Synthesis method and application of urolithin A and urolithin B derivatives

Abstract

The invention discloses a synthesis method and application of urolithin A and urolithin B derivatives, and discloses several novel urolithin A and urolithin B derivatives and related activity information. A series of novel urolithin A and urolithin B derivatives are obtained by respectively taking urolithin A and urolithin B as parent bodies and carrying out alkylation reaction on the parent bodies and halogenated hydrocarbon. In an alkaline environment, the urolithin and halogenated hydrocarbon are subjected to an alkylation reaction to generate a target derivative. And further purifying and separating a crude product obtained by the reaction, and determining a target structure by using nuclear magnetic spectrum. The invention discloses a synthesis method of two types of urolithin derivatives, structural information and anti-inflammatory and anti-oxidation effect results on ZEA-OGCs (Zinc Ethylenediamine-Oriented Growth Cells). A reference is provided for exploring wider structural modification of urolithin substances; rich structure types provide a structural basis for activity research of urolithin substances.

Description

TECHNICAL FIELD

[0001] The present application relates to a synthesis method of urolithin A and urolithin B derivatives and its application, in particular to a synthesis method of urolithin A and urolithin B derivatives and its anti-inflammatory and antioxidant effect in ZEA-OGCs, belonging to the technical field of drug structure modification and activity research. BACKGROUND

[0002] Urolithins (Uro) are metabolites derived from Ellagic acid (EA) produced by gut microbiota. Generally, the ellagitannins (ETs) entering the body are first hydrolyzed to form Ellagic acid, and then lose one lactone ring and gradually lose hydroxyl groups to form urolithins, which are absorbed, and they may be the material basis for the biological activity of ellagitannins in the body. Urolithins are a class of diphenylpyran-6-one derivatives containing different phenolic hydroxyl groups, and have biological activities such as anti-inflammatory injury, antioxidant stress, and anti-apoptosis. Recent studies have also shown that they have anti-SARS-CoV-2 and anticancer effects.

[0003] In recent years, urolithins have attracted attention due to their wide range of biological activities and health benefits, and more and more studies have revealed the mechanism of action of their biological activities. Urolithin B also shows great potential in anticancer activity, especially in bladder cancer and colon cancer. However, the bioavailability of natural urolithin B is low, and its water solubility is poor and its intestinal absorption is limited. In 2019, Andreux et al. first conducted a human-based drug safety study on urolithin A, and no toxicity was observed in subjects after taking urolithin, indicating good oral safety. In order to improve and overcome the solubility and metabolic stability problems, structural modifications have been made to urolithins and their derivatives, and some compounds have been found to show promising results and potential for further research. Studies have focused on the rigid structure of the urolithin mother ring, and structural modifications have mainly focused on the hydroxyl and lactone groups to enhance the stability and biological activity of the compounds. However, the types of alkylated structures are not rich, and the corresponding activity studies are also rarely reported. SUMMARY

[0004] The purpose of the present application is to provide a synthesis method of urolithin A and urolithin B derivatives and its application. The present application enriches the structural types of such compounds, introduces alkyl groups on the molecules of urolithin A and urolithin B to effectively regulate the biological activity of the molecules, improve the physicochemical properties, and optimize the pharmacokinetic properties, thereby improving the development potential. On the other hand, the synthesis method is optimized, and the two types of urolithin alkylated derivatives are expected to promote the study of biological activity.

[0005] The technical solution of the present application is a synthesis method of urolithin A and urolithin B derivatives, which comprises the following steps:

[0006] (1) Put a certain amount of urolithin A or urolithin B, potassium carbonate into a reaction bottle, take a certain amount of acetone or N,N-dimethylformamide as the solvent, stir the reaction, the feeding ratio of urolithin B to potassium carbonate is 1:1 in terms of the amount of substance, the feeding ratio of urolithin A to potassium carbonate is 1:2 in terms of the amount of substance, and the mass ratio of urolithin to acetone or N,N-dimethylformamide is 1:80-100;

[0007] (2) After the urolithin raw material in the reaction bottle in step (1) is completely dissolved, halogenated hydrocarbon, dimethyl carbonate or diethyl carbonate is added to continue stirring the reaction, the feeding ratio of urolithin B to halogenated hydrocarbon, dimethyl carbonate or diethyl carbonate is 1:2 in terms of the amount of substance, and the feeding ratio of urolithin A to halogenated hydrocarbon, dimethyl carbonate or diethyl carbonate is 1:4 in terms of the amount of substance;

[0008] (3) The reaction process is monitored by thin layer chromatography, and the reaction can be stopped when the light blue raw material fluorescence spot disappears;

[0009] (4) The purification method is as follows: after the reaction is completely monitored in step (3), the reaction liquid is concentrated to obtain a crude product;

[0010] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 14:1, 10:1, 8:1 is used as the chromatographic mobile phase in sequence to obtain the corresponding urolithin derivative.

[0011] In the aforementioned synthesis method of a urolithin A or urolithin B derivative, when the solvent is acetone, the stirring reaction temperature in steps (1) and (2) is 50°C; when the solvent is N,N-dimethylformamide, the stirring reaction temperature in steps (1) and (2) is 80-120°C.

[0012] In the aforementioned synthesis method of a urolithin A or urolithin B derivative, when the solvent is N,N-dimethylformamide, the purification method in step (4) is as follows: the reaction liquid after the reaction is completely extracted with ethyl acetate and saturated brine, the organic phase is collected and concentrated, and the obtained crude product is purified by column chromatography.

[0013] In the aforementioned synthesis method of a urolithin A or urolithin B derivative, when the raw material used for synthesis is urolithin B, the halogenated hydrocarbon used is bromopropene, bromoheptane, bromopropane, n-bromobutane, 1-bromo-3-phenylpropane, 3-bromo-1-propanol, 3-bromopropyl methyl ether, n-bromooctane, bromomethane or bromoethane; when the raw material used for synthesis is urolithin A, the halogenated hydrocarbon used is bromopropene, bromoheptane, bromopropane, n-bromobutane, 1-bromo-3-phenylpropane, 3-bromopropyl methyl ether, bromomethane or bromoethane.

[0014] In the aforementioned synthesis method of the urolithin A, urolithin B derivative, when urolithin B is used as a raw material for synthesis, the structures of the obtained products are sequentially shown in 3-12, wherein when dimethyl carbonate or diethyl carbonate is added, the structures of the obtained products are shown in 11 and 12, respectively:

[0015]

[0016] When urolithin A is used as a raw material for synthesis, the structures of the obtained products are sequentially shown in 13-20, wherein when dimethyl carbonate or diethyl carbonate is added, the structures of the obtained products are shown in 19 and 20, respectively:

[0017] Structure of urolithin A derivative

[0018]

[0019] In the aforementioned synthesis method of the urolithin A, urolithin B derivative, in step (3), the thin layer development system is petroleum ether: ethyl acetate = 3:1-4:1.

[0020] In the aforementioned synthesis method of the urolithin A, urolithin B derivative, the derivative of urolithin B has anti-inflammatory and antioxidant activities on OGCs (ovarian granulosa cells) after ZEA (Zeralenone) damage, and improves the healing activity and cell proliferation activity of OGCs.

[0021] The beneficial effects of the present application are as follows:

[0022] 1. The synthesis route selected by the present application is simple, and the synthesis of urolithin derivatives and the reaction mechanism are alkylization of phenolic hydroxyl group (Williamson ether synthesis reaction). The weak base potassium carbonate is partially dissolved in a polar aprotic solvent (acetone, DMF), which promotes the deprotonation of phenolic hydroxyl group (-OH) to generate phenolic oxygen anion (Ar-O - ), and then the nucleophilic substitution reaction of bromoalkane is carried out to obtain the alkylated derivative.

