Cordycepin salt as well as preparation method and application thereof

Abstract

The invention discloses cordycepin salt as well as a preparation method and application thereof. The cordycepin salt comprises cordycepin nucleotide salt, cordycepin natural organic acid salt, cordycepin amino acid salt, cordycepin vitamin salt and cordycepin inorganic acid salt. The functional salt of cordycepin provided by the invention has a good effect on preventing and treating related diseases caused by cell functional damage variation in mammals. For parent drugs, the functional salt of cordycepin has better solubility, the in-vivo bioavailability can be increased, and the in-vivo drug effect of the drug is better.

Description

Technical Field

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

[0002] This section merely provides background information related to the present disclosure and is not necessarily prior art.

[0003] Cordycepin (3'-deoxyadenosine) is an effective natural ingredient isolated from Cordyceps sinensis. It is an adenosine analog. Many studies have shown that it has the potential to treat or assist in the treatment of various diseases, mainly including inflammatory diseases and cancer. After entering the cell, cordycepin is phosphorylated in the body into monophosphate derivatives, diphosphate derivatives or triphosphate derivatives. Among them, the triphosphate derivative can inhibit the synthesis of mRNA and ultimately affect the synthesis of the corresponding protein to inhibit tumor growth. In addition, the physiological effects of cordycepin in the body's metabolic process are mainly to cause cell death, prevent cell growth, inhibit the expression of multiple inflammatory genes, reduce cell migration, etc. Clinical studies have shown that cordycepin can prevent the occurrence of inflammation and reduce tumor occurrence. The molecular mechanism of the biological effects of cordycepin includes regulating the expression of genes related to inflammation and tumor occurrence, 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, and cordycepin is one of the main ways to prepare nucleoside anti-tumor drugs. However, the nucleoside compounds prepared by cordycepin have poor solubility, which affects the subsequent dosage form preparation and human absorption.

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

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

[0007] Therefore, the present invention provides cordycepin salt and its preparation method and application to further improve its anti-tumor efficacy. Summary of the invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to provide cordycepin salt and its preparation method and application in view of the deficiencies in the prior art.

[0009] In order to solve the above technical problems, the present invention discloses the following technical solutions:

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

[0011] In some embodiments, the salt is a pharmaceutically acceptable functional salt, including nucleotide salts, natural product organic acid salts, food organic acid 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 nucleotide salt is selected from uridylate, cytidylate, adenylate, cyclophosphate adenylate, guanylate, inosinate, or xanthate.

[0013] In some embodiments, the natural product organic acid salt is selected from chlorogenic acid, caffeic acid, sialic acid, hibiscus acetate, glycyrrhizic acid, glycyrrhizic acid, oleanolic acid, ursolic acid, shikimic acid, salvianolic acid, quinic acid, deoxycholate, betulinic acid, gambogic acid, or rhein.

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

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

[0016] In some embodiments, the vitamin salt is 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 salt is selected from hydrochloride, phosphate, phosphite, or borate; the amino acid salt is 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, comprising dissolving cordycepin and any one or more of inorganic acids, amino acids, vitamins, nucleotides, food organic acids and natural product organic acids in a solvent, filtering or directly filtering after crystallization to obtain the cordycepin salt.

[0019] In some embodiments, the molar ratio of any one of the nucleotides, natural product organic acids, food organic acids, amino acids, vitamins and inorganic acids to cordycepin is 0.1 to 10:1, preferably 1-2:0.6-3, 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 comprises: in a first solvent, stirring and dissolving cordycepin and any one or more of inorganic acid, amino acid, vitamin, nucleotide, food organic acid, and natural product organic acid to obtain a solution, cooling to room temperature, adding a second solvent for crystallization, that is, stopping stirring and filtering after the solution becomes a suspension, and drying the filter residue to obtain the product.

[0023] In some embodiments, the molar ratio of the cordycepin to any one or more of the inorganic acid, amino acid, vitamin, nucleotide, food organic acid, and natural product organic acid is 1-2:1.

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

[0025] In some embodiments, the ratio of cordycepin to 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 a reverse solvent, specifically alcohol; in some embodiments, the alcohol is any one or a combination 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 comprises: dissolving excess cordycepin and any one or more of inorganic acid, amino acid, vitamin, nucleotide, food organic acid and natural product organic acid in water, filtering to remove excess cordycepin, and freeze-drying the filtrate to obtain the product.

[0031] In some embodiments, the molar ratio of cordycepin to any one or more of the inorganic acid, amino acid, vitamin, nucleotide, food organic acid, and natural product organic acid is 1-2:0.6-3.

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

[0033] Furthermore, 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 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, pellets, syrups, disintegrants, injections, sustained-release preparations, or kits.

[0038] In a fourth aspect, the present invention provides the use of the salt of the above cordycepin or the above pharmaceutical composition in the preparation of a product for preventing and treating diseases related to mutation of cellular functional damage in mammals.

[0039] In some embodiments, the disease associated with the cellular functional damage and mutation is a tumor.

[0040] In some embodiments, the mammal comprises a human.

[0041] In some embodiments, the tumor comprises liver cancer, gastric cancer, small cell lung cancer, colon cancer, ovarian cancer, thyroid cancer, human glioma, or a combination thereof.

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

[0043] The present invention combines cordycepin with acid to form a salt, which can not only increase the solubility, improve the absorption and efficacy of the drug, but also promote the anti-tumor effect of cordycepin as a strengthening or auxiliary treatment through the metabolic effect in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0045] Figure 1 This is the NMR spectrum of the functional salt formed by cordycepin and hydrochloric acid.

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

[0047] Figure 3 This is the NMR spectrum of the functional salt formed by cordycepin and niacin.

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

[0049] Figure 5 This is the NMR spectrum of the functional salt formed by cordycepin and folic acid.

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

[0051] Figure 7 This is the NMR spectrum of the functional salt formed by cordycepin and n-butyric acid.

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

[0053] Fig. 9 This is the NMR spectrum of the functional salt formed by cordycepin and malic acid.

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

[0055] Fig.11 This is the NMR spectrum of the functional salt formed by cordycepin and shikimic acid.

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

[0057] Fig.13 The present invention is an experimental effect of cordycepin or its functional salt on anti-tumor proliferation of small cell lung cancer.

[0058] Fig.14 The present invention is an experimental study on the anti-tumor proliferation effect of cordycepin or its functional salt on colon cancer.

[0059] Fig.15 The present invention relates to the experimental effect of cordycepin or its functional salt on the inhibition of ovarian cancer.

[0060] Fig.16 This is the experimental effect of cordycepin or its functional salt on the inhibition of thyroid cancer.

[0061] Fig.17 This is the TG-DSC spectrum of cordycepin. DETAILED DESCRIPTION

[0062] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0063] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0064] The anti-cancer related cell and animal model experiments of cordycepin or its functional salts in the following examples are not all effective experimental results, but are only related experiments listed to illustrate the effects, and the drug evaluation experiments including cell model and animal model experiments follow ethical rules.

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

[0066] Example 1: Preparation of cordycepin inorganic acid salt

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

[0068] The cordycepin hydrochloride functional salt prepared in Example 1 1 H NMR images Figure 1 The obtained functional salt 1 The HNMR spectrum is the sum of the characteristic peaks of cordycepin and hydrochloric acid, which indicates that these two components exist in the new phase. 1 The H NMR chemical distribution is as follows: 1H NMR(400MHz, Deuterium Oxide)δ8.37(s,1H),8.27(s,1H),6.00(d,J=1.8Hz,1H),4.72(d,J=5.7Hz,13H),4.54(dtd,J=11.5,6.3,5.4,2.8Hz,1H),3.84 (dd,J=12.7,2.8Hz,1H),3.64(dd,J=12.7,4.7Hz,1H),2.20(ddd,J=13.9,9.6,5.6Hz,1H),2.08(ddd,J=13.9,6.3,2.6Hz,1H).

[0069] The TG-DSC curve of cordycepin hydrochloride functional salt prepared in Example 1 is as follows: Figure 2 As shown. The sample lost about 4.6% of its weight in the temperature range of 24-43°C, and it is speculated that it contains solvent. According to the DSC curve analysis, cordycepin hydrochloride has endothermic peaks at 123°C and 163°C. The sample began 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 prepare 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 glutamate 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 prepare 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, and 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. Stirring was stopped when the solution became a suspension, and the obtained suspension was filtered. The filter residue was dried in vacuo at 50°C to obtain a nicotinic acid salt as a functional salt 4 (such as Figure 3 , Figure 4 ) and folate as functional salts 5 (such as Figure 5 , Figure 6 ).

[0074] The functional salt of cordycepin nicotinic acid prepared in Example 3 1 H NMR images Figure 3 The obtained functional salt 1The HNMR spectrum is the sum of the characteristic peaks of cordycepin and nicotinic acid, which indicates that these two components exist in the new phase. 1 The H NMR chemical distribution is as follows: 1 H NMR(400MHz, Deuterium Oxide)δ8.98(d,J=1.9Hz,2H),8.78–8.59(m,5H),8.22(s,1H),8.10(s,1H),7.90(dd,J=8.0,5.7Hz,2H),5.93(d,J=2.2 Hz,1H),4.70(s,26H),4.59–4.47(m,0H),3.83(dd,J=12.7,2.8Hz,1H),3.62(dd,J=12.7,4.5Hz,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 follows: Figure 4 As shown. The sample lost about 2.5% of its weight in the temperature range of 37-63.0℃, and it is speculated that it contains solvent. According to the DSC curve analysis, cordycepin nicotinate has endothermic peaks at 107℃ and 174℃ respectively. The sample began to decompose at 150℃.

[0076] The cordycepin folic acid functional salt prepared in Example 3 1 H NMR images Figure 5 The obtained functional salt 1 The HNMR spectrum is the sum of the characteristic peaks of cordycepin and folic acid, which indicates that these two components exist in the new phase. 1 The H NMR chemical distribution is as follows: 1 H NMR(400MHz, Deuterium Oxide)δ8.18(s,1H),8.05(s,1H),6.02–5.89(m,1H),4.53(s,1H),3.84(d,J=11.0Hz,1H),3.63(dd,J=11.5,3.6Hz,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 follows: Figure 6 As shown. The sample lost about 6.2% of its weight in the temperature range of 40-89℃, and it is speculated that it contains solvent. According to the DSC curve analysis, cordycepin folate has endothermic peaks at 115℃ and 172℃ respectively. The sample began to decompose at 206℃.

[0078] Example 4: Preparation of cordycepin nucleotide salt

[0079] 2.51 g (10 mmol) of cordycepin and 30 mL of water were added to a flask to prepare a suspension, and 3.24 g (10 mmol) of uridylic acid and 3.29 g (10 mmol) of cyclophosphate adenylic acid were added to the suspension respectively. The reaction was stirred at 50°C for 2 h to completely dissolve the cordycepin in the water. After the reaction was cooled to room temperature, 90 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 uridylic acid salt as functional salt 6 and cyclophosphate adenylic acid salt as functional salt 7.

[0080] Example 5: Preparation of cordycepin food organic acid salt (fatty acid salt)

[0081] 7.53 g (30 mmol) of cordycepin and 20 mL of water were added to a flask to prepare a suspension, 1.20 g (20 mmol) of acetic acid and 1.76 g (20 mmol) of butyric acid were added to the suspension, and the reaction was stirred at room temperature for 2 h to dissolve most of the cordycepin in a water bath at room temperature. The remaining suspension was filtered, and the filtrate was freeze-dried to obtain acetate as functional salt 8 and butyrate as functional salt 9 (such as Figure 7 , Figure 8 ).

[0082] The cordycepin butyrate functional salt prepared in Example 5 1 H NMR images Figure 7 The obtained functional salt 1 The HNMR spectrum is the sum of the characteristic peaks of cordycepin and butyric acid, which indicates that these two components exist in the new phase. 1 H NMR chemical distribution is 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 Example 5 is as follows: 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. Through DSC curve analysis, it can be seen that cordycepin butyrate has multiple endothermic peaks, at 76°C, 134°C, 205°C and 221°C, and the sample begins to decompose at 100°C. During the whole process, the sample first loses solvent and then decomposes, and after decomposition to a certain extent, melting occurs; this shows that in the DSC curve, a series of endothermic peaks are caused by decomposition and melting.

[0084] Example 6: Preparation of organic acid salt of cordycepin food

[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 added to the suspension respectively, and the reaction was stirred at 60°C for 2 h to completely dissolve the cordycepin in the 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, and the obtained suspension was filtered. The residue was dried in vacuo at 60°C to obtain a malate as a functional salt 10 (such as Fig. 9 , Fig.10 ) and fructose-1,6-diphosphate as functional salts11.

[0086] The cordycepin malic acid functional salt prepared in Example 6 1 H NMR images Fig. 9 The obtained functional salt 1 The H NMR spectrum is the sum of the characteristic peaks of cordycepin and malic acid, which indicates that these two components exist in the new phase. 1 The H NMR chemical distribution is as follows: 1 H NMR(400MHz, Deuterium Oxide)δ8.31(d,J=2.3Hz,2H),8.22–8.16(m,2H),5.96(d,J=2.0Hz,2H),4.38(ddd,J=7.2,4.4,1.9Hz,3H),3.82(dd,J=12.6,2 .8Hz,2H),3.62(dd,J=12.6,4.7Hz,2H),2.82–2.75(m,1H),2.75(dd,J=4.5,1.0Hz,2H),2.70–2.59(m,3H),2.11–2.01(m,1H).

[0087] The TG-DSC curve of the cordycepin malic acid functional salt prepared in Example 6 is as follows: Fig.10As shown. There is no solvent in the sample. According to the DSC curve analysis, cordycepin malate has endothermic peaks at 140℃ and 178℃. The sample begins to decompose at 177℃.

[0088] Example 7: Preparation of organic acid salt of cordycepin natural product

[0089] 15.06 g (60 mmol) of cordycepin and 100 mL of water were added to a flask to prepare a suspension, and 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 hibiscus acetic acid, 8.23 ​​g (10 mmol) of glycyrrhizic acid, 4.57 g (10 mmol) of oleanolic acid, 1.74 g (10 mmol) of shikimic acid, and 1.6 g (10 mmol) of rhein were added to the suspension. 2.84 g (10 mmol) and 6.29 g (10 mmol) of gambogic acid, all the reactions were stirred at room temperature for 1 h to dissolve most of the cordycepin in a water bath at room temperature, the remaining suspension was filtered, and the filtrate was freeze-dried to obtain chlorogenic acid salt as functional salt 12, caffeic acid salt as functional salt 13, sialic acid salt as functional salt 14, hibiscus acetate as functional salt 15, glycyrrhizic acid salt as functional salt 16, oleanolic acid salt as functional salt 17, shikimate as functional salt 18 (such as Fig.11 , Fig.12 ), rhein salt is functional salt 19, and gamboge salt is functional salt 20.

[0090] The functional salt of cordycepin shikimic acid prepared in Example 7 1 H NMR images Fig.11 The obtained functional salt 1 The H NMR spectrum is the sum of the characteristic peaks of cordycepin and shikimic acid, which indicates that these two components exist in the new phase. 1 The H NMR chemical distribution is as follows: 1H NMR (400 MHz, Deuterium Oxide)δ8.24(s,2H),8.10(s,2H),6.62(dd,J=4.1,1.9Hz,3H),5.93(d,J=2.0Hz ,2H),4.51(dddd,J=9.3,6.7,4.4,2.8Hz,2H),4.33(t,J=4.3Hz,3H),3.91(ddd,J =8.4,6.8,5.2Hz,3H),3.84(d,J=2.8Hz,1H),3.68–3.61(m,3H),3.65–3.58(m,2 H),2.63(ddt,J=18.0,5.3,1.6Hz,3H),2.11(d,J=6.9Hz,1H),2.09–2.04(m,2H).

[0091] The TG-DSC curve of the cordycepin shikimic acid functional salt prepared in Example 7 is as follows: Fig.12 As shown. There is no solvent in the sample. According to the DSC curve analysis, cordycepin shikimate has endothermic peaks at 170℃ and 179℃ respectively. The sample begins to decompose at 160℃.

[0092] Application Example 1: Experimental effect of functional salt of cordycepin on anti-tumor proliferation of liver cancer

[0093] The MTT method was used to determine the proliferation inhibitory activity of the functional salt of cordycepin on liver cancer cells: Hepa 1-6 and Hep G2 liver cancer cells in good exponential growth phase were taken, and 0.25% trypsin digestion solution was added to make the adherent cells fall off, and about 2×10 4 / mL, make liver cancer cell suspension. Take the cell suspension and inoculate it on a 96-well plate, 90μL / well, and culture it in a constant temperature CO2 incubator for 24h. Add cordycepin, or its functional salt 10μL / well and culture it for 72h. Add MTT reagent 10μL / well to the 96-well plate, react in the incubator for 4h, aspirate the supernatant, add 100μL / well dimethyl sulfoxide, dissolve it, and use an enzyme-linked immunosorbent assay to measure the absorbance of each well at a wavelength of 570nm, and calculate the cell inhibition rate to obtain the IC of the corresponding compound. 50 By calculating the half inhibitory concentration (IC 50 ) are summarized in Table 1. The results of in vitro anti-tumor experiments show that compared with cordycepin, the effective concentration of most cordycepin functional salts on tumor cells is greatly reduced.

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

[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: Experimental effect of cordycepin or its functional salt on anti-tumor proliferation of gastric cancer

[0097] Establishment of mouse gastric cancer transplantation model and drug evaluation: 2.5×10 5 AGS gastric cancer cells (ATCC CRL-1739 cells) were inoculated on the outer side of the left thigh of nude mice (C57BL / 6j); about 1 week later, the transplanted tumor reached about 100 mm 3 The mice were randomly divided into model group, cordycepin control group and functional salt drug treatment group (functional salt 1, 2, 3, 5, 6, 8, 11, 15) with 10 mice in each group. The model group was gavaged with corn syrup and other nutrients and DMSO as solvent control every other day, and the cordycepin control group and functional salt drug treatment group were gavaged with drugs (500 μg / time / mouse) every other day for 14 consecutive days. The changes in the size of gastric cancer tumors in mice in each group were observed. After the experiment, the mice were killed, blood and corresponding tissue specimens were collected, and the tumors were immediately photographed and weighed. 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), respectively. The experimental results of gastric cancer tumor volume changes during mouse drug evaluation are shown in Fig.13 The experimental results showed that some functional salt drug groups could significantly inhibit the growth of mouse tumors, and the effects were better than those of the cordycepin control group. Among them, functional salt 11 and functional salt 15 had excellent anti-gastric cancer effects (1040 mm 3 Reduce to <100mm 3 )(P<0.001), indicating that in addition to the efficacy of killing gastric cancer cells and activating immunity, fructose-1,6-diphosphate plays an important auxiliary role in the treatment of cordycepin.

[0098] Application Example 3: Experimental effect of cordycepin or its functional salt on anti-tumor proliferation of small cell lung cancer

[0099] The MTT assay was used to analyze the antitumor dose-effect curves of cordycepin and its functional salts 1-20 in small cell lung cancer cell lines H1048, H446 and H69, and the half inhibitory concentration (IC 50 ) are shown in Table 2. The results of in vitro antitumor experiments showed that compared with cordycepin, the effective concentrations of most cordycepin functional salts on tumor cells were lower, and compared with cordycepin, functional salts 6, 7, 11, 14, 15 and 17-20 had stronger in vitro killing effects on three small cell lung cancer cells.

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

[0101]

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

[0103] Establishment of mouse transplanted colon cancer model and drug evaluation: 2.5×10 6 MC-38 colon cancer cells were inoculated into the outer side of the left thigh of nude mice (C57BL / 6j); one week later, the transplanted tumor reached about 100 mm 3 The mice were randomly divided into groups, each with 10 mice, including the model group, cordycepin control group and functional salt drug treatment group (functional salt 2, 11, 15); the model group was gavaged with DMSO as a solvent control every 3 days, and the cordycepin control group and functional salt drug treatment group were gavaged with DMSO every 3 days (500μg / time / mouse), respectively, and observed for 21 consecutive days. After the experiment, the mice were killed, blood and corresponding tissue specimens were collected, the tumor was immediately photographed and weighed, and 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), respectively. The changes in the size of colon cancer tumors in each group of mice were observed. The results are as follows Fig.14 After 21 days, the experimental results showed that cordycepin had an anti-colon cancer proliferation effect (size 2150mm 3 Down to 640mm 3 ), functional salt 11 and functional salt 15 have a more significant anti-tumor effect than cordycepin (size 2150mm 3 Down to <300mm 3 ).

[0104] Application Example 5: Experimental Effect of Cordycepin or Its Functional Salt on Inhibition of Ovarian Cancer

[0105] Establishment of mouse transplanted ovarian cancer model and drug evaluation: 2.5×10 6 ID8 ovarian cancer cells were inoculated into the outer side of the left thigh of C57BL / 6j nude mice. After about one week, the transplanted tumor reached about 100 mm. 3The mice were randomly divided into groups of 10 each, namely, the model group, the cordycepin group, the functional salt 11 group, the PD-1 + PD-L1 antibody treatment group, and the functional salt 11 drug + PD-1 + PD-L1 antibody treatment group. Model control group: daily gavage of immunoglobulin G (IgG) (500 μg / time / mouse) and PBS; cordycepin group: daily gavage of cordycepin (500 μg / time / mouse) and prepared with PBS; functional salt 11 drug group: daily gavage of functional salt 11 drug (500 μg / time / mouse) and prepared with PBS; PD-1+PD-L1 antibody treatment group: daily gavage of antibody PD-1+PD-L1 (500 μg / time / mouse, respectively) every 3 days; functional salt 11 drug + PD-1+PD-L1 antibody treatment group: daily gavage of immunoglobulin and functional salt 11 drug (500 μg / time / mouse), and daily gavage of antibody PD-1+PD-L1 (500 μg / time / mouse, respectively); the above-mentioned administration method was continued for 21 days. The size changes of ovarian tumors in each group of mice were observed every two days. 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 Fig.15 The experimental results showed that cordycepin had an anti-proliferation effect on ovarian cancer after 21 days (size 2850mm 3 Down to 950mm 3 ), functional salt 11 used alone has a more significant anti-tumor effect than cordycepin (size 2850mm 3 Down to <550mm 3 ), immune checkpoint inhibitors PD-1 and PD-L1, when used alone, also have significant anti-tumor growth effects (size 2850mm 3 Reduced to <1500mm 3 ), functional salt 11 and immune checkpoint inhibitors PD-1 and PD-L1 can significantly enhance the anti-tumor effect when used simultaneously (size 2850mm 3 Reduced to <500mm 3 ).

[0106] Application Example 6: Experimental effect of cordycepin or its functional salt on the inhibition of human glioma cells

[0107] The inhibitory activity of cordycepin and its functional salt 1-20 on the proliferation of transplanted human glial tumor cells in rats was evaluated by MTT method: the half inhibitory concentration (IC50 ) are summarized in Table 3. The results of in vitro antitumor experiments showed that compared with cordycepin, the effective concentrations of most cordycepin functional salts on tumor cells were lower, among which functional salts 13 and 20 had strong in vitro killing effects on four types of human glial tumor cells.

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

[0109]

[0110] Application Example 7: Inhibitory Effect of Cordycepin and Its Functional Salts on Small Cell Lung Cancer in Zebrafish

[0111] Three zebrafish small cell lung cancer xenograft models were established: 1×10 6 mL concentration of H69, H446, H1048 cