A cordycepin derivative, and a preparation method and use thereof

Abstract

The application discloses a cordycepin derivative, a preparation method and application thereof. The structural general formula of the cordycepin derivative is provided in the application. The cordycepin derivative is modified by a special group, so that the stability of the cordycepin derivative is improved, and the biological activities such as antibacterial activity, anti-inflammatory activity, antioxidant activity and antitumor activity are also significantly improved. In addition, the cordycepin derivative in the application has certain selectivity in the aspect of antibacterial activity, especially for some beneficial bacteria, so that the application range of the cordycepin derivative is expanded.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a cordycepin derivative, its preparation method, and its uses. Background Technology

[0002] Nucleoside analogues exert their therapeutic effects by blocking the growth of RNA or DNA chains. Cordycepin is the first nucleoside antibiotic isolated from fungi, possessing a variety of pharmacological activities, including antibacterial, anti-inflammatory, antioxidant, antitumor, immunomodulatory, and hyperlipidemia prevention. Cordycepin and its derivatives have received widespread attention and exploration in the biomedical field, but the types of cordycepin derivatives developed so far are relatively limited. Furthermore, due to their specific structures, cordycepin and its derivatives exhibit poor chemical stability, hindering their widespread application. Wei et al. synthesized N-acyl cordycepin derivatives that can protect their amino groups and enhance their biological activity; Shimada et al. reported that replacing the hydrogen at the 4' position with fluorine can delay its metabolism and reduce its cytotoxicity. Currently, the types of cordycepin derivatives obtained are limited, and while adding certain side groups to cordycepin can delay deamination, it still cannot completely block the deamination of adenosine deaminase (ADA enzyme). Therefore, it is still necessary to develop cordycepin derivatives with higher biological activity and chemical stability. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a cordycepin derivative, its preparation method, and its uses. The cordycepin derivative provided by this invention exhibits both strong stability and high biological activity, offering more drug options for clinical applications.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] On the one hand, the present invention provides a cordycepin derivative, the general structural formula of which is shown in formula (I):

[0006]

[0007] In formula (I):

[0008] R1 and R2 are each selected in isolation from either alkoxycarbonyl or H;

[0009] R3 is selected from any one of alkoxycarbonyl, silyl, substituted benzenesulfonyl and H;

[0010] R4 is selected from any one of thiol-substituted heterocycles, thiol-substituted aromatic rings, and H;

[0011] R5 can be either silicon-based or H; R1 to R5 cannot all be H at the same time.

[0012] In the technical solution of this invention, the structural formula of the alkoxycarbonyl group is as follows: The Z1 group is selected from any one of the following: substituted or unsubstituted alkyl groups with 1 to 10 carbon atoms, substituted or unsubstituted cycloalkyl groups with 3 to 10 carbon atoms, substituted or unsubstituted cycloalkenyl groups with 3 to 10 carbon atoms, substituted or unsubstituted aryl groups with 6 to 30 carbon atoms, substituted or unsubstituted aryloxy groups with 6 to 30 carbon atoms, and substituted or unsubstituted arylthio groups with 6 to 30 carbon atoms, preferably any one of the following: substituted or unsubstituted alkyl groups with 1 to 10 carbon atoms.

[0013] In the technical solution of this invention, the silicon-based structural formula is as follows: The Z2, Z3, and Z4 groups are each isolatedly selected from any one of hydrogen, -F, -Cl, -Br, hydroxyl, cyano, nitro, amido, hydrazine, hydrazone, alkyl with 1 to 10 carbon atoms, alkenyl with 2 to 10 carbon atoms, alkoxy with 2 to 10 carbon atoms, alkynyl with 1 to 10 carbon atoms, cycloalkyl with 3 to 10 carbon atoms, cycloalkenyl with 3 to 10 carbon atoms, aryl with 6 to 30 carbon atoms, aryloxy with 6 to 30 carbon atoms, and arylthio with 6 to 30 carbon atoms, preferably any one of alkyl with 1 to 10 carbon atoms.

