Cordycepin salts, their preparation methods and applications

Abstract

The present invention discloses cordycepin salts, their preparation methods and applications. The cordycepin salts include cordycepin nucleosides, cordycepin natural organic salts, cordycepin amino acid salts, cordycepin vitamin salts and cordycepin inorganic salts. The functional salts of cordycepin provided by the present invention have good effects on preventing and treating diseases related to the functional damage and mutation of cells in mammals. For the parent drug, the functional salts of cordycepin have better solubility, can increase the bioavailability in vivo, and make the drug have better efficacy in vivo.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to cordycepin salts, a preparation method thereof, and an application thereof in the preparation of products for preventing cell damage-related diseases. Background Art

[0002] The information provided in this section is only background information related to the present disclosure, and it is not necessarily prior art.

[0003] Cordycepin (3'-deoxyadenosine) is an effective natural component isolated from Cordyceps sinensis. It is an adenosine analog. Many studies have shown its potential for treating or assisting in the treatment of various diseases, mainly including inflammatory diseases and cancers. After entering cells, cordycepin is phosphorylated in vivo into monophosphate derivatives, diphosphate derivatives, or triphosphate derivatives. Among them, the triphosphorylated derivative can inhibit the synthesis of mRNA and ultimately affect the synthesis of the corresponding protein, thereby playing a role in inhibiting tumor growth. In addition, the physiological effects of cordycepin in the body's metabolic process mainly include causing cell death, preventing cell growth, inhibiting the expression of multiple inflammatory genes, and reducing cell migration. Clinical studies have shown that cordycepin can prevent the occurrence of inflammation and reduce the occurrence of tumors. The molecular mechanism of the biological effects of cordycepin includes regulating the expression of genes related to inflammation and tumorigenesis, inhibiting the phosphorylation of many important kinase substrates, or directly affecting protein synthesis.

[0004] As an adenosine analog, cordycepin plays an important role in the screening of anti-tumor drugs. Among them, cordycepin is one of the main ways to prepare nucleoside anti-tumor drugs. However, the nucleoside compounds prepared from cordycepin have poor solubility, which affects the subsequent dosage form preparation and human absorption.

[0005] In the prior art, the following strategies are often adopted to provide solubility: 1. Generating soluble salts. This method usually has good chemical stability, and different acid-base pairs can be selected to generate various salts to find the salt with the best solubility; 2. Changing the crystal form of the drug / forming solvates or co-crystals. This method may cause crystal form transformation during storage or production, affecting product quality; 3. Using surfactants for solubilization, which may affect the bioavailability of the drug and has certain limitations in the scope of application; 4. Solid dispersion systems. The selection and optimization of polymer carriers in this method are complex, and interactions may occur between the drug and the carrier; 5. Cyclodextrin inclusion complexes. It is difficult to ensure uniform distribution of the drug in the cyclodextrin inclusion complex. Although cyclodextrin inclusion complexes can control drug release, the prediction and control of release kinetics are complex; 6. Prodrug design is a promising drug development strategy that can solve many challenges faced by traditional drugs, but at the same time brings new scientific and regulatory issues; 6. Co-solvents may be toxic or irritating to patients, especially during injection administration, and some safety and cost challenges need to be overcome; 7. Changing the pH of the formulation. Changing the pH value may affect drug stability, especially for some drugs sensitive to pH changes. At the same time, extreme pH values will also cause irritation or damage to the patient's oral cavity, gastrointestinal tract or other tissues; 8. Micronization can improve the stability of some drugs, but for heat-sensitive or mechanically stress-sensitive drugs, the micronization process may cause degradation; 9. Liposomes, as a drug delivery system, have significant advantages in improving drug efficacy and reducing side effects, but challenges such as cost, stability and large-scale production need to be overcome in actual production.

[0006] Although forming salts is a common method to improve drug solubility, it does not always succeed in improving solubility in all cases. During the drug development process, experiments are needed to determine the optimal salt form and conditions.

[0007] Therefore, the present invention provides cordycepin salts, their preparation methods and applications to further improve their anti-tumor efficacy. Summary of the Invention

[0008] Object of the Invention: The technical problem to be solved by the present invention is to provide cordycepin salts, their preparation methods and applications in view of the deficiencies of the prior art.

[0009] To solve the above technical problems, the present invention discloses the following technical solutions:

[0010] In the first aspect, the present invention discloses functional salts of cordycepin.

[0011] In some embodiments, the salt is a pharmaceutically acceptable functional salt, including nucleoside salts, natural product organic salts, food organic salts, amino acid salts, vitamin salts, or inorganic acid salts; the salt does not include hydrochloride, sulfate, phosphate, maleate, tartrate, oxalate, and fumarate.

[0012] In some embodiments, the nucleoside salts are selected from uridylate, cytidylate, adenosine monophosphate, cyclic adenosine monophosphate, guanylate, inosinate, or xanthylate.

[0013] In some embodiments, the natural product organic salts are selected from chlorogenate, caffeate, sialate, pseudolaric acid A, glycyrrhizinate, glycyrrhetate, oleanolate, arbutinate, anisate, salvianolate, quinate, deoxycholate, betulinate, gambogate, or rheinate.

[0014] In some embodiments, the food organic salts are selected from acetate, malonate, butyrate, valerate, oleate, palmitate, stearate, laurate, maleate, malate, citrate, tartrate, fructose-1,6-diphosphate, succinate, gluconate, lactobionate, laurylsulfonate, or isethionate.

[0015] In some embodiments, the amino acid salts are selected from aspartate or glutamate.

[0016] In some embodiments, the vitamin salts are selected from vitamin B3 (niacin) salt, vitamin B5 (pantothenic acid) salt, vitamin C (ascorbic acid) salt, or vitamin M (folic acid) salt.

[0017] In some embodiments, the inorganic acid salts are selected from hydrochloride, phosphate, phosphite, or borate; the amino acid salts are selected from aspartate or glutamate.

[0018] In a second aspect, the present invention provides a method for preparing the cordycepin salt described in the first aspect above. In a solvent, cordycepin is dissolved with any one or several acids selected from inorganic acids, amino acids, vitamins, nucleotides, food organic acids, and natural product organic acids, and after crystallization, it is filtered or directly filtered to obtain the product.

[0019] In some embodiments, the molar ratio of any one of the nucleotide, natural product organic acid, food organic acid, amino acid, vitamin, and inorganic acid to cordycepin is 0.1 to 10:1, preferably 1-2:0.6-3, more preferably 0.1-2:1, preferably 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, or 9.5:1.

[0020] In the present invention, the preparation method of the cordycepin salt specifically includes the following two methods:

[0021] Method 1:

[0022] The method includes: in a first solvent, cordycepin is stirred and dissolved with any one or several of inorganic acids, amino acids, vitamins, nucleotides, food organic acids, and natural product organic acids to obtain a solution, cooled to room temperature, and a second solvent is added for crystallization. That is, when the solution becomes a suspension, stirring is stopped, filtration is carried out, and the filter residue is dried to obtain the product.

[0023] In some embodiments, the molar ratio of the cordycepin to any one or several of the inorganic acids, amino acids, vitamins, nucleotides, food organic acids, and natural product organic acids is 1-2:1.

[0024] In some embodiments, the first solvent is a solvent that can dissolve cordycepin, specifically water.

[0025] In some embodiments, the dosage ratio of cordycepin to the solvent is 1 mmol:0.5-5 mL.

[0026] In some embodiments, the dissolution temperature is 20-80 °C, such as 45 °C, 50 °C, 55 °C, 60 °C, 65 °C.

[0027] In some embodiments, the second solvent is an anti-solvent, specifically an alcohol; in some embodiments, the alcohol is any one or several combinations of methanol, ethanol, isopropanol, and cyclohexanol.

[0028] In some embodiments, the volume ratio of the first solvent to the second solvent is 1:1-10, such as 1:2, 1:3, 1:4, 1:5, 1:6.

[0029] Method 2:

[0030] The method includes: dissolving an excess of cordycepin with any one or several of inorganic acids, amino acids, vitamins, nucleotides, food organic acids, and natural product organic acids in water, filtering to remove the excess cordycepin, and freeze-drying the filtrate to obtain the product.

[0031] In some embodiments, the molar ratio of cordycepin to any one or several of the inorganic acids, amino acids, vitamins, nucleotides, food organic acids, and natural product organic acids is 1-2:0.6-3.

[0032] In some embodiments, the dosage ratio of cordycepin to water is 1 mmol:0.5-2 mL.

[0033] Further, when the salt is a functional salt with good solubility and poor recrystallization effect, Method 2 is selected for preparation.

[0034] In a third aspect, the present invention provides a pharmaceutical composition comprising a) the cordycepin salt described in the foregoing first aspect, and b) other active substances and / or pharmaceutically acceptable excipients.

[0035] In some embodiments, the other active substance is an immune checkpoint inhibitor; in some embodiments, the immune checkpoint inhibitor is PD-1 and / or PD-L1.

[0036] In some embodiments, the mass ratio of the cordycepin salt to the other active substance is 2-4:1, preferably 3:1.

[0037] In some embodiments, the dosage form of the pharmaceutical composition is selected from tablets, pills, capsules, dripping pills, syrups, disintegrants, injections, sustained-release agents, or kits.

[0038] In a fourth aspect, the present invention provides the application of the above-mentioned salt of cordycepin, or the above-mentioned pharmaceutical composition in the preparation of products for preventing and treating diseases related to the occurrence and variation of cell functional damage in mammals.

[0039] In some embodiments, the diseases related to the occurrence and variation of cell functional damage are tumors.

[0040] In some embodiments, the mammals include humans.

[0041] In some embodiments, the tumors include liver cancer, gastric cancer, small cell lung cancer, colon cancer, ovarian cancer, thyroid cancer, human glioma, or a combination of the foregoing several.

[0042] Beneficial effects: Compared with the prior art, the present application has the following advantages:

[0043] By combining cordycepin with an acid to form a salt, the present invention can not only improve solubility, enhance drug absorption and efficacy, but also, through metabolic action in vivo, perform combined superposition, and can promote the anti-tumor effect of cordycepin as a way of enhancing or adjuvant treatment. Description of the Drawings

[0044] The following further specifically describes the present invention in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0045] Figure 1 It is the nuclear magnetic resonance spectrum of the functional salt formed by cordycepin and hydrochloric acid.

[0046] Figure 2 It is the TG-DSC spectrum of the functional salt formed by cordycepin and hydrochloric acid.

[0047] Figure 3 It is the nuclear magnetic resonance spectrum of the functional salt formed by cordycepin and nicotinic acid.

[0048] Figure 4 It is the TG-DSC spectrum of the functional salt formed by cordycepin and nicotinic acid.

