Dihydroxybenzaldehyde cysteine condensate with tyrosinase inhibitory activity

Abstract

The invention provides dihydroxy benzaldehyde cysteine condensation compounds (epimer mixtures 1-6), a preparation method thereof and application of the dihydroxy benzaldehyde cysteine condensation compounds in preparation of cosmetics and medicines. Belongs to the technical field of medicines and cosmetics. According to the invention, six groups of reaction products are prepared from one of raw materials of o-dihydroxy, m-dihydroxy and p-dihydroxy benzaldehyde and a condensation compound of D-cysteine or L-cysteine, and the reaction products are a mixture formed by generating a pair of epimers when dihydroxy benzaldehyde is condensed. A tyrosinase inhibition activity screening experiment is carried out on the novel condensation compounds, and the tyrosinase inhibition ratios of a condensation compound (1) of 2, 3-dihydroxy benzaldehyde and L-cysteine, a condensation compound (2) of 2, 3-dihydroxy benzaldehyde and D-cysteine and a condensation compound (3) of 2, 4-dihydroxy benzaldehyde and L-cysteine are respectively 52.45%, 60.34% and 60.34%. 53.89% of the compound shows that the compound has relatively remarkable tyrosinase inhibitory activity. The novel dihydroxy benzaldehyde and cysteine condensate mixture has obvious tyrosinase inhibition activity, and can be applied to preparation of medicines and cosmetics for beautifying and whitening.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics and medicines, and specifically relates to a dihydroxybenzaldehyde condensation product with tyrosinase inhibitory activity, which is generated by condensing dihydroxybenzaldehyde with cysteine, and a preparation method thereof. The mixture is a new type of functional compound with potential whitening function, and the application of such compound in the preparation of beauty and whitening cosmetics and in the preparation of tyrosinase inhibitors. Technical Background

[0002] Whitening cosmetics are products with great demand in the current market. Most of the best-selling cosmetics use natural plant functional ingredients as raw materials. Among the whitening functional ingredients of natural origin, kojic acid, niacinamide, arbutin, and vitamin C are all common whitening ingredients in the cosmetics market. The natural product silver pinus pyrifolia (Phenylethyl Resorcinol, PR) found in the cones of European red pine has tyrosinase inhibitory activity, but because silver pinus pyrifolia is unstable, it was subjected to structural derivatization and modification. Finally, German whitening experts screened out a new and highly effective whitening active ingredient similar to dihydrosilver pinus pyrifolia - phenylethyl resorcinol (and named it 377). Phenylethyl resorcinol ( 377) has therefore become the ceiling of tyrosinase inhibitory activity and one of the indicators of whitening functional ingredients.

[0003] Recently, the inventors found some simple structures in wild Ganoderma lucidum when conducting trace components in Ganoderma lucidum. However, in the zebrafish embryo melanin formation inhibition activity experiment, it was found that 2,4-hydroxyacetophenone has very significant activity and is a natural product with great whitening function prospects. Therefore, similar structures of dihydroxyacetophenone have been prepared in large quantities. Among them, a type of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) with a very simple synthesis preparation method is a new type of derivative structure that has not been reported. Therefore, the inventors prepared 6 groups of reaction products by combining and translating one of the raw materials of ortho-dihydroxy, meta-dihydroxy, and p-dihydroxybenzaldehyde with D-cysteine ​​and L-cysteine. This type of reaction product is that when dihydroxybenzaldehyde is condensed, a pair of diastereomers are produced to form a mixture. A tyrosinase inhibitory activity screening experiment was conducted on this type of condensation product. It was found that the o-dihydroxy derivative mixture 2,3-dihydroxybenzaldehyde and L-cysteine ​​condensation product (1), 2,3-dihydroxybenzaldehyde and D-cysteine ​​condensation product (2) showed relatively significant tyrosinase inhibitory activity, while the m-dihydroxy and p-dihydroxybenzaldehyde cysteine ​​condensation products showed very low tyrosinase inhibitory activity. It can be inferred that the o-dihydroxybenzaldehyde cysteine ​​condensation product should be a product of the reaction with phenethyl resorcinol ( 377) have novel tyrosinase inhibitory activity with different mechanisms of action.

[0004] So far, there is no report on o-dihydroxybenzaldehyde cysteine ​​condensate and its activity in the prior art. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a novel o-dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) having significant tyrosinase inhibitory activity as a whitening functional molecule. The application of such compounds in the preparation of beauty and whitening cosmetics and in the preparation of tyrosinase inhibitors. The preparation methods thereof are also provided.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0007] Dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) as shown in the following structural formula,

[0008]

[0009]

[0010] The preparation method of the dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) shown in the structural formula comprises the following steps:

[0011]

[0012] D (or L) -cysteine ​​hydrochloride (0.7 mol-1.0 mol), NaHCO3 (0.7 mol), dihydroxybenzaldehyde (1 mol) and 4 L 50% ethanol are added to a reaction kettle. The reaction materials can be adjusted according to the above mol ratio. Stirring is carried out at room temperature for 24 hours to generate an insoluble precipitated compound. The insoluble precipitated compound is filtered out by suction filtration. The compound is rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The compound is naturally dried at room temperature to obtain a condensation product of dihydroxybenzaldehyde and D (or L) -cysteine ​​(C-2 diastereomer mixture).

