 2H), 6.78-6.68 (m, 2H), 5.00 (s, 1H), 3.76 (s, 2H);

[0111] 13 C NMR (100 MHz, DMSO-d6) δ 181.51, 156.12, 130.72, 128.81, 124.29, 119.34, 115.65, 50.32;

[0112] HRMS (ESI) calcd for [M+Na] + C8H 11 N3OSNa + : 220.0515, found: 220.0522. Example 10:

[0113] Preparation of 2-(3,5-dichloro-2-hydroxybenzyl)hydrazine-1-thioamide (Compound 10)

[0114] Example 1 except that only 4-methoxybenzaldehyde was replaced by 3,5-dichloro-2- hydroxybenzaldehyde.

[0115] The structural formula of compound 10 is shown as follows:

[0116]

[0117] wherein compound 10 is a light yellow solid with a yield of 47%; melting point: 159-162 °C; and the data of the nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and high resolution mass spectrum of compound 10 are as follows:

[0118] 1 H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.63 (s, 1H), 7.46 (s, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.26 (d, J = 2.8 Hz, 2H), 5.24 (s, 1H), 3.83 (s, 2H);

[0119] 13 C NMR (100 MHz, DMSO-d6) δ 181.56, 150.63, 129.50, 129.04, 128.12, 123.73, 122.27, 49.78;

[0120] HRMS (ESI) calcd for [M-H] - C8H8Cl2N3OS - : 265.9916, found: 265.9926. Example 11:

[0121] Preparation of 2-(2-hydroxy-5-methylbenzyl)hydrazine-1-thioamide (compound 11)

[0122] Example 1 except that only 4-methoxybenzaldehyde was replaced by 2-hydroxy-5- methylbenzaldehyde.

[0123] The structural formula of compound 11 is shown as follows:

[0124]

[0125] wherein compound 11 is a yellow solid with a yield of 23%; melting point: 137-140 °C; and the data of the nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and high resolution mass spectrum of compound 10 are as follows:

[0126] 1H NMR (400 MHz, DMSO-d6) δ 9.11 (s, 1H), 8.56 (s, 1H), 7.51 (s, 1H), 7.27 (s, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.84 (dd, J = 8.0, 2.4 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 4.95 (s, 1H), 3.73 (d, J = 6.0 Hz, 2H), 2.14 (s, 3H);

[0127] 13 C NMR (100 MHz, DMSO-d6) δ 181.53, 153.82, 131.30, 129.12, 127.68, 123.94, 115.54, 50.46, 20.64;

[0128] HRMS (ESI) calcd for [M-H] - C9H 12 N3OS - : 210.0707, found: 210.0699.

[0129] Example 12:

[0130] Preparation of 2-(2-hydroxy-5-nitrobenzyl)hydrazine-1-carbothioamide (Compound 12)

[0131] The difference between Example 1 and this example is that only 4-methoxybenzaldehyde is replaced by 2-hydroxy-5-nitrobenzaldehyde.

[0132] The structural formula of Compound 12 is shown below:

[0133]

[0134] Compound 12 is a golden yellow powder with a yield of 28%, a melting point of 182-187°C, and the data of its nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and high resolution mass spectrum are shown below:

[0135] 1 H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.68 (s, 1H), 8.14 (d, J = 2.8 Hz, 1H), 8.02 (dd, J = 8.8, 2.8 Hz, 1H), 7.53 (s, 1H), 7.22 (s, 1H), 6.94 (d, J = 8.8 Hz, 1H), 5.28 (s, 1H), 3.83 (s, 2H);

[0136] 13C NMR (100 MHz, DMSO-d6) δ 181.66, 162.65, 140.04, 126.68, 125.76, 125.30, 115.75, 49.20;

[0137] HRMS (ESI) calcd for [M-H] - C8H9N4O3S - : 241.0401, found: 241.0396.

[0138] Example 13:

[0139] Preparation of 2-(5-chloro-2-hydroxybenzyl)hydrazine-1-thioamide (Compound 13)

[0140] The difference between Example 1 and this example is that only 4-methoxybenzaldehyde is replaced by 2-hydroxy-5-chlorobenzaldehyde.

[0141] The structural formula of Compound 13 is as follows:

[0142]

[0143] Compound 13 is a white solid with a yield of 20%, a melting point of 176-180°C, and the data of its 1H NMR, 13C NMR and high resolution mass spectrum are as follows:

[0144] 1 H NMR (400 MHz, DMSO-d6) δ 9.67 (s, 1H), 8.61 (s, 1H), 7.51 (s, 1H), 7.24 (d, J = 2.8 Hz, 2H), 7.08 (dd, J = 8.4, 2.8 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 5.12 (s, 1H), 3.75 (d, J = 6.0 Hz, 2H);

[0145] 13 C NMR (100 MHz, DMSO-d6) δ 181.55, 154.93, 129.99, 128.25, 126.67, 122.84, 117.06, 49.54;

[0146] HRMS (ESI) calcd for [M-H]- C8H9ClN3OS-: 232.0306, found: 232.0317.

