A kojic acid-coumarin tyrosinase inhibitor and its preparation method and application

Abstract

The present invention discloses a kojic acid-coumarin tyrosinase inhibitor and a preparation method and application thereof. The chemical structural formula of the tyrosinase inhibitor is shown in formula (I): The tyrosinase inhibitor provided by the present invention has good tyrosinase inhibition activity and can be used as a lead compound for novel anti-tyrosinase research.

Description

Technical Field

[0001] The invention belongs to the technical field of new drug development and drug synthesis, and specifically relates to a kojic acid-coumarin tyrosinase inhibitor, a preparation method thereof, and an application thereof in the preparation of an anti-tyrosinase inhibitor. Background Art

[0002] Tyrosinase is a key rate-limiting enzyme in the synthesis of melanin. It catalyzes the ortho-hydroxylation of monophenols and the subsequent oxidation of diphenols to quinones, and then undergoes a series of steps to finally generate melanin. Melanin has many functions, but its most important function is to protect cells. However, abnormal pigmentation in human skin can cause chloasma, freckles, post-inflammatory melanosis, pigmented acne scars and malignant melanoma. In addition, tyrosinase also affects insect molting and wound healing, as well as enzymatic browning of fruits and vegetables.

[0003] Tyrosinase inhibitors can reduce the production of melanin by reducing the catalytic efficiency of tyrosinase. Nowadays, tyrosinase inhibitors come from a wide range of sources, including artificial synthesis, microbial fermentation, and natural plant sources. Among them, the commonly used tyrosinase inhibitors are arbutin, vitamin C, tropolone, hydroquinone, etc., but these additives have shown low efficiency and low stability in long-term use, and there is a lack of safety regulations to regulate their use.

[0004] Therefore, the development of safe, non-toxic, long-term stable and highly effective tyrosinase inhibitors has very broad application prospects in the fields of medical cosmetology, preservatives, new pesticides, etc. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a kojic acid-coumarin tyrosinase inhibitor and a preparation method thereof in view of the problems existing in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A kojic acid-coumarin tyrosinase inhibitor having a structure as shown in formula (I):

[0008]

[0009] Wherein, R in the general formula I is at least one of hydrogen, alkoxy, C1-C3 alkyl, and phenyl.

[0010] Specifically, the structural formula of the kojic acid-coumarin tyrosinase inhibitor is as follows:

[0011]

[0012]

[0013] Furthermore, the preparation method of the kojic acid-coumarin tyrosinase inhibitor specifically comprises the following steps:

[0014] Step 1: Kojic acid, potassium carbonate and 4-methoxybenzyl chloride are dissolved in N,N-dimethylformamide, followed by reflux at 80° C. for 8 hours, and cooled and purified to obtain 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one.

[0015]

[0016] Step 2: The product obtained in step 1 was dissolved in dichloromethane and stirred at 0°C for 3 minutes. Then triethylamine, 4-toluenesulfonyl chloride and 4-dimethylaminopyridine were added in sequence and stirred at room temperature for 6 hours. After the reaction was completed, dichloromethane was added to dilute the mixture, and the solution was extracted three times with 5% dilute hydrochloric acid. After combining, the solution was purified by chromatographic column to obtain 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one.

[0017]

[0018] Step 3: The product obtained in step 2, coumarin with different substitutions and potassium carbonate are refluxed in DMF for 6 hours. After cooling and filtration, the kojic acid-coumarin derivative containing the PMB protective group is obtained.

[0019]

[0020] Step 4: trifluoroacetic acid is slowly added to the dichloromethane solution containing the product obtained in step 3, and the mixture is reacted at 24° C. for 30 minutes. The extract is purified by chromatography to obtain a compound of formula (I).

[0021]

[0022] Preferably, in step 1, the molar ratio of kojic acid to 4-methoxybenzyl chloride and potassium carbonate is 1:1-2:1-3, and 1-5 ml of DMF is added for every 1 mmol of kojic acid.

[0023] Preferably, in step 2, the molar ratio of 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one to triethylamine, 4-toluenesulfonyl chloride and 4-dimethylaminopyridine is 1:1 to 2:1 to 2, and the amount of dichloromethane added per 1 mmol of 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one is 5 to 10 ml.

