L-phenylalanine derivative tyrosinase inhibitor and preparation method thereof

Abstract

The present invention belongs to the field of pharmaceutical synthesis and specifically discloses an L-phenylalanine derivative tyrosinase inhibitor and its preparation method. The tyrosinase inhibitor comprises a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, prodrug, or isotopic variant thereof. The compound represented by formula (I) of the present invention has tyrosinase inhibitory activity superior to that of kojic acid, can inhibit excessive melanin accumulation, and has great application potential in inhibiting mushroom tyrosinase. Therefore, it can be used as a mushroom preservative. The compound represented by formula (I) of the present invention exerts tyrosinase inhibitory activity by acting on amino acid residues and copper ions within the active site and is a reversible competitive inhibitor. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of drug synthesis, and in particular relates to an L-phenylalanine derivative tyrosinase inhibitor and a preparation method thereof. Background Art

[0002] Tyrosinase, with its binuclear copper ion structure, is a key metalloenzyme in the synthesis of melanin and is closely associated with many pigmentation-related skin diseases. Tyrosinase catalyzes the hydroxylation of tyrosine, initiating a series of biochemical reactions to produce two melanin pigments. Tyrosinase plays a key role in the synthesis of neuromelanin and is a key factor in dopamine neurotoxicity, thereby influencing the induction and progression of many important neurodegenerative diseases, such as Parkinson's disease. Furthermore, tyrosinase promotes insect development and defense, playing a crucial role in melanin formation, wound healing, anti-parasitic activities, and the physiological processes of skin keratinization. Tyrosinase is also a key enzyme in the enzymatic browning of fruits and vegetables.

[0003] Tyrosinase inhibitors derived from natural plant ingredients have attracted widespread attention in the whitening cosmetic market, such as licorice extract, bearberry extract, natural vitamin C, animal placenta extract, and aloe vera extract. However, most tyrosinase inhibitors used as cosmetic additives have drawbacks to varying degrees. For example, while vitamin C inhibits tyrosinase, it is unstable and has a short shelf life; fruit acid is highly irritating to human skin; animal placenta extract and aloe vera extract are expensive, contain complex ingredients, and are easily perishable; and structurally modified small molecule inhibitors require more market samples to confirm their safety. Therefore, the research and development of effective and safe tyrosinase inhibitors is becoming increasingly important in the pharmaceutical, cosmetic, agricultural, and food industries. Summary of the Invention

[0004] In order to overcome at least one technical problem existing in the above-mentioned prior art, one of the objects of the present invention is to provide a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, prodrug or isotopic variant thereof;

[0005]

[0006] A second object of the present invention is to provide a method for preparing the compound represented by formula (I).

[0007] A third object of the present invention is to provide a tyrosinase inhibitor.

[0008] A fourth object of the present invention is to provide a product.

[0009] A fifth object of the present invention is to provide the use of the compound represented by the above formula (I) or its pharmaceutically acceptable salts, esters, stereoisomers, solvates, prodrugs or isotopic variants in the preparation of products that inhibit tyrosinase.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, prodrug or isotopic variant thereof;

[0012]

[0013] wherein each R1 is independently selected from halogen, nitro;

[0014] R2 is selected from

[0015] Each R3 is independently selected from halogen, nitro;

[0016] n and m are independently selected from 0, 1, 2, 3, 4 or 5.

[0017] In some embodiments of the present invention, each R3 is independently selected from F, nitro.

[0018] In some embodiments of the present invention, n is selected from 1, 2 or 3.

[0019] In some embodiments of the present invention, m is selected from 0, 1, 2 or 3.

[0020] In some embodiments of the present invention, R2 is selected from 1-methyl-4-nitro-phenyl, 1-methyl-4-fluoro-phenyl, 5-hydroxy-4-oxyylidenepyran-2-methyl.

[0021] In some embodiments of the present invention, the compound represented by formula (I) is selected from

[0022] The second aspect of the present invention provides a method for preparing the compound represented by formula (I) according to the first aspect of the present invention, comprising the following steps:

[0023] S1: Make L-phenylalanine and Reaction generation

[0024] S2: Make and Reaction to obtain the compound represented by formula (I).

[0025] In some embodiments of the present invention, the step S1 is: L-phenylalanine and React under alkaline conditions at 60-70°C for 5-7h, then cool to -5-5°C and react with sodium borohydride or potassium borohydride for 1-4h, then acidify to obtain

[0026]

[0027] In some embodiments of the present invention, the solvent in step S1 is selected from at least one of water and ethanol; in some embodiments of the present invention, the solvent in step S1 is selected from a mixture of water and ethanol.

[0028] In some embodiments of the present invention, in step S1, the molar volume ratio of L-phenylalanine to ethanol is: 1 mmol: (1-5) mL.

[0029] In some embodiments of the present invention, in step S1, the molar ratio of L-phenylalanine to the base is 1:(1-2).

[0030] In some embodiments of the present invention, the base in step S1 is selected from at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.

[0031] In some embodiments of the present invention, in step S1, L-phenylalanine and The molar ratio is 1:(1.5~2).

[0032] In some embodiments of the present invention, in step S1, the ratio of the molar amount of L-phenylalanine to the total molar amount of sodium borohydride or potassium borohydride is 1:(2-3).

[0033] In some embodiments of the present invention, the acidification step refers to adjusting the pH to 4-6 using acid.

[0034] In some embodiments of the present invention, the acid is selected from at least one of hydrochloric acid, nitric acid, hypochlorous acid, and sulfuric acid. In some embodiments of the present invention, the acid is hydrochloric acid.

[0035] In some embodiments of the present invention, step S2 is: and The catalyst, base and solvent are reacted at 60-80° C. for 1-4 hours to prepare the compound represented by formula (I).

[0036] In some embodiments of the present invention, and The molar ratio is 1:(1~1.5).

