Cordycepin amino acid derivative, and preparation method and application thereof

Abstract

The application discloses a cordycepin amino acid derivative, a preparation method and application thereof. 1 The application discloses a cordycepin amino acid derivative, a preparation method and application thereof. The application discloses a cordycepin amino acid derivative, a preparation method and application thereof.

Description

Technical Field

[0001] This invention belongs to the field of organic compounds, specifically relating to a cordycepin amino acid derivative, its preparation method, and its application. Background Technology

[0002] Hyperuricemia, caused by long-term purine metabolism disorders, is characterized by elevated uric acid levels and subsequent crystal deposition, leading to diseases such as gout, cardiovascular events, and kidney damage, posing a significant threat to health, especially in developed countries where its prevalence is high. Current treatment options are limited and have safety concerns, resulting in poor treatment outcomes for symptomatic hyperuricemia patients and a lack of drug interventions for asymptomatic cases. For example, allopurinol, a xanthine oxidase inhibitor, is a commonly used drug for the clinical treatment of hyperuricemia. However, its use is controversial, mainly due to potential nephrotoxicity and the risk of inducing Stevens-Johnson syndrome. Another xanthine oxidase inhibitor, febuxostat, has been found to carry a risk of cardiovascular complications. Uric acid excretion stimulants, such as benzbromarone and probenecid, lower serum uric acid levels by acting on renal transport proteins to promote uric acid excretion. However, these drugs also face challenges related to adverse reactions, including enhanced 6-mercaptopurine toxicity, allergic reactions, and kidney damage. To avoid these adverse reactions, more and more researchers are focusing on natural active substances with uric acid-lowering effects.

[0003] Cordyceps sinensis is an entomopathogenic fungus belonging to the Ascomycota phylum. For the past 300 years, it has been a commonly used medicine in Traditional Chinese Medicine. Cordycepin is the main active ingredient in Cordyceps sinensis. In recent years, research on the positive effects of cordycepin on various diseases and health problems has flourished. Scientific studies have shown that cordycepin possesses anti-inflammatory, immunomodulatory, anti-diabetic, anti-hyperuricemia, antioxidant, cardiovascular disease prevention, and anti-cancer effects. However, the uric acid-lowering activity of cordycepin alone is relatively weak, requiring high doses to effectively reduce uric acid levels. Therefore, improving its uric acid-lowering effect through chemical modification is of significant research value. Summary of the Invention

[0004] To address the above technical problems, this invention provides a cordycepin amino acid derivative, its preparation method, and its applications. The cordycepin derivative of this invention retains the basic structure of cordycepin, exhibits excellent biocompatibility, reduces adverse reactions in organisms, and its structural modification significantly enhances its regulatory effect on uric acid.

[0005] The cordycepin amino acid derivative of this invention has the following general structural formula:

[0006] ;

[0007] Where R 1It is a group formed by the condensation reaction between the carboxyl group and the amino group in BOC-L-amino acids.

[0008] The BOC-L-amino acid is one of BOC-L-phenylalanine, BOC-L-valine, BOC-L-leucine, BOC-L-isoleucine, BOC-L-tyrosine, BOC-L-glycine, BOC-L-methionine, BOC-L-threonine, BOC-L-alanine, and BOC-L-serine.

[0009] The method for preparing cordycepin amino acid derivatives of the present invention includes the following steps:

[0010] Step 1: After mixing and dissolving cordycepin 1 and compound 2, react fully in the presence of an alkaline catalyst to generate intermediate 3;

[0011] Step 2: After intermediate 3 and BOC-L-amino acid are mixed and dissolved, they react fully in the presence of condensing agent and base catalyst to generate compounds C1-C10, namely cordycepin derivatives.

[0012] In step 1, the molar ratio of cordycepin to compound 2 is 1:(1.5-1.8); for example, 1.0:1.5, 1.0:1.6, 1.0:1.7, etc.

[0013] In step 1, the reaction between cordycepin and compound 2 is carried out in the presence of imidazole. Preferably, the amount of imidazole used is 1.5-1.8 times the molar amount of cordycepin; for example, 1.5 times, 1.6 times, 1.0 times, 1.7 times, etc.

[0014] In step 1, the reaction solvent is N,N-dimethylformamide.