[0049] Figure 5 It is the nuclear magnetic resonance spectrum of the functional salt formed by cordycepin and folic acid.

[0050] Figure 6 It is the TG-DSC spectrum of the functional salt formed by cordycepin and folic acid.

[0051] Figure 7 It is the nuclear magnetic resonance spectrum of the functional salt formed by cordycepin and n-butyric acid.

[0052] Figure 8 It is the TG-DSC spectrum of the functional salt formed by cordycepin and n-butyric acid.

[0053] Figure 9 It is the nuclear magnetic resonance spectrum of the functional salt formed by cordycepin and malic acid.

[0054] Figure 10 It is the TG-DSC spectrum of the functional salt formed by cordycepin and malic acid.

[0055] Figure 11 It is the nuclear magnetic resonance spectrum of the functional salt formed by cordycepin and shikimic acid.

[0056] Figure 12 It is the TG-DSC spectrum of the functional salt formed by cordycepin and shikimic acid.

[0057] Figure 13 It is the anti-tumor proliferation experimental effect of cordycepin or its functional salt on small cell lung cancer.

[0058] Figure 14 It is the anti-tumor proliferation experimental effect of cordycepin or its functional salt on colon cancer.

[0059] Figure 15 It is the inhibitory experimental effect of cordycepin or its functional salt on ovarian cancer.

[0060] Figure 16 The inhibitory experimental effect of cordycepin or its functional salt on thyroid cancer.

[0061] Figure 17 TG-DSC spectrum of cordycepin. Detailed implementation manners

[0062] According to the following embodiments, the present invention can be better understood. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims.

[0063] In the following embodiments, unless otherwise specified, the experimental methods are all conventional methods; unless otherwise specified, the reagents and materials can all be obtained from commercial channels.

[0064] In the following embodiments, the cell and animal model experiments related to the anti-cancer effect of cordycepin or its functional salt are not all effective experimental results. Only the relevant experiments listed for illustrative purposes are presented, and the drug evaluation experiments, including cell models and animal models, follow ethical rules.

[0065] The preparation methods of the functional salts mentioned in the following embodiments are not for all compounds. For the sake of convenience of description, only the preparation processes and characterization results of several representative functional salts are listed here.

[0066] Example 1: Preparation of inorganic salts of cordycepin

[0067] 25.1 g (100 mmol) of cordycepin and 100 mL of water were added to a flask to prepare a suspension. 10 mL (120 mmol) of 12 M hydrochloric acid and 10 mL (140 mmol) of 14 M phosphoric acid were respectively added to the suspension. Most of the cordycepin was stirred and dissolved in a water bath at room temperature. After dissolution, the suspension was filtered, and the filtrates were freeze-dried to obtain hydrochloride as functional salt 1 (such as Figure 1 , Figure 2 ) and phosphate as functional salt 2.

[0068] The 1 H NMR spectrum of the cordycepin hydrochloride functional salt prepared in this Example 1 is as shown in Figure 1 . The 1 HNMR spectrum of the obtained functional salt is the sum of the characteristic peaks of cordycepin and hydrochloric acid, indicating the presence of these two components in the new phase. The 1 H NMR chemical shifts of the cordycepin hydrochloride functional salt are as follows: 11H NMR (400 MHz, Deuterium Oxide) δ 8.37 (s, 1H), 8.27 (s, 1H), 6.00 (d, J = 1.8 Hz, 1H), 4.72 (d, J = 5.7 Hz, 13H), 4.54 (dtd, J = 11.5, 6.3, 5.4, 2.8 Hz, 1H), 3.84 (dd, J = 12.7, 2.8 Hz, 1H), 3.64 (dd, J = 12.7, 4.7 Hz, 1H), 2.20 (ddd, J = 13.9, 9.6, 5.6 Hz, 1H), 2.08 (ddd, J = 13.9, 6.3, 2.6 Hz, 1H).

[0069] The TG-DSC curve of the cordycepin hydrochloride functional salt prepared in Example 1 is as Figure 2 shown. The sample lost about 4.6% of its weight in the temperature range of 24 - 43 °C, presumably containing solvent. It can be seen from the DSC curve analysis that cordycepin hydrochloride has endothermic peaks at 123 °C and 163 °C respectively. The sample starts to decompose at 168 °C.

[0070] Example 2: Preparation of cordycepin amino acid salt

[0071] 2.51 g (10 mmol) of cordycepin and 10 mL of water were added to a flask to form a suspension. 4.43 g (30 mmol) of glutamic acid was added to the suspension, and the cordycepin was dissolved in a water bath at room temperature. The remaining suspension was filtered, and the filtrate was freeze-dried to obtain the glutamate salt as functional salt 3.

[0072] Example 3: Preparation of cordycepin vitamin salt

[0073] 2.51 g (10 mmol) of cordycepin and 20 mL of water were added to a flask to form a suspension. 1.23 g (10 mmol) of nicotinic acid and 4.41 g (10 mmol) of folic acid were added to the suspension respectively. The reaction was stirred at 50 °C for 2 h to completely dissolve the cordycepin in the solution. After the reaction was cooled to room temperature, 60 mL of anti-solvent ethanol was slowly added thereto. When the solution became a suspension, the stirring was stopped. The obtained suspension was filtered, and the filter residue was dried in vacuo at 50 °C to obtain the nicotinate as functional salt 4 (as Figure 3 , Figure 4 ) and the folate as functional salt 5 (as Figure 5 , Figure 6 ).