[0037] In some embodiments of the present invention, and The molar ratio is 1:(1~1.5).

[0038] In some embodiments of the present invention, The molar ratio to the catalyst is 1:(0.1~0.5).

[0039] In some embodiments of the present invention, The molar ratio of the alkali is 1: (1.5-2.0).

[0040] In some embodiments of the present invention, the catalyst is selected from at least one of potassium iodide and sodium iodide.

[0041] In some embodiments of the present invention, the solvent in step S2 includes N,N-dimethylformamide.

[0042] In some embodiments of the present invention, the base in step S2 is selected from at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate.

[0043] The reaction scheme of the compound represented by formula (I) of the present invention is:

[0044]

[0045] The third aspect of the present invention provides a tyrosinase inhibitor, comprising the compound represented by formula (I) according to the first aspect of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, prodrug or isotope variant thereof.

[0046] The fourth aspect of the present invention provides a product comprising the compound represented by formula (I) according to the first aspect of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, prodrug or isotope variant thereof; the product is selected from cosmetics, preservatives or pesticides.

[0047] The fifth aspect of the present invention provides the use of the compound represented by formula (I) or its pharmaceutically acceptable salt, ester, stereoisomer, solvate, prodrug or isotope variant described in the first aspect of the present invention in the preparation of a product that inhibits tyrosinase.

[0048] Terms and descriptions:

[0049] The term "pharmaceutically acceptable salt" as used herein refers to a salt formed by an acidic functional group (e.g., -COOH, -OH, etc.) present in a compound with a suitable inorganic or organic cation (base), including salts formed with alkali metals or alkaline earth metals, ammonium salts, and salts formed with nitrogen-containing organic bases. These salts can be prepared during the synthesis, isolation, and purification of the compound, or by reacting the purified free form of the compound with a suitable acid or base.

[0050] The term "solvate" as used herein includes hydrates. The compounds of the present invention can exist in unsolvated as well as solvated forms, including hydrates. In general, solvated forms are equivalent to unsolvated forms and are also encompassed within the scope of the present invention.

[0051] "Esters" as used herein refer to products formed by the dehydration of an acid and an alcohol. When a -COOH group is present in the structure of a compound of the present invention, it can dehydrate with a pharmaceutically acceptable alcohol compound to form an ester. When a -OH group is present in the structure of a compound of the present invention, it can dehydrate with a pharmaceutically acceptable organic or inorganic acid compound to form an ester. These ester compounds can produce the active compounds of the present invention in vivo through metabolism or hydrolysis. These esters may exhibit similar biological activity to the free form in vitro, or they may have no or weak biological activity.

[0052] The compounds of the present invention exist in stereoisomers. Specific examples of the "isomers" according to the present invention include cis-trans isomers, enantiomers, diastereomers, tautomers, and racemic mixtures and other mixtures thereof. All of these mixtures fall within the scope of the present invention.

[0053] The term "enantiomer" refers to stereoisomers that are mirror images of one another.

[0054] The term "tautomer" refers to a class of functional group isomers that have different points of attachment via the displacement of one or more double bonds, for example, a ketone and its enol form are keto-enol tautomers.

[0055] The term "diastereomer" refers to stereoisomers that have two or more chiral centers and are not mirror images of each other.

[0056] The term "cis-trans isomers" refers to different configurations formed by different spatial arrangements of double bonds or single bonds of ring carbon atoms in a molecule that cannot rotate freely.

[0057] The beneficial effects of the present invention are as follows: the compound represented by formula (I) of the present invention has a tyrosinase inhibitory activity superior to that of kojic acid, can inhibit excessive accumulation of melanin, and has great application potential in inhibiting mushroom tyrosinase. Therefore, it can be used as a mushroom preservative. The compound represented by formula (I) of the present invention has a significant inhibitory effect on mushroom tyrosinase, and its IC 50 The values ​​ranged from 4.86±0.026μmol / L to 20.64±0.273μmol / L, all of which were superior to the positive control kojic acid. The compound of formula (I) exerts tyrosinase inhibitory activity by acting on amino acid residues and copper ions within the active site and is a reversible competitive inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is a diagram of the tyrosinase inhibition mechanism of compound 10 in Example 10;

[0059] Figure 2 is the Lineweaver-Burk double reciprocal curve plot of compound 10 in Example 10;

[0060] Figure 3 This is a molecular docking model diagram of compound 10 in Example 10. DETAILED DESCRIPTION

[0061] The specific implementation of the present invention will be further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are any processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be purchased commercially.

[0062] Example 1

[0063] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(2-chlorophenyl)methyl]-L-phenylalanine-(5-hydroxy-4-oxyylidenepyran-2-yl)methyl ester, denoted as Compound 1. The structural formula of Compound 1 is shown below:

[0064]

[0065] The tyrosinase inhibitor in this example is prepared by a preparation method comprising the following steps:

[0066] Step 1: Under magnetic stirring, commercially available L-phenylalanine (5 mmol) and potassium hydroxide (5 mmol) were dissolved in ethanol (15 mL) and water (2 mL). The mixture was stirred until completely dissolved and cooled to room temperature. 7.5 mmol of 2-chlorobenzaldehyde was then added to the mixture and stirred at 65°C for 7 h. Cooled to 0°C in an ice bath, sodium borohydride (10 mmol) was added and stirred for 2 h. The mixture was acidified to pH 5 with 5% by volume dilute hydrochloric acid and purified by crystallization from dichloromethane / ethanol to obtain the intermediate N-[(2-chlorophenyl)methyl]-L-phenylalanine.