[0013] The preparation method of the dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) shown in the structural formula comprises the following steps:

[0014] The preparation steps of mixture 1 are:

[0015]

[0016] L-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 3,4-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate compound. The insoluble precipitate compound was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The compound was naturally dried at room temperature to obtain a condensation product of 2,3-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture) (1);

[0017] The preparation steps of mixture 2 are:

[0018]

[0019] D-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 3,4-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate compound. The insoluble precipitate compound was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The compound was naturally dried at room temperature to obtain a condensation product of 2,3-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture) (2);

[0020] The preparation steps of mixture 3 are:

[0021]

[0022] In a 10 mL round-bottom flask, add L-cysteine ​​hydrochloride (1 mmol, 175 mg), NaHCO3 (1 mmol, 84 mg), 2,4-dihydroxybenzaldehyde (0.5 mmol, 69 mg) and 4 mL of 50% ethanol, and stir at room temperature for 24 hours to generate an insoluble precipitate compound. Filter out the insoluble precipitate compound by suction filtration, rinse the compound with pure methanol several times and then with ethyl acetate several times, and dry it naturally at room temperature to obtain a condensation product of 2,4-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture) (3);

[0023] The preparation steps of mixture 4 are:

[0024]

[0025] D-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 2,4-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The mixture was naturally dried at room temperature to obtain a condensation product of 2,4-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture) (4);

[0026] The preparation steps of mixture 5 are:

[0027]

[0028] L-cysteine ​​hydrochloride (0.7mmol, 122.5mg), NaHCO3 (0.7mmol, 58.8mg), 2,5-dihydroxybenzaldehyde (1mmol, 138mg) and 4mL 50% ethanol were added to a 10mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate compound. The insoluble precipitate compound was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The compound was naturally dried at room temperature to obtain a condensation product of 2,5-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture) (5);

[0029] The preparation steps of mixture 6 are:

[0030]

[0031] D-cysteine ​​hydrochloride (0.7mmol, 122.5mg), NaHCO3 (0.7mmol, 58.8mg), 2,5-dihydroxybenzaldehyde (1mmol, 138mg) and 4mL 50% ethanol were added to a 10mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction, and the compound was rinsed multiple times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain a condensation product of 2,5-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture) (6).

[0032] The application of the dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) shown in the structural formula in the preparation of cosmetics.

[0033] A cosmetic containing the dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) shown in the structural formula, which is composed of an effective amount of any one or any combination of the dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) and common cosmetic auxiliary materials.

[0034] A tyrosinase inhibitor containing the dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) shown in the structural formula, containing an effective amount of any one of the dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) or any combination thereof.

[0035] The application of the dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) shown in the structural formula in the preparation of tyrosinase inhibitors.

[0036] When the dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) of the present invention is used to prepare medicine or cosmetics, the content of the dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) accounts for 0.1-99% of the mass of the active ingredient, preferably 0.5-90% of the active ingredient, and the rest is pharmaceutical excipients or carriers that are acceptable in pharmacology and pharmaceutical science and non-toxic to humans and animals.

[0037] The preparations are in various dosage forms that can be used clinically, such as capsules, granules, pills, tablets, injections, ointments, tinctures, oral liquids, etc. The dosage forms of the pharmaceutical composition are lyophilized powders, oral liquids, and capsules, etc. The dosage forms of the cosmetics are creams, facial masks, and hydrogels, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the general formula for the synthesis of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers).

[0039] Figure 2 The process of melanin formation.

[0040] Figure 3 Schematic diagram of the structure of dihydroxybenzaldehyde and D (or L)-cysteine ​​condensation product (C-2 diastereomer mixture) 1-6. DETAILED DESCRIPTION

[0041] The substantial content of the present invention is further described below with reference to the accompanying drawings using embodiments of the present invention, but is not limited thereto.

[0042] Example 1

[0043] 1. Synthesis and preparation of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers).

[0044] D (or L) -cysteine ​​hydrochloride (0.7 mol-1.0 mol), NaHCO3 (0.7 mol), dihydroxybenzaldehyde (1 mol) and 4L 50% ethanol (the reaction materials can be adjusted according to the above mol ratio) are added to a round-bottom flask (reactor). Stir at room temperature for 24 hours to generate an insoluble precipitated compound, filter out the insoluble precipitated compound by suction filtration, rinse the compound with pure methanol for multiple times and then rinse it with ethyl acetate for multiple times, dry it naturally at room temperature, and obtain dihydroxybenzaldehyde and D (or L) -cysteine ​​condensate (C-2 diastereomer mixture). In general, the yield can reach 40%-80%, and the purity of the mixture can reach 97%. The general formula for the synthesis and preparation of dihydroxybenzaldehyde cysteine ​​condensate (diastereomer mixture) is expressed as follows using the reaction formula of mixture 1 and 2 with significant tyrosinase inhibitory activity:

[0045]

[0046] 2. Dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) and its preparation:

[0047] 1,2,3-Dihydroxybenzaldehyde and L-cysteine ​​condensation product (C-2 diastereomer mixture) (1)

[0048]

[0049] 1) Synthetic preparation:

[0050] L-cysteine ​​hydrochloride (0.7mmol, 122.5mg), NaHCO3 (0.7mmol, 58.8mg), 3,4-dihydroxybenzaldehyde (1mmol, 138mg) and 4mL 50% ethanol were added to a 10mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction, and the compound was rinsed multiple times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain 107mg (1) of condensation product of 2,3-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture).

[0051]

[0052] Yield: 63.4%; purity: 97% (R:S at C-2 is close to 1:1).

[0053] 2) Structural characterization

[0054] Molecular formula: C 10 H 11 NO4S; molecular weight: 241.0409

[0055] HR-EIS:242.0422([M+H] + )(Calculated value: 242.0482)

[0056] 1 H NMR (600MHz, DMSO-d6) δ (ppm): 8.99 (2H, brs, OH × 2), 8.89 (2H, brs, OH × 2), 6.88 (1H, d, J = 2.2Hz, 5′-H), 6.83 (1H, d, J = 2.2Hz, 5′-H) , 6.73 (1H, dd, J = 2.2, 8.1Hz, 6′-H), 6.68 (1H, dd, J = 2.7, 8.1Hz, 6′-H), 6.65 (1H, s, 2′-H), 6.64 (1H, s, 2′-H), 5.46 (1H, s, benzylic H), 5.32(1H, s, benzylic H), 4.21 (1H, dd, J=4.1, 7.2Hz, 3-H×2), 3.82 (1H, t, J=7.9Hz, 4-H×2), 3.33 (1H, dd, J=7.3, 10.2Hz, 5-H a ), 3.24 (1H, dd, J=7.2, 10.3Hz, 5-H a ), 3.11 (1H, dd, J=4.0, 10.3Hz, 5-H b ), 3.01 (1H, dd, J=8.5, 10.2Hz, 5-H b ).

[0057] 13 C NMR (150MHz, DMSO-d6) δ (ppm): 173.16 (COOH), 172.47 (COOH), 145.47 (C4′), 145.23(C4′), 144.99(C3′), 144.92(C3′), 131.42(C1′), 129.36(C1′), 118. 30(C6′), 118.05(C6′), 115.31(C5′), 115.07(C5′), 114.56(C2′), 114.44(C 2′), 72.04(C2), 71.43(C2), 65.02(C4), 64.74(C4), 38.50(C5), 37.75(C5).

[0058] 2.2,3-Dihydroxybenzaldehyde and D-cysteine ​​condensation product (C-2 diastereomer mixture) (2)

[0059]

[0060] 1) Synthetic preparation:

[0061] D-cysteine ​​hydrochloride (0.7mmol, 122.5mg), NaHCO3 (0.7mmol, 58.8mg), 3,4-dihydroxybenzaldehyde (1mmol, 138mg) and 4mL 50% ethanol were added to a 10mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction and rinsed several times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain 102mg (2) of condensation product of 2,3-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture).

[0062]

[0063] Yield: 60.4%; purity: 97% (R:S at C-2 is close to 1:1).

[0064] 2) Structural characterization

[0065] Molecular formula: C 10 H 11 NO4S; molecular weight: 241.0409

[0066] HR-EIS:242.0626([M+H] + )(Calculated value: 242.0482)

[0067] 1 H NMR (600MHz, DMSO-d6) δ (ppm): 8.99 (2H, brs, Ar-OH×2), 8.89 (2H, brs, Ar-OH×2), 6.88 (1H, d, J=2.2Hz, 5′-H), 6.83 (1H, d, J=2.2Hz, 5′ -H), 6.73 (1H, dd, J = 2.2, 8.2Hz, 6′-H), 6.68 (1H, dd, J = 2.5, 8.2Hz, 6′-H), 6.65 (1H, s, 2′-H), 6.64 (1H, s, 2′-H), 5.46 (1H, s, benzylic H), 5.32(1H, s, benzylic H), 4.21 (1H, dd, J=4.1, 7.2Hz, 3-H×2), 3.82 (1H, t, J=7.9Hz, 4-H×2), 3.33 (1H, dd, J=7.3, 10.1Hz, 5-Ha), 3 .24 (1H, dd, J=7.2, 10.3Hz, 5-Ha), 3.11 (1H, dd, J=4.1, 10.3Hz, 5-Hb), 3.01 (1H, dd, J=8.5, 10.1Hz, 5-Hb).