[0015] In step 1, the reaction is carried out at room temperature for 8-24 hours; for example, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, etc. Preferably, cordycepin and imidazole are dissolved first under ultrasonic assistance, and then compound 2 is added in batches and stirred to react.

[0016] After the reaction in step 1 is completed, the solvent is concentrated under vacuum, and the organic layer is separated by extraction with water and ethyl acetate. The organic layer is then purified to obtain compound 3.

[0017] In step 2, the BOC-L-amino acid is one of BOC-L-phenylalanine, BOC-L-valine, BOC-L-leucine, BOC-L-isoleucine, BOC-L-tyrosine, BOC-L-glycine, BOC-L-methionine, BOC-L-threonine, BOC-L-alanine, and BOC-L-serine.

[0018] In step 2, the molar ratio of compound 3 to BOC-L-amino acid is 1:(1.1-1.3); for example, 1.0:1.1, 1.0:1.15, 1.0:1.2, etc.

[0019] In step 2, the reaction between compound 3 and the BOC-L amino acid is carried out in the presence of 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU). Preferably, the amount of TBTU used is 1.1-1.3 times the molar amount of compound 3; for example, 1.1, 1.15, 1.2, etc.

[0020] In step 2, the reaction between compound 3 and the BOC-L amino acid is carried out in the presence of triethylamine. Preferably, the amount of triethylamine used is 1.5-2.0 times the molar amount of compound 3; for example, 1.5 times, 1.55 times, 1.6 times, etc.

[0021] In step 2, the reaction solvent is dichloromethane.

[0022] In step 2, the reaction temperature is 35-60℃; for example, 35℃, 40℃, 45℃, etc. The reaction time is 6-24 h; for example, 6 h, 8 h, 10 h, 12 h, etc.

[0023] After the reaction in step 2 is completed, the mixture is cooled to room temperature, and the solvent is concentrated under vacuum. Using ethyl acetate as the organic phase, the mixture is washed stepwise with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively. The organic layer is then separated and purified to obtain cordycepin amino acid derivatives (C1-C10).

[0024] In this invention, the specific purification methods for the products in steps 1 and 2 are not limited in any way. They can be any one or more of the commonly used methods in the art, such as distillation, water washing, recrystallization, and column chromatography, or a combination of them with known separation and purification methods (such as extraction, rotary evaporation, etc.).

[0025] The synthetic route of this invention is shown below:

[0026]

[0027] The present invention relates to the application of cordycepin amino acid derivatives in the preparation of uric acid-lowering drug formulations.

[0028] Cordycepin is one of the main active ingredients in Cordyceps sinensis and Cordyceps militaris, and is similar to adenosine analogues. The cordycepin amino acid derivative provided by this invention not only significantly improves the uric acid-lowering level of cordycepin, but also has almost zero biotoxicity and good biocompatibility, making it a promising candidate for clinical use as a uric acid-lowering drug or preparation without causing serious adverse reactions. Detailed Implementation

[0029] The present invention will be further illustrated below with specific embodiments. These embodiments are merely illustrative and do not limit the scope of the invention.

[0030] Unless otherwise specified, the raw materials and reagents used in the following embodiments of the present invention are all derived from commercially available finished products.

[0031] The following Examples 1-10 prepared cordycepin derivatives through the following reaction process:

[0032] Example 1: Preparation of compound C1

[0033] The synthetic steps of compound C1 are as follows:

[0034] 1. Under ultrasonic assistance, cordycepin (10.0 mmol) and imidazole (17.0 mmol) were added to 15 mL of dry N,N-dimethylformamide, and compound 2 (17.0 mmol) was added in portions. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixture was concentrated under vacuum. The organic phase was extracted with water and ethyl acetate, and purified by column chromatography to give a pale yellow intermediate compound 3.

[0035] 2. Compound 3 (1.0 mmol), BOC-L-phenylalanine (1.15 mmol), and TBTU (1.15 mmol) were added to 8 mL of dichloromethane, followed by the addition of triethylamine (1.6 mmol). The mixture was refluxed at 45 °C and stirred for 10 h to obtain the reaction solution. After the reaction was complete, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 1:1, V / V) to obtain the cordycepin derivative compound C1.

[0036] Compound C1: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-phenylpropionyl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0037]

[0038] Compound C1 is a white solid with a yield of 63%.