[0023] 2. The reaction conditions are mild, the raw materials are cheap and easy to obtain, the experimental operation is simple, and the column chromatography technology is used to purify the substances, which is a common method for purification in the synthesis field. The product has high purification degree and high yield, and the excess raw material with high purity can be recycled.

[0024] 3. Different urolithin substances are named according to the number of phenolic hydroxyl groups. The synthesis method of urolithin A, B derivative is also applicable to other structures, which provides a reference for the derivatization of other urolithins.

[0025] 4. In the synthesis of the two types of urolithin derivatives, the alkyl modification is not rich, and the structure type of the derivatives is few; the present application obtains 12 kinds of unpublished compounds by using alkyl modification, which enriches the structure of the derivatives and provides a basis for further activity research.

[0026] 5. The synthesis of urolithin A and B derivatives promotes the development of urolithin production. In addition, urolithin is a product obtained by metabolic decomposition of EA (ellagic acid) in vivo, which is rich in types and has high activity, and provides a reference for the synthesis and modification of similar drugs, and is expected to promote the production and development of synthesis research work.

[0027] 6. The partial activity test results of urolithin and its derivatives show that the urolithin series derivatives exhibit good antioxidant and anti-inflammatory effects on OGCs after ZEA damage, which provides a reference for subsequent more extensive activity research, and the test data illustrates the novelty and practicality of urolithin and its derivatives in ovary protection. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The synthesis route map of the two types of urolithin derivatives of the present application;

[0029] Figure 2 The hydrogen spectrum of the raw material urolithin B;

[0030] Figure 3 、 4 The hydrogen spectrum and high-resolution mass spectrum of 3-allyloxy-urolithin B in the urolithin B derivative;

[0031] Figure 5 、 6 The hydrogen spectrum and high-resolution mass spectrum of 3-heptyloxy-urolithin B in the urolithin B derivative;

[0032] Figure 7 、 8 The hydrogen spectrum and high-resolution mass spectrum of 3-propyloxy-urolithin B in the urolithin B derivative;

[0033] Figure 9 、 10 The hydrogen spectrum and high-resolution mass spectrum of 3-butyloxy-urolithin B in the urolithin B derivative;

[0034] Figure 11 、 12 The hydrogen spectrum and high-resolution mass spectrum of 3-phenylpropyloxy-urolithin B in the urolithin B derivative;

[0035] Figure 13 、 14 The hydrogen spectrum and high-resolution mass spectrum of 3-hydroxy-propyloxy-urolithin B in the urolithin B derivative;

[0036] Figure 15 、16 Hydrogen spectrum, high resolution mass spectrum of 3-methoxypropoxy-elliptacin B, which is an elliptacin B derivative;

[0037] Figure 17 , 18 Hydrogen spectrum, high resolution mass spectrum of 3-octyloxy-elliptacin B, which is an elliptacin B derivative;

[0038] Figure 19 , 20 Hydrogen spectrum, high resolution mass spectrum of 3-methoxy-elliptacin B, which is an elliptacin B derivative;

[0039] Figure 21 , 22 Hydrogen spectrum, high resolution mass spectrum of 3-ethoxy-elliptacin B, which is an elliptacin B derivative;

[0040] Figure 23 Hydrogen spectrum of raw material elliptacin A;

[0041] Figure 24 , 25 Hydrogen spectrum, high resolution mass spectrum of 3,8-bis(allyloxy)-elliptacin A, which is an elliptacin A derivative;

[0042] Figure 26 , 27 Hydrogen spectrum, high resolution mass spectrum of 3,8-bis(heptyloxy)-elliptacin A, which is an elliptacin A derivative;

[0043] Figure 28 , 29 Hydrogen spectrum, high resolution mass spectrum of 3,8-dipropoxy-elliptacin A, which is an elliptacin A derivative;

[0044] Figure 30 , 31 Hydrogen spectrum, high resolution mass spectrum of 3,8-dibutoxy-elliptacin A, which is an elliptacin A derivative;

[0045] Figure 32 , 33 Hydrogen spectrum, high resolution mass spectrum of 3,8-bis(3-phenylpropoxy)-elliptacin A, which is an elliptacin A derivative;

[0046] Figure 34 , 35 Hydrogen spectrum, high resolution mass spectrum of 3,8-bis(3-methoxypropoxy)-elliptacin A, which is an elliptacin A derivative;

[0047] Figure 36 , 37 Hydrogen spectrum, high resolution mass spectrum of 3,8-dimethoxy-elliptacin A, which is an elliptacin A derivative;

[0048] Figure 38 , 39Hydrogen spectrum, high resolution mass spectrum of 3,8-diethoxy-urobilin A in urobilin A derivatives;

[0049] Figure 40 Scratch map of 1 μmol / L of UB3 on OGCs in the present application;

[0050] Figure 41 Scratch map of 1 μmol / L of EA on OGCs in the present application;

[0051] Figure 42 Scratch map of 1 μmol / L of EA, UB and derivatives on ZEA-OGCs in the present application;

[0052] Figure 43 Difference in cell survival rate of ZEA-OGCs treated with EA, UB and derivatives thereof;

[0053] Figure 44 Inflammatory indicators after ZEA-OGCs were treated with 1 μmol / L of UB3 and EA;

[0054] Figure 45 ROS concentration indicators after ZEA-OGCs were treated with 1 μmol / L of UB3 and EA. DETAILED DESCRIPTION

[0055] The present application is further described below in conjunction with the accompanying drawings and examples, but is not limited to the basis of the present application.

[0056] The materials, reagents and the like used in the following examples can be obtained commercially unless otherwise specified, urobilin A and urobilin B were prepared in the laboratory, and the synthesis method is given below.

[0057] Example 1

[0058] Synthesis of urobilin B:

[0059] Take 10 g of o-bromobenzoic acid in a 100 mL round-bottom flask, add 20 mL of ultrapure water and stir with a magnetic stirrer, add 10 mL of 10 mol / L sodium hydroxide solution, stir until the o-bromobenzoic acid is completely dissolved, then add 6.03 g of resorcinol, stir at 60°C. After 0.5 h, add 2 mL of copper chloride solution (1 g / 10 mL), heat to 80°C, and reflux for 3 h. (PE:EA = 3:1 to monitor the reaction)

[0060] Treatment: after the reaction is completed, the reaction solution is cooled to room temperature, filtered, washed with pure water until the filtrate is clear, and dried. The obtained solid is dissolved with low-temperature ethyl acetate, extracted with ethyl acetate and saturated brine, the organic phase is collected, concentrated, and half of the solvent is concentrated by rotary evaporator. The solid is precipitated and analyzed at room temperature. Filtration obtains 4.04 g of white crystal urolithin B, molecular formula C 13 H8O3, number: UB.

[0061] The product obtained by purification in Example 1 is analyzed by nuclear magnetic resonance hydrogen spectrum, and the results are as follows: Figure 2

[0062] 1 H NMR (600 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 8.17 (dd, J = 15.2, 8.3 Hz, 2H), 7.88 (t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.5 Hz, 1H), 6.84 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H).

[0063] Example 2

[0064] Synthesis of 3-allyloxy-urolithin B:

[0065] (1) A certain amount of urolithin B and potassium carbonate are put into a reaction bottle, a certain amount of acetone is used as a solvent, and the reaction is stirred at 50°C. The molar ratio of urolithin B to potassium carbonate is 1:1, and the mass ratio of urolithin B to acetone is 1:80-100.

[0066] (2) After the urolithin B raw material in the reaction bottle of step (1) is completely dissolved, bromine propylene is added, and the reaction is continued at 50°C. The molar ratio of urolithin B to bromine propylene is 1:2.