[0014] In the technical solution of this invention, the structural formula of the substituted benzenesulfonyl group is as follows: C is any one of aryl groups with 6 to 30 carbon atoms or heteroaryl groups with 5 to 30 carbon atoms; Z5 group is selected from hydrogen, -F, -Cl, -Br, hydroxyl, cyano, nitro, mercapto, amidine, hydrazine, hydrazone, alkyl with 1 to 10 carbon atoms, alkenyl with 2 to 10 carbon atoms, alkynyl with 2 to 10 carbon atoms, alkoxy with 1 to 10 carbon atoms, cycloalkyl with 3 to 10 carbon atoms, heterocycloalkyl with 1 to 10 carbon atoms, cycloalkenyl with 3 to 10 carbon atoms, and cycloalkyl with 1 to 10 carbon atoms. The Z5 group may be any one of heterocyclic alkenyl, aryl with 6 to 30 carbon atoms, aryloxy with 6 to 30 carbon atoms, arylthio with 6 to 30 carbon atoms, heteroaryl with 1 to 30 carbon atoms, heteroaryloxy with 1 to 30 carbon atoms, or heteroarylthio with 1 to 30 carbon atoms, preferably any one of alkyl with 1 to 10 carbon atoms or alkoxy with 1 to 10 carbon atoms; the number of substitutions of the Z5 group may be 1 to 2, and the substitution position is not particularly limited; when the Z5 group is polysubstituted, they may be the same or different from each other.

[0015] In the technical solution of this invention, the structural formula of the thiol-substituted heterocycle is as follows: A is any one of heteroaryl or fused heterocyclic compounds with 5 to 30 carbon atoms; the heteroatom is any one or more of O, S, and N; Z6 is hydrogen, -F, -Cl, -Br, hydroxyl, cyano, nitro, mercapto, amido, hydrazine, hydrazone, alkyl with 1 to 10 carbon atoms, alkenyl with 2 to 10 carbon atoms, alkynyl with 2 to 10 carbon atoms, alkoxy with 1 to 10 carbon atoms, cycloalkyl with 3 to 10 carbon atoms, heterocyclic alkyl with 1 to 10 carbon atoms, or alkyl with 3 to 10 carbon atoms. The Z6 group can be any one of the following: cycloalkenyl, heterocyclic alkenyl with 1 to 10 carbon atoms, aryl with 6 to 30 carbon atoms, aryloxy with 6 to 30 carbon atoms, arylthio with 6 to 30 carbon atoms, heteroaryl with 1 to 30 carbon atoms, heteroaryloxy with 1 to 30 carbon atoms, and heteroarylthio with 1 to 30 carbon atoms, preferably any one of mercapto and H; the number of substitutions of the Z6 group can be 1 to 2, and the substitution position is not particularly limited; when the Z6 group is polysubstituted, they can be the same or different from each other.

[0016] In the technical solution of this invention, the structural formula of the thiol-substituted aromatic ring is as follows: B is any one of the aryl groups having 6 to 30 carbon atoms; hydrogen, -F, -Cl, -Br, hydroxyl, cyano, mercapto, amidine, hydrazine, hydrazone, alkyl with 1 to 10 carbon atoms, alkenyl with 2 to 10 carbon atoms, alkynyl with 2 to 10 carbon atoms, alkoxy with 1 to 10 carbon atoms, cycloalkyl with 3 to 10 carbon atoms, heterocyclic alkyl with 1 to 10 carbon atoms, cycloalkenyl with 3 to 10 carbon atoms, heterocyclic alkenyl with 1 to 10 carbon atoms, carbon atom The Z7 group is any one of aryl groups with 6 to 30 carbon atoms, aryloxy groups with 6 to 30 carbon atoms, arylthio groups with 6 to 30 carbon atoms, heteroaryl groups with 1 to 30 carbon atoms, heteroaryloxy groups with 1 to 30 carbon atoms, and heteroarylthio groups with 1 to 30 carbon atoms, preferably any one of alkyl groups with 1 to 10 carbon atoms and H; the number of substitutions of the Z7 group can be 1 to 2, and the substitution position is not particularly limited; when the Z7 group is multi-substituted, they can be the same or different from each other.

[0017] In some specific embodiments, the cordycepin derivatives may include:

[0018]

[0019] In another aspect, the present invention provides a method for preparing the above-mentioned cordycepin derivative;

[0020] (i) R1 and R2 are alkoxycarbonyl groups, and R3, R4, and R5 are H groups; the preparation method is shown in formula (II):

[0021]

[0022] Specifically, the following steps are included:

[0023] S1: Cordycepin and tert-butyldimethylchlorosilane (TBSCl) are reacted via a substitution reaction to prepare...

[0024] S2: Will Prepared by amidation reaction

[0025] S3: The protecting group on the product of step S2 is removed by tetrabutylammonium fluoride (TBAF) to obtain

[0026] In a preferred embodiment, in step S1, the substitution reaction is carried out in an organic solvent; the organic solvent is selected from any one or more of CH2Cl2, tetrahydrofuran (THF) or N,N-dimethylformamide (DMF);

[0027] Preferably, in step S1, the substitution reaction is carried out under the action of an organic base; the organic base is selected from any one of imidazole, pyridine, and Et3N;

[0028] In some specific embodiments, Et3N is used as the organic base, and the substitution reaction also requires a catalyst; the catalyst is 4-dimethylaminopyridine (DMAP); the equivalent amount of the catalyst is 1% to 20%;

[0029] Preferably, the molar ratio of cordycepin to tert-butyldimethylchlorosilane is 1:2 to 15;

[0030] In some specific embodiments, the concentration of cordycepin is 0.05–1 M;

[0031] Preferably, the substitution reaction does not require heating and the reaction time is 8–12 hours.

[0032] In a preferred embodiment, in step S2, the amidation reaction is carried out in an organic solvent; the organic solvent is selected from any one or more of N,N-dimethylformamide (DMF), dichloromethane (DCM), and tetrahydrofuran (THF);

[0033] Preferably, in step S2, the amidation reaction is carried out under the action of an organic base; the organic base is selected from any one of imidazole, pyridine, and Et3N;

[0034] Preferably, in step S2, the amidation reaction is... The amidation reaction is carried out with R1-O-R2; The molar ratio of R1-O-R2 is 1:10 to 14;

[0035] Preferably, in step S2, the The molar ratio with organic bases is 1:0.1 to 2;

[0036] In some specific implementations, the The concentration is 0.01–0.5 M;

[0037] Preferably, the amidation reaction is carried out at a temperature of 20–80°C for a time of 10–14 h.

[0038] In a preferred embodiment, the reaction in step S3 is carried out in an organic solvent; the organic solvent is selected from any one of N,N-dimethylformamide (DMF), acetonitrile, and tetrahydrofuran (THF);

[0039] Preferably, in step S3, the molar ratio of tetrabutylammonium fluoride (TBAF) to the product of step S2 is 1:40-60.

[0040] Preferably, the reaction temperature in step S3 is 20–80°C, and the reaction time is 3–5 h.

[0041] (ii) R3 is a substituted benzenesulfonyl group, and R1, R2, R4, and R5 are H; the preparation method includes the following steps:

[0042] Cordycepin and sulfonyl chloride compounds were prepared via a substitution reaction to obtain...

[0043] In a preferred embodiment, the substitution reaction is carried out under the action of an organic base; the organic base is selected from either imidazole or pyridine.

[0044] Preferably, in the substitution reaction, the molar ratio of cordycepin to sulfonyl chloride is 1:1 to 5;

[0045] Preferably, the temperature of the substitution reaction is 20–80°C;

[0046] In some specific embodiments, the concentration of cordycepin is 0.05–1 M.

[0047] (III) R4 is a thiol-substituted heterocyclic ring or a thiol-substituted aromatic ring, and R1, R2, R3, and R5 are H; the preparation method is shown in formula (III):

[0048]

[0049] Specifically, the following steps are included:

[0050] Step i: Synthesis of 2',3'-adenosine (3):

[0051] Will Prepared by substitution reaction with 2-acetoxyisobutyryl bromide

[0052]

[0053] Step ii: Prepare the product from step i by epoxidation reaction.

[0054] Step iii: R4H was prepared by substitution reaction to obtain

[0055] In a preferred embodiment, in step i, the substitution reaction is carried out in a solvent; the solvent is water and / or acetonitrile;

[0056] Preferably, the substitution reaction is carried out at a temperature of 20–80°C for a time of 10–14 hours.

[0057] Preferably, The molar ratio of 2-acetoxyisobutyryl bromide is 1:3 to 5;

[0058] Preferably, the The concentration is 0.01–1 M.

[0059] In a preferred embodiment, in step ii, the epoxidation reaction is carried out by the product of step i under the oxidation of a strong base anion exchange resin; the strong base anion exchange resin is Amberlite IRA402 resin.