[0024] Preferably, in step 3, the molar ratio of the 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one, the substituted coumarin and potassium carbonate is 1:1.5:3-5, and the amount of DMF added per 1 mmol of 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one is 5-10 ml.

[0025] Preferably, in step 4, the amount of trifluoroacetic acid and dichloromethane added per 1 mmol of the kojic acid-coumarin derivative containing the PMB protecting group is 1-2 ml and 5-10 ml respectively.

[0026] In addition, the present invention also claims to protect the use of the above-mentioned kojic acid-coumarin tyrosinase inhibitor in the preparation of anti-tyrosinase agents.

[0027] And, the use of the kojic acid-coumarin tyrosinase inhibitor in the preparation of anti-tyrosinase active drugs.

[0028] Furthermore, the kojic acid-coumarin tyrosinase inhibitor is used in medical cosmetology, preservatives, and pesticides.

[0029] Compared with the prior art, the kojic acid-coumarin tyrosinase inhibitor disclosed in the present invention and its preparation method and application have the following advantages:

[0030] 1. The kojic acid-coumarin tyrosinase inhibitors provided by the present invention have good tyrosinase inhibition activity and can be used as a lead compound for new anti-tyrosinase research;

[0031] 2. The preparation method of the kojic acid-coumarin type compound disclosed in the present invention is simple, the synthetic route is short, it is easy to prepare in large quantities, and the price is low;

[0032] 3. The kojic acid-coumarin tyrosinase inhibitor provided by the present invention has low toxicity to normal human cells and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0034] Figure 1The present invention provides a preparation route for the kojic acid-coumarin tyrosinase inhibitors; wherein: (a) 4-methoxybenzyl chloride, K2CO3, DMF, 80°C, 8h, 58°C, 6h; (b) triethylamine, TsCl, CH2Cl2, DMAP, room temperature, 6h; (c) K2CO3, DMF, 90°C, 6h; (d) THF, CH2Cl2, room temperature, 0.5h.

[0035] Figure 2 A diagram showing the effect of a kojic acid-coumarin tyrosinase inhibitor on the browning of fresh lotus root slices provided in an embodiment of the present invention; wherein (A) is a photo; (B) is a change in L* value; and (C) is a change in ΔE value.

[0036] Figure 3 This is the hydrogen nuclear magnetic resonance spectrum of compound 1 provided in an embodiment of the present invention.

[0037] Figure 4 This is the carbon nuclear magnetic resonance spectrum of compound 1 provided in an embodiment of the present invention.

[0038] Figure 5 This is a high-resolution mass spectrum of compound 1 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] In order to better understand the present invention, the present invention is further specifically described below through the following examples, but it should not be understood as a limitation of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content are also considered to fall within the protection scope of the present invention.

[0041] The present invention discloses a kojic acid-coumarin tyrosinase inhibitor having a general structural formula as shown in formula (I):

[0042]

[0043] Wherein, R in the general formula I is at least one of hydrogen, alkoxy, C1-C3 alkyl, and phenyl.

[0044] like Figure 1 As shown, the present invention provides a method for preparing the kojic acid-coumarin tyrosinase inhibitor of the above general formula I, the method comprising the following steps:

[0045] Step 1: Kojic acid, potassium carbonate and 4-methoxybenzyl chloride were dissolved in DMF, followed by reflux at 80° C. for 8 hours, and cooled and purified to obtain 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one.

[0046] Step 2: The product obtained in step 1 was dissolved in dichloromethane and stirred at 0°C for 3 minutes. Then triethylamine, 4-toluenesulfonyl chloride and 4-dimethylaminopyridine were added in sequence and stirred at room temperature for 6 hours. The solution was extracted three times with 5% dilute hydrochloric acid, combined and purified by chromatographic column to obtain 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one.

[0047] Step 3: The product obtained in step 2, coumarin with different substitutions and potassium carbonate are refluxed in DMF for 6 hours. After cooling and filtration, the kojic acid-coumarin derivative containing the PMB protective group is obtained.

[0048] Step 4: Slowly add trifluoroacetic acid to the dichloromethane solution containing the product obtained in step 3, and react at 24° C. for 30 minutes. The extract is purified by chromatography to prepare the target compound I.

[0049] The technical content to be protected by the present invention will be explained below in conjunction with specific embodiments.

[0050] Embodiment 1:

[0051] Preparation of 7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-4-propylcoumarin (1)

[0052] The structural formula of compound 1 is shown below:

[0053]

[0054] The specific preparation steps are as follows:

[0055] Step 1: Under magnetic stirring, the purchased kojic acid (1mmol), potassium carbonate (1mol) and 4-methoxybenzyl chloride (2mol) were dissolved in DMF (5ml). The mixed solution was then refluxed at 80°C for 8 hours. The reaction was completed by spot plate monitoring, the reaction solution was cooled, and then diluted with an appropriate amount of water. The resulting precipitate was purified by silica gel column chromatography to obtain 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one.

[0056] Step 2: First, 1 mmol of 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one was dissolved in 5 ml of dichloromethane and stirred continuously at 0°C for 3 minutes. Then, triethylamine (1.5 mmol), 4-toluenesulfonyl chloride (1.5 mol) and 4-dimethylaminopyridine (1.5 mmol) were added to the mixed solution in sequence, and the reaction was stirred continuously at room temperature for 6 hours after the ice bath was removed. After the reaction was completed, 20 ml of dichloromethane was added to dilute the mixed solution, and the solution was extracted three times with 5% dilute hydrochloric acid. Finally, the collected organic layer solutions were combined and dried, and then purified by silica gel chromatography to obtain 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one.

[0057] Step 3: 2-(Chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one (1mmol), 7-hydroxycoumarin (1.5mmol) and potassium carbonate were added to 5mL DMF, and then refluxed at 90°C for 6 hours. After the reaction was completed by TLC, the mixed solution was cooled and 30mL of water was added. At this time, the solid precipitate was filtered and dried to obtain 7-((5-((4-methoxybenzyl)oxy)-4-oxo-4H-pyran-2-yl)methoxy)-4-propyl-coumarin.

[0058] Step 4: Slowly add 1 mL of trifluoroacetic acid to a dichloromethane solution containing the compound 7-((5-((4-methoxybenzyl)oxy)-4-oxo-4H-pyran-2-yl)methoxy)-4-propyl-coumarin, and continue stirring the reaction solution at room temperature for half an hour. After the reaction is completed by spot plate monitoring, a large amount of dichloromethane solution is poured into the reaction solution for dilution, and then extracted three times with a saturated sodium bicarbonate solution. The organic layers are combined and dried by spin drying, and finally purified by a chromatographic column to obtain the target compound 1.

[0059] Compound 1 is a white solid with a yield of 33% and a melting range of 293-297°C; Figure 3-Figure 5 As shown, the data of H NMR spectrum, C NMR spectrum and mass spectrum of compound 1 are as follows:

[0060] 1HNMR(400MHz,DMSO-d6)δ:9.25(s,1H,pyranone-OH),8.09(s,2H,Ar-H),7.63(d,1H,J=8.8Hz,Ar-H),7.10(d,1H,J=2.4Hz,Ar- H),7.09(s,1H,pyranone-H),7.02(d,1H,J=8.4Hz),6.56(s,1H,pyranone-H),6.29(d,1H,J=9.6Hz,Ar-H),5.10(s,2H,CH2O);

[0061] 13 CNMR(100MHz,DMSO-d6)δ:174.1,162.2,161.0,160.6,155.8,146.6,144.6,140.5,130.2,113.7,113.3,102.4,66.5;

[0062] HRMS (ESI) calculation for [M+Na] + C 15 H 10 NaO6 + :309.0370,found:309.0382.

[0063] The preparation methods of the following examples are similar to those of Example 1, and the ratio of raw materials used is the same as that of Example 1, except that the 7-hydroxycoumarin in Example 1 is replaced with other corresponding substituted coumarins.

[0064] Embodiment 2:

[0065] Preparation of 4-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)coumarin (2)

[0066] The structural formula of compound 2 is shown below:

[0067]

[0068] Compound 2 is a reddish brown solid with a yield of 30% and a melting range of 292-300°C. The H-NMR spectrum and C-NMR spectrum of compound 2 are shown below:

[0069] 1HNMR(400MHz,DMSO-d6)δ:9.31(s,1H,pyranone-OH),8.13(s,1H,pyranone-H),7.82(d,1H,J=8.0Hz,Ar-H),7.64 (t,1H,J=8.0Hz,Ar-H),7.40-7.34(m,2H,Ar-H),6.67(s,1H,pyranone-H),6.05(s,1H,Ar-H),5.24(s,2H,CH2O);

[0070] 13 CNMR(100MHz,DMSO-d6)δ:174.1,164.5,161.8,160.9,153.3,146.7,140.6,133.5,124.9,123.5,117.0,115.4,113.7,92.3,67.1;

[0071] HRMS (ESI) calculation for [M+Na] + C 15 H 10 NaO6 + :309.0370,found:309.0380.

[0072] Embodiment three:

[0073] Preparation of 7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-4-methylcoumarin (3)

[0074] The structural formula of compound 3 is shown below:

[0075]

[0076] Compound 3 is a white solid with a yield of 32% and a melting range of 298-301°C. The H-NMR spectrum and C-NMR spectrum of compound 3 are shown below:

[0077] 1 HNMR(400MHz,DMSO-d6)δ:9.25(s,1H,pyranone-OH),8.08(s,1H,pyranone-H),7.73(d,1H,J=12.0Hz,Ar-H),7.09(d,1H,J =2.4Hz,Ar-H),7.05-7.02(m,1H,Ar-H),6.56(s,1H,pyranone-H),6.21(s,1H,Ar-H),5.11(s,2H,CH2O),2.36(s,3H,CH3);

[0078] 13 CNMR(100MHz,DMSO-d6)δ:174.2,162.1,161.0,155.7,153.3,147.6,146.6,142.1,140.5,120.3,113.6,110.8,110.3,109.0,67.4,13.5;

[0079] HRMS (ESI) calculation for [M+Na] + C 16 H 12 NaO6 + :323.0526,found:323.0543.

[0080] Embodiment 4:

[0081] Preparation of 7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-3,4,5-trimethylcoumarin (4)

[0082] The structural formula of compound 4 is shown below:

[0083]

[0084] Compound 4 is a reddish brown solid with a yield of 40% and a melting range of 204-210°C. The H-NMR spectrum and C-NMR spectrum of compound 4 are shown below:

[0085] 1 HNMR(400MHz,DMSO-d6)δ:9.30(s,1H,pyranone-OH),8.12(s,1H,pyranone-H),6.83(d,2H,J=1 8.0Hz,Ar-H),6.58(s,1H,pyranone-H),5.08(s,2H,CH2O),2.32(s,3H,CH3),2.02(s,3H,CH3);

[0086] 13 CNMR(100MHz,DMSO-d6)δ:162.2,160.9,160.6,155.1,153.8,140.5,127.2,114.5,113.3,133.0,112.2,102.4,66.4,55.3,18.6,18.5;

[0087] HRMS(ESI)calcdfor[MH] - C 18 H 15 O6 -:327.0874,found:327.0871.

[0088] Embodiment five:

[0089] Preparation of 4-ethyl-7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)coumarin (5)

[0090] The structural formula of compound 5 is shown below:

[0091]

[0092] Compound 5 is a yellow solid with a yield of 58.2% and a melting range of 234-238°C. The H-NMR spectrum and C-NMR spectrum of compound 5 are shown below:

[0093] 1 HNMR(400MHz,DMSO-d6)δ:9.23(s,1H,pyranone-OH),8.09(s,1H,pyranone-H),7.73(d,1H,J=8.8Hz,Ar-H),7.10(s,1H ,Ar-H),7.03(d,1H,J=8.4Hz,Ar-H),6.56(s,1H,pyranone-H),6.15(s,1H,Ar-H),5.10(s,2H,CH2O),2.80-2.74(m,2H, CH2 CH3),1.18(t,3H,J=7.2Hz,CH2 CH3 );

[0094] 13 CNMR(100MHz,DMSO-d6)δ:174.1,162.2,160.8,158.6,155.3,146.6,140.5,126.8,113.7,113.3,113.1,110.4,102.7,102.6,66.4,24.6,12.8;

[0095] HRMS(ESI)calcdfor[MH] - C 17 H 13 O6 - :313.0718,found:313.0716.

[0096] Embodiment six:

[0097] Preparation of 7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-4-propylcoumarin (6)

[0098] The structural formula of compound 6 is shown below:

[0099]

[0100] Compound 6 is a reddish brown solid with a yield of 33% and a melting range of 225-234°C. The H-NMR spectrum and C-NMR spectrum of compound 6 are shown below:

[0101] 1 HNMR(400MHz,DMSO-d6)δ:9.27(s,1H,pyranone-OH),8.10(s,1H,pyranone-H),7.74(d,1H,J=8.8Hz,Ar-H),7.10(d,1H,J=2.4Hz ,Ar-H),7.02(dd,1H,J=2.8,8.8Hz,Ar-H),6.56(s,1H,pyranone-H),6.16(s,1H,Ar-H),5.10(s,2H,CH2O),2.70(t,2H,J=7.6Hz, CH2 CH2CH3),1.62-1.56(q,2H,J=7.2Hz,CH2 CH2 CH3),0.93(t,3H,J=6.4Hz,CH2CH2 CH3 );

[0102] 13 CNMR(100MHz,DMSO-d6)δ:174.1,162.2,160.8,160.6,157.1,155.4,146.6 ,140.5,126.9,113.7,113.3,113.0,111.3,102.6,66.4,33.3,21.8,14.1;

[0103] HRMS(ESI)calcdfor[MH] - C 18 H 15 O6 - :327.0874,found:327.0870.

[0104] Embodiment seven:

[0105] Preparation of 7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-3,4-dimethylcoumarin (7)

[0106] The structural formula of compound 7 is shown below:

[0107]

[0108] Compound 7 is a reddish brown solid with a yield of 84% and a melting range of 260-270°C. The H-NMR spectrum and C-NMR spectrum of compound 7 are shown below:

[0109] 1 HNMR(400MHz,DMSO-d6)δ:9.23(s,1H,pyranone-OH),8.09(s,1H,pyranone-H),7.69(d,1H,J=8.8Hz,Ar-H),7.06(s,1 H,Ar-H),7.01(d,1H,J=8.8Hz,Ar-H),6.55(s,1H,pyranone-H),5.09(s,2H,CH2O),2.33(s,3H,CH3),3.40(s,3H,CH3);

[0110] 13 CNMR(100MHz,DMSO-d6)δ:174.1,162.3,161.6,159.9,153.4,147.1,146.6,140.5,126.8,119.0,115.0,113.3,112.9,102.2,66.4,15.4,13.4;

[0111] HRMS(ESI)calcdfor[MH] - C 17 H 13 O6 - :313.0718,found:313.0717.

[0112] Embodiment eight:

[0113] Preparation of 7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-4,5-dimethylcoumarin (8)

[0114] The structural formula of compound 8 is shown below:

[0115]

[0116] Compound 8 is a reddish brown solid with a yield of 84% and a melting range of 260-270°C. The H-NMR spectrum and C-NMR spectrum of compound 8 are shown below:

[0117] 1HNMR(400MHz,DMSO-d6)δ:7.60(s,1H,pyranone-H),6.84(d,2H,J=16Hz,Ar-H),6.37(s,1 H,pyranone-H),6.10(s,1H,Ar-H),4.99(s,2H,CH2O),2.43(s,3H,CH3),2.33(s,3H,CH3);

[0118] 13 CNMR(100MHz,DMSO-d6)δ:159.9,156.6,155.2,154.0,143.7,139.2,113.8,112.1,110.8,110.1,108.4,67.9,24.1,21.9;

[0119] HRMS(ESI)calcdfor[MH] - C 17 H 13 O6 - :313.0718,found:313.0719.

[0120] Embodiment nine:

[0121] Preparation of 7-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-4,8-dimethylcoumarin (9)

[0122] The structural formula of compound 9 is shown below:

[0123]

[0124] Compound 9 is a yellow solid with a yield of 47% and a melting range of 293-301°C. The H-NMR spectrum and C-NMR spectrum of compound 9 are shown below:

[0125] 1 HNMR(400MHz,DMSO-d6)δ:9.25(s,1H,pyranone-OH),8.10(s,1H,pyranone-H),7.59(d,1H,J=12.0Hz,Ar-H),7.11(d,1H,J =8Hz,Ar-H),6.51(s,1H,pyranone-H),6.21(d,1H,J=4.0Hz,Ar-H),5.14(s,2H,CH2O),2.36(s,3H,CH3),2.19(s,3H,CH3);

[0126] 13CNMR(100MHz,DMSO-d6)δ:174.1,162.7,160.5,158.5,153.9,152.6,146.6,140.5,124.1,114.6,113.8,112.9,112.1,109.3,66.7,18.7,8.6;

[0127] HRMS(ESI)calcdfor[MH] - C 17 H 13 O6 - :313.0718,found:313.0716.

[0128] Embodiment ten:

[0129] Preparation of 4-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-6,8-dimethylcoumarin (10)

[0130] The structural formula of compound 10 is shown below:

[0131]

[0132] Compound 10 is a white solid with a yield of 93% and a melting range of 315-327°C. The H-NMR spectrum and C-NMR spectrum of compound 10 are shown below:

[0133] 1 HNMR(400MHz,DMSO-d6)δ:9.35(s,1H,pyranone-OH),8.14(s,1H,pyranone-H),7.43(s,1H,Ar-H),7 .34(s,1H,Ar-H),6.67(s,1H,pyranone-H),6.01(s,1H,Ar-H),5.21(s,2H,CH2O),2.29(s,6H,CH3);

[0134] 13 CNMR(100MHz,DMSO-d6)δ:174.2,164.8,161.9,160.9,149.8,146.8,140.7,135.5,133.6,125.6,120.5,114.8,113.8,91.9,67.1,20.8,15.6.;

[0135] HRMS(ESI)calcdfor[MH] - C 17 H 13 O6 -:313.0718,found:313.0720.

[0136] Embodiment eleven:

[0137] Preparation of 4-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-7,8-benzocoumarin (11)

[0138] The structural formula of compound 11 is shown below:

[0139]

[0140] Compound 11 is a white solid with a yield of 98% and a melting range of 268-281°C. The H-NMR spectrum and C-NMR spectrum of compound 11 are shown below:

[0141] 1 HNMR(400MHz,DMSO-d6)δ:9.35(s,1H,pyranone-OH),8.08-8.02(m,2H,pyranone-H,Ar-H),7.82(d,2H,J =4.0Hz,Ar-H),7.72-7.69(m,2H,Ar-H),6.66(s,1H,pyranone-H),6.13(s,1H,Ar-H),5.28(s,2H,CH2O);

[0142] 13 CNMR(100MHz,DMSO-d6)δ:174.1,165.4,161.8,160.9,150.6,146.7,140.7,135 .4,129.5,128.6,128.0,124.6,122.6,122.3,119.0,113.8,110.8,91.8,67.3;

[0143] HRMS(ESI)calcdfor[MH] - C 19 H 11 O6 - :335.0561,found:335.0561.

[0144] Embodiment 12:

[0145] Preparation of 4-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-7-methylcoumarin (12)

[0146] The structural formula of compound 12 is shown below:

[0147]

[0148] Compound 12 is a reddish brown solid with a yield of 94% and a melting range of 312-320°C. The H-NMR spectrum and C-NMR spectrum of compound 12 are shown below:

[0149] 1 HNMR(400MHz,DMSO-d6)δ:9.17(s,1H,pyranone-OH),8.09(s,1H,pyranone-H),7.69(d,1H,J=4.0Hz,Ar-H) ,7.19(t,2H,J=10.8Hz,Ar-H),6.63(s,1H,pyranone-H),5.95(s,1H,Ar-H),5.21(s,2H,CH2O),2.39(s,3H);

[0150] 13 CNMR(100MHz,DMSO-d6)δ:174.2,162.1,161.0,155.7,153.3,147.6,146.6,142.1,140.5,120.3,113.6,110.8,110.3,109.0,67.4,13.5;

[0151] HRMS(ESI)calcdfor[MH] - C 16 H 11 O6 - :299.0561,found:299.0559.