[0067] Step 2: Dissolve N-[(2-chlorophenyl)methyl]-L-phenylalanine (3 mmol) and potassium carbonate (4.5 mmol) obtained in step 1 in N,N-dimethylformamide and stir until completely dissolved. Add 3.6 mmol of 2-(chloromethyl)-5-hydroxy-4H-pyran-4-one and 0.3 mmol of potassium iodide sequentially. Heat under reflux at 70°C for 2 h. After completion of the reaction, add 150 mL of water and stir. Then, acidify with 5% (v / v) dilute hydrochloric acid to a pH of 7, filter, and extract the filtrate with 300 mL of ethyl acetate. Finally, combine the organic layers, dry over Na2SO4, and remove the solvent. The residue is purified on a silica gel column to obtain N-[(2-chlorophenyl)methyl]-L-phenylalanine-(5-hydroxy-4-oxyylidenepyran-2-yl)methyl ester.

[0068] Compound 1 is a white solid with a yield of 34.0% and a melting range of 90-91°C. The H-NMR, C-NMR, and high-resolution mass spectrometry data are shown below: 1 H NMR (400MHz, deuterated chloroform) δ: 7.78(s,1H,CH),7.31(m,1H,ArH),7.28(s,1H,ArH),7.24(s,1H,ArH),7.19(s,1H,ArH),7.17(m,2H,ArH),7.15(d,J=1 .6Hz,2H,ArH),7.13(s,1H,ArH),6.35(s,1H,CH),4.80(s,2H,CH2),3.85(m,2H,CH2),3.63(t,J=7.2Hz,1H,CH),3.00(p,J=6.4Hz,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 173.7, 173.5, 162.2, 145.8, 137.7, 136.7, 136.6, 133.8, 130.0, 129.5, 129.1 (2C), 128.6 (2C), 128.5, 127.0, 126.8, 111.5, 62.2, 61.6, 49.5, 39.7. HRMS (ESI) calculated for C22 H 21 ClNO5[M+H] + 413.11028, detected 414.10905.

[0069] Example 2

[0070] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-nitrophenyl)methyl]-L-phenylalanine-(5-hydroxy-4-oxyylidenepyran-2-yl)methyl ester, denoted as Compound 2. The structural formula of Compound 2 is shown below:

[0071]

[0072] Compound 2 is a yellow solid with a yield of 43.5% and a melting range of 93-95°C. The data of H-NMR, C-NMR, and high-resolution mass spectrometry are shown below: 1 H NMR (400MHz, deuterated chloroform) δ: 8.15 (d, J = 8.4Hz, 2H, ArH), 7.83 (d, J = 1.2Hz, 1H, CH), 7.39 (d ,J=8.0Hz,2H,ArH),7.32(s,1H,ArH),7.30(s,1H,ArH),7.28(s,1H,ArH),7.17(d, J=7.6Hz,2H,ArH),6.38(s,1H,CH),4.92(s,2H,CH2),4.01(d,J=14.4Hz,1H,CH2), 3.71(d,J=14.4Hz,1H,CH2), 3.60(t,J=7.2Hz,1H,CH), 3.04(d,J=6.8Hz,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 173.8, 173.4, 162.0, 147.2, 147.0, 146.0, 138.0, 136.5, 129.2 (2C), 128.6 (2C), 128.6 (2C), 127.1, 123.6 (2C), 111.7, 62.1, 61.7, 51.1, 39.7. HRMS (ESI) calculated for C 22 H 21 N2O7[M+H] + 425.13433, detection found 425.13351.

[0073] Example 3

[0074] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-bromophenyl)methyl]-L-phenylalanine-(5-hydroxy-4-oxyylidenepyran-2-yl)methyl ester, denoted as Compound 3. The structural formula of Compound 3 is shown below:

[0075]

[0076] Compound 3 is a light yellow solid with a yield of 41.8% and a melting range of 142-143°C. The H-NMR spectrum, C-NMR spectrum, and high-resolution mass spectrometry data are shown below: 1 H NMR (400 MHz, deuterated chloroform) δ: 7.79 (s, 1H, CH), 7.40 (d, J = 8.4 Hz, 2H, ArH), 7.26-7.23 (m, 4H, ArH), 7.17-7.05 (m, 3H, ArH), 6.33 (s, 1H, CH), 4.84 (s, 2H, CH2), 3.78 (d, J = 13.6 Hz, 1H, CH2), 3.63 (s, 1H, CH2), 3.59 (t, J = 7.2 Hz, 1H, CH), 2.99 (t, J = 6.8 Hz, 2H, CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 173.4, 145.9, 137.9, 136.5, 131.5 (2C), 129.9 (2C), 129.2 (2C), 128.6 (3C), 127.1 (3C), 121.1, 111.6, 61.8, 61.6, 51.2, 39.6. HRMS (ESI) calculated for C 22 H 21 BrNO5[M+H] + 458.09949, detection found 458.05976.

[0077] Example 4

[0078] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(2,4-dichlorophenyl)methyl]-L-phenylalanine-(5-hydroxy-4-oxyylidenepyran-2-yl)methyl ester, denoted as Compound 4. The structural formula of Compound 4 is shown below:

[0079]

[0080] Compound 4 is a white solid with a yield of 50.1% and a melting range of 98-99°C. The data of H-NMR, C-NMR, and high-resolution mass spectrometry are shown below: 1H NMR (400MHz, deuterated chloroform) δ: 7.80 (s, 1H, CH), 7.33 (d, J = 8.4Hz, 1H, ArH), 7.30 (s, 1H, ArH) ),7.28(s,1H,ArH),7.24(s,1H,ArH),7.14(s,1H,ArH),7.12(s,1H,ArH),7.03(s ,1H,ArH),7.01(s,1H,ArH),6.35(s,1H,CH),4.86(s,2H,CH2),3.71(d,J=14.0Hz ,1H,CH2),3.61(s,1H,CH2),3.58(t,J=8.0Hz,1H,CH),2.98(d,J=7.2Hz,2H,CH2). 13 CNMR (101 MHz, deuterated chloroform) δ: 173.6 (2C), 162.32, 146.0, 137.9, 136.7, 135.6, 134.5, 133.7, 130.9, 129.4, 129.4 (2C), 128.8 (2C), 127.3, 127.3, 111.8, 62.3, 61.9, 49.0, 39.9. HRMS (ESI) calculated for C 22 H 20 Cl2NO5[M+H] + 447.07130, detection found 448.07025.