[0074] The 1 1H NMR spectrum of the cordycepin nicotinic acid functional salt prepared in this Example 3 is as Figure 3 shown. The 1The HNMR spectrum is the sum of the characteristic peaks of cordycepin and nicotinic acid, indicating the presence of these two components in the new phase. Cordycepin nicotinic acid functional salt 1 The chemical shifts of 1H NMR are as follows: 1 1H NMR (400 MHz, Deuterium Oxide) δ8.98 (d, J = 1.9 Hz, 2H), 8.78–8.59 (m, 5H), 8.22 (s, 1H), 8.10 (s, 1H), 7.90 (dd, J = 8.0, 5.7 Hz, 2H), 5.93 (d, J = 2.2 Hz, 1H), 4.70 (s, 26H), 4.59–4.47 (m, 0H), 3.83 (dd, J = 12.7, 2.8 Hz, 1H), 3.62 (dd, J = 12.7, 4.5 Hz, 1H), 2.34–2.11 (m, 1H).

[0075] The TG-DSC curve of the cordycepin nicotinic acid functional salt prepared in Example 3 is as shown in Figure 4 the figure. The sample loses about 2.5% of its weight in the temperature range of 37 - 63.0 °C, presumably containing solvent. It can be seen from the DSC curve analysis that the cordycepin nicotinate has endothermic peaks at 107 °C and 174 °C respectively. The sample starts to decompose at 150 °C.

[0076] The 1 1H NMR spectrum of the cordycepin folic acid functional salt prepared in Example 3 is as shown in Figure 5 the figure. The 1 HNMR spectrum of the obtained functional salt is the sum of the characteristic peaks of cordycepin and folic acid, indicating the presence of these two components in the new phase. Cordycepin folic acid functional salt 1 The chemical shifts of 1H NMR are as follows: 1 1H NMR (400 MHz, Deuterium Oxide) δ8.18 (s, 1H), 8.05 (s, 1H), 6.02–5.89 (m, 1H), 4.53 (s, 1H), 3.84 (d, J = 11.0 Hz, 1H), 3.63 (dd, J = 11.5, 3.6 Hz, 1H), 2.21–2.04 (m, 2H).

[0077] The TG-DSC curve of the cordycepin folic acid functional salt prepared in Example 3 is as shown in Figure 6 the figure. The sample loses about 6.2% of its weight in the temperature range of 40 - 89 °C, presumably containing solvent. It can be seen from the DSC curve analysis that the cordycepin folate has endothermic peaks at 115 °C and 172 °C respectively. The sample starts to decompose at 206 °C.

[0078] Example 4: Preparation of Cordycepin Nucleotide Salt

[0079] Add cordycepin 2.51 g (10 mmol) and 30 mL of water to a flask to prepare a suspension. Add uridylic acid 3.24 g (10 mmol) and cyclic adenosine monophosphate 3.29 g (10 mmol) to the suspension respectively. React and stir for 2 h at 50 °C to completely dissolve cordycepin in water. Wait for the reaction to cool to room temperature, slowly add 90 mL of ethanol to it. Stop stirring after the solution completely becomes a suspension. Filter the obtained suspension, and the filter residue is dried in vacuo at 60 °C to obtain uridylate as functional salt 6 and cyclic adenosine monophosphate salt as functional salt 7.

[0080] Example 5: Preparation of Cordycepin Food Organic Acid Salts (Fatty Acid Salts)

[0081] Add cordycepin 7.53 g (30 mmol) and 20 mL of water to a flask to prepare a suspension. Add acetic acid 1.20 g (20 mmol) and butyric acid 1.76 g (20 mmol) to the suspension respectively. React and stir for 2 h at room temperature to dissolve most of the cordycepin in a water bath at room temperature. Filter the remaining suspension, and the filtrate is freeze-dried to obtain acetate as functional salt 8 and butyrate as functional salt 9 (as Figure 7 , Figure 8 ).

[0082] The 1 H NMR spectrum of the cordycepin butyrate functional salt prepared in this Example 5 is as Figure 7 shown. The 1 HNMR spectrum of the obtained functional salt is the sum of the characteristic peaks of cordycepin and butyric acid, indicating the presence of these two components in the new phase. The 1 H NMR chemical shifts of the cordycepin butyrate functional salt are as follows: 1H NMR (400 MHz, Deuterium Oxide) δ 8.21 (s, 1H), 8.11 (s, 1H), 5.96 (d, J = 2.4 Hz, 1H), 4.70 (s, 50H), 3.84 (dd, J = 12.7, 2.8 Hz, 1H), 3.63 (dd, J = 12.7, 4.5 Hz, 1H), 2.23 (ddd, J = 14.5, 8.9, 5.9 Hz, 1H), 2.14–2.03 (m, 1H).

[0083] The TG-DSC curve of the cordycepin butyrate functional salt prepared in this Example 5 is as Figure 8As shown. The sample has two weight loss steps. The first weight loss step is at 67 °C with a weight loss of about 11%. The second weight loss step is around 100 °C. It can be known from the DSC curve analysis that cordycepin butyrate has multiple endothermic peaks at 76 °C, 134 °C, 205 °C and 221 °C respectively, and the sample starts to decompose at 100 °C. During the whole process, the sample first loses the solvent and then decomposes. After decomposing to a certain extent, melting occurs; this indicates that in the DSC curve, a series of endothermic peaks are caused by both decomposition and melting.