[0039] 1H NMR (400 MHz, Chloroform-d) δ 9.80 (s, 1H), 8.72 (s, 1H), 8.59 (s,1H), 7.27 (d, J = 6.0 Hz, 2H), 7.21 (d, J = 3.9 Hz, 1H), 7.18 (d, J = 7.0 Hz,2H), 6.11 (s, 1H), 5.43 – 5.37 (m, 1H), 4.69 – 4.62 (m, 3H), 4.12 (dd, J =11.6, 2.6 Hz, 1H), 3.76 (dd, J = 11.6, 2.5 Hz, 1H), 3.31 – 3.25 (m, 1H), 3.14– 3.03 (m, 1H), 2.98 (d, J = 9.1 Hz, 1H), 2.32 (dd, J = 13.7, 8.6 Hz, 1H), 2.07 (dd, J = 11.8, 7.4 Hz, 1H), 1.37 (s, 9H), 0.90 (s, 9H), 0.10 (s, 6H). 13 CNMR (101 MHz, Chloroform-d) δ 171.15, 152.20, 150.37, 148.91, 141.55, 136.47,136.39, 129.49, 128.59, 126.92, 122.28, 92.79, 82.06, 79.80, 76.20, 63.72,56.33, 38.67, 32.33, 29.74, 28.30, 26.00, -5.36, -5.41.

[0040] Example 2: Preparation of compound C2

[0041] 1. Step 1 is the same as in Example 1.

[0042] 2. Compound 3 (1.0 mmol), BOC-L-valine (1.1 mmol), and TBTU (1.1 mmol) were added to 8 mL of dichloromethane, followed by the addition of triethylamine (1.5 mmol). The mixture was refluxed at 45 °C and stirred for 8 h to obtain the reaction solution. After the reaction was complete, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution stepwise using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 1:1, V / V) to obtain the cordycepin derivative compound C2.

[0043] Compound C2: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[(6-{[(2S)-2-{[(tert-butoxy)carbonyl]amino}-3-methylbutyryl]amino}-9H-purin-9-yl)tetrahydrofuran-3-ol, with the following structural formula:

[0044]

[0045] Compound C2 is a white solid with a yield of 61%.

[0046] 1 H NMR (400 MHz, Chloroform-d) δ 9.98 (s, 1H), 8.73 (s, 1H), 8.62 (s,1H), 6.12 (s, 1H), 5.44 (d, J = 8.8 Hz, 1H), 5.33 (d, J = 9.0 Hz, 1H), 4.77(s, 1H), 4.70 – 4.63 (m, 2H), 4.13 (dd, J = 11.7, 2.5 Hz, 1H), 3.76 (dd, J =11.6, 2.4 Hz, 1H), 2.81 -2.78 (m, 1H), 2.26 (dd, J = 12.3, 6.0 Hz, 1H), 2.11– 2.04 (m, 1H), 1.44 (s, 9H), 1.08 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H), 0.90 (s, 9H), 0.10 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 170.48,154.79, 151.26, 149.20, 148.04, 140.41, 120.99, 91.77, 81.06, 78.92, 75.76,62.60, 59.08, 37.61, 31.16, 30.09, 27.30, 24.92, 18.60, 17.46, -6.45, -6.49.

[0047] Example 3: Preparation of compound C3

[0048] 1. Step 1 is the same as in Example 1.

[0049] 2. Compound 3 (1.0 mmol), BOC-L-leucine (1.2 mmol), and TBTU (1.2 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (1.8 mmol). The mixture was refluxed at 45 °C and stirred for 14 h to obtain the reaction solution. After the reaction was completed, the organic layer was washed stepwise with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 1:1, V / V) to obtain the cordycepin derivative compound C3.

[0050] Compound C3: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S)-2-[(tert-butoxycarbonyl)amino]-4-methylpentanoyl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0051]

[0052] Compound C3 is a white solid with a yield of 60%.

[0053] 1 H NMR (400 MHz, Chloroform-d) δ 9.84 (s, 1H), 8.67 (s, 1H), 8.56 (s,1H), 6.09 (s, 1H), 5.31 (d, J = 8.8 Hz, 1H), 4.92 (s, 1H), 4.68 – 4.56 (m,3H), 4.09 (dd, J = 11.7, 2.3 Hz, 1H), 3.74 (dd, J = 11.6, 2.5 Hz, 1H), 2.30(t, J = 8.3 Hz, 1H), 2.09 – 2.03 (m, 1H), 1.86 – 1.64 (m, 2H), 1.56 (t, J =9.9 Hz, 1H), 1.41 (s, 9H), 0.99 (dd, J = 6.5, 2.5 Hz, 3H), 0.94 (d, J = 6.2Hz, 3H), 0.87 (s, 9H), 0.07 (s, 6H). 13C NMR (101 MHz, Chloroform-d) δ 176.15,155.89, 152.08, 150.25, 149.04, 141.44, 122.20, 92.74, 82.00, 79.63, 76.70,63.65, 53.96, 41.17, 32.23, 29.69, 28.33, 25.94, 24.90, 23.26, -5.42, -5.48.

[0054] Example 4: Preparation of compound C4

[0055] 1. Step 1 is the same as in Example 1.

[0056] 2. Compound 3 (1.0 mmol), BOC-L-isoleucine (1.2 mmol), and TBTU (1.2 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (1.8 mmol). The mixture was refluxed at 45 °C and stirred for 14 h to obtain the reaction solution. After the reaction was completed, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 1:1, V / V) to obtain the cordycepin derivative compound C4.

[0057] Compound C4: ((2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S,3S)-2-[(tert-butoxycarbonyl)amino]-3-methylpentanoyl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0058]

[0059] Compound C4 is a white solid with a yield of 58%.

[0060] 1H NMR (400 MHz, Chloroform-d) δ 9.73 (s, 1H), 8.71 (s, 1H), 8.58 (s,1H), 6.11 (s, 1H), 5.40 (d, J = 9.1 Hz, 1H), 4.66 (dd, J = 11.9, 3.6 Hz, 3H),4.13 – 4.09 (m, 1H), 3.78 – 3.74 (m, 1H), 2.36 – 2.30 (m, 1H), 2.12 – 1.93(m, 3H), 1.60 (td, J = 6.8, 6.1, 3.4 Hz, 1H), 1.44 (s, 9H), 1.06 (d, J = 6.8Hz, 3H), 0.91 (d, J = 7.3 Hz, 3H), 0.90 (s, 9H), 0.10 (s, 6H). 13 C NMR (101MHz, Chloroform-d) δ 170.10, 155.01, 151.10, 149.29, 147.86, 140.44, 121.43,91.65, 80.93, 79.07, 75.69, 62.62, 58.87, 36.41, 31.23, 28.67, 27.30, 24.91,23.11, 14.80, 10.39, -6.44, -6.51.

[0061] Example 5: Preparation of compound C5

[0062] 1. Step 1 is the same as in Example 1.

[0063] 2. Compound 3 (1.0 mmol), BOC-L-glycine (1.1 mmol), and TBTU (1.1 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (1.5 mmol). The mixture was refluxed at 40 °C and stirred for 8 h to obtain the reaction solution. After the reaction was complete, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 1:1.2, V / V) to obtain the cordycepin derivative compound C5.

[0064] Compound C5: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({2-[(tert-butoxycarbonyl)amino]acetyl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0065]

[0066] Compound C5 is a white solid with a yield of 65%.

[0067] 1 H NMR (400 MHz, Chloroform-d) δ 10.12 (s, 1H), 8.67 (s, 1H), 8.50 (s, 1H), 6.12 (s, 1H), 5.36 (d, J = 8.8 Hz, 1H), 5.13 (s, 1H), 4.57 – 0.95 (s, 9H), 0.10 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 170.70, 153.88, 151.83, 149.08, 146.72,142.14, 121.65, 92.79, 81.13, 78.58, 76.47, 64.22, 46.37, 35.89, 31.38,27.96, 25.62, -6.12, -6.18.

[0068] Example 6: Preparation of compound C6

[0069] 1. Step 1 is the same as in Example 1.

[0070] 2. Compound 3 (1.0 mmol), BOC-L-alanine (1.2 mmol), and TBTU (1.2 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (1.8 mmol). The mixture was refluxed at 45 °C and stirred for 12 h to obtain the reaction solution. After the reaction was complete, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 1:1.1, V / V) to obtain the cordycepin derivative compound C6.

[0071] Compound C6: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S)-2-[(tert-butoxycarbonyl)amino]propionyl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0072]

[0073] Compound C6 is a white solid with a yield of 63%.

[0074] 1 H NMR (400 MHz, Chloroform-d) δ 9.95 (s, 1H), 8.71 (s, 1H), 8.60 (s,1H), 6.10 (s, 1H), 5.39 (d, J = 8.8 Hz, 1H), 5.33 (d, J = 9.0 Hz, 1H), 4.68 – 4.59 (m, 3H), 4.10 (dd, J = 11.7, 2.3 Hz, 1H), 3.74 (dd, J = 11.6, 2.5 Hz, 1H), 2.28 (t, J = 8.4 Hz, 1H), 2.10 – 2.02 (m, 1H), 1.48 (d, J = 6.4 Hz, 3H),1.43 (s, 9H), 0.92 (s, 9H), 0.08 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ172.45, 155.43, 152.30, 148.87, 147.52, 141.34, 121.20, 93.35, 80.16, 77.42,75.86, 63.48, 52.23, 36.78, 30.85, 27.58, 25.32, 17.41, -5.83, -5.88.

[0075] Example 7: Preparation of compound C7

[0076] 1. Step 1 is the same as in Example 1.

[0077] 2. Compound 3 (1.0 mmol), BOC-L-tyrosine (1.3 mmol), and TBTU (1.3 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (2.0 mmol). The mixture was refluxed at 60 °C and stirred for 16 h to obtain the reaction solution. After the reaction was complete, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 2:1, V / V) to obtain the cordycepin derivative compound C7.

[0078] Compound C7: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-(4-hydroxyphenyl)propionyl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0079]

[0080] Compound C7 is a white solid with a yield of 52%.

[0081] 1 H NMR (400 MHz, Chloroform-d) δ 10.23 (s, 1H), 9.65 (s, 1H), 8.75 (s, 1H), 8.62 (s, 1H), 7.00 (d, J = 6.0 Hz, 2H), 6.82 (d, J = 7.0 Hz, 2H), 6.10 (s, 1H), 5.40 (s, 1H), 4.73 – 4.64 (m, 3H), 4.09 (dd, J = 11.5, 2.4 Hz, 1H), 3.70 (dd, J = 11.5, 2.5 Hz, 1H), 3.33 – 3.27 (m, 1H), 3.12 – 3.05 (m,1H), 2.96 (d, J = 9.0 Hz, 1H), 2.35 (dd, J = 13.5, 8.4 Hz, 1H), 2.09 (dd, J =11.7, 7.5 Hz, 1H), 1.39 (s, 9H), 0.91 (s, 9H), 0.09 (s, 6H). 13C NMR (101 MHz, Chloroform-d) δ 172.03, 154.53, 154.50, 151.12, 149.23, 141.75, 136.47,129.97, 129.02, 123.18, 114.62, 95.15, 81.36, 80.10, 77.71, 64.42, 57.24, 38.91, 33.11, 30.16, 28.28, 26.07, -5.31, -5.38.

[0082] Example 8: Preparation of compound C8

[0083] 1. Step 1 is the same as in Example 1.

[0084] 2. Compound 3 (1.0 mmol), BOC-L-threonine (1.2 mmol), and TBTU (1.2 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (1.8 mmol). The mixture was refluxed at 50 °C and stirred for 14 h to obtain the reaction solution. After the reaction was completed, the organic layer was washed stepwise with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 2.5:1, V / V) to obtain the cordycepin derivative compound C8.

[0085] Compound C8: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S,3R)-2-[(tert-butoxycarbonyl)amino]-3-hydroxybutyryl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0086]

[0087] Compound C8 is a white solid with a yield of 55%.

[0088] 1H NMR (400 MHz, Chloroform-d) δ 9.95 (s, 1H), 8.69 (s, 1H), 8.58 (s,1H), 6.11 (s, 1H), 5.44 (s 1H), 5.38 (d, J = 7.0 Hz, 2H), 4.75 – 4.70 (m,2H), 4.66 – 4.60 (m, 2H), 4.03 (dd, J = 11.4, 2.3 Hz, 1H), 3.75 (dd, J =11.5, 2.5 Hz, 1H), 2.25 – 2.20 (m, 1H), 2.09 – 2.05 (m, 1H), 1.40 (s, 9H),1.14 (d, J = 6.5 Hz, 3H), 0.95 (s, 9H), 0.08 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 171.26, 153.96, 151.77, 149.82, 148.24, 141.01, 121.53,94.31, 81.53, 79.27, 76.05, 66.67, 63.11, 60.48, 33.64, 30.36, 26.41, 25.42,18.88, -6.02, -6.08.

[0089] Example 9: Preparation of compound C9

[0090] 1. Step 1 is the same as in Example 1.

[0091] 2. Compound 3 (1.0 mmol), BOC-L-serine (1.2 mmol), and TBTU (1.2 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (1.8 mmol). The mixture was refluxed at 50 °C and stirred for 14 h to obtain the reaction solution. After the reaction was completed, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution stepwise using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 2.5:1, V / V) to obtain the cordycepin derivative compound C9.

[0092] Compound C9: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-hydroxypropionyl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0093]

[0094] Compound C9 is a white solid with a yield of 54%.

[0095] 1 H NMR (400 MHz, Chloroform-d) δ 10.23 (s, 1H), 8.64 (s, 1H), 8.42 (s, 1H), 6.09 (s, 1H), 5.72. (s, 1H), 5.35 (s, 1H), 5.02 (s, 1H), 4.59 – 4.50(m, 2H), 4.43 – 4.31 (m, 3H), 4.05 (dd, J = 11.5, 2.6 Hz, 1H), 3.80 (dd, J =11.6, 2.6 Hz, 1H), 2.21 – 2.19 (m, 1H), 2.08 – 2.03 (m, 1H), 1.41 (s, 9H), 0.94 (s, 9H), 0.10 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 171.82, 154.74,152.06, 150.57, 149.66, 140.48, 122.62, 93.24, 80.53, 78.89, 76.85, 65.76,61.93, 60.79, 33.87, 30.52, 27.82, 24.55, -5.92, -5.98.

[0096] Example 10: Preparation of compound C10

[0097] 1. Step 1 is the same as in Example 1.

[0098] 2. Compound 3 (1.0 mmol), BOC-L-methionine (1.3 mmol), and TBTU (1.3 mmol) were added to 8 mL of dichloromethane, followed by triethylamine (2.0 mmol). The mixture was refluxed at 55 °C and stirred for 14 h to obtain the reaction solution. After the reaction was complete, the organic layer was separated by washing with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively, using ethyl acetate as the organic phase. The organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The solution was then purified by column chromatography (ethyl acetate: petroleum ether = 1.5:1, V / V) to obtain the Cordyceps militaris derivative compound C10.

[0099] Compound C10: (2R,3R,5S)-5-({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-[6-({(2S)-2-[(tert-butoxycarbonyl)amino]-4-(methylthio)butyryl}amino)-9H-purin-9-yl]tetrahydrofuran-3-ol, with the following structural formula:

[0100]

[0101] Compound C10 is a white solid with a yield of 55%.

[0102] 1 H NMR (400 MHz, Chloroform-d) δ 9.95 (s, 1H), 8.62 (s, 1H), 8.37 (s,1H), 6.14 (s, 1H), 5.36 (s, 1H), 4.97 (s, 1H), 4.50 – 4.43 (m, 2H), 4.26 – 4.20 (m, 1H), 4.03 (dd, J = 11.6, 2.5 Hz, 1H), 3.78 (dd, J = 11.5, 2.5 Hz, 1H), 2.64 – 2.58 (m, 2H), 2.08 (s, 2H), 2.06 (s, 3H),2.05 – 2.00 (m, 1H),1.92 (m, 1H), 1.40 (s, 9H), 0.97 (s, 9H), 0.13 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 170.99, 155.05, 152.09, 150.83, 149.92, 140.15, 123.10,91.74, 81.04, 79.43, 75.96, 65.33, 58.43, 34.37, 31.62, 30.63, 29.75, 27.87,25.72, 15.1, -6.12, -6.17.

[0103] Application Example 1:

[0104] I. Cytotoxicity Test

[0105] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group used culture medium containing cordycepin derivative C1 prepared in Example 1, while the control group used culture medium containing cordycepin. Results showed that at drug concentrations of 0.5–20 μmol / L, the OD values ​​at 450 nm were not significantly different between the experimental and control groups at the same concentration.

[0106] II. Determination of Uric Acid Lowering Effect

[0107] (1) Animal preparation: The experimental animals were adult male SD rats weighing 150-170 g each, provided by Anhui Medical University. During the experiment, the rats were kept in a 12-hour light / dark cycle at 21±2 ℃ with free access to food and water. The welfare of the experimental animals and the experimental procedures were strictly carried out in accordance with the relevant provisions of the guidelines for the husbandry and use of experimental animals, and every effort was made to minimize the suffering of the animals.

[0108] (2) Animal grouping and feeding: The rats were divided into 7 groups, namely blank group, model group, low-dose experimental group, medium-dose experimental group, high-dose experimental group, positive control group and raw material control group; each group consisted of 10 rats.

[0109] (3) Drug preparation: First, prepare a 0.8 wt% CMC-Na solution. Dissolve the cordycepin derivative C1 prepared in Example 1 in the CMC-Na solution to prepare low-dose experimental group solutions, medium-dose experimental group solutions, and high-dose experimental group solutions with drug concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively. Then, dissolve benzbromarone in the CMC-Na solution to prepare a positive control group solution with a drug concentration of 1 mg / mL. Dissolve cordycepin in the CMC-Na solution to prepare a raw material control group solution with a drug concentration of 1 mg / mL.

[0110] (4) Drug administration experiment: Rats were administered the drug via gastric administration at a dose of 10 mL / kg per day for 7 consecutive days according to their body weight. The specific administration methods were as follows: the blank group and the model group were given 0.8 wt% CMC-Na solution; the low-dose experimental group, the medium-dose experimental group, and the high-dose experimental group were given experimental drug solutions with concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL, respectively; the positive control group was given positive control drug solution with a concentration of 1 mg / mL; and the raw material control group was given raw material control drug solution with a concentration of 1 mg / mL.

[0111] (5) Sample processing and sampling: 30 minutes after the last administration on day 7, except for the blank group, all other groups were intraperitoneally injected with 50 mg / ml hypoxanthine solution (solvent: 0.8 wt% CMC-Na solution) to establish a hyperuricemia model. The dosage was 1000 mg / kg. 30 minutes after administration, the eyeballs were immediately enucleated and blood was collected. After serum exudation, the sample was centrifuged at 15000 rpm for 4 minutes. The supernatant serum was collected and placed in a capped sample tube. The volume was prepared with physiological saline and the serum uric acid content was measured on a Beckman LX20 fully automated biochemical analyzer. The data are expressed as mean ± standard deviation. The test results are shown in Table 1.

[0112]

[0113] Application Example 2:

[0114] I. Cytotoxicity Test

[0115] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C2 prepared in Example 2, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental group and the control group at 450 nm for the same concentration.

[0116] II. Determination of Uric Acid Lowering Effect

[0117] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C2, and the test results are shown in Table 2.

[0118]

[0119] Application Example 3:

[0120] I. Cytotoxicity Test

[0121] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C3 prepared in Example 3, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental group and the control group at 450 nm for the same concentration.

[0122] II. Determination of Uric Acid Lowering Effect

[0123] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C3, and the test results are shown in Table 3.

[0124]

[0125] Application Example 4:

[0126] I. Cytotoxicity Test

[0127] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was cultured in a medium containing cordycepin derivative C4 prepared in Example 4, while the control group was cultured in a medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental and control groups at 450 nm for the same concentration.

[0128] II. Determination of Uric Acid Lowering Effect

[0129] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C4, and the test results are shown in Table 4.

[0130]

[0131] Application Example 5:

[0132] I. Cytotoxicity Test

[0133] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C5 prepared in Example 5, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental and control groups at 450 nm for the same concentration.

[0134] II. Determination of Uric Acid Lowering Effect

[0135] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C5, and the test results are shown in Table 5.

[0136]

[0137] Application Example 6:

[0138] I. Cytotoxicity Test

[0139] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C6 prepared in Example 6, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental and control groups at 450 nm for the same concentration.

[0140] II. Determination of Uric Acid Lowering Effect

[0141] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C6, and the test results are shown in Table 6.

[0142]

[0143] Application Example 7:

[0144] I. Cytotoxicity Test

[0145] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C7 prepared in Example 7, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental group and the control group at 450 nm for the same concentration.

[0146] II. Determination of Uric Acid Lowering Effect

[0147] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C7, and the test results are shown in Table 7.