[0081] Example 5

[0082] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-chloro-3-nitrophenyl)methyl]-L-phenylalanine-(5-hydroxy-4-oxyylidenepyran-2-yl)methyl ester, denoted as Compound 5. The structural formula of Compound 5 is shown below:

[0083]

[0084] Compound 5 is a yellow solid with a yield of 47.3% and a melting range of 110-112°C. The data of H-NMR, C-NMR, and high-resolution mass spectrometry are shown below: 1H NMR (400 MHz, deuterated chloroform) δ: 7.82 (s, 1H, CH), 7.70 (d, J = 2.0 Hz, 1H, ArH), 7.42 (d, J = 8.4 Hz, 1H, ArH), 7.33 (dd, J = 8.4, 2.0 Hz, 1H, ArH), 7.29 (s, 1H, ArH), 7.27 (s, 1H, ArH), 7.25 (s, 1H, ArH), 7.15 (d, J =2.0Hz,1H,ArH),7.13(t,J=1.6Hz,1H,ArH),6.37(s,1H,CH),4.91(s,2H,CH2),3.91(d,J=14.4 Hz,1H,CH2),3.70(d,J=14.4Hz,1H,CH2),3.56(t,J=7.2Hz,1H,CH),3.01(q,J=7.6Hz,2H,CH2), 13 C NMR (101 MHz, deuterated chloroform) δ: 192.0, 173.8, 162.1, 146.0, 138.0, 136.4 (2C), 132.7, 131.8, 129.3 (3C), 128.8 (3C), 127.3, 124.9, 111.7, 62.0, 60.6, 50.4, 39.7. HRMS (ESI) calculated for C 22 H 20 ClN2O7[M+H] + 459.09536, detected 459.09283.

[0085] The preparation methods of the tyrosinase inhibitors in Examples 2 to 5 are substantially the same as those in Example 1, with the only difference being that other corresponding substituted benzaldehydes are used in place of the 2-chloro-benzaldehyde in Example 1 in Examples 2 to 5.

[0086] Example 6

[0087] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(2-chlorophenyl)methyl]-L-phenylalanine-(4-nitrophenyl)methyl ester, denoted as Compound 6. The structural formula of Compound 6 is shown below:

[0088]

[0089] Compound 6 is a yellow oil with a yield of 67.0%. The data of H NMR spectrum, C NMR spectrum and high-resolution mass spectrum are shown below: 1H NMR (400MHz, deuterated chloroform) δ:7.37-7.31(m,1H,ArH),7.30(s,1H,ArH),7.28(s,1H, ArH),7.24(s,1H,ArH),7.21(d,J=1.2Hz,2H,ArH),7.20-7.18(m,2H,ArH),7 .17(d,J=2.2Hz,2H,ArH),7.15(s,1H,ArH),7.05(m,2H,ArH),5.04(s,2H,C H2),3.85(m,2H,CH2),3.61(t,J=7.2Hz,1H,CH),3.02(d,J=7.2Hz,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 174.2, 137.0, 133.8, 130.5, 130.4, 129.9, 129.4, 129.3 (2C), 128.5 (2C), 128.4, 126.8, 126.8, 115.6, 115.4, 65.8, 62.3, 49.4, 39.8. HRMS (ESI) calculated for C 23 H 22 ClN2O4[M+H] + 425.12626, detected 425.12537.

[0090] Example 7

[0091] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-nitrophenyl)methyl]-L-phenylalanine-(4-nitrophenyl)methyl ester, denoted as Compound 7. The structural formula of Compound 7 is shown below:

[0092]

[0093] Compound 7 is a yellow oil with a yield of 64.3%. The data of H NMR spectrum, C NMR spectrum and high-resolution mass spectrum are shown below: 1H NMR (400 MHz, deuterated chloroform) δ: 8.11 (d, J = 8.8 Hz, 2H, ArH), 7.56 (d, J = 8.8 Hz, 1H, ArH), 7.34 (d, J = 8.4 Hz, 2H, ArH), 7.28 (s, 2H, ArH), 7.26 (s, 1H, ArH), 7.24-7.21 (m, 1H, ArH), 7.15-7. 11(m,2H,ArH),7.05(t,J=8.4Hz,2H,ArH),5.08(d,J=2.4Hz,2H,CH2),3.84(d,J=14.4 Hz,1H,CH2),3.60(d,J=14.8Hz,1H,CH2),3.51(t,J=6.8Hz,1H,CH),2.91(m,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 174.2, 161.5, 164.0, 147.5, 147.1, 137.1, 130.6, 130.5, 129.3 (2C), 128.6 (2C), 128.5 (2C), 126.9, 123.5 (2C), 115.6, 115.4, 65.9, 62.2, 51.1, 39.8. HRMS (ESI) calculated for C 23 H 22 N3O6[M+H] + 436.15031, detection found 436.14975.