[0060] Preferably, the epoxidation reaction is carried out in an organic solvent; the organic solvent is selected from either methanol or ethanol.

[0061] In some specific implementations, the The concentration is 0.01–0.5 M.

[0062] In a preferred embodiment, in step iii, the substitution reaction is carried out under the catalysis of an organic base; the organic base is selected from either sodium methoxide or sodium tert-butoxide.

[0063] Preferably, in step iii, the substitution reaction is carried out in an organic solvent; the organic solvent is selected from either methanol or tert-butanol.

[0064] Preferably, in step iii, the The molar ratio of R4H to R4H is 1:1-3.

[0065] Preferably, in step iii, the temperature of the substitution reaction is 20–100°C;

[0066] In some specific implementations, the The concentration is 0.05–1 M.

[0067] In another aspect, the present invention provides the use of the above-mentioned cordycepin derivatives in the preparation of antibacterial drugs, antioxidant drugs or cosmetics, and drugs for treating and / or preventing cancer.

[0068] In the technical solution of the present invention, the bacteria include Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Lactobacillus, Bifidobacterium and Candida albicans.

[0069] In the technical solution of this invention, the cancer includes leukemia, breast cancer, stomach cancer, and lung cancer.

[0070] Compared with the prior art, the present invention has the following advantages:

[0071] This invention addresses the problem of poor stability of cordycepin and its derivatives in existing technologies by providing a cordycepin derivative. This derivative employs special groups to structurally modify cordycepin, which not only improves its stability but also significantly enhances its antibacterial, anti-inflammatory, antioxidant, and antitumor biological activities. Furthermore, the cordycepin derivative of this invention exhibits selectivity for certain strains, particularly beneficial bacteria, thus expanding the application range of cordycepin derivatives. Attached Figure Description

[0072] Figure 1 The product S3-1 obtained in Example 1 1 H NMR spectrum.

[0073] Figure 2 The product S2-4 obtained in Example 10 1 H NMR spectrum.

[0074] Figure 3 The graph shows the free radical scavenging rate of compound S3-2 in Example 13 as a function of concentration. Detailed Implementation

[0075] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0076] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0077] Preparation Example 1:

[0078]

[0079] The preparation method of compound 3 (2',3'-deoxynucleosides) is based on non-patent literature 1 "Hansske F, Robins MJ. Regiospecific and stereoselective conversion of ribonucleosides to 3′-deoxynucleosides. A high yield three-stage synthesis of cordycepin from adenosine. [J]. Tetrahedron Letters, 1985, 26(36): 4295-4298, DOI: 10.1016 / S0040-4039(00)98716-1", the specific process is as follows:

[0080] Adenosine (a, 267 mg) was suspended in wet acetonitrile (acetonitrile:water = 100:1) (7.25 mL), and 2-acetoxyisobutyryl bromide (b, 0.60 mL) was added dropwise at 0 °C. 20 mL of ultra-dry acetonitrile was added at room temperature, and the solution became clear after stirring for 1 h. After complete consumption of the starting material adenosine (a), the reaction was quenched, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with a combination of Na₂CO₃ aqueous solution and water, dried with anhydrous Na₂SO₄, and concentrated by rotary evaporation to obtain a yellow oily crude product (728.30 mg).

[0081] Activating Amberlite IRA402(c) resin: Add twice the volume of resin to 4% HCl and soak for 4-8 hours. Wash with water until the pH is approximately 3-5. Further rinse the resin with four times the volume of 4% NaOH aqueous solution. Immerse the resin in an equal volume of 4% NaOH aqueous solution for 4-8 hours. Wash with water until the pH is neutral and store in water for later use.

[0082] In a dry flask, the crude product obtained in the previous step (728.30 mg) and 10 mL of methanol were added, followed by 7 mL of freshly activated resin (c). The mixture was stirred at room temperature for 1 hour. The reaction solution was collected, and the resin was washed with sufficient methanol and collected. The crude product was concentrated by rotary evaporation. Column chromatography yielded 236.70 mg of a white solid (3), with a yield of 95%.