[0084] Example 6: Preparation of Cordycepin Food Organic Acid Salt

[0085] 5.02 g (20 mmol) of cordycepin and 40 mL of water were added to a flask to prepare a suspension. 1.34 g (10 mmol) of malic acid and 3.40 g (10 mmol) of fructose-1,6-diphosphate were respectively added to the suspension. The reaction was stirred at 60 °C for 2 h to completely dissolve cordycepin in water. After the reaction was cooled to room temperature, 120 mL of ethanol was slowly added thereto. Stirring was stopped after the solution completely became a suspension. The obtained suspension was filtered, and the filter residue was vacuum dried at 60 °C to obtain malate as functional salt 10 (such as Figure 9 , Figure 10 ) and fructose-1,6-diphosphate as functional salt 11.

[0086] The 1 1H NMR spectrum of the cordycepin malate functional salt prepared in this Example 6 is as Figure 9 shown. The 1 1H NMR spectrum of the obtained functional salt is the sum of the characteristic peaks of cordycepin and malic acid, indicating the presence of these two components in the new phase. The 1 1H NMR chemical shifts of the cordycepin malate functional salt are as follows: 1 1H NMR (400 MHz, Deuterium Oxide) δ8.31 (d, J = 2.3 Hz, 2H), 8.22–8.16 (m, 2H), 5.96 (d, J = 2.0 Hz, 2H), 4.38 (ddd, J = 7.2, 4.4, 1.9 Hz, 3H), 3.82 (dd, J = 12.6, 2.8 Hz, 2H), 3.62 (dd, J = 12.6, 4.7 Hz, 2H), 2.82–2.75 (m, 1H), 2.75 (dd, J = 4.5, 1.0 Hz, 2H), 2.70–2.59 (m, 3H), 2.11–2.01 (m, 1H).

[0087] The TG-DSC curve of the cordycepin malate functional salt prepared in this Example 6 is as Figure 10As shown. There is no solvent in the sample. It can be known from the DSC curve analysis that cordycepin malate has endothermic peaks at 140 °C and 178 °C respectively. The sample starts to decompose at 177 °C.

[0088] Example 7: Preparation of Organic Salts of Cordycepin Natural Products

[0089] Add 15.06 g (60 mmol) of cordycepin and 100 mL of water into a flask to prepare a suspension. Add 3.54 g (10 mmol) of chlorogenic acid, 1.80 g (10 mmol) of caffeic acid, 3.09 g (10 mmol) of sialic acid, 4.32 g (10 mmol) of pseudolaric acid A, 8.23 g (10 mmol) of glycyrrhizic acid, 4.57 g (10 mmol) of oleanolic acid, 1.74 g (10 mmol) of shikimic acid, 2.84 g (10 mmol) of rhein, and 6.29 g (10 mmol) of gambogic acid to the suspension respectively. Stir all reactions at room temperature for 1 h to dissolve most of the cordycepin in a water bath at room temperature. Filter the remaining suspension. The filtrate is freeze-dried to obtain chlorogenate as functional salt 12, caffeate as functional salt 13, sialate as functional salt 14, pseudolaricate as functional salt 15, glycyrrhizate as functional salt 16, oleanolate as functional salt 17, shikimate as functional salt 18 (as Figure 11 , Figure 12 ), rheinate as functional salt 19, and gambogate as functional salt 20.

[0090] The 1 1H NMR spectrum of the cordycepin shikimate functional salt prepared in this Example 7 is as Figure 11 shown. The 1 1H NMR spectrum of the obtained functional salt is the sum of the characteristic peaks of cordycepin and shikimic acid, indicating the presence of these two components in the new phase. The 1 1H NMR chemical shifts of the cordycepin shikimate functional salt are as follows: 11H NMR (400 MHz, Deuterium Oxide) δ 8.24 (s, 2H), 8.10 (s, 2H), 6.62 (dd, J = 4.1, 1.9 Hz, 3H), 5.93 (d, J = 2.0 Hz, 2H), 4.51 (dddd, J = 9.3, 6.7, 4.4, 2.8 Hz, 2H), 4.33 (t, J = 4.3 Hz, 3H), 3.91 (ddd, J = 8.4, 6.8, 5.2 Hz, 3H), 3.84 (d, J = 2.8 Hz, 1H), 3.68–3.61 (m, 3H), 3.65–3.58 (m, 2H), 2.63 (ddt, J = 18.0, 5.3, 1.6 Hz, 3H), 2.11 (d, J = 6.9 Hz, 1H), 2.09–2.04 (m, 2H).

[0091] The TG-DSC curves of the cordycepin shikimic acid functional salt prepared in Example 7 are as Figure 12 shown. There is no solvent in the sample. It can be seen from the DSC curve analysis that the cordycepin shikimate has endothermic peaks at 170 °C and 179 °C respectively. The sample starts to decompose at 160 °C.

[0092] Application Example 1: Anti-tumor proliferation experimental effect of the functional salt of cordycepin on liver cancer

[0093] The MTT method was used to determine the proliferation inhibitory activity of the functional salt of cordycepin on liver cancer tumor cells: Take Hepa 1-6 and Hep G2 liver cancer cells in the exponential growth phase with good condition, and add 0.25% trypsin digestive solution respectively to make the adherent cells detach. Count about 2×10 4 cells / mL to make a liver cancer cell suspension. Take the cell suspension and inoculate it on a 96-well plate, 90 μL / well, and incubate it in a constant temperature CO 2 incubator for 24 h. Add cordycepin or its functional salt 10 μL / well and culture for 72 h. Add 10 μL / well of MTT reagent to the 96-well plate, react in the incubator for 4 h, aspirate the supernatant, add 100 μL / well of dimethyl sulfoxide, dissolve it, and use an enzyme-linked immunosorbent detector to measure the absorbance value of each well at a wavelength of 570 nm, and calculate the cell inhibition rate to obtain the IC 50 of the corresponding compound. By calculating the results of the half-maximal inhibitory concentration (IC 50 ), the summary is shown in Table 1 below. The results of the in vitro anti-tumor experiment show that compared with cordycepin, the effective concentration of most of the cordycepin functional salts is significantly reduced for tumor cells.