[0094] Example 8

[0095] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-bromophenyl)methyl]-L-phenylalanine-(4-nitrophenyl)methyl ester, denoted as Compound 8. The structural formula of Compound 8 is shown below:

[0096]

[0097] Compound 8 is a yellow oil with a yield of 71.6%. The data of H NMR spectrum, C NMR spectrum and high-resolution mass spectrum are shown below: 1H NMR (400 MHz, deuterated chloroform) δ: 7.40 (d, J = 8.4 Hz, 2H, ArH), 7.23-7.18 (m, 3H, ArH), 7.14 (dd, J = 7.2, 2.4 Hz, 3H, ArH), 7.11 (s, 1H, ArH), 7.09 (s, 1H, ArH), 7.05 (t, J = 8.8 Hz, 3H, ArH), 5.06 (s, 2H, CH2), 3.79 (d, J = 13.6 Hz, 1H, CH2), 3.64 (s, 1H, CH2), 3.56 (d, J = 6.8 Hz, 1H, CH), 3.00 (d, J = 7.2 Hz, 2H, CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 174.4, 164.0, 161.5, 138.7, 137.2, 131.5 (2C), 130.5, 130.5, 129.9 (2C), 129.4 (2C), 128.5 (2C), 126.8, 120.9, 115.6, 115.4, 65.8, 62.0, 51.3, 39.8. HRMS (ESI) calculated for C 23 H 22 BrN2O4[M+H] + 469.07575, detected 469.07510.

[0098] Example 9

[0099] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(2,4-dichlorophenyl)methyl]-L-phenylalanine-(4-nitrophenyl)methyl ester, denoted as Compound 9. The structural formula of Compound 9 is shown below:

[0100]

[0101] Compound 9 is a yellow oil with a yield of 68.3%. The data of H NMR spectrum, C NMR spectrum and high-resolution mass spectrum are shown below: 1H NMR (400 MHz, deuterated chloroform) δ: 7.36 (d, J = 2.0 Hz, 1H, ArH), 7.31 (s, 1H, ArH), 7.30 (s, 2H, ArH), 7.28 (s, 1H, ArH), 7.27 (s, 1H, ArH), 7.24 (d, J = 2.4 Hz, 1H, ArH), 7.22 (s, 1H, ArH), 7.18 (s, 1H, ArH), 7.18 (s, 1H, ArH), 7.08 (t, J = 8.8Hz, 2H, ArH), 5.09 (s, 2H, CH2), 3.81 (d, J = 14. 8Hz,1H,CH2),3.65(d,J=14.8Hz,1H,CH2),3.59(t,J=7.2Hz,1H,CH),3.04(qd,2H,CH2), 13 C NMR (101 MHz, deuterated chloroform) δ: 174.2, 164.0, 161.5, 137.1, 135.8, 134.3, 133.3, 131.5, 131.4, 130.6, 130.5, 130.4, 129.3, 129.1, 128.5, 127.0, 126.8, 115.6, 115.4, 65.89, 62.3, 48.8, 39.8. HRMS (ESI) calculated for C 23 H 21 Cl2N2O4[M+H] + 459.08729, detected 459.08652.

[0102] Example 10

[0103] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-chloro-3-nitrophenyl)methyl]-L-phenylalanine-(4-nitrophenyl)methyl ester, denoted as Compound 10. The structural formula of Compound 10 is shown below:

[0104]

[0105] Compound 10 was a yellow oil with a yield of 72.3%. The H-NMR, C-NMR, and high-resolution mass spectrometry data are shown below: 1H NMR (400MHz, deuterated chloroform) δ:8.24-8.07(m,2H,ArH),7.71(d,J=2.0Hz,1H,ArH),7.41(d,J=8.4Hz,1H,A rH),7.35(s,1H,ArH),7.33(s,1H,ArH),7.30-7.19(m,4H,ArH),7.15(d,J=7.2Hz,2H,ArH),5.25 -5.14(m,2H,CH2),3.91(d,J=14.4Hz,1H,CH2),3.68(d,J=14.4Hz,1H,CH2),3.57(t,J=7.2Hz,1H,CH),3.05-2.96(m,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 174.0, 147.9, 147.8, 142.6, 140.5, 136.8, 132.5, 131.6, 129.3 (2C), 128.6 (4C), 127.1, 125.2, 124.7, 123.8 (2C), 65.2, 62.1, 50.4, 39.9. HRMS (ESI) calculated for C 23 H 21 ClN3O6[M+H] + 470.11134, detected 470.11069.

[0106] Example 11

[0107] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(2-chlorophenyl)methyl]-L-phenylalanine-(4-fluorophenyl)methyl ester, denoted as Compound 11. The structural formula of Compound 11 is shown below:

[0108]

[0109] Compound 11 was obtained as a white oil with a yield of 67.0%. The H-NMR spectrum, C-NMR spectrum, and high-resolution mass spectrometry data are shown below: 1H NMR (400MHz, deuterated chloroform) δ: 8.18 (d, J = 8.8Hz, 2H, ArH), 7.34-7.32 (m, 1H, ArH), 7. 32(s,1H,ArH),7.31(s,1H,ArH),7.28(s,2H,ArH),7.21(s,1H,ArH),7.21-7 .19(m,2H,ArH),7.18(d,J=2.4Hz,2H,ArH),7.16(s,1H,ArH),5.12(m,2H,C H2),3.87(m,2H,CH2),3.67(t,J=7.2Hz,1H,CH),3.03(d,J=7.2Hz,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 173.8, 147.7, 142.7, 136.9, 136.7, 133.8, 130.1, 129.5, 129.3 (2C), 128.6 (2C), 128.5 (2C), 126.9, 126.9, 123.7 (2C), 123.6, 65.0, 62.32, 49.5, 39.8. HRMS (ESI) calculated for C 23 H 22 ClFNO2[M+H] + 398.13176, detection found 398.13132.