[0152] Embodiment 13:

[0153] Preparation of 4-((5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy)-7-methoxycoumarin (13)

[0154] The structural formula of compound 13 is shown below:

[0155]

[0156] Compound 13 is a reddish brown solid with a yield of 92% and a melting range of 283-295°C. The H-NMR spectrum and C-NMR spectrum of compound 13 are shown below:

[0157] 1HNMR(400MHz,DMSO-d6)δ:9.17(s,1H,pyranone-OH),8.09(s,1H,pyranone-H),7.71(d,1H,J=8.8Hz,Ar-H), 7.96-6.91(m,2H,Ar-H),6.63(s,1H,pyranone-H),5.86(s,1H,Ar-H),5.20(s,2H,CH2O),2.46(s,3H,OCH3);

[0158] 13 CNMR(100MHz,DMSO-d6)δ:174.1,164.9,163.7,162.2,161.0,155.2,146.7,140.6,124.6,113.7,112.8,108.5,101.2,89.56,67.0,56.5;

[0159] HRMS(ESI)calcdfor[MH] - C 16 H 11 O7 - :315.0510,found:315.0509.

[0160] In order to further verify the excellent effect of the present invention, the inventors also conducted the following experimental comparisons:

[0161] Experiment 1

[0162] First, add 0.1 ml of inhibitors of different concentrations (dissolved in DMSO), 2.8 ml of substrate solution (0.5 mM), and 0.1 ml of tyrosinase solution (200 U / ml) to a quartz cuvette. After rapid mixing, use an ultraviolet spectrophotometer (UV-2700) to measure the growth curve of the absorbance of the mixed solution at a wavelength of 475 nm over time, and calculate the IC 50 The results are shown in Table 1.

[0163] Table 1. Inhibitory activity of kojic acid-coumarin compounds against tyrosinase (IC 50 ):

[0164]

[0165] a Values ​​are the mean of three replicates ± SD. 50 : The inhibitory concentration of the test compound or kojic acid required to inhibit the enzyme activity by 50%.

[0166] b IC 50Values ​​were determined from dose response curves. All concentrations are final test compound or kojic acid in the reaction mixture.

[0167] The experimental results are listed in Table 1. Compared with the positive control kojic acid (16.38±0.57μM), these 13 compounds showed stronger anti-tyrosinase activity, among which compounds 5 and 6 had the most significant activity, with IC 50 The values ​​were 0.97 ± 0.10 μM and 0.88 ± 0.10 μM, respectively.

[0168] Experiment 2

[0169] The cytotoxicity of compound 6 on the human normal embryonic kidney cell line HEK-293 was evaluated using the CCK-8 method. The cells were seeded in a 96-well plate. After incubation for 24 hours, the cells were treated with different concentrations of the compound. After 24 hours, CCK-8 (10 μL) solution was added for 2 hours. The absorbance value of the solution at 450nm was then recorded using a multifunctional microplate reader, and the survival rate of the compound on normal cells was calculated using SPSS software.

[0170] Table 2. Effect of compound 6 on the viability of HEK-293 cells:

[0171]

[0172] The experimental results show that compound 6 has low toxicity and high safety.

[0173] Experiment 3

[0174] First, rinse the soil off the surface of the lotus root, cut it into thin slices of uniform size, and then completely immerse it in the test solution for 10 minutes. After treatment, the samples were sealed with sterile sampling bags and stored at room temperature. The test solutions were water (control group), kojic acid (0.0025%) and compound 6 (0.0025%). The potato slices were photographed regularly within 24 hours, and the L* value (brightness), a* value (red and green) and b* value (yellow and blue) were recorded using a colorimeter. The degree of browning of the lotus root slices was determined by calculating ΔE (total color difference).

[0175] Depend on Figure 2 (A) It can be seen that compound 6 can make lotus root slices store at room temperature for 24 hours without obvious browning. Figure 2 (B) It can be seen that the initial L* value has no statistically significant difference and shows a downward trend over time. The blank group has almost no anti-browning effect on lotus root slices at room temperature, while the anti-browning effect of compound 6 group is more obvious than that of kojic acid group. The results of the color difference parameter ΔE value are consistent with the L* value, such as Figure 2 (C). Therefore, it is shown that compound 6 can effectively delay the browning of fresh-cut lotus root slices.