[0068] 13 C NMR (150MHz, DMSO-d6) δ (ppm): 173.16 (COOH), 172.47 (COOH), 145.48 (C4′), 145.24(C4′), 144.99(C3′), 144.91(C3′), 131.42(C1′), 129.36(C1′), 118. 30(C6′), 118.05(C6′), 115.31(C5′), 115.07(C5′), 114.57(C2′), 114.44(C 2′), 72.04(C2), 71.43(C2), 65.02(C4), 64.74(C4), 38.49(C5), 37.75(C5).

[0069] 3.2,4-Dihydroxybenzaldehyde and L-cysteine ​​condensation product (C-2 diastereomer mixture) (3)

[0070]

[0071] 1) Synthetic preparation:

[0072] In a 10 mL round-bottom flask, L-cysteine ​​hydrochloride (1 mmol, 175 mg), NaHCO3 (1 mmol, 84 mg), 2,4-dihydroxybenzaldehyde (0.5 mmol, 69 mg) and 4 mL of 50% ethanol were added and stirred at room temperature for 24 hours to generate an insoluble precipitated compound. The insoluble precipitated compound was filtered out by suction, and the compound was rinsed several times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain 110 mg (3) of condensation product of 2,4-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture).

[0073]

[0074] Yield: 45.6%; purity: 97% (R:S at C-2 is close to 1:1).

[0075] 2) Structural characterization:

[0076] Molecular formula: C 10 H 11 NO4S; molecular weight: 241.0409

[0077] HR-EIS:242.0499([M+H] + )(Calculated value: 242.0482)

[0078] 1H NMR (600MHz, DMSO-d6) δ (ppm): 9.34 (2H, brs, Ar-OH×2), 9.22 (2H, brs, Ar-OH), 7.10 (1H, d, J = 8.3Hz, 6′-H), 7.08 (1H, d, J = 8.5Hz, 6′-H), 6.27 ( 1H, d, J=2.5Hz, 5′-H), 6.24 (1H, d, J=2.0Hz, 5′-H), 6.20 (1H, dd, J=2.5, 8.5Hz, 3′-H), 6.17 (1H, dd, J=2.5, 8.0Hz, 3′-H), 5.74 (1H, s, benzylic H),5.57(1H,s,benzylic H), 4.23 (1H, dd, J=4.4, 7.2Hz, 3-H×2), 3.76 (1H, t, J=8.0Hz, 4-H×2), 3.30 (1H, dd, J=7.0, 10.2Hz, 5-H a ), 3.19 (1H, dd, J=7.0, 10.3Hz, 5-H a ), 3.06 (1H, dd, J=4.5, 10.2Hz, 5-H b ), 2.93 (1H, t, J=9.4Hz, 5-H b ).

[0079] 13 C NMR (150MHz, DMSO-d6) δ (ppm): 173.11 (COOH), 172.61 (COOH), 158.19 (C-4′), 15 7.60(C-4), 156.23(C-2), 155.68(C-2), 128.84(C-6′), 127.47(C-6′), 117.39(C -1′), 114.67(C-1′), 106.41(C-5′), 106.08(C-5′), 102.71(C-3′), 102.31(C-3′ ), 68.03(C-4), 65.97(C-4), 65.15(C-2), 64.62(C-2), 38.35(C-5), 37.04(C-5).

[0080] 4.2,4-Dihydroxybenzaldehyde and D-cysteine ​​condensation product (C-2 diastereomer mixture) (4)

[0081]

[0082] 1) Synthetic preparation:

[0083] D-cysteine ​​hydrochloride (0.7mmol, 122.5mg), NaHCO3 (0.7mmol, 58.8mg), 2,4-dihydroxybenzaldehyde (1mmol, 138mg) and 4mL of 50% ethanol were added to a 10mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction and rinsed several times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain 40mg (4) of condensation product of 2,4-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture).

[0084]

[0085] Yield: 23.7%; purity: 97% (R:S at C-2 is close to 1:1).

[0086] 2) Structural characterization:

[0087] Molecular formula: C 10 H 11 NO4S; molecular weight: 241.0409

[0088] HR-EIS:242.0442([M+H] + )(Calculated value: 242.0482)

[0089] 1 H NMR (600MHz, DMSO-d6) δ (ppm): 9.33 (2H, brs, Ar-OH×2), 9.21 (2H, brs, Ar-OH×2), 7.10 (1H, d, J=8.3Hz, 6′-H), 7.07 (1H, d, J=8.4Hz, 6′-H), 6.26 (1H, d, J = 2.5Hz, 5′-H), 6.23 (1H, d, J = 2.5Hz, 5′-H), 6.19 (1H, dd, J = 2.5, 8.3Hz, 3′-H), 6.16 (1H, dd, J = 2.6, 8.3Hz, 3′-H), 5.73 (1H, s, benzylic H), 5.56 (1H, s, benzylic H), 4.23 (1H, dd, J=4.4, 7.1Hz, 3-H×2), 3.76 (1H, t, J=8.0Hz, 4-H×2), 3.30 (1H, dd, J=7.0, 10.0Hz, 5-H a ), 3.18 (1H, dd, J=6.9, 10.2Hz, 5-H a ), 3.05 (1H, dd, J=4.4, 10.2Hz, 5-H b), 2.93 (1H, t, J=9.4Hz, 5-H b ).

[0090] 13 C NMR (150MHz, DMSO-d6) δ (ppm): 173.09 (COOH), 172.56 (COOH), 158.18 (C4′), 157.59(C4′), 156.21(C2′), 155.65(C2′), 128.84(C6′), 127.45(C6′), 117. 37(C1′), 114.63(C1′), 106.40(C5′), 106.07(C5′), 102.69(C3′), 102.29(C 3′), 68.03(C4), 65.94(C4), 65.02(C2), 64.57(C2), 38.28(C5), 37.02(C5).

[0091] 5.2,5-Dihydroxybenzaldehyde and L-cysteine ​​condensation product (C-2 diastereomer mixture) (5)

[0092]

[0093] 1) Synthetic preparation:

[0094] L-cysteine ​​hydrochloride (0.7mmol, 122.5mg), NaHCO3 (0.7mmol, 58.8mg), 2,5-dihydroxybenzaldehyde (1mmol, 138mg) and 4mL 50% ethanol were added to a 10mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction and rinsed several times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain 124mg (5) of a condensation product of 2,5-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture).

[0095]

[0096] Yield: 73.5%; purity: 97% (R:S at C-2 is close to 1:1).

[0097] 2) Structural characterization:

[0098] Molecular formula: C 10 H 11 NO4S; molecular weight: 241.0409

[0099] HR-EIS:242.0280([M+H] + )(Calculated value: 242.0482)

[0100] 1 H NMR (600MHz, DMSO-d6) δ (ppm): 8.76 (2H, brs, Ar-OH×2), 8.64 (2H, brs, Ar-OH×2), 6.74 (1H, d, J=2.9Hz, 6′-H), 6.73 (1H, d, J=3.0Hz, 6′-H), 6.61 (1H, d, J = 8.6Hz, 3′-H), 6.55 (1H, d, J = 8.5Hz, 3′-H), 6.53 (1H, dd, J = 2.9, 8.5Hz, 4′-H), 6.45 (1H, dd, J = 3.0, 8.5Hz, 4′-H), 5.76 (1H, s, benzylic H),5.55(1H,s,benzylic H), 4.14 (1H, t, J=6.1Hz, 3-H×2), 3.79 (1H, dd, J=7.0, 8.9Hz, 4-H×2), 3.32 (1H, dd, J=7.0, 10.0Hz, 5-H a ), 3.18 (1H, dd, J=6.7, 10.2Hz, 5-H a ), 2.97 (1H, dd, J=5.9, 10.4Hz, 5-H b ), 2.93 (1H, t, J=5.0Hz, 5-H b ).

[0101] 13 C NMR (150MHz, DMSO-d6) δ (ppm): 172.90 (COOH), 172.52 (COOH), 149.81 (C5′), 149.66(C5′), 147.52(C2′), 146.99(C2′), 128.35(C1′), 124.61(C1′), 116. 41(C3′), 115.66(C3′), 115.57(C4′), 114.39(C4′), 114.03(C6′), 112.72(C 6′), 67.65(C4), 65.70(C4), 65.05(C2), 64.85(C2), 38.21(C5), 37.07(C5).

[0102] 6.2,5-Dihydroxybenzaldehyde and D-cysteine ​​condensate (C-2 epimer mixture) (6)

[0103]

[0105] 1) Synthetic preparation:

[0106] D-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 2,5-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction and then rinsed several times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain 119 mg (6) of a condensation product of 2,5-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture).

[0107]

[0108] Yield: 70.5%; purity: 97% (R:S at C-2 is close to 1:1).

[0109] 2) Structural characterization:

[0110] Molecular formula: C 10 H 11 NO4S; molecular weight: 241.0409

[0111] HR-EIS:242.0286([M+H] + )(Calculated value: 242.0482)

[0112] 1 H NMR (600MHz, DMSO-d6) δ (ppm): 8.76 (2H, brs, Ar-OH×2), 8.64 (2H, brs, Ar-OH×2), 6.74 (1H, d, J=2.9Hz, 6′-H), 6.73 (1H, d, J=3.0Hz, 6′-H), 6.61 (1H, d, J = 8.6Hz, 3′-H), 6.55 (1H, d, J = 8.5Hz, 3′-H), 6.53 (1H, dd, J = 2.9, 8.6Hz, 4′-H), 6.45 (1H, dd, J = 3.0, 8.5Hz, 4′-H), 5.76 (1H, s, benzylic H),5.55(1H,s,benzylic H), 4.15 (1H, t, J=6.1Hz, 3-H×2), 3.80 (1H, dd, J=7.0, 8.9Hz, 4-H×2 / , 3.32 (1H, dd, J=7.1, 10.1Hz, 5-H a ), 3.18 (1H, dd, J=6.7, 10.1Hz, 5-H a ), 2.97 (1H, dd, J=5.8, 10.4Hz, 5-H b), 2.93 (1H, t, J=4.8Hz, 5-H b ).