[0083] Example 1:

[0084] The synthetic steps of compound (S3-1) are as follows:

[0085]

[0086] In a dry flask, sodium methoxide (86.40 mg) and 4-methylbenzyl mercaptan (4,298.09 mg) were suspended in methanol (3.20 mL) under nitrogen protection and stirred at room temperature for 10 minutes. 2',3'-O-adenosine (3,86.40 mg) obtained in Preparation Example 1 was added, and the mixture was refluxed at 75 °C for 3 h. After cooling, the mixture was concentrated by rotary evaporation to obtain the crude product, which was purified by column chromatography to obtain 80 mg of blue solid (compound S3-1), with a yield of 67%.

[0087] Figure 1 The image shows compound S3-1. 1 H NMR spectrum: 1 H NMR (400MHz, DMSO-d6) δ8.36(s,1H),8.15(s,1H),7.40(s,2H),7.36(d,J=8.2Hz,2H),7.14(d,J=8.0Hz,2H),6.09(d,J=5.8Hz, 1H),5.85-5.71(m,2H),4.78-4.62(m,1H),4.52(dt,J=8.1,2.8Hz,1H),3.92(t,J=8.3Hz,1H),3.83-3.67(m,2H),2.27(s,3H).

[0088] Example 2:

[0089] The synthetic steps of compound (S3-2) are as follows:

[0090]

[0091] In a dry flask, 2,5-dimercapto-1,3,4-thiadiazole (5,66.10 mg) was dissolved in THF (5 mL), and sodium tert-butoxide was added to form a white suspension. The suspension was stirred for 10 minutes, and 2',3'-O-adenosine (3,100 mg) obtained in Preparation Example 1 was added. The mixture was heated at 55 °C for 6 h. After cooling, the crude product was concentrated by rotary evaporation and purified by column chromatography to obtain 46.50 mg of a yellow oily product (compound S3-2), with a yield of 29%.

[0092] Example 3:

[0093] The synthetic steps of compound (S3-3) are as follows:

[0094]

[0095] In a dry flask, sodium methoxide (207.92 mg) and 2-mercaptopyridine (6,213.92 mg) were dissolved in methanol (8 mL) and stirred for 10 minutes. 2',3'-O-adenosine (3,200 mg) obtained in Preparation Example 1 was added, followed by the addition of sodium tert-butoxide to form a white suspension. The suspension was refluxed overnight at 75 °C. After cooling, the crude product was concentrated by rotary evaporation and purified by column chromatography to give 166 mg of a white solid compound (S3-3), with a yield of 57%.

[0096] Example 4:

[0097] The synthetic steps of compound (S3-4) are as follows:

[0098]

[0099] In a dry flask, sodium methoxide (312.14 mg) and 6-mercaptopurine (7,491.85 mg) were dissolved in methanol (3 mL) and stirred for 10 minutes. Then, 2',3'-O-adenosine (3,240 mg) obtained in Preparation Example 1 was added, and the mixture was refluxed overnight at 75 °C. After cooling, the mixture was concentrated by rotary evaporation to obtain the crude product. Purification by column chromatography yielded 367 mg of a blue solid (S3-4), with a yield of 95%.

[0100] Example 5:

[0101] The synthetic steps of compound (S1-1) are as follows:

[0102]

[0103] Synthesis of 6,6-bis(tert-butyloxycarbonyl)-3'-deoxyadenosine (S1-1):

[0104] In a dry flask, cordycepin (1,460 mg), imidazole (996.70 mg), and DMAP (22.36 mg) were added. Under nitrogen protection, DMF (16 mL) and TBSCl (1.10 g) were added at 0 °C. The mixture was stirred at room temperature for 15 hours. The reaction was quenched with saturated NH4Cl aqueous solution. The mixture was extracted three times with EtOAc. The combined organic layers were washed with H2O and saturated NaCl aqueous solution, respectively, and concentrated by rotary evaporation to obtain the crude product. Purification by chromatography yielded 605 mg of a white solid, 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-deoxyadenosine (d), in 69% yield.

[0105] In a dry flask, 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-deoxyadenosine (S4, 600 mg, 1.25 mmol) and DMAP (30.56 mg) were added. THF (10 mL) was added at 0 °C, followed by dropwise addition of Boc₂O (3.60 mL). The mixture was stirred at room temperature for 12 hours. After cooling to room temperature, the mixture was washed with a saturated NaCl aqueous solution, dehydrated with anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain the crude product. Purification by column chromatography yielded a colorless oily product of 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-deoxyadenosine (2,786 mg), in 92% yield.