[0110] Example 12

[0111] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-nitrophenyl)methyl]-L-phenylalanine-(4-fluorophenyl)methyl ester, denoted as Compound 12. The structural formula of Compound 12 is shown below:

[0112]

[0113] Compound 12 was a yellow oil with a yield of 66.2%. The H NMR spectrum, C NMR spectrum, and high-resolution mass spectrometry data are shown below: 1H NMR (400 MHz, deuterated chloroform) δ: 8.17 (d, J = 8.8 Hz, 2H, ArH), 8.10 (d, J = 8.8 Hz, 2H, ArH), 7.53 (d, J = 8.4 Hz, 1H, ArH), 7.36 (d, J = 8.4 Hz, 2H, ArH), 7.31 (d, J = 8.4 Hz, 2H, ArH), 7.26 (s, 1H, ArH), 7.25 (s ,1H,ArH),7.14(dd,J=6.8,2.8Hz,2H,ArH),5.17(q,J=13.2Hz,2H,CH2),3.93(d,J=14.4Hz, 1H, CH2), 3.70 (d, J = 14.4Hz, 1H, CH2), 3.58 (t, J = 7.2Hz, 1H, CH), 3.00 (d, J = 7.2Hz, 2H, CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 174.1, 147.8, 147.3, 147.1, 142.6, 136.8, 129.3 (2C), 128.6 (4C), 128.5 (2C), 127.0, 123.8 (2C), 123.6 (2C), 65.1, 62.2, 51.2, 39.9. HRMS (ESI) calculated for C 23 H 22 FN2O4[M+H] + 409.15581, detected 409.15527.

[0114] Example 13

[0115] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-bromophenyl)methyl]-L-phenylalanine-(4-fluorophenyl)methyl ester, denoted as Compound 13. The structural formula of Compound 13 is shown below:

[0116]

[0117] Compound 13 was obtained as a white oil with a yield of 68.4%. The H-NMR spectrum, C-NMR spectrum, and high-resolution mass spectrometry data are shown below: 1H NMR (400MHz, deuterated chloroform) δ: 8.16 (d, J=8.4Hz, 2H, ArH), 7.39 (d, J=8.0Hz, 2H, ArH), 7.28 (s, 1H, ArH), 7.25-7.20 (m, 4H, ArH), 7.17-7.12 (m, 2H, ArH), 7.09 (d,J=8.4Hz,2H,ArH),5.13(q,J=13.2Hz,2H,CH2),3.78(d,J=13.6Hz,1H,CH2),3.64(s,1H,CH2),3.62-3.56(m,1H,CH),3.00(d,J=7.2Hz,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 174.2, 147.7, 142.7, 138.4, 136.9, 131.5 (2C), 129.8 (2C), 129.3 (2C), 128.5 (2C), 128.5 (2C), 126.9, 123.7 (2C), 121.0, 64.9, 62.1, 51.3, 39.9. HRMS (ESI) calculated for C 23 H 22 BrFNO2[M+H] + 442.08125, detected 442.08066.

[0118] Example 14

[0119] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(2,4-dichlorophenyl)methyl]-L-phenylalanine-(4-fluorophenyl)methyl ester, denoted as Compound 14. The structural formula of Compound 14 is shown below:

[0120]

[0121] Compound 14 was obtained as a white oil with a yield of 67.7%. The H-NMR spectrum, C-NMR spectrum, and high-resolution mass spectrometry data are shown below: 1H NMR (400 MHz, deuterated chloroform) δ: 8.18 (d, J = 8.4 Hz, 2H, ArH), 7.32 (d, J = 8.0 Hz, 2H, ArH), 7.29 (s, 1H, ArH), 7.28 (s, 1H, ArH), 7.26 (s, 1H, ArH), 7.24 (s, 1H, ArH), 7.22 (s, 1H, ArH), 7.16 (s, 2H ,ArH),7.15(d,J=2.4Hz,1H,ArH),5.19–5.07(m,2H,CH2),3.88(d,J=14.4Hz,1H,CH2),3 .77(d,J=14.4Hz,1H,CH2),3.61(t,J=7.2Hz,1H,CH),3.02(qd,J=13.6,7.2Hz,2H,CH2). 13 C NMR (101 MHz, deuterated chloroform) δ: 174.0, 147.7, 142.7, 136.9, 135.6, 134.3, 133.3, 130.6, 129.3 (2C), 129.1, 128.5 (2C), 128.5 (2C), 127.1, 126.9, 123.7 (2C), 65.0, 62.3, 48.8, 39.9. HRMS (ESI) calculated for C 23 H 21 Cl2FNO2[M+H] + 432.09279, detected 432.09235.

[0122] Example 15

[0123] This example provides a tyrosinase inhibitor, whose chemical name is: N-[(4-chloro-3-nitrophenyl)methyl]-L-phenylalanine-(4-fluorophenyl)methyl ester, denoted as Compound 15. The structural formula of Compound 15 is shown below:

[0124]

[0125] Compound 15 was obtained as a yellow oil with a yield of 70.9%. The H-NMR spectrum, C-NMR spectrum, and high-resolution mass spectrometry data are shown below: 1H NMR (400 MHz, deuterated chloroform) δ: 7.68 (d, J = 2.0 Hz, 1H, ArH), 7.40 (d, J = 8.4 Hz, 1H, ArH), 7.31 (d, J = 2.0 Hz, 1H, ArH), 7.28 (s, 1H, ArH), 7.25 (s, 1H, ArH), 7.24 (s, 1H, ArH), 7.12 (dd, J = 7.2, 2.4 Hz, 3H, ArH), 7.09 -6.98(m,3H,ArH),5.10(d,J=2.0Hz,2H,CH2),3.88(d,J=14.4Hz,1H,CH2),3.63(d,J=14.4Hz,1H,CH2),3.52-3.45(m,1H,CH),3.06-2.88(m,2H,CH2). 13 CNMR (101 MHz, deuterated chloroform) δ: 174.2, 164.2, 161.7, 148.1, 140.6, 137.0, 132.5, 131.7, 130.7, 130.6, 129.4 (2C), 128.6 (2C), 127.1, 125.3, 124.8, 115.8, 115.6, 66.2, 62.1, 50.9, 39.9. HRMS (ESI) calculated as C 23 H 21 ClFN2O4[M+H] + 443.11684, detected 443.11624.

[0126] The preparation methods of the tyrosinase inhibitors in Examples 6 to 15 are substantially the same as that in Example 1, except that other corresponding substituted benzaldehydes are used in place of the 2-chloro-benzaldehyde in Example 1, and other corresponding 4-substituted benzyl chlorides are used in place of the 2-(chloromethyl)-5-hydroxy-4H-pyran-4-one in Example 1.

[0127] Performance Testing

[0128] (1) In vitro tyrosinase activity inhibition experiment

[0129] Experimental methods

[0130] Dissolve compounds 1-15 in DMSO to prepare a 200 μmol / L stock solution for later use. Prepare a 50 mmol / L PBS buffer solution at pH 6.8. Dissolve lyophilized tyrosinase powder and substrate (L-DOPA) in PBS to prepare a 1 mg / mL mushroom tyrosinase solution and a 0.5 mmol / L L-DOPA substrate solution, respectively. Kojic acid was used as a positive control.

[0131] Take a 96-well culture plate and add 176 μL of buffer and 2 μL of DMSO to the first row (columns 2-12). Add 88 μL of buffer and 2 μL of DMSO to each of the other rows.

[0132] Take 2 μL of each stock sample compound 1-15 and add it to each well in the first row (columns 2-12). Gently stir using a multichannel pipette. Add 90 μL to the second row and gently stir again. Add 90 μL to the third row and gently stir again. Add 90 μL to the fourth row, and so on, stirring gently until the eighth row. After gently stirring, discard 90 μL. Then, use an 8-channel pipette to add 90 μL of buffer to each well, bringing the volume in each well to 180 μL. Add 10 μL of enzyme solution using an 8-channel pipette, leaving wells A, B, C, and D in the first row without adding enzyme solution. Gently shake the plate and place it in a constant-temperature incubator for 20 minutes. Then, use an 8-channel pipette to add 10 μL of substrate solution. At this point, the volume of each well was 200 μL, and the sample concentration was divided into eight steps. The first row of wells had a sample concentration of 100 μmol / L, and the remaining rows had a decreasing concentration gradient of 50 μmol / L, 25 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 3.125 μmol / L, 1.5625 μmol / L, and 0.78125 μmol / L. The samples were detected 2-3 times at 490 nm (1 minute interval between each step).

[0133] The enzyme catalytic activity was calculated by subtracting the previous absorbance from the latter absorbance. The average value of the EFGH wells without compound addition was defined as 100%.

[0134] The inhibitory activity was calculated as follows:

[0135] Inhibition rate (%) = (blank absorbance - sample absorbance) / blank absorbance × 100%

[0136] Absorbance: The absorbance value of the last time minus the absorbance value of the previous time

[0137] The inhibitory activities of compounds 1 to 15 against tyrosinase measured according to the above test method are shown in Table 1 below.

[0138] Table 1 Tyrosinase inhibitory activity data of compounds 1 to 15

[0139] Compound <![CDATA[IC 50 (μmol / L) a ]]> Compound <![CDATA[IC 50 (μmol / L) a ]]> Compound 1 20.64±0.273 Compound 9 6.78±0.114 Compound 2 13.43±0.293 Compound 10 4.86±0.026 Compound 3 15.26±0.336 Compound 11 18.20±0.409 Compound 4 10.35±0.199 Compound 12 11.77±0.356 Compound 5 11.65±0.229 Compound 13 16.07±0.137 Compound 6 16.99±0.192 Compound 14 8.33±0.207 Compound 7 10.58±0.141 Compound 15 6.11±0.288 Compound 8 11.95±0.030 Kojic acid 29.12±0.060

[0140] IC 50 (μmol / L) a Assay: Mushroom tyrosinase was used. Values ​​are means of three separate experiments. IC50 = mean ± SEM, where SEM is the standard error of the mean.

[0141] As shown in Table 1, compounds 1 to 15 all showed more effective inhibitory activity than the positive control group, kojic acid, with IC 50 The values ​​ranged from 4.86 ± 0.026 μmol / L to 20.64 ± 0.273 μmol / L, among which compound 10 was found to be the most potent tyrosinase inhibitor with IC 50 The value was 4.86±0.026μmol / L. Compounds 1, 6, and 11, which carry a chloro group at the 2-position of the benzene ring, exhibited weak tyrosinase inhibition, while the corresponding compounds with a nitro or bromine group on the secondary amino group showed more potent tyrosinase inhibitory activity. It is worth noting that when the R2 group remains consistent, their disubstituted analogs exhibit more significant tyrosinase inhibition compared to the monosubstituted compounds with the secondary amino group attached to the benzene ring. For example, the tyrosinase inhibitory activity of compounds 9-10 was significantly stronger than that of compounds 6-8. These results indicate that the simultaneous introduction of substituents at the 3- and 4-positions of the benzene ring on the secondary amino group is beneficial to the inhibitory activity against tyrosinase, and that the 4-nitro-benzyl group as the R2 group is more favorable for tyrosinase inhibition.

[0142] (2) Study on the tyrosinase activity inhibition mechanism of compound 10

[0143] First, 20 μmol / L, 40 μmol / L, and 80 μmol / L solutions of compound 10 were prepared with N,N-dimethyl sulfoxide using PBS buffer at pH 6.8 and 50 mmol / L, respectively. 0.5 mmol / L L-dopa substrate solutions were prepared with PBS buffer at pH 6.8 and 50 mmol / L, and 0.5 mg / mL mushroom tyrosinase solution was diluted to 0.15 mg / ml, 0.125 mg / mL, 0.10 mg / mL, 0.075 mg / mL, and 0.05 mg / mL using PBS buffer solution. The total reaction system was 200 μL, that is, 178 μL of PBS buffer solution, 2 μL of DMSO or different concentrations of compound 10, 10 μL of 0.5 mmol / L L-DOPA substrate solution, and 10 μL of different concentrations of mushroom tyrosinase solution were added to a 96-well plate. It was pre-incubated at 25°C for 10 minutes, and then 10 μL of substrate solution was added. It was pre-incubated again at 25°C for 5 minutes (L-DOPA). The absorbance was immediately measured at 490 nm using a microplate reader. The measurement was repeated three times. The specific test results are as follows: Figure 1 As shown. Figure 1 It can be seen that Figure 1 The test lines in the graph are all straight lines passing through the origin. As the inhibitor concentration increases, the slope of the curve decreases, indicating that compound 10 is a reversible inhibitor.

[0144] (3) Study on the tyrosinase activity inhibition type of compound 10

[0145] First, compound 10 sample solutions with concentrations of 20 μmol / L, 40 μmol / L, and 80 μmol / L were prepared with N,N-dimethyl sulfoxide; L-DOPA substrate solutions with concentrations of 0.5 mmol / L, 0.3125 mmol / L, 0.25 mmol / L, 0.1875 mmol / L, and 0.125 mmol / L and mushroom tyrosinase solution with concentrations of 1 mg / mL were prepared with 50 mmol / L PBS buffer at pH 6.8; The total system was 200 μL, i.e., 178 μL of PBS, 2 μL of sample solution, 10 μL of L-DOPA substrate solution, and 10 μL of tyrosinase solution were added to a 96-well plate and pre-incubated at 25°C for 10 min. Then, 10 μL of substrate solution was added and pre-incubated again at 25°C for 1 min (L-DOPA). The absorbance was immediately measured at 490 nm using a microplate reader, with PBS solvent as the blank control. The test was performed 2-3 times (1 min interval each time), and the measurement was repeated three times in parallel. The enzymatic reaction rate was obtained by linear regression of time (t)-absorbance (A). Subsequently, 1 / V (V represents the L-DOPA reaction rate, the unit is OD / min, representing the absorbance change per minute) was plotted against 1 / [S] ([S] represents the final concentration of L-DOPA in the well, the unit is μM). No compound 10 was added as a control, and the Lineweaver-Burk enzyme kinetic equation of compound 10 was obtained. The Vm and Km values ​​in the Michaelis-Menten equation were calculated, as shown in the following example: Figure 2 As shown. Figure 2 It can be seen that the Lineweaver-Burk double reciprocal curve of compound 10 intersects the Y axis of the coordinate axis, and its slope increases with the increase of the compound concentration. Figure 2 The enzyme kinetic parameters of compound 10 on tyrosinase are as follows: with the increase of the concentration of compound 10, its Km value decreases and its Vm value remains unchanged, indicating that the inhibition type of compound 10 on tyrosinase activity is competitive inhibition.

[0146] The molecular docking model of compound 10 is shown in Figure 2. Figure 3 As shown by Figure 3 It can be seen that compound 10 partially enters the catalytic active center pocket of tyrosinase and forms interactions with the surrounding amino acid residues. Figure 3 It showed that the amino acid residues His 85 and Asn81 formed three hydrogen bonds with the nitro group of the 3-nitro-4-chlorobenzyl group connected by the ester bridge of compound 10, and the bond lengths were His85, as the histidine residue coordinating with the copper ion in the active center of mTYR, forms two key hydrogen bonds with the nitro group; in particular, the nitro group forms a bond length of His244 forms a bond with the oxygen atom on the ester bridge with a length of This indicates that compound 10 competitively inhibits mushroom tyrosinase, which is consistent with the experimental results.

[0147] (4) Drug similarity studies of compounds 4-5, compounds 9-10, and compounds 14-15

[0148] Computer software was used to simulate the pharmacokinetic properties and drug similarity of the absorption, distribution, metabolism and excretion (ADME) of compounds 4-5, compounds 9-10, compounds 14-15 and ascorbic acid. The specific test results are shown in Table 2 below.

[0149] Table 2 ADME pharmacokinetic properties and drug similarity test results

[0150]

[0151]

[0152] In Table 2, TPSA: topological polar surface area (20-130); HBA: number of hydrogen bond acceptors (<5); HBD: number of hydrogen bond donors (<10); LogS(ESOL): water solubility (-6 to -2); ilogP: octanol-water partition coefficient (-0.7 to 5); BBB: blood-brain barrier penetration; GI: gastrointestinal absorption capacity; B Score: bioavailability score (≥0.55); RO5: drug-like five rule.

[0153] As shown in Table 2, the ADME results demonstrate that the compounds in the examples of the present invention possess impressive ADME properties and drug-like properties. All compounds adhere to the five drug-like principles, indicating good drug-like properties, favorable lipid-water partition coefficients, and good oral bioavailability and absorption characteristics. In general, further optimization of these compounds could enhance their drug-like properties and make them safer and more effective tyrosinase inhibitors.

[0154] In summary, the L-phenylalanine derivatives of the present invention have excellent tyrosinase inhibitory activity and can serve as lead compounds for novel anti-tyrosinase research. They also have a simple preparation method, a short synthetic route, are easy to prepare in large quantities, and are inexpensive. Computer simulation analysis shows that these compounds bind well to the active site of mushroom tyrosinase and have excellent drug-like properties.

[0155] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable salt thereof; in, Each R1 is independently selected from halogen, nitro; R2 is selected from Each R3 is independently selected from halogen, nitro; n and m are independently selected from 1, 2, 3, 4 or 5.

2. The compound of formula (I) according to claim 1, characterized in that: Each R3 is independently selected from F, nitro.

3. The compound of formula (I) according to claim 1, characterized in that: n is selected from 1, 2 or 3; and / or m is selected from 1, 2 or 3.

4. The compound of formula (I) according to claim 1, characterized in that: The compound represented by formula (I) is selected from 5. A tyrosinase inhibitor, characterized in that: The invention comprises a compound represented by formula (I) according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

6. A product characterized by: The invention comprises a compound represented by formula (I) according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof; the product is selected from cosmetics, preservatives or pesticides.

7. Use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of a product for inhibiting tyrosinase.

Images