[0113] 13 C NMR (150MHz, DMSO-d6) δ (ppm): 172.91 (COOH), 172.52 (COOH), 149.81 (C5′), 149.66(C5′), 147.52(C2′), 146.99(C2′), 128.35(C1′), 124.61(C1′), 116. 41(C3′), 115.66(C3′), 115.57(C4′), 114.39(C4′), 114.03(C6′), 112.72(C 6′), 67.65(C4), 65.70(C4), 65.05(C2), 64.85(C2), 38.21(C5), 37.07(C5).

[0114] 3. Tyrosinase Inhibition Activity Test

[0115] 1) Experimental principle

[0116] Tyrosinase is a key enzyme in the formation of melanin. Inhibiting the activity of tyrosinase can reduce the production of melanin, thereby achieving the purpose of whitening. Tyrosine generates DOPA under the action of tyrosinase, and DOPA is further oxidized by tyrosinase to DOPA quinone, and further oxidized to generate melanin. This experiment uses L-Dopa as a substrate, and the activity of tyrosinase is determined by detecting the production of the product DOPA quinone. Figure 2 .

[0117] 2) Experimental procedures

[0118] The drug to be tested was mixed with tyrosinase (final concentration 25 U / mL), L-Dopa was added to start the reaction, 3 replicate wells were set, and blank control and positive control without drug were set at the same time. The OD value was measured by microplate reader at room temperature for 20 minutes, and the detection wavelength was 490 nm. The tyrosinase activity inhibition rate was calculated.

[0119] 3) Result calculation

[0120] Tyrosinase activity inhibition rate (%) = (1 - sample well OD 490nm / OD of experimental control well 490nm )×100%

[0121] 4) Experiment on the inhibitory activity of dihydroxybenzaldehyde cysteine ​​condensate (mixture of isomers) (1-6) on tyrosinase

[0122] The tyrosinase inhibitory activity test of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) was performed, and the results are as follows:

[0123] Table 1 Tyrosinase inhibition rate of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers)

[0124]

[0125] The above tyrosinase inhibition activity test results show that the 2,3-dihydroxybenzaldehyde and L-cysteine ​​condensate (1) and the 2,3-dihydroxybenzaldehyde and D-cysteine ​​condensate (2) of the ortho-dihydroxy derivative mixture have tyrosinase inhibition rates of 52.45% and 60.34% respectively, showing relatively significant tyrosinase inhibition activity, while the 2,4-dihydroxybenzaldehyde and L-cysteine ​​condensate (3) of the meta-dihydroxybenzaldehyde and cysteine ​​condensate have tyrosinase inhibition rates of 53.89% respectively, showing relatively high activity. This shows that the dihydroxybenzaldehyde and cysteine ​​condensate is a type of tyrosinase inhibitor with a promising development prospect for whitening effect. However, the 2,4-dihydroxybenzaldehyde and D-cysteine ​​condensate (4) and the p-dihydroxybenzaldehyde and cysteine ​​condensate have very low tyrosinase inhibition activity. It shows that the mechanism of action of dihydroxybenzaldehyde and cysteine ​​condensation products on tyrosinase inhibitory activity is quite complex and worthy of in-depth study. It can not only explain the stage details of tyrosinase's mechanism of melanin production, but also play a very positive role in the development of efficient and harmless whitening functional molecules.

[0126] The natural product argentin, phenylethyl resorcinol (PR), found in the cones of European pine, has tyrosinase inhibitory activity. However, due to the instability of argentin, it was subjected to structural derivatization and modification. Finally, German whitening experts screened out a new and highly effective whitening active ingredient similar to dihydroargentin - phenylethyl resorcinol (named 377). Phenylethyl resorcinol ( 377) has therefore become the ceiling of tyrosinase inhibitory activity and one of the indicators of whitening functional ingredients. When studying the tyrosinase inhibitory activity of dihydroxybenzaldehyde and cysteine ​​condensates, it was found that the o-dihydroxy derivative mixture 2,3-dihydroxybenzaldehyde and L-cysteine ​​condensate (1), 2,3-dihydroxybenzaldehyde and D-cysteine ​​condensate (2), showed significant tyrosinase inhibitory activity, which was the condensate with resorcinol fragment structural units; while the cysteine ​​condensate with resorcinol and hydroquinone fragment structural units showed very low tyrosinase inhibitory activity. It can be inferred that the o-dihydroxybenzaldehyde cysteine ​​condensate should be a condensate with phenylethylresorcinol ( 377) have novel tyrosinase inhibitory activity with different mechanisms of action.

[0127] Example 2

[0128] Preparation of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) cleansing milk.

[0129] 1. Materials

[0130] The dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) obtained by the same method as in Example 1 uses cosmetic grade or food grade raw materials.