[0094] Table 1 IC 50 (μM) of cordycepin and cordycepin modified compounds in liver cancer cells

[0095] Functional salt Hepa 1-6 Hep G2 Functional salt Hepa 1-6 Hep G2 Cordycepin 0.278 24.1 11 0.073 1.37 1 0.251 20.7 12 0.143 10.4 2 0.244 19.4 13 0.197 13.1 3 0.253 21.6 14 0.083 1.79 4 0.214 16.7 15 0.108 3.54 5 0.184 18.4 16 0.146 3.1 6 0.103 3.28 17 0.092 1.92 7 0.094 0.943 18 0.174 7.46 8 0.268 23.4 19 0.139 13.7 9 0.249 22.5 20 0.115 16.3 10 0.219 23.9

[0096] Application Example 2: Anti-tumor proliferation experimental effect of cordycepin or its functional salts on gastric cancer

[0097] Establishment of mouse gastric cancer transplantation model and drug evaluation: Suspend 2.5×10 5 AGS gastric cancer cells (ATCC CRL-1739 cells) in 100 μL of PBS and inoculate them on the outer side of the left lower limb thigh of nude mice (C57BL / 6j); After about 1 week, when the transplanted tumor reaches about 100 mm 3 in size, randomly divide the mice into groups of 10 each, namely the model group, cordycepin control group, and functional salt drug treatment groups (functional salts 1, 2, 3, 5, 6, 8, 11, 15); The model group was intragastrically administered nutritional agents such as corn steep liquor and DMSO as a solvent control every other day, and the cordycepin control group and the functional salt drug treatment groups were intragastrically administered drugs (500 μg / time / animal) every other day for 14 consecutive days; Observe the changes in the size of gastric cancer tumors in each group of mice. After the experiment, sacrifice the mice, collect blood and corresponding tissue specimens, immediately take pictures and weigh the tumor mass, measure the three perpendicular diameters (length, width, and height) of the transplanted tumor with a caliper every two days, calculate the size of the transplanted tumor, and calculate the volume according to the formula: Tumor volume (mm 3 ) = 0.5236×A×B×C, where A, B, and C respectively represent the three diameters of the tumor (cm). The experimental results of the changes in the volume of gastric cancer tumors during the mouse drug evaluation process are shown in Figure 13 . The experimental results show that some functional salt drug groups can significantly inhibit the growth of mouse tumors, and the effects are better than those of the cordycepin control group. Among them, functional salts 11 and 15 have excellent anti-gastric cancer tumor effects (from 1040 mm 3 reduced to <100 mm 3 ) (P<0.001), indicating that in addition to having the effects of killing gastric cancer tumor cells and activating immunity, fructose-1,6-diphosphate plays an important auxiliary role in the treatment process of cordycepin.

[0098] Application Example 3: Anti-tumor proliferation experimental effect of cordycepin or its functional salts on small cell lung cancer

[0099] The MTT assay was used to analyze the anti-tumor dose-effect relationship curves of cordycepin and its functional salts 1-20 in small cell lung cancer cell lines H1048, H446, and H69, and the results of calculating the half-maximal inhibitory concentration (IC 50 ) are shown in Table 2 below. The in vitro anti-tumor experimental results show that compared with cordycepin, the effective concentrations of most cordycepin functional salts on tumor cells are reduced. Compared with cordycepin, functional salts 6, 7, 11, 14, 15, and 17-20 have strong in vitro killing effects on the three small cell lung cancer cells.

[0100] Table 2: IC of the compound in three small cell lung cancer cells 50 (μM)

[0101]

[0102] Application Example 4: Anti-tumor proliferation experimental effect of cordycepin or its functional salt on colon cancer

[0103] Establishment of mouse transplanted colon cancer model and drug evaluation: Suspend 2.5×10 6 MC-38 colon cancer cells in 100 μL of PBS and inoculate them on the outer side of the left lower thigh of nude mice (C57BL / 6j); after 1 week, when the transplanted tumor reaches about 100 mm 3 in size, randomly divide the mice into groups, with 10 mice in each group, namely the model group, the cordycepin control group and the functional salt drug treatment group (functional salts 2, 11, 15); among them, the model group is given DMSO by gavage every 3 days as a solvent control, and the cordycepin control group and the functional salt drug treatment group are given drugs by gavage (500 μg / time / animal) every 3 days respectively, and observe continuously for 21 days. After the experiment, the mice are sacrificed, blood and corresponding tissue specimens are collected, the tumor mass is immediately photographed and weighed, and the three perpendicular diameters (length, width and height) of the transplanted tumor are measured with a caliper every two days to calculate the size of the transplanted tumor, and the volume is calculated according to the formula: Tumor volume (mm 3 ) = 0.5236×A×B×C, where A, B and C respectively represent the three diameters of the tumor (cm), observe the changes in the size of colon cancer tumors in each group of mice, and the results are as Figure 14 shown. After 21 days, the experimental results show that cordycepin has the effect of anti-colon cancer proliferation (the size decreases from 2150 mm 3 to 640 mm 3 ), and functional salts 11 and 15 have a significantly more effective anti-tumor growth effect than cordycepin (the size decreases from 2150 mm 3 to <300 mm 3 ).