[0067] (3) TLC (thin layer chromatography) petroleum ether: ethyl acetate = 3:1 is used for monitoring, and the reaction is stopped when the light blue raw material fluorescence spot disappears.

[0068] (4) Purification method: after the reaction is completely monitored in step (3), the reaction solution is concentrated to obtain the reaction crude product.

[0069] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 is used as the chromatography mobile phase in turn to obtain 3-allyloxy-urolithin B, molecular formula C 16 H 12 O3. Number: UB1.

[0070] The product obtained by purification in Example 2 is analyzed by nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum, and the results are as follows: Figure 3 ​, 4 :

[0071] 1 H NMR (600 MHz, DMSO-d6) δ 8.31 (d, J = 8.2 Hz, 1H), 8.25 (d, J = 9.4 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.58 (s, 1H), 7.01 (d, J = 7.6 Hz, 2H), 6.05 (d, J = 6.7 Hz, 1H), 5.43 (dd, J = 17.3, 1.7 Hz, 1H), 5.29 (dd, J = 10.5, 1.6 Hz, 1H), 4.68 (d, J = 5.3 Hz, 2H).

[0072] HRMS (ESI) m / z [M+H] + calcd for C 16 H 13 O3: 253.0864, found: 253.0849.

[0073] Example 3

[0074] Synthesis of 3-heptyloxy-urobilin B:

[0075] (1) A certain amount of urobilin B and potassium carbonate were put into a reaction bottle, a certain amount of acetone was used as a solvent, and the reaction was stirred at 50°C. The molar ratio of urobilin B to potassium carbonate was 1:1, and the mass ratio of urobilin B to acetone was 1:80-100.

[0076] (2) After the urobilin B raw material in the reaction bottle of step (1) was completely dissolved, bromoheptane was added, and the reaction was continued at 50°C. The molar ratio of urobilin B to bromoheptane was 1:2.

[0077] (3) The reaction was stopped when the light blue raw material fluorescence spot disappeared, which was monitored by thin layer chromatography (TLC) with petroleum ether: ethyl acetate = 3:1.

[0078] (4) After the reaction in step (3) was completed, the reaction liquid was concentrated to obtain the reaction crude product.

[0079] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 was used as the chromatography mobile phase in turn to obtain 3-heptyloxy-urobilin B, molecular formula C 20 H 22 O3. Number: UB2.

[0080] The product obtained by purification in Example 3 was analyzed by nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrometry, and the results are shown in the following table: Figure 5 ,6 :

[0081] 1 H NMR (600 MHz, DMSO-d6) δ 8.29 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 9.5 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H), 7.88 (t, J = 7.7 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 6.97 - 6.93 (m, 2H), 4.04 (t, J = 6.6 Hz, 2H), 1.71 (p, J = 6.6 Hz, 2H), 1.40 (p, J = 7.0 Hz, 2H), 1.34 - 1.23 (m, 6H), 0.85 (t, J = 6.9 Hz, 3H).

[0082] HRMS (ESI) m / z [M+H] + calcd for C 20 H 23 O3: 311.1647, found: 311.1628.

[0083] Example 4

[0084] Synthesis of 3-propoxy-urobilin B:

[0085] (1) A certain amount of urobilin B and potassium carbonate were put into a reaction bottle, a certain amount of acetone was used as a solvent, and the reaction was stirred at 50°C. The molar ratio of urobilin B to potassium carbonate was 1:1, and the mass ratio of urobilin B to acetone was 1:80-100.

[0086] (2) After the urobilin B raw material in the reaction bottle of step (1) was completely dissolved, bromo-n-propyl was added, and the reaction was continued to be stirred at 50°C. The molar ratio of urobilin B to bromo-n-propyl was 1:2.

[0087] (3) TLC (petroleum ether: ethyl acetate = 3:1) was used for monitoring, and the reaction was stopped when the light blue raw material fluorescence spot disappeared.

[0088] (4) After the reaction in step (3) was completely monitored, the reaction liquid was concentrated to obtain a crude product.

[0089] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 12:1, 8:1 was used as the chromatographic mobile phase in turn to obtain 3-propoxy-urobilin B, molecular formula C 16 H 14 O3. Number: UB3.

[0090] The product purified in Example 4 was analyzed by nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry, and the results are shown in the following table:Figure 7 、 8 :

[0091] 1 H NMR (600 MHz, CDC13) δ 8.33 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 6.89 (dd, J = 8.8, 3.3 Hz, 1H), 6.83 (d, J = 3.1 Hz, 1H), 3.98 (t, J = 6.6 Hz, 2H), 1.85 (h, J = 7.1 Hz, 2H), 1.06 (t, J = 7.4 Hz, 3H).

[0092] HRMS (ESI) m / z [M+H] + calcd for C 16 H 15 O3: 255.1021, found: 255.1013.

[0093] Example 5

[0094] Synthesis of 3-butoxy-hydrastine B:

[0095] (1) A certain amount of hydrastine B and potassium carbonate were put into a reaction bottle, a certain amount of acetone was used as a solvent, and the reaction was stirred at 50°C. The molar ratio of hydrastine B to potassium carbonate was 1:1, and the mass ratio of hydrastine B to acetone was 1:80-100.

[0096] (2) After the raw material hydrastine B in the reaction bottle of step (1) was completely dissolved, bromo-n-butane was added, and the reaction was continued at 50°C. The molar ratio of hydrastine B to bromo-n-butane was 1:2.

[0097] (3) The reaction was stopped when the light blue raw material fluorescence spot disappeared, which was monitored by thin layer chromatography (TLC) with petroleum ether: ethyl acetate = 3:1.

[0098] (4) After the reaction was completely monitored in step (3), the reaction liquid was concentrated to obtain the crude product.

[0099] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 12:1, 8:1 was used as the chromatographic mobile phase in turn to obtain 3-butoxy-hydrastine B, molecular formula C 17 H 16 O3.

[0100] The product obtained by purification in Example 5 was analyzed by nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrometry, and the results are shown in the following table.Figure 9 、 10 :

[0101] 1 H NMR (600 MHz, CDC13) δ 8.34 (dd, J = 8.0, 1.6 Hz, 1H), 7.99 (d, J = 7.1 Hz, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.79 - 7.75 (m, 1H), 7.51 - 7.47 (m, 1H), 6.90 (dd, J = 8.9, 2.6 Hz, 1H), 6.84 (d, J = 2.6 Hz, 1H), 4.02 (t, J = 6.5 Hz, 2H), 1.83 - 1.78 (m, 2H), 1.52 (h, J = 7.5 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H).

[0102] HRMS (ESI) m / z [M + H] + calcd for C 17 H 17 O3: 269.1177, found: 269.1169.

[0103] Example 6

[0104] Synthesis of 3-phenylpropoxy-urobilin B:

[0105] (1) A certain amount of urobilin B and potassium carbonate were put into a reaction bottle, a certain amount of DMF (N,N-dimethylformamide) was used as a solvent, and stirring reaction was carried out at 80-120°C. The feeding ratio of urobilin B to potassium carbonate was 1:1 in terms of the amount of substance, and the feeding ratio of urobilin B to DMF was 1:80-100 in terms of mass.

[0106] (2) After the urobilin B raw material in the reaction bottle of step (1) was completely dissolved, 1-bromo-3-phenylpropane was added, and stirring reaction was continued at 80-120°C. The feeding ratio of urobilin B to 1-bromo-3-phenylpropane was 1:2 in terms of the amount of substance.