[0131] 2. Formulation

[0132] Dihydroxybenzaldehyde cysteine ​​condensate (mixture of isomers) (1-6) 7g, glycerin 2.0g, butylene glycol 4.0g, propylene glycol 0.5g, sodium EDTA 0.1g, guar gum 0.3g, zinc dioxide 2.0g, C12-15 alcohol benzoate 4.0g, C12-20 alkyl glucoside 3.0g, C14-22 alcohol 0.5g, cetearyl alcohol 1.2g, paraben 0.2g, sodium stearate 0.3g, polydimethylsiloxane alcohol 0.5g, polysorbate 0.2g, deionized water 55g.

[0133] 3. Preparation process

[0134] Weigh the prescribed amount of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6), add purified water to completely dissolve; take glycerol, butylene glycol, propylene glycol, sodium EDTA, guar gum, zinc dioxide, sodium stearate, C12-15 alcohol benzoate, C12-20 alkyl glucoside, C14-22 alcohol, cetearyl alcohol, add purified water to completely dissolve; take paraben, polydimethylsiloxane alcohol, polysorbate, add purified water to completely dissolve. Mix the above-prepared solutions, stir well, filter with a 0.22μm microporous membrane, can the filtrate in 30mL per bottle, seal and sterilize to obtain.

[0135] Example 3

[0136] Preparation of facial mask of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6).

[0137] 1. Materials

[0138] The dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) is obtained in the same manner as in Example 1, and other materials are cosmetic grade or food grade raw materials.

[0139] 2. Formulation

[0140] Dihydroxybenzaldehyde cysteine ​​condensate (mixture of isomers) (1-6) 12g, xanthan gum 3.5g, hydrolyzed hyaluronic acid 2.0g, sodium hyaluronate 2.0g, propylene glycol 0.3g, carbomer 0.5g, paraben 0.1, PPG-10 methyl glucose ether 0.25g, glyceryl glucoside 1.0g, deionized water 45g.

[0141] 3. Mask preparation process

[0142] Step 1: Add deionized water, xanthan gum, hydrolyzed hyaluronic acid, sodium hyaluronate, propylene glycol, carbomer and paraben into an emulsifier and heat to 75°C while stirring. After reaching the temperature, keep warm and continue stirring until the solution is transparent and uniform. Then keep warm for 20 minutes and then cool down to 42°C.

[0143] Step 2: Adjust pH = 7.6 ± 0.2.

[0144] Step 3: Continue to cool to 35°C, add PPG-10 methyl glucose ether, dihydroxybenzaldehyde cysteine ​​condensate (mixture of isomers) (1-6), and glycerol glucoside, continue to stir evenly, and discharge the material after passing the inspection. Coating is performed on a non-woven fabric substrate, with a coating amount of 2.1 g per square centimeter.

[0145] Example 4

[0146] Preparation of capsules of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6).

[0147] 1. Materials

[0148] The dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) obtained by the same method as in Example 1, and food or pharmaceutical grade starch.

[0149] 2. Prescription

[0150]

[0151] 3. Preparation process

[0152] Weigh the prescribed amount of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) and starch, stir and mix thoroughly, add an appropriate amount of talc, wet granulate with ethanol, sieve and dry, and then put into No. 2 capsule shells, with each capsule body filled with 60 mg of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6), to prepare dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) capsules.

[0153] Example 5

[0154] Preparation of oral solution of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6).

[0155] 1. Materials

[0156] The dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) is obtained in the same manner as in Example 1, and the food or pharmaceutical grade flavoring agent is and caramel flavor.

[0157] 2. Prescription

[0158]

[0159]

[0160] 3. Preparation process

[0161] Weigh the prescribed amount of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6), sucrose and caramel essence, add purified water to completely dissolve, filter through a 0.22 μm microporous filter membrane, and fill the filtrate into 2 mL bottles using an oral liquid filling machine. Sterilize after sealing to obtain the product.

[0162] Example 6

[0163] Preparation of lyophilized powder injection of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6).

[0164] 1. Materials

[0165] The dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) obtained by the same method as in Example 1, pharmaceutical grade sodium chloride.

[0166] 2. Prescription

[0167]

[0168] 3. Preparation process

[0169] Weigh the prescribed amount of dihydroxybenzaldehyde cysteine ​​condensate (mixture of diastereomers) (1-6) and sodium chloride, add water for injection to the full amount, stir to dissolve completely, and sterilize by intermittent hot pressing. Add 0.3% medicinal activated carbon and stir for 20 minutes; use a Buchner funnel and a 3.0μm microporous filter membrane to remove the heat source by decarbonization filtration. After the content is qualified, filter with a 0.22μm microporous filter membrane; divide into controlled vials, 0.5mL per bottle, half-stopper, place in a freeze drying box, freeze-dry according to the set freeze-drying curve, stopper, unbox, roll cover, visually inspect and package to obtain the finished product.