[0147] In order to further verify the excellent effect of the present application, the inventors also carried out the following comparative experiments:

[0148] Experiment 1

[0149] To evaluate the inhibitory effect of the compound on diphenolase, levodopa was used as the reaction substrate, and all reagents were prepared according to the principle of present preparation. In a 3 mL reaction system, 2800 μL of a dopa solution (0.5 mM), 100 μL of a sample solution with different concentrations, and 100 μL of a tyrosinase solution (200 U / mL) were sequentially added. Then, the absorbance value of the mixed solution was measured by a UV spectrophotometer (UV-2401PC) at a wavelength of 475 nm, and the growth curve of the absorbance value with time was determined. Three parallel tests were performed for each concentration, and the slope of the straight line was used to calculate the enzyme activity. The tyrosinase activity inhibition rate was calculated according to the following formula: tyrosinase activity inhibition rate (%) = (k 空白 -k 样品 ) / k 空白 × 100%, k 空白 is the slope of the blank group; k 样品 is the slope after adding the sample. The sample concentration (μM) was used as the horizontal coordinate, and the inhibition rate (%) was used as the vertical coordinate to draw a curve, and the IC 50 value of the sample on tyrosinase diphenolase was calculated. The results are shown in Table 1.

[0150] Table 1. Tyrosinase inhibitory activity (IC 50 ) of aminothiourea compounds:

[0151]

[0152] As can be seen from Table 1, the compound synthesized according to the present application has good tyrosinase inhibitory activity. Compound 6 has the strongest tyrosinase activity, with an IC 50 of 1.21 ± 0.02 μM, which is significantly better than the positive control drug kojic acid (26.57 ± 0.69 μM).

[0153] The inventors of the present application conducted water solubility experiments, melanin production inhibition experiments, and safety experiments on compounds 1-13, and the results showed that: ① Compared with the respective double bond reduction intermediates, the water solubility of each compound was significantly improved after double bond reduction; ② Each compound significantly inhibited the melanin production of zebrafish; ③ Each compound had low toxicity and high safety. Taking compound 6 as an example, some experiments are disclosed below.

[0154] Experiment two

[0155] To explore whether the water solubility of compound 6 is increased after the double bond is reduced, compound 6 and the intermediate before the double bond is reduced are respectively dissolved in the same volume of pure water to prepare a saturated solution. After centrifugation at 10000 rpm for 5 minutes, the supernatant is measured. The water solubility of the sample is measured by Thermo HPLC (Ultimate 3000) high performance liquid chromatograph, the sample injection amount is 20 μL, the detection wavelength is 254 nm, the mobile phase is acetonitrile-water (40 / 60, v / v), the flow rate is 1.0 mL / min, and the solubility is calculated by peak area. From the results, the solubility of compound 6 is about 70 times that of the intermediate before the double bond is reduced, indicating that the water solubility of compound 6 is significantly changed after the double bond is reduced, which is suitable for practical application. The results are shown in Table 1. Figure 2 .

[0156] Experiment three

[0157] Compound 6 is used to explore its effect on melanin production of zebrafish embryos.

[0158] In this experiment, the purchased adult male and female zebrafish are first classified and acclimated for one week. The day before taking the eggs, the male and female zebrafish are put into the same mating box according to the proportion (female / male = 1:2) and separated by a partition. The next day, a small amount of food is fed, and when the light starts at 8 am, the partition is removed, the male fish start to chase the female fish and collide with the female fish body, the female fish start to lay eggs, and after the end of egg laying, the fish eggs are immediately sucked out, the unfertilized white eggs are picked out, and the remaining fish eggs are randomly divided into blank control group and different drug groups, 20 in each group. After washing, they are put into culture dishes containing Holt buffer culture solution and placed in a constant temperature incubator at 28.5°C for culture. The fertilization is given after 6 hours, and the photograph is taken after 72 hours.

[0159] From Figure 3 It can be seen that as the concentration of compound gradually increases, the black spots on the head and back of the zebrafish in the drug group gradually decrease, and the body of the zebrafish tends to be transparent, indicating that compound 6 can significantly inhibit the generation of melanin in zebrafish.

[0160] Experiment four

[0161] HEK-293 is cultured according to 4x10 5The cells were seeded in sterile 96-well plates at a density of 1 cell / well, and incubated in a 37°C, 5% CO2 incubator for 24 hours. Then, different concentrations of compound 6 prepared with complete culture medium were added. After 24 hours of incubation with the compound, 10-20 μL of 5 mg / mL sterile MTT solution was added and incubated for 1-4 hours. After the incubation, the supernatant was discarded, 150 μL of DMSO was added, and the mixture was shaken on a shaker until the crystals were completely dissolved. Then, the absorbance value was measured at 490 nm using a multifunctional enzyme labeler. The experimental results showed that when the concentration of compound 6 was 96 μM, the inhibition rate of compound 6 on normal cells was 86.25%, indicating that compound 6 has low toxicity and high safety.

[0162] Finally, it should be noted that the above examples are only representative of the present application. Obviously, the technical solutions of the present application are not limited to the above examples, and there can be many variations. All variations that can be directly derived or inferred from the content disclosed in the present application by those of ordinary skill in the art should be considered as falling within the scope of protection of the present application.

Claims

1. Use of an aminothiourea tyrosinase inhibitor for the preparation of a tyrosinase inhibiting medicament and a whitening product, characterized in that, The inhibitor has a structure shown in the following general formula (I): R in the general formula I is any one or more of hydrogen, halogen, C1-C5 alkyl, C1-C5 alkoxy, nitro, and hydroxyl.

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