[0148]

[0149] Application Example 8:

[0150] I. Cytotoxicity Test

[0151] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C8 prepared in Example 8, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental and control groups at 450 nm for the same concentration.

[0152] II. Determination of Uric Acid Lowering Effect

[0153] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C8, and the test results are shown in Table 8.

[0154]

[0155] Application Example 9:

[0156] I. Cytotoxicity Test

[0157] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C9 prepared in Example 9, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental group and the control group at 450 nm for the same concentration.

[0158] II. Determination of Uric Acid Lowering Effect

[0159] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C9, and the test results are shown in Table 9.

[0160]

[0161] Application Example 10:

[0162] I. Cytotoxicity Test

[0163] The cytotoxicity of PC12 cells was tested using the CCK-8 assay. The experimental group was a culture medium containing cordycepin derivative C10 prepared in Example 10, while the control group was a culture medium containing cordycepin. The results showed that at drug concentrations of 0.5-20 μmol / L, there was no significant change in the OD values ​​of the experimental and control groups at 450 nm for the same concentration.

[0164] II. Determination of Uric Acid Lowering Effect

[0165] The difference from Application Example 1 is that cordycepin derivative C1 was replaced with cordycepin derivative C10, and the test results are shown in Table 10.

[0166]

[0167] The above tests show that the cordycepin derivatives C3, C4, and C10 provided by this invention are comparable in efficacy to some existing uric acid-lowering drugs at the same dosage; cordycepin derivatives C1 and C7 are better than some existing uric acid-lowering drugs at the same dosage; and their uric acid-lowering levels at the same dosage are significantly better than the control group using cordycepin alone. These results indicate that this type of drug is expected to contribute to future uric acid-lowering products.

[0168] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A cordycepin amino acid derivative, characterized in that... Its general structural formula is as follows: ； Where R 1 It is a group formed by the condensation reaction between the carboxyl group and the amino group in BOC-L-amino acids; The BOC-L-amino acid is one of BOC-L-phenylalanine, BOC-L-valine, BOC-L-leucine, BOC-L-isoleucine, BOC-L-tyrosine, BOC-L-glycine, BOC-L-methionine, BOC-L-threonine, BOC-L-alanine, and BOC-L-serine.

2. The method for preparing the cordycepin amino acid derivative according to claim 1, characterized in that... Includes the following steps: Step 1: After mixing and dissolving cordycepin 1 and compound 2, react fully in the presence of an alkaline catalyst to generate intermediate 3; Step 2: After intermediate 3 and BOC-L-amino acid are mixed and dissolved, they are reacted fully in the presence of a condensing agent and an alkaline catalyst to obtain cordycepin derivatives. The synthesis route is shown below: 。 3. The preparation method according to claim 2, characterized in that: In step 1, the reaction between cordycepin and compound 2 is carried out in the presence of imidazole, wherein the amount of imidazole is 1.5-1.8 times the molar amount of cordycepin.

4. The preparation method according to claim 2, characterized in that: In step 2, the BOC-L-amino acid is one of BOC-L-phenylalanine, BOC-L-valine, BOC-L-leucine, BOC-L-isoleucine, BOC-L-tyrosine, BOC-L-glycine, BOC-L-methionine, BOC-L-threonine, BOC-L-alanine, and BOC-L-serine.

5. The preparation method according to claim 2 or 4, characterized in that: In step 2, the molar ratio of compound 3 to BOC-L-amino acid is 1:(1.1-1.3).

6. The preparation method according to claim 2, characterized in that: In step 2, the reaction of compound 3 and BOC-L amino acid is carried out in the presence of 2-(1H-benzotriazo-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate.

7. The preparation method according to claim 2 or 6, characterized in that: In step 2, the reaction between compound 3 and the BOC-L amino acid is carried out in the presence of triethylamine.

8. The preparation method according to claim 2, characterized in that: After the reaction in step 2 was completed, the mixture was cooled to room temperature, and the solvent was concentrated under vacuum. Using ethyl acetate as the organic phase, the mixture was washed stepwise with saturated ammonium chloride solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively. The organic layer was separated and purified to obtain cordycepin amino acid derivatives.

9. The use of the cordycepin amino acid derivative of claim 1 in the preparation of uric acid-lowering drug formulations.

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