[0106] In a dry flask, 2,393 mg of 2',5'-bis-O-(tert-butyldimethylsilyl)-3'-deoxyadenosine was added to 3 mL of THF, and 0.90 mL of TBAF was added dropwise at 0 °C. The mixture was stirred at room temperature for 4 h. The solution was purified by column chromatography to obtain 239 mg of white solid 6,6-bis(tert-butyloxycarbonyl)-3'-deoxyadenosine (S1-1), with a yield of 92%.

[0107] Example 6:

[0108] The synthetic steps of compound (S1-2) are as follows:

[0109]

[0110] In a dry flask, 6,6-bis(tert-butyloxycarbonyl)-3'-deoxyadenosine (S1-1, 80 mg) was dissolved in 3 mL of deionized water and left at room temperature for more than 4 days. The compound (S1-2) was purified by column chromatography to obtain 47 mg of compound (S1-2) with a yield of 76%.

[0111] Example 7:

[0112] The synthetic steps of compound (S2-1) are as follows:

[0113]

[0114] In a dry flask, cordycepin (1,300 mg) was dissolved in pyridine (2 mL) at 0 °C, and TsCl (8,409.76 mg) was added. The mixture was stirred overnight at room temperature. The product was purified by column chromatography to obtain 106.50 mg of a pale yellow solid (S2-1), with a yield of 16%.

[0115] Example 8:

[0116] The synthetic steps of compound (S2-2) are as follows:

[0117]

[0118] In a dry flask, cordycepin (1,200 mg) was dissolved in pyridine (2 mL) at 0 °C, and then 4-methoxybenzenesulfonyl chloride (9,296.10 mg) was added and stirred overnight at room temperature; 108 mg of pale yellow solid (S2-2) was obtained by column chromatography, with a yield of 32%.

[0119] Example 9:

[0120] The synthetic steps of compound (S2-3) are as follows:

[0121]

[0122] In a dry flask, a THF (6 mL) solution of cordycepin (1,500 mg) and DMAP (48.60 mg) was added to Boc2O (10,362 μL) at 0 °C. The reaction was heated to 65 °C and stirred for 12 hours. Post-treatment: The reaction flask was cooled to room temperature, the organic phase was washed with saturated saline solution, dehydrated with anhydrous sodium sulfate, and concentrated under vacuum. 46 mg of white solid (S2-3) was obtained by column chromatography, with a yield of 11%.

[0123] Example 10:

[0124] The synthetic steps of compound (S2-4) are as follows:

[0125]

[0126] In a dry flask, a solution of d (300 mg, 0.63 mmol) and TFA / H₂O (4.60 mL, 1:1, 50 eq.) in THF (6.30 mL, 0.1 M) was stirred for 1 hour at 0 °C. Post-treatment: the mixture was washed with saturated sodium bicarbonate solution, extracted three times with ethyl acetate, dehydrated with anhydrous sodium sulfate, and concentrated under vacuum. Purification by column chromatography yielded 111 mg of a white solid (S₂-₄), with a yield of 49%.

[0127] Characterization data: 1 ¹H NMR (400MHz, CDCl₃) δ 8.30 (s, 1H), 7.84 (s, 1H), 6.36 (s, 2H), 6.26 (s, 1H), 5.61 (d, J = 5.8 Hz, 1H), 5.04–4.94 (m, 1H), 4.52–4.42 (m, 1H), 3.97 (dd, J = 12.8, 1.7 Hz, 1H), 3.54 (d, J = 11.8 Hz, 1H), 2.52 (ddd, J = 10.8, 7.2, 3.4 Hz, 1H), 2.24–2.09 (m, 1H), 0.76 (s, 9H), -0.15 (s, 3H), -0.27 (s, 3H). (See detailed spectrum for...) Figure 2 )

[0128] Example 11 Evaluation of antibacterial effect:

[0129] This embodiment uses a two-fold dilution method to assess the effect of the compound on the growth inhibition of the selected strains for 16–24 h. The procedure is as follows: 100 μL of blank liquid culture medium is added to a 96-well plate; 100 μL of the test compound solution is added to the experimental group; the compound is continuously diluted twofold; finally, 10 μL of bacterial inoculum is added; the positive control group only adds 100 μL of blank liquid culture medium and 10 μL of bacterial inoculum, without adding the test compound solution; the negative control group only adds 110 μL of blank liquid culture medium; the 96-well plates are placed in a water-insulated incubator at 37°C and incubated on a shaker for 16–24 h, and then the OD is measured using a microplate reader. 600 Values; all tests were performed independently three to five times. The test results are shown in Table 1-3.