[0104] Application Example 5: Inhibitory experimental effect of cordycepin or its functional salt on ovarian cancer

[0105] Establishment of mouse transplanted ovarian cancer model and drug evaluation: Suspend 2.5×10 6 ID8 ovarian cancer cells in 100 μL of PBS and inoculate them on the outer side of the left lower thigh of C57BL / 6j nude mice. After about one week, the transplanted tumor reaches about 100 mm 3When determining the size, the mice were randomly divided into groups of 10 each, namely the model group, cordycepin group, functional salt 11 group, PD-1 + PD-L1 antibody treatment group, and functional salt 11 drug + PD-1 + PD-L1 antibody treatment group. Model control group: Immunoglobulin G (IgG) (500 μg / time / animal) and PBS were administered by gavage every day; Cordycepin group: Cordycepin (500 μg / time / animal) was administered by gavage every day and prepared with PBS; Functional salt 11 drug group: Functional salt 11 drug (500 μg / time / animal) was administered by gavage every day and prepared with PBS; PD-1 + PD-L1 antibody treatment group: Antibodies PD-1 + PD-L1 (500 μg / time / animal respectively) were administered by gavage every 3 days; Functional salt 11 drug + PD-1 + PD-L1 antibody treatment group: Immunoglobulin and functional salt 11 drug (500 μg / time / animal) were administered by gavage every day, and antibodies PD-1 + PD-L1 (500 μg / time / animal respectively) were administered by gavage every 3 days; The above-mentioned administration methods were carried out continuously for 21 days. The changes in the size of ovarian tumors in each group of mice were observed every two days. After the experiment, the mice were sacrificed, blood and corresponding tissue specimens were collected, the tumor masses were immediately photographed and weighed, and some tumor tissues were fixed in formalin solution for further detection. The three perpendicular diameters (length, width, and height) of the transplanted tumors were measured every two days with a caliper, the size of the transplanted tumors was calculated, and the volume was calculated according to the formula: Tumor volume (mm 3 ) = 0.5236 × A × B × C, where A, B, and C represent the three diameters (cm) of the tumor respectively. The experimental results are shown in Figure 15 . The experimental results showed that after 21 days, cordycepin had the effect of inhibiting the proliferation of ovarian cancer (the size decreased from 2850 mm 3 to 950 mm 3 ), functional salt 11 alone had a more significant anti-tumor growth effect than cordycepin (the size decreased from 2850 mm 3 to <550 mm 3 ), the immune checkpoint inhibitors PD-1 and PD-L1 alone also had a significant anti-tumor growth effect (the size decreased from 2850 mm 3 to <1500 mm 3 ), and when functional salt 11 and the immune checkpoint inhibitors PD-1 and PD-L1 were used simultaneously, the anti-tumor effect could be greatly improved (the size decreased from 2850 mm 3 to <500 mm 3 ).

[0106] Application Example 6: Inhibitory experimental effect of cordycepin or its functional salt on human glioma cells

[0107] The inhibitory activity of cordycepin and its functional salts 1 - 20 on the proliferation of transplanted human glioma cells in rats was determined by the MTT method: By calculating the half-maximal inhibitory concentration (IC50 ) The results are summarized in Table 3 below. The in vitro anti-tumor experimental results show that compared with cordycepin, the effective concentration of most cordycepin functional salts on tumor cells decreases, and among them, functional salts 13 and 20 have strong in vitro killing effects on four human glioma cells.

[0108] Table 3: IC of cordycepin and its functional salts in four human glioma cells 50 (μM)

[0109]

[0110] Application Example 7: Inhibitory effects of cordycepin and its functional salts on small cell lung cancer in zebrafish

[0111] Zebrafish xenograft models of small cell lung cancer with three types of H69, H446, and H1048 cells were established respectively: 1×10 6 mL of H69, H446, and H1048 cell suspensions with a concentration were placed in serum-free cell culture medium. 5 mL of red fluorescent dye (CM-DiI) cell labeling solution was added to each milliliter of cell suspension, gently mixed, incubated at 37 °C for 20 min, centrifuged at 1500 rpm for 5 min, and then the supernatant was removed. Serum-free medium was added again for resuspension, and then the above steps were repeated twice to obtain the required labeled cell suspension. The cell suspension was transplanted into the zebrafish yolk sac by microinjection, with about 100 cells transplanted into each fish, to establish a zebrafish-human small cell lung cancer sensitive strain xenograft model. The zebrafish injected with ovarian cancer cells were placed in an incubator at 35 °C and cultured until 3 dpf. To test the proliferative effects of cordycepin and the prepared functional salts on anti-small cell lung cancer in zebrafish: When the tumor-bearing zebrafish model reached 3 dpf, zebrafish with relatively consistent xenografts were selected under a microscope and randomly assigned to 6-well plates, with 30 fish in each well. At 3 dpf, the above compounds were diluted according to the MTD dose, dissolved in DMSO and added to each well. At the same time, a normal control group, a model control group, and a solvent control group (DMSO) were set up, and the volume of fish water in each well was 3 mL. After the zebrafish in each experimental group were continuously cultured at 35 °C until 5 dpf, 10 zebrafish were randomly selected from each experimental group and observed, photographed, and the pictures were saved under a fluorescence microscope. The Nikon NIS-Elements l3.10 advanced image processing software was used for image analysis to calculate the fluorescence intensity (S) of zebrafish xenografts. The inhibition results are shown in Table 3 below. The growth inhibitory effects of Xiaoaiping injection and paclitaxel alone on the zebrafish-human ovarian cancer sensitive strain were calculated based on the overall fluorescence intensity, and the formula is as follows: Tumor growth inhibition (%) = [S(model control group) - S(drug group)] / S(model control group) × 100%.