[0170] Freeze-drying process: put the sample into the box, lower the partition temperature to -40℃, keep it for 4 hours; lower the cold trap to -50℃, and start to evacuate to 250μbar. Start sublimation: uniformly increase the temperature to -20℃ in 1 hour, keep it for 3 hours; uniformly increase the temperature to -10℃ in 3 hours, keep it for 8 hours, and keep the vacuum at 100~250μbar; then dry: increase the temperature to -5℃ in 2 hours, keep it for 2 hours, and keep the vacuum at 150~200μbar; increase the temperature to 10℃ in 0.5 hours, keep it for 2 hours, and keep the vacuum at 80~100μbar; increase the temperature to 40℃ in 0.5 hours, keep it for 4 hours, and evacuate to the lowest vacuum.

[0171] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0172] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) as shown in the following structural formula, 2. A method for preparing the dihydroxybenzaldehyde cysteine ​​condensate (epimer mixture 1-6) shown in the structural formula of claim 1, characterized in that: The method comprises the following steps: D (or L) -cysteine ​​hydrochloride (0.7 mol-1.0 mol), NaHCO3 (0.7 mol), dihydroxybenzaldehyde (1 mol) and 4 L 50% ethanol are added to a reaction kettle. The reaction materials can be adjusted according to the above mol ratio. Stirring is carried out at room temperature for 24 hours to generate an insoluble precipitated compound. The insoluble precipitated compound is filtered out by suction filtration. The compound is rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The compound is naturally dried at room temperature to obtain a condensation product of dihydroxybenzaldehyde and D (or L) -cysteine ​​(C-2 diastereomer mixture).

3. A method for preparing the dihydroxybenzaldehyde cysteine ​​condensate (epimer mixture 1-6) shown in the structural formula of claim 1, characterized in that: The method comprises the following steps: The preparation steps of mixture 1 are: L-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 3,4-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate compound. The insoluble precipitate compound was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The compound was naturally dried at room temperature to obtain a condensation product of 2,3-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture) (1); The preparation steps of mixture 2 are: D-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 3,4-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate compound. The insoluble precipitate compound was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The compound was naturally dried at room temperature to obtain a condensation product of 2,3-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture) (2); The preparation steps of mixture 3 are: In a 10 mL round-bottom flask, add L-cysteine ​​hydrochloride (1 mmol, 175 mg), NaHCO3 (1 mmol, 84 mg), 2,4-dihydroxybenzaldehyde (0.5 mmol, 69 mg) and 4 mL of 50% ethanol, and stir at room temperature for 24 hours to generate an insoluble precipitate compound. Filter out the insoluble precipitate compound by suction filtration, rinse the compound with pure methanol several times and then with ethyl acetate several times, and dry it naturally at room temperature to obtain a condensation product of 2,4-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture) (3); The preparation steps of mixture 4 are: D-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 2,4-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The mixture was naturally dried at room temperature to obtain a condensation product of 2,4-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture) (4); The preparation steps of mixture 5 are: L-cysteine ​​hydrochloride (0.7 mmol, 122.5 mg), NaHCO3 (0.7 mmol, 58.8 mg), 2,5-dihydroxybenzaldehyde (1 mmol, 138 mg) and 4 mL of 50% ethanol were added to a 10 mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction, and the compound was rinsed with pure methanol for multiple times and then with ethyl acetate for multiple times. The mixture was naturally dried at room temperature to obtain a condensation product of 2,5-dihydroxybenzaldehyde and L-cysteine ​​(C-2 diastereomer mixture) (5); The preparation steps of mixture 6 are: D-cysteine ​​hydrochloride (0.7mmol, 122.5mg), NaHCO3 (0.7mmol, 58.8mg), 2,5-dihydroxybenzaldehyde (1mmol, 138mg) and 4mL 50% ethanol were added to a 10mL round-bottom flask and stirred at room temperature for 24 hours to generate an insoluble precipitate. The insoluble precipitate was filtered out by suction, and the compound was rinsed multiple times with pure methanol and then with ethyl acetate. The mixture was naturally dried at room temperature to obtain a condensation product of 2,5-dihydroxybenzaldehyde and D-cysteine ​​(C-2 diastereomer mixture) (6).

4. Use of the dihydroxybenzaldehyde cysteine ​​condensate (different isomer mixture 1-6) shown in the structural formula of claim 1 in the preparation of cosmetics.

5. A cosmetic containing the dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) represented by the structural formula of claim 1, characterized in that: The cosmetics are composed of an effective amount of any one or any combination of dihydroxybenzaldehyde cysteine ​​condensates (different isomer mixtures 1-6) and common cosmetic auxiliary materials.

6. A tyrosinase inhibitor comprising the dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) represented by the structural formula of claim 1, characterized in that: The tyrosinase inhibitor contains an effective amount of any one of dihydroxybenzaldehyde cysteine ​​condensates (diamer mixtures 1-6) or any combination thereof.

7. Use of the dihydroxybenzaldehyde cysteine ​​condensate (diamer mixture 1-6) represented by the structural formula according to claim 1 in the preparation of tyrosinase inhibitors.

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