[0130] Table 1: Antibacterial rate test data of 4800 μg / mL test compound solution

[0131]

[0132] Table 2: Antibacterial rate test data of 300 μg / mL test compound solution

[0133]

[0134] Table 3. Antibacterial properties of the compounds at the maximum test concentration.

[0135]

[0136] In Table 3, "+" and "-" indicate the degree of inhibition: "+++" indicates clear bacterial solution, "++" indicates slight turbidity, "+" indicates turbidity, and "-" indicates very turbidity.

[0137] Non-patent literature 2, “Qu, Shuhao, et al. Design, synthesis, antibacterial / antitumor activity and in vitro stability of novel cordycepin derivatives with unsaturated fatty acid chain. European Journal of Pharmaceutical Sciences 187(2023):106466,” discloses cordycepin derivative 1b and cordycepin derivative 1d, with the following structures:

[0138]

[0139] In vitro antibacterial activity of cordycepin derivatives 1b and 1d:

[0140]

[0141] As can be seen from the table above, the cordycepin derivatives S1-1, S2-3, and S2-4 in this invention exhibit good antibacterial activity at the maximum tested concentration. Some cordycepin derivatives, such as S1-1, show increased inhibitory activity against pathogenic bacteria while exhibiting selectivity for beneficial bacteria. Cordycepin derivatives 1b and 1d in Reference 1 require concentrations of 512 and 1024 μg / mL, respectively, to inhibit bacterial growth and fail to kill bacteria. In comparison, compounds S2-4, S2-3, S3-1, and S3-2 in this application all show significant bactericidal effects at a concentration of 300 μg / mL. S2-4 achieves a 65% bactericidal rate against Staphylococcus aureus, and compounds S1-1, S1-2, and S3 exhibit stronger antibacterial effects at higher concentrations.

[0142] Furthermore, the cordycepin derivatives in this application have antibacterial effects against certain pathogenic bacteria comparable to antibiotics, while being more favorable to probiotics.

[0143] Example 12 Evaluation of the inhibitory effect on tumor cells:

[0144] In this embodiment, the CCK-8 assay was used to detect cell viability. Cells in the logarithmic growth phase were aspirated from the culture medium, digested with trypsin, and the culture medium was added to stop the growth. Cells were gently pipetted, counted, and seeded at a density of 2500 cells / well (100 μL). After overnight adhesion, the supernatant was discarded, and fresh culture medium containing a specific concentration of the compound was added. Two replicates were set up, with each concentration added to the corresponding well. Cells were incubated at 37°C with 5% CO2 for 72 hours. Afterward, the supernatant was discarded, and a 10:1 mixture of fresh culture medium and CCK-8 was added. After incubation at 37°C for approximately 2 hours, the absorbance at 450 nm (L1) was measured using a Thermo K3, with a reference wavelength of 630 nm (L2). The relative cell viability was calculated by subtracting L1 from L2, thus determining the effect of the compound on the cells. The results are shown in Table 4. Cordycepin (1) and compound (S2-3) have certain inhibitory effects on cancer cells K562 (human chronic myeloid leukemia cells) and MGC-803 (gastric cancer cell line). The half-maximal inhibitory concentrations (WMCs) of cordycepin (1) against K562 and MGC-803 were 37.32 μM and 16.10 μM, respectively, and the WMCs of S2-3 against K562 and MGC-803 were 42.21 μM and 27.79 μM, respectively.

[0145] Table 4

[0146] compound K562 MGC-803 Cordycepin 37.32μM 16.10μM S2-3 42.21μM 27.79μM

[0147] Example 13 Antioxidant performance evaluation:

[0148] This embodiment tested the antioxidant activity of the above derivatives. The specific procedure was as follows: 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) solution (0.3 mmol / L, maximum absorption wavelength around 517 nm) was used as the substrate, and four groups were formed: a positive control group, a negative control group, a sample group, and a sample control group. The positive control group consisted of 160 μL of vitamin C solution and 40 μL of DPPH; the negative control group A consisted of 160 μL of methanol and 40 μL of DPPH; and the sample group consisted of 160 μL of the compound solution and 40 μL of DPPH. A multi-functional microplate reader was used, with the wavelength set to 517 nm, and measurements were performed in triplicate. The test results are shown in Table 5, where the free radical scavenging rate of compound (S3-2) as a function of concentration is shown in the graph. Figure 3 As shown.

[0149] Table 5

[0150]

[0151]

[0152] Example 14 Stability Evaluation

[0153] This embodiment tested the stability of the above-mentioned cordycepin derivatives. The test procedure was as follows: 0.007 mmol of the test drug was weighed and dissolved in 40 μL of DMSO, and diluted to 10 mL with phosphate buffer solution at pH 7.4. At least 0.5 mL of the solution was then transferred to a centrifuge tube. 1 mg of adenosine deaminase (64.8 U) was weighed and dissolved in 10 mL of PBS solution at pH 7.4. 1 mL of this solution was diluted to 10 mL, and 50 μL of this solution was added to a centrifuge tube containing 0.5 mL of the test drug, and diluted to 9 mL. The experiment was conducted on a UV analyzer. In the presence of 3.5 mU / mL adenosine deaminase, the absorption spectrum was scanned every 15 min at a resolution of 2 nm in the 230–300 nm range for 6–7 h.

[0154] Tables 6-8 show the transformation of cordycepin (1), cordycepin derivative S1-1, and cordycepin derivative S2-3 over time under the action of adenosine deaminase (ADA enzyme):

[0155] Table 6

[0156] time Percentage of remaining unconverted portion 0min 100.00％ 15min 53.31％ 30min 34.80％ 45min 12.90％ 60min 2.72％ 75min 0.51％ 90min 0.00％

[0157] Table 7

[0158]

[0159]

[0160] Table 8

[0161] time Percentage of remaining unconverted portion 0min 100.00％ 15min 79.10％ 30min 57.63％ 45min 50.28％ 60min 23.73％ 75min 9.04％ 90min 0.00％

[0162] As can be seen from the table above, the stability of cordycepin derivatives S1-1 and S2-3 in this application under the action of adenosine deaminase is improved to varying degrees compared with cordycepin.

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cordycepin derivative, characterized by, The structure is shown as formula (I):

2. The method of claim 1, wherein the cordycepin derivative is prepared by the steps of, The preparation method is shown as formula (III): Specifically comprising the following steps: Step i: synthesis of 2', 3'-anhydro-adenosine (3): Preparation of 2-acetyloxyisobutyryl bromide by substitution reaction and 2-acetyloxyisobutyryl bromide by substitution reaction Step ii: The product of step i is subjected to an epoxidation reaction to prepare Step iii: Preparation of R4H by substitution reaction and R4H are prepared by substitution reaction R4H is 3. The preparation method according to claim 2, characterized in that, In step i, the substitution reaction is carried out in a solvent; the solvent is water and / or acetonitrile.

4. The preparation method according to claim 2, characterized in that, In step i, the temperature of the substitution reaction is 20-80°C, and the time is 10-14 h.

5. The preparation method according to claim 2, characterized in that, in step i, and the molar ratio of 2-acetyloxyisobutyryl bromide is 1:3-5.

6. The preparation method according to claim 2, characterized in that, In step i, the concentration of the compound of formula (I) is 0.01 to 1 M. 0.01 to 1 M.

7. The preparation method according to claim 2, characterized in that, In step ii, the epoxidation reaction is carried out by oxidation of the product of step i by a strong base type anion resin; the strong base type anion resin is Amberlite IRA402 resin.

8. The preparation method according to claim 2, characterized in that, In step ii, the epoxidation reaction is carried out in an organic solvent; the organic solvent is selected from any one of methanol and ethanol.

9. The preparation method according to claim 2, characterized in that, In step iii, the substitution reaction is carried out under catalysis of an organic base; the organic base is selected from any one of sodium methoxide and sodium tert-butoxide.

10. The method of claim 2, wherein, In step iii, the substitution reaction is carried out in an organic solvent; the organic solvent is selected from any one of methanol and tert-butanol.

11. The method of claim 2, wherein, In step iii, the and R4H in a molar ratio of 1 : 1 to 3.

12. The method of claim 2, wherein, In step iii, the temperature of the substitution reaction is 20-100°C.

13. Use of the cordycepin derivative according to claim 1 in the preparation of an antibacterial drug, an antioxidant drug, characterized in that, The bacteria are Escherichia coli.

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