[0107] (3) TLC (thin layer chromatography) was monitored with petroleum ether: ethyl acetate = 3:1, and the reaction was stopped when the light blue raw material fluorescence spot disappeared.

[0108] (4) After the reaction was completely monitored in step (3), the reaction liquid was extracted with ethyl acetate and saturated brine, the organic phase was collected, and concentrated to obtain a crude product.

[0109] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 was used as the chromatography mobile phase in turn to obtain 3-phenylpropoxy-urobilin B, molecular formula C 22 H18 O3.

[0110] The purified product of Example 6 was subjected to nuclear magnetic resonance hydrogen spectrum, high-resolution mass spectrometry analysis, and the results are shown in the following table. Figure 11 、 12 :

[0111] 1 H NMR (600 MHz, CDC13) δ 8.34 (dd, J = 7.9, 1.4 Hz, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.77 (ddd, J = 8.3, 7.2, 1.4 Hz, 1H), 7.51 - 7.47 (m, 1H), 7.31 (t, J = 7.5 Hz, 2H), 7.24 - 7.20 (m, 3H), 6.90 (dd, J = 8.8, 2.6 Hz, 1H), 6.82 (d, J = 2.5 Hz, 1H), 4.02 (t, J = 6.3 Hz, 2H), 2.84 (t, J = 7.6 Hz, 2H), 2.18 - 2.13 (m, 2H).

[0112] HRMS (ESI) m / z [M + H] + calcd for C 22 H 19 O3: 331.1334, found: 331.1316.

[0113] Example 7

[0114] Synthesis of 3-hydroxy-propyloxy-urotensin B:

[0115] (1) A certain amount of urotensin B and potassium carbonate were put into a reaction bottle, a certain amount of DMF (N, N-dimethylformamide) was used as a solvent, and stirring reaction was carried out at 80-120°C. The feeding ratio of urotensin B to potassium carbonate was 1:1 in terms of the amount of substance, and the feeding ratio of urotensin B to DMF was 1:80-100 in terms of mass.

[0116] (2) After the urotensin B raw material in the reaction bottle of step (1) was completely dissolved, 3-bromo-1-propanol was added, and stirring reaction was continued at 80-120°C. The feeding ratio of urotensin B to 3-bromo-1-propanol was 1:1 in terms of the amount of substance.

[0117] (3) The reaction was stopped after the disappearance of the light blue raw material fluorescence spot was monitored by thin layer chromatography (TLC) with petroleum ether: ethyl acetate = 3:1.

[0118] (4) After the reaction was completely monitored in step (3), the reaction liquid was extracted with ethyl acetate and saturated brine, the organic phase was collected, and concentrated to obtain the reaction crude product.

[0119] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 was used as the eluent to obtain 3-hydroxy-propoxy-urapidil B, molecular formula C 16 H 14 O4.

[0120] The product obtained by purification in Example 7 was subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry analysis, and the results are shown in the following table. Figure 13 、 14 :

[0121] 1 H NMR (600 MHz, CDC13) δ 8.34-8.31 (m, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.76 (dd, J = 15.3, 1.5 Hz, 1H), 7.50-7.46 (m, 1H), 6.87 (dd, J = 8.8, 2.6 Hz, 1H), 6.83 (d, J = 2.5 Hz, 1H), 4.18 (t, J = 6.0 Hz, 2H), 3.89 (t, J = 5.9 Hz, 2H), 2.09 (p, J = 5.9 Hz, 2H), 0.89-0.82 (m, 1H).

[0122] HRMS (ESI) m / z [M+H] + calcd for C 16 H 15 O4: 271.0970, found: 271.0957.

[0123] Example 8

[0124] Synthesis of 3-methoxypropoxy-urapidil B:

[0125] (1) A certain amount of urapidil B and potassium carbonate were put into a reaction bottle, a certain amount of DMF (N, N-dimethylformamide) was used as the solvent, and stirring reaction was carried out at 80-120°C. The molar ratio of urapidil B to potassium carbonate was 1:1, and the mass ratio of urapidil B to DMF was 1:80-100.

[0126] (2) After the urapidil B raw material in the reaction bottle of step (1) was completely dissolved, 3-bromopropyl methyl ether was added, and stirring reaction was continued at 80-120°C. The molar ratio of urapidil B to 3-bromopropyl methyl ether was 1:2.

[0127] (3) TLC (thin layer chromatography) petroleum ether: ethyl acetate = 3:1 was used for monitoring, and the reaction was stopped when the light blue raw material fluorescence spot disappeared.

[0128] (4) Purification method: after the reaction in step (3) is monitored to be complete, the reaction solution is extracted with ethyl acetate and saturated brine, the organic phase is collected, concentrated, and the reaction crude product is obtained.

[0129] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 is used as the chromatographic mobile phase in sequence to obtain 3-methoxypropoxy-urotensin B, molecular formula C 17 H 16 O4.

[0130] The product obtained by purification in Example 8 is subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum analysis, and the results are as follows: Figure 15 、 16 :

[0131] 1 H NMR (600 MHz, CDC13) δ 8.33 (dd, J = 7.9, 1.5 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 8.9 Hz, 1H), 7.78-7.73 (m, 1H), 7.49-7.45 (m, 1H), 6.89 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 4.11 (t, J = 6.3 Hz, 2H), 3.57 (t, J = 6.1 Hz, 2H), 3.37 (s, 3H), 2.08 (p, J = 6.2 Hz, 2H).

[0132] HRMS (ESI) m / z [2M+H] + calcd for C 34 H 33 O8: 569.2176, found: 569.1332.

[0133] Example 9

[0134] Synthesis of 3-octyloxy-urotensin B:

[0135] (1) A certain amount of urotensin B and potassium carbonate are put into a reaction bottle, a certain amount of acetone is used as the solvent, and the reaction is stirred at 50°C. The molar ratio of urotensin B to potassium carbonate is 1:1, and the mass ratio of urotensin B to acetone is 1:80-100.

[0136] (2) After the urotensin B raw material in the reaction bottle in step (1) is completely dissolved, bromo-n-octane is added, and the reaction is continued at 50°C. The molar ratio of urotensin B to bromo-n-octane is 1:2.

[0137] (3) TLC (Thin Layer Chromatography) with petroleum ether: ethyl acetate = 3:1 as mobile phase, the reaction was stopped until the blue fluorescent spot of the starting material disappeared.

[0138] (4) Purification method: after the reaction was completed by TLC monitoring in step (3), the reaction solution was concentrated to obtain the crude product.

[0139] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 was used as the mobile phase in turn to obtain 3-octyloxy-urobilin B with the molecular formula C 21 H 24 O3.

[0140] The product obtained by purification in Example 9 was subjected to nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrum analysis, and the results are shown in the following table. Figure 17 、 18 :

[0141] 1 H NMR (600 MHz, CDCl3) δ 8.33 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 6.89 (dd, J = 8.8, 2.6 Hz, 1H), 6.83 (d, J = 2.6 Hz, 1H), 4.00 (t, J = 6.6 Hz, 2H), 1.84-1.78 (m, 2H), 1.47 (p, J = 7.0 Hz, 2H), 1.39-1.28 (m, 8H), 0.89 (t, J = 7.0 Hz, 3H).

[0142] HRMS (ESI) m / z [M+H] + calcd for C 21 H 25 O3: 325.1803, found: 325.1791.

[0143] Example 10

[0144] Synthesis of 3-methoxy-urobilin B:

[0145] (1) A certain amount of urobilin B and potassium carbonate were put into a reaction bottle, and a certain amount of DMF (N,N-dimethylformamide) was used as a solvent, and the reaction was stirred at 90°C. The molar ratio of urobilin B to potassium carbonate was 1:1.5, and the mass ratio of urobilin B to DMF was 1:80-100.

[0146] (2) After the raw material of Urolithin B in the reaction bottle of step (1) is completely dissolved, dimethyl carbonate (DMC) is added to it, and halogen methane can be used instead of dimethyl carbonate (DMC), and the stirring reaction is continued at 90°C. The feeding ratio of Urolithin B to dimethyl carbonate is 1:2 in terms of the amount of substance.

[0147] (3) The reaction is stopped when the light blue raw material fluorescence spot disappears, which is monitored by thin layer chromatography (TLC) with petroleum ether: ethyl acetate = 3:1.

[0148] (4) After the reaction is completely monitored in step (3), the reaction liquid is extracted with ethyl acetate and saturated brine, and the organic phase is collected and concentrated to obtain the crude product of the reaction.

[0149] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 14:1, 8:1 is used as the chromatography mobile phase in turn to obtain 3-methoxy-urrolithin B, molecular formula C 14 H 10 O3.

[0150] The product obtained by purification in Example 10 is subjected to nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrum analysis, and the results are as follows: Figure 19 、 20 :

[0151] 1 H NMR (600 MHz, CDCl3) δ 8.35 (dd, J = 7.9, 1.6 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.78 (ddd, J = 8.3, 7.2, 1.5 Hz, 1H), 7.51-7.48 (m, 1H), 6.91 (dd, J = 8.8, 2.6 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 3.88 (s, 3H).

[0152] HRMS (ESI) m / z [M+H] + calcd for C 14 H 11 O3:227.0663, found:227.0698.

[0153] Example 11

[0154] 3-ethoxy-urrolithin B synthesis:

[0155] (1) A certain amount of Urolithin B and potassium carbonate are put into a reaction bottle, a certain amount of acetone is used as a solvent, and the stirring reaction is carried out at 50°C. The feeding ratio of Urolithin B to potassium carbonate is 1:1.5 in terms of the amount of substance, and the mass ratio of Urolithin B to acetone is 1:80-100.

[0156] (2) After the raw material of Urolithin B in the reaction bottle of step (1) is completely dissolved, bromoethane is added to it, and halogenated ethane can be replaced with diethyl carbonate (DEC), and the reaction is continued with stirring at 50°C. The feeding ratio of Urolithin B to bromoethane is 1:2 in terms of the amount of substance.

[0157] (3) TLC (petroleum ether: ethyl acetate = 3:1) monitoring is performed, and the reaction is stopped when the light blue raw material fluorescence spot disappears.

[0158] (4) After the reaction in step (3) is completed, the reaction liquid is concentrated to obtain a crude product.

[0159] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 is used as the chromatographic mobile phase in sequence to obtain 3-ethoxy- Urolithin B, molecular formula C 15 H 12 O3.

[0160] The product obtained by purification in Example 11 is subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry analysis, and the results are as follows: Figure 21 、 22 :

[0161] 1 H NMR (600 MHz, CDCl3) δ 8.32 (dd, J = 7.9, 1.4 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.75 (ddd, J = 8.5, 7.2, 1.5 Hz, 1H), 7.47 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H), 6.87 (dd, J = 8.8, 2.5 Hz, 1H), 6.81 (d, J = 2.5 Hz, 1H), 4.08 (q, J = 7.0 Hz, 2H), 1.45 (t, J = 7.0 Hz, 3H).

[0162] HRMS (ESI) m / z [M+H] + calcd for C 15 H 13 O3: 241.0820, found: 241.0851.

[0163] Example 12

[0164] Synthesis of Urolithin A:

[0165] To a solution of 2-bromo-5-hydroxybenzoic acid (1.08 g, 5 mmol) and resorcinol (3.2 g, 30 mmol) in 20 mL of 5.0% NaOH solution, reflux for 1 h. Then add 10 mL (1 g / 10 ml) of CuCl2 solution to the reaction solution, continue to reflux for 0.5 h, monitor by TLC (PE:EA = 3:1), after the reaction is completed, cool to room temperature, a large amount of powder is generated, filter and water wash the filter residue. Dissolve the filter residue in water again, adjust the pH to 3 with 1 M HC1, extract the solvent with DCM, dry with anhydrous Na2SO4, concentrate the organic layer, leave half of the residue, stir at room temperature to crystallize, filter to obtain a light yellow solid of urolithin A, molecular formula C 13 H8O4.

[0166] The product obtained by purification in Example 12 was analyzed by nuclear magnetic resonance hydrogen spectrum, and the results are shown in the following table: Figure 23

[0167] 1 H NMR (600 MHz, DMSO-d6) δ 10.20 (s, 2H), 8.10 (d, J = 8.9 Hz, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.50 (d, J = 2.7 Hz, 1H), 7.31 (dd, J = 8.8, 2.8 Hz, 1H), 6.80 (dd, J = 8.7, 2.4 Hz, 1H), 6.72 (d, J = 2.5 Hz, 1H).

[0168] Example 13

[0169] Synthesis of 3,8-bis(allyloxy)-urolithin A:

[0170] (1) A certain amount of urolithin A and potassium carbonate were put into a reaction bottle, a certain amount of acetone was added as a solvent, and the reaction was stirred at 50°C. The molar ratio of urolithin A to potassium carbonate was 1:2, and the mass ratio of urolithin A to acetone was 1:80-100.

[0171] (2) After the urolithin A raw material in the reaction bottle of step (1) was completely dissolved, bromoallyl was added, and the reaction was continued at 50°C. The molar ratio of urolithin A to bromoallyl was 1:4.

[0172] (3) TLC (petroleum ether: ethyl acetate = 4:1) was used to monitor the reaction, and the reaction was stopped when the light blue raw material fluorescence spot disappeared.

[0173] (4) Purification method: after the reaction was completed in step (3), the reaction solution was concentrated to obtain the reaction crude product.

[0174] ​(5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 14:1, 8:1 was used as the eluent in sequence to obtain 3,8-bis(allyloxy)-urolithin A, molecular formula C 19 H 16 O4.

[0175] The product obtained by purification in Example 13 was subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrometry analysis, and the results are shown in the following table. Figure 24 、 25 :

[0176] 1 H NMR (600 MHz, CDC13) δ 7.90 (d, J = 8.9 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 2.8 Hz, 1H), 7.38 (dd, J = 8.9, 2.8 Hz, 1H), 6.91 (dd, J = 8.9, 2.6 Hz, 1H), 6.85 (d, J = 2.6 Hz, 1H), 6.06 (ddt, J = 22.6, 10.4, 5.2 Hz, 2H), 5.46 (ddd, J = 17.3, 6.8, 1.6 Hz, 2H), 5.34 (d, J = 10.5 Hz, 2H), 4.64 (d, J = 5.3 Hz, 2H), 4.59 (d, J = 5.3 Hz, 2H).

[0177] HRMS (ESI) m / z [M + Na] + calcd for C 19 H 16 O4Na: 331.0947, found: 331.0942.

[0178] Example 14

[0179] Synthesis of 3,8-bis(heptyloxy)urolithin A:

[0180] (1) A certain amount of urolithin A and potassium carbonate were put into a reaction bottle, a certain amount of acetone was added as a solvent, and stirring was carried out at 50°C. The molar ratio of urolithin A to potassium carbonate was 1:2, and the mass ratio of urolithin A to acetone was 1:80-100.

[0181] (2) After the urolithin A raw material in the reaction bottle of step (1) was completely dissolved, bromoheptane was added, and stirring was continued at 50°C. The molar ratio of urolithin A to bromoheptane was 1:4.

[0182] (3) The reaction was stopped when the light blue raw material fluorescence spot disappeared, which was monitored by thin layer chromatography (TLC) with petroleum ether: ethyl acetate = 4:1.

[0183] (4) Purification method: after the reaction in step (3) is monitored to be complete, the reaction solution is concentrated to obtain a crude product.

[0184] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 12:1, 8:1 is used as the chromatographic mobile phase in turn to obtain 3,8-bis (heptyloxy) urolithin A, molecular formula C 27 H 36 O4.

[0185] The product obtained by purification in Example 14 is subjected to nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrum analysis, and the results are as follows: Figure 26 、 27 :

[0186] 1 H NMR (600 MHz, CDCl3) δ 7.90 (d, J = 8.9 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 2.8 Hz, 1H), 7.35 (dd, J = 8.8, 2.7 Hz, 1H), 6.88 (dd, J = 8.8, 2.6 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 4.00 (t, J = 6.6 Hz, 2H), 1.82 (h, J = 6.7 Hz, 4H), 1.47 (p, J = 7.4 Hz, 4H), 1.39 - 1.23 (m, 13H), 0.93 - 0.86 (m, 6H).

[0187] HRMS (ESI) m / z [M + Na] + calcd for C 27 H 36 O4Na: 447.2512, found: 447.2930.

[0188] Example 15

[0189] Synthesis of 3,8-dipropoxy-urolithin A:

[0190] (1) A certain amount of urolithin A and potassium carbonate are put into a reaction bottle, a certain amount of acetone is added as a solvent, and the reaction is stirred at 50°C. The molar ratio of urolithin A to potassium carbonate is 1:2, and the mass ratio of urolithin A to acetone is 1:80-100.

[0191] (2) After the urolithin A raw material in the reaction bottle in step (1) is completely dissolved, bromopropane is added, and the reaction is continued at 50°C. The molar ratio of urolithin A to bromopropane is 1:4.

[0192] (3) TLC (petroleum ether: ethyl acetate = 4:1) monitoring, the reaction was stopped until the light blue raw material fluorescence spot disappeared.

[0193] (4) Purification method: after the reaction was completed in step (3), the reaction solution was concentrated to obtain the crude product.

[0194] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 12:1, 8:1 was used as the chromatographic mobile phase in turn to obtain 3,8-dipropoxy-urotensin A, molecular formula C 19 H 20 O4.

[0195] The product obtained by purification in Example 15 was subjected to nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrum analysis, and the results are shown in the following table. Figure 28 、 29 :

[0196] 1 H NMR (600 MHz, CDCl3) δ 7.89 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.73 (d, J = 2.8 Hz, 1H), 7.35 (dd, J = 8.7, 2.8 Hz, 1H), 6.88 (dd, J = 8.8, 2.6 Hz, 1H), 6.83 (d, J = 2.6 Hz, 1H), 4.03 (t, J = 6.6 Hz, 2H), 3.96 (t, J = 6.6 Hz, 2H), 1.85 (hept, J = 7.0 Hz, 4H), 1.06 (t, J = 7.4 Hz, 6H).

[0197] HRMS (ESI) m / z [M + Na] + calcd for C 19 H 20 O4Na: 335.1259, found: 335.1255.

[0198] Example 16

[0199] Synthesis of 3,8-dibutoxy-urotensin A:

[0200] (1) A certain amount of urotensin A and potassium carbonate were put into a reaction bottle, a certain amount of DMF was added as a solvent, and the reaction was stirred at 50°C. The molar ratio of urotensin A to potassium carbonate was 1:2, and the mass ratio of urotensin A to DMF was 1:80-100.

[0201] (2) After the urotensin A raw material in the reaction bottle of step (1) was completely dissolved, n-butyl bromide was added, and the reaction was continued at 50°C. The molar ratio of urotensin A to n-butyl bromide was 1:4.

[0202] (3) TLC (Thin Layer Chromatography) Petroleum ether: ethyl acetate = 4:1 monitoring, stop the reaction when the light blue raw material fluorescence spot disappears.

[0203] (4) Purification method: after the reaction in step (3) is completed, the reaction solution is extracted with ethyl acetate and saturated brine, the organic phase is collected and concentrated to obtain the reaction crude product.

[0204] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 14:1, 8:1 is used as the chromatographic mobile phase in turn to obtain 3,8-dibutoxy-urotensin A, molecular formula C 21 H 24 O4.

[0205] The product obtained by purification in Example 16 is subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum analysis, and the results are as follows: Figure 30 、 31 :

[0206] 1 H NMR (600MHz, CDCl3) δ 7.89 (d, J = 8.9 Hz, 1H), 7.83 (d, J = 8.9 Hz, 1H), 7.73 (d, J = 2.8 Hz, 1H), 7.34 (dd, J = 8.9, 2.8 Hz, 1H), 6.88 (dd, J = 8.8, 2.6 Hz, 1H), 6.83 (d, J = 2.6 Hz, 1H), 4.07 (t, J = 6.6 Hz, 2H), 4.01 (t, J = 6.5 Hz, 2H), 1.80 (dt, J = 13.9, 6.8 Hz, 4H), 1.52 (h, J = 7.4 Hz, 4H), 0.99 (td, J = 7.5, 1.7 Hz, 6H).

[0207] HRMS (ESI) m / z [M + Na] + calcd for C 21 H 24 O4Na: 363.1573, found: 363.1567.

[0208] Example 17

[0209] Synthesis of 3,8-bis(3-phenylpropoxy)-urotensin A:

[0210] (1) A certain amount of urotensin A and potassium carbonate are put into a reaction bottle, a certain amount of DMF is added as a solvent, and the reaction is stirred at 50°C. The molar ratio of urotensin A to potassium carbonate is 1:2, and the mass ratio of urotensin A to DMF is 1:80-100.

[0211] (2) After the raw material of Urolithin A in the reaction bottle of step (1) is completely dissolved, 1-bromo-3-phenylpropane is added, and the stirring reaction at 50°C is continued. The feeding ratio of Urolithin A to 1-bromo-3-phenylpropane is 1:4 in terms of the amount of substance.

[0212] (3) The reaction is stopped after the light blue raw material fluorescence spot disappears, which is monitored by thin layer chromatography (TLC) with petroleum ether: ethyl acetate = 4:1.

[0213] (4) After the reaction is completely monitored in step (3), the reaction solution is extracted with ethyl acetate and saturated brine, and the organic phase is collected and concentrated to obtain the reaction crude product.

[0214] (5) The crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 14:1, 8:1 is used as the chromatography mobile phase in sequence to obtain 3,8-bis(3-phenylpropoxy)-urolithin A, molecular formula C 31 H 28 O4.

[0215] The product obtained by purification in Example 17 is subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum analysis, and the results are as follows: Figure 32 、 33 :

[0216] 1 H NMR (600 MHz, CDCl3) δ 7.91 (d, J = 9.0 Hz, 1H), 7.85 (dd, J = 8.8, 2.5 Hz, 1H), 7.74 (d, J = 2.7 Hz, 1H), 7.37 (dd, J = 8.8, 2.7 Hz, 1H), 7.33-7.29 (m, 4H), 7.25-7.19 (m, 6H), 6.90 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 4.02 (t, J = 6.3 Hz, 2H), 2.84 (t, J = 7.6 Hz, 4H), 2.19-2.13 (m, 4H).

[0217] HRMS (ESI) m / z [M + Na] + calcd for C 31 H 28 O4Na: 487.1886, found: 487.1872.

[0218] Example 18

[0219] Synthesis of 3,8-bis(3-methoxypropoxy)-urolithin A:

[0220] (1) A certain amount of urolithin A and potassium carbonate were put into a reaction bottle, a certain amount of DMF was added as a solvent, and the reaction was stirred at 50°C. The molar ratio of urolithin A to potassium carbonate was 1:2, and the mass ratio of urolithin A to DMF was 1:80-100.

[0221] (2) After the urolithin A raw material in the reaction bottle of step (1) was completely dissolved, 3-bromopropyl methyl ether was added, and the reaction was continued at 50°C. The molar ratio of urolithin A to 3-bromopropyl methyl ether was 1:4.

[0222] (3) Thin layer chromatography (TLC) was used for monitoring, and the reaction was stopped when the light blue raw material fluorescence spot disappeared.

[0223] (4) After the reaction was completely monitored in step (3), the reaction liquid was extracted with ethyl acetate and saturated brine, the organic phase was collected, and concentrated to obtain the crude product.

[0224] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 14:1, 8:1 was used as the chromatography mobile phase in turn to obtain 3,8-bis(3-methoxypropoxy)-urolithin A, molecular formula C 21 H 24 O6.

[0225] The product obtained by purification in Example 18 was subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum analysis, and the results are shown in the following table. Figure 34 、 35 :

[0226] 1 H NMR (600 MHz, CDCl3) δ 7.88 (d, J = 9.0 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 2.8 Hz, 1H), 7.35-7.33 (m, 1H), 6.87 (dd, J = 8.8, 2.5 Hz, 1H), 6.84 (d, J = 2.5 Hz, 1H), 4.16 (t, J = 6.3 Hz, 2H), 4.10 (t, J = 6.3 Hz, 2H), 3.56 (t, J = 6.1 Hz, 4H), 3.36 (s, 6H), 2.08 (dt, J = 8.4, 6.3 Hz, 4H).

[0227] HRMS (ESI) m / z [M+Na] + calcd for C 21 H 24 O6Na: 395.1471, found: 395.1464.

[0228] Example 19

[0229] 3,8-Dimethoxy-urotensin A synthesis:

[0230] (1) A certain amount of urotensin A, potassium carbonate was put into a reaction bottle, a certain amount of DMF was used as solvent, and the reaction was stirred at 90°C. The molar ratio of urotensin A to potassium carbonate was 1:2, and the mass ratio of urotensin A to DMF was 1:80-100.

[0231] (2) After the urotensin A raw material in the reaction bottle of step (1) was completely dissolved, dimethyl carbonate was added, dimethyl carbonate (DMC) could also be replaced with halogenated methane, and the reaction was continued at 90°C. The molar ratio of urotensin A to dimethyl carbonate was 1:4.

[0232] (3) TLC (petroleum ether: ethyl acetate = 3:1) was used for monitoring, and the reaction was stopped when the light blue raw material fluorescence spot disappeared.

[0233] (4) Purification method: after the reaction was completely monitored in step (3), the reaction liquid was extracted with ethyl acetate and saturated brine, the organic phase was collected, and concentrated to obtain the crude product of the reaction.

[0234] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 was used as the chromatographic mobile phase in turn to obtain 3,8-dimethoxy-urotensin A, molecular formula C 15 H 12 O4.

[0235] The product obtained by purification in Example 19 was subjected to nuclear magnetic resonance hydrogen spectrum and high resolution mass spectrum analysis, and the results are shown in the following table: Figure 36 、 37 :

[0236] 1 H NMR (600 MHz, CDCl3) δ 7.93 (d, J = 8.8 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 2.8 Hz, 1H), 7.38 (dd, J = 8.8, 2.8 Hz, 1H), 6.91 (dd, J = 8.8, 2.6 Hz, 1H), 6.87 (d, J = 2.6 Hz, 1H), 3.93 (s, 3H), 3.88 (s, 3H).

[0237] HRMS (ESI) m / z [M+H] + calcd for C 15 H 13 O4: 257.0769, found: 257.0805.

[0238] Example 20

[0239] 3,8-Diethoxy-urotensin II synthesis:

[0240] (1) A certain amount of urotensin II, potassium carbonate was put into a reaction bottle, a certain amount of acetone was used as solvent, and the reaction was stirred at 50°C. The feeding ratio of urotensin II to potassium carbonate was 1:2 in terms of the amount of substance, and the feeding ratio of urotensin II to acetone was 1:80-100 in terms of mass.

[0241] (2) After the urotensin II raw material in the reaction bottle of step (1) was completely dissolved, bromoethane was added, and the reaction was continued to be stirred at 50°C. The feeding ratio of urotensin II to bromoethane was 1:4 in terms of the amount of substance.

[0242] (3) TLC (petroleum ether: ethyl acetate = 3:1) was used for monitoring, and the reaction was stopped when the light blue raw material fluorescence spot disappeared.

[0243] (4) After the reaction was completely monitored in step (3), the reaction liquid was concentrated to obtain a crude product.

[0244] (5) The crude product obtained in step (4) was purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 10:1, 8:1 was used as the chromatographic mobile phase in turn to obtain 3,8-diethoxy-urotensin II, molecular formula C 17 H 16 O4.

[0245] The product obtained by purification in Example 20 was subjected to nuclear magnetic resonance hydrogen spectrum and high-resolution mass spectrum analysis, and the results are shown in the following table. Figure 38 、 39 :

[0246] 1 H NMR (600 MHz, CDCl3) δ 7.85 (d, J = 8.9 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 2.8 Hz, 1H), 7.31 (dd, J = 8.8, 2.8 Hz, 1H), 6.84 (dd, J = 8.8, 2.6 Hz, 1H), 6.78 (d, J = 2.5 Hz, 1H), 4.11 (q, J = 7.0 Hz, 2H), 4.05 (q, J = 7.0 Hz, 2H), 1.44 (td, J = 7.0, 5.0 Hz, 6H).

[0247] HRMS (ESI) m / z [M+H] + calcd for C 17 H 17 O4: 285.1082, found: 285.1111.

[0248] Example 21

[0249] Use of urolithin and its derivatives in anti-inflammatory and antioxidant activity

[0250] Specifically, the anti-inflammatory and antioxidant activity of urolithin B and its derivatives in treating OGCs (ovarian granulosa cells) after ZEA (Zeralenone) damage.

[0251] The healing ability of urolithin B and its derivatives on OGCs (non-injured cells) was evaluated by cell scratch test; the effect of urolithin B and its derivatives on the proliferation of ZEA-OGCs was determined by CCK-8 method; the effect of several compounds on the healing rate of ZEA-OGCs was tested by cell scratch test to select the best test drug; the anti-inflammatory and antioxidant activity of urolithin B and its derivatives was evaluated by testing the antioxidant indicators (such as ROS level) and inflammatory indicators (such as IL-6, IL-1β, TNF-α) of the selected drug in treating ZEA-OGCs damage.

[0252] 1. Ovarian granulosa cell separation: The Guizhou black goats used in this test were from the Maiping black goat breeding base of Guizhou Institute of Animal Husbandry and Veterinary Medicine. The goats were slaughtered strictly in accordance with the national operating procedures and technical requirements, and the ovarian tissue was collected immediately and transported back to the laboratory within 1 hour. The goat ovarian GCs were identified and used after culture.

[0253] 2. ZEA damage model: OGCs were divided into control group, ZEA group (10 μM ZEA treatment for 24 hours), UB group (ZEA+10 μM UB), UB1 group (ZEA+10 μM 3-allyl UB), UB2 group (ZEA+10 μM 3-heptyl UB), and UB3 group (ZEA+10 μM 3-propyl UB)

[0254] 3. Detection index and method

[0255] (1) Cell viability: The results of cell scratch test 0-24h with or without serum showed that UB3 had the best wound healing effect on OGCs cells at the same concentration (1 μmol / L), better than the positive control drug EA (ellagic acid) and the other three drugs; CCK-8 method showed that the survival rate of OGCs treated with ZEA in the UB3 group was significantly higher than that in the EA group (P<0.001), and the cell survival rate in the UB and UB1 groups tended to be similar to that in the EA group, with no significant difference. Cell scratch test (0-48h) showed that the healing ability of UB3 group was slightly higher than that of EA (P<0.05) at the same concentration, and the healing ability of UB and UB1 groups tended to be similar to that of EA, with no significant difference. Figures 40-42 .

[0256] The results of CCK-8 cell proliferation test after the construction of the damage model are shown in Table 1. Figure 43 At the same concentration (1 μmol / L), the effect of UB and UBI is closest to that of EA; and the cell proliferation and migration ability of UB3 is significantly better than that of EA (p<0.001, n=4).

[0257] (2) Inflammatory factor secretion: The IL-6, IL-1β and TNF-α levels are detected by ELISA. The results show that UB3 and EA can improve the main anti-inflammatory indicators of OGCs after ZEA damage, and the effect of UB3 in inhibiting IL-6 secretion is slightly lower than that of EA (P<0.05, n=7), as shown in Table 2. Figure 44 .

[0258] (3) ROS level: After ZEA damage, EA and UB3 are used for treatment for 24 hours, and then the ROS concentration in OGCs is immediately measured. The results show that the ROS activity in the UB3 group is significantly lower than that in the EA group and the control group (P<0.05, n=7), as shown in Table 3. Figure 45 .

[0259] 4. Conclusion

[0260] The data of the examples show that urolithin B and derivatives have strong activity on OGCs, and can significantly improve the cell healing ability of OGCs whether the cells are damaged or not, and the effect of UB3 is significantly better than that of EA. It can be seen that the derivatives show good anti-inflammatory and antioxidant effects on OGCs after ZEA damage, and the effect of UB3 is better than that of other three types of drugs, and the anti-inflammatory and antioxidant effects are significantly better than those of EA. However, compared with EA and the positive drug, the effect of the derivatives in inhibiting IL-6 secretion is slightly weaker, which provides a reference for the research of urolithin substances on OGCs.

[0261] In summary, the present application provides a synthesis method of urolithin A and B derivatives, and discloses several new urolithin A and B derivatives and related activity information. A series of new urolithin B derivatives, such as 3-allyloxy-urolithin B, 3-heptyloxy-urolithin B, 3-propyloxy-urolithin B, 3-phenylpropyloxy-urolithin B, 3-hydroxy-propyloxy-urolithin B, 3-methoxypropyloxy-urolithin B and 3-octyloxy-urolithin B, are obtained by alkylation reaction of urolithin B and halogenated hydrocarbon. Urolithin A derivatives, such as 3,8-bis(allyloxy)-urolithin A, 3,8-bis(heptyloxy)-urolithin A, 3,8-dipropoxy-urolithin A, 3,8-dibutoxy-urolithin A, 3,8-bis(3-phenylpropyloxy)-urolithin A and 3,8-bis(3-methoxypropyloxy)-urolithin A, are obtained by alkylation reaction of urolithin A and halogenated hydrocarbon. Urolithin is reacted with halogenated hydrocarbon by alkylation reaction to generate target derivatives in an alkaline environment, and the reaction formula is as follows:

[0262]

[0263] The reaction obtains a crude product which is further purified and separated, and the structure of the target product is determined by nuclear magnetic spectrum. The application discloses a synthesis method and structural information of two types of urolithin derivatives and results of anti-inflammatory and antioxidant effects on ZEA-OGCs. The application provides a reference for exploring more extensive structural modification of urolithin substances, and rich structural types provide structural basis for activity research of urolithin substances.

Claims

1. A method for synthesizing a derivative of Urolithin A, Urolithin B, characterized by: The synthesis method comprises the following steps: (1) a certain amount of urolithin A or urolithin B and potassium carbonate are put into a reaction bottle, a certain amount of acetone or N,N-dimethylformamide is used as a solvent, and stirring reaction is performed, the feeding ratio of urolithin B to potassium carbonate is 1:1 in terms of the amount of substance, the feeding ratio of urolithin A to potassium carbonate is 1:2 in terms of the amount of substance, and the mass ratio of urolithin to acetone or N,N-dimethylformamide is 1:80-100; (2) after the urolithin raw material in the reaction bottle in step (1) is completely dissolved, halogenated hydrocarbon, dimethyl carbonate or diethyl carbonate is added, and stirring reaction is continuously performed, the feeding ratio of urolithin B to halogenated hydrocarbon, dimethyl carbonate or diethyl carbonate is 1:2 in terms of the amount of substance, and the feeding ratio of urolithin A to halogenated hydrocarbon, dimethyl carbonate or diethyl carbonate is 1:4 in terms of the amount of substance; (3) reaction progress is monitored by using thin layer chromatography, and the reaction can be stopped when the light blue raw material fluorescence spot disappears; (4) after the reaction in step (3) is completely monitored, the reaction liquid is concentrated to obtain a reaction crude product; (5) the crude product obtained in step (4) is purified by column chromatography, and petroleum ether: ethyl acetate = 1:0, 14:1, 10:1 and 8:1 are used as chromatographic mobile phases in sequence to obtain corresponding urolithin derivatives.

2. The method for synthesizing urolithin A and urolithin B derivatives according to claim 1, characterized in that: In the case that the solvent is acetone, the stirring reaction temperature in steps (1) and (2) is 50 DEG C; in the case that the solvent is N,N-dimethylformamide, the stirring reaction temperature in steps (1) and (2) is 80-120 DEG C.

3. The method for synthesizing urolithin A and urolithin B derivatives according to claim 1, characterized in that: In the case that the solvent is N,N-dimethylformamide, the purification method in step (4) is specifically as follows: after the reaction is completely performed, the reaction liquid is extracted with ethyl acetate and saturated brine, the organic phase is collected, concentrated, and the obtained crude product is purified by column chromatography.

4. The method for synthesizing urolithin A and urolithin B derivatives according to claim 1, characterized in that: In the case that the raw material used for synthesis is urolithin B, the halogenated hydrocarbon used is bromine propylene, bromine heptane, bromine propane, bromine n-butane, 1-bromo-3-phenylpropane, 3-bromo-1-propanol, 3-bromopropyl methyl ether, bromine n-octane, bromomethane or bromoethane; in the case that the raw material used for synthesis is urolithin A, the halogenated hydrocarbon used is bromine propylene, bromine heptane, bromine propane, bromine n-butane, 1-bromo-3-phenylpropane, 3-bromopropyl methyl ether, bromomethane or bromoethane.

5. The method for synthesizing urolithin A and urolithin B derivatives according to claim 4, characterized in that: In the case that the raw material used for synthesis is urolithin B, the obtained product structures are shown in 3-12 in sequence, wherein in the case that dimethyl carbonate or diethyl carbonate is added, the obtained product structures are shown in 11 and 12, respectively; In the case that the raw material used for synthesis is urolithin A, the obtained product structures are shown in 13-20 in sequence, wherein in the case that dimethyl carbonate or diethyl carbonate is added, the obtained product structures are shown in 19 and 20, respectively: Urolithin A derivative structure 6. The method for synthesizing urolithin A and urolithin B derivatives according to claim 4, characterized in that: In the step (3), the thin layer development system is petroleum ether: ethyl acetate = 3:1-4:

1.

7. Use of a derivative of Urolithin A, Urolithin B according to any one of claims 1 to 6, characterized in that: The urolithin B derivative has anti-inflammatory and antioxidant activities on OGCs after ZEA damage, and improves the healing activity and cell proliferation activity of OGCs.

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