[0176] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the technical content disclosed above, or modify it into an equivalent embodiment of equivalent changes without departing from the scope of the technical solution of the present invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention should fall within the scope of protection of the technical solution of the present invention.

[0177] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kojic acid-coumarin tyrosinase inhibitor, characterized in that: It has the general structural formula shown in formula (I): ； Wherein, R in the general formula I is hydrogen, alkoxy, or C1-C3 alkyl, Or, the structural formula of the kojic acid-coumarin tyrosinase inhibitor is .

2. A method for preparing the kojic acid-coumarin tyrosinase inhibitor according to claim 1, characterized in that: The method specifically comprises the following steps: Step 1: dissolving kojic acid, potassium carbonate and 4-methoxybenzyl chloride in N,N-dimethylformamide, followed by reflux at 80° C. for 8 hours, cooling and purification to obtain 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one; Step 2: Dissolve the 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one obtained in step 1 in dichloromethane, and stir at 0°C for 3 minutes; then add triethylamine, 4-toluenesulfonyl chloride and 4-dimethylaminopyridine in sequence and stir at room temperature for 6 hours, add dichloromethane to dilute the mixed solution, and then extract the solution three times with 5% dilute hydrochloric acid. After combining, purify by chromatographic column to obtain 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one; Step 3: reflux the 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one obtained in step 2, substituted coumarin and potassium carbonate in DMF for 6 hours, and obtain a kojic acid-coumarin derivative containing a PMB protective group after cooling and filtration; The structure of the substituted coumarin is ; Step 4: slowly add trifluoroacetic acid to the dichloromethane solution containing the kojic acid-coumarin derivative obtained in step 3, react at 24° C. for 30 minutes, extract and purify by chromatography to prepare a compound of formula (I), i.e., the kojic acid-coumarin tyrosinase inhibitor.

3. The method for preparing a kojic acid-coumarin tyrosinase inhibitor according to claim 2, characterized in that: In step 1, the molar ratio of kojic acid to 4-methoxybenzyl chloride and potassium carbonate is 1: 1-2: 1-3, and 1-5 ml of DMF is added for every 1 mmol of kojic acid.

4. The method for preparing a kojic acid-coumarin tyrosinase inhibitor according to claim 2, characterized in that: In step 2, the molar ratio of 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one to triethylamine, 4-toluenesulfonyl chloride and 4-dimethylaminopyridine is 1: 1~2: 1~2: 1~2, and the amount of dichloromethane added per 1 mmol of 2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one is 5~10 ml.

5. The method for preparing a kojic acid-coumarin tyrosinase inhibitor according to claim 2, characterized in that: In step 3, the molar ratio of 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one, substituted coumarin and potassium carbonate is 1:1.5:3-5, and the amount of DMF added per 1 mmol of 2-(chloromethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one is 5-10 ml.

6. The method for preparing a kojic acid-coumarin tyrosinase inhibitor according to claim 2, characterized in that: In step 4, the amounts of trifluoroacetic acid and dichloromethane added per 1 mmol of the kojic acid-coumarin derivative containing a PMB protecting group are 1-2 ml and 5-10 ml, respectively.

7. Use of the kojic acid-coumarin tyrosinase inhibitor according to claim 1 or the kojic acid-coumarin tyrosinase inhibitor prepared by the method according to any one of claims 2 to 6 in the preparation of an anti-tyrosinase preparation.

8. Use of the kojic acid-coumarin tyrosinase inhibitor according to claim 1 or the kojic acid-coumarin tyrosinase inhibitor prepared by the method according to any one of claims 2 to 6 in the preparation of an anti-tyrosinase active drug.

9. Use of the kojic acid-coumarin tyrosinase inhibitor according to claim 1 or the kojic acid-coumarin tyrosinase inhibitor prepared by the method according to any one of claims 2 to 6 in the preparation of medical cosmetic preparations, preservatives, and pesticides.

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