[0112] Table 4: Effects of each compound on the proliferation of three cell lines in the zebrafish model

[0113]

[0114] Application Example 8: Experimental effect of cordycepin or its functional salt on anti-tumor proliferation of thyroid cancer cells

[0115] Establishment of mouse transplanted thyroid cancer cell model and drug evaluation: 2×10 7 TPC-1 thyroid cancer cells were inoculated subcutaneously on the back of BALB / c nude mice. Five days after the injection, an obvious mass was observed, which was considered a successful model. The mice were randomly divided into 10 groups, namely the model group, the Cordyceps group, the functional salt 12 drug group, the PD-1+PD-L1 antibody treatment group, and the functional salt 12 drug+PD-1+PD-L1 antibody treatment group. Cordyceps group: 1 ml of cordycepin (500 μg / time / mouse) was intraperitoneally injected every day and prepared with PBS; functional salt 12 drug group: 1 ml of functional salt 12 drug (500 μg / time / mouse) was intraperitoneally injected every day and prepared with PBS; PD-1+PD-L1 antibody treatment group: antibody PD-1+PD-L1 (500 μg / time / mouse) was intraperitoneally injected every 3 days; functional salt 12 drug + PD-1+PD-L1 antibody treatment group: immunoglobulin and functional salt 12 drug (500 μg / time / mouse) were intraperitoneally injected every day, and antibody PD-1+PD-L1 (500 μg / time / mouse, respectively) was gavage every 3 days; the above administration method was continued for 21 days. The size changes of thyroid cancer in each group of mice were observed every day. After the experiment, the mice were killed, and blood and corresponding tissue specimens were collected. The tumors were immediately photographed and weighed. Part of the tumor tissue was fixed in formalin solution for further testing. The three vertical diameters (length, width and height) of the transplanted tumor were measured with a caliper every two days to calculate the size of the transplanted tumor, and the volume was calculated according to the formula: tumor volume (mm 3 )=0.5236×A×B×C, where A, B and C represent the three diameters of the tumor (cm). The experimental results are shown in Figure 16 The experimental results showed that cordycepin had an anti-thyroid cancer proliferation effect after 21 days (size 2504mm 3 Down to 1025mm 3 ), functional salt 12 used alone has a more significant anti-tumor effect than cordycepin (size 2504mm 3 Down to 928mm 3 ), immune checkpoint inhibitors PD-1 and PD-L1, when used alone, also have significant anti-tumor growth effects (size 2504mm 3 Down to 1520mm 3) When the functional salt 12 and the immune checkpoint inhibitors PD-1 and PD-L1 are used simultaneously respectively, the anti-tumor effect can be greatly improved (the size is reduced from 2504 mm 3 to 602 mm 3 ).

[0116] The above embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims

1. A cordycepin salt, characterized in that: The salt is a nucleotide salt; the nucleotide salt is selected from uridylate, cyclophosphoadenylate, cytidylate, adenylate, guanylate, inosinate, or xanthate.

2. The method for preparing the cordycepin salt according to claim 1, characterized in that: The method comprises dissolving nucleotides and cordycepin in a solvent, filtering, and obtaining the cordycepin salt; or, The method comprises dissolving nucleotides and cordycepin in a solvent, crystallizing, and filtering to obtain the cordycepin salt.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the nucleotide to cordycepin is 0.1 to 10:

1.

4. The preparation method according to claim 2, characterized in that: The molar ratio of the nucleotide to cordycepin is 1-2:0.6-3.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the nucleotide to cordycepin is 0.1-2:

1.

6. The preparation method according to claim 2, characterized in that: The molar ratio of the nucleotide to cordycepin is 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, or 9.5:

1.

7. A pharmaceutical composition, characterized in that The invention comprises a) the cordycepin salt according to claim 1, and b) other active substances and / or pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that: The other active substance is an immune checkpoint inhibitor.

9. The pharmaceutical composition according to claim 8, characterized in that: The immune checkpoint inhibitor is PD-1 and / or PD-L1.

10. The pharmaceutical composition according to claim 7, characterized in that: The mass ratio of the cordycepin salt to the other active substances is 2-4:

1.

11. The pharmaceutical composition according to claim 7, characterized in that: The mass ratio of the cordycepin salt to the other active substances is 3:

1.

12. The pharmaceutical composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is selected from tablets, pills, capsules, dripping pills, syrups, disintegrants, injections, sustained-release preparations, or kits.

13. Use of the cordycepin salt according to claim 1 or the pharmaceutical composition according to any one of claims 7 to 12 in the preparation of a product for preventing and treating mammalian tumors.

14. The use according to claim 13, characterized in that: The tumor includes liver cancer, gastric cancer, small cell lung cancer, colon cancer, ovarian cancer, thyroid cancer, human glioma, or a combination of the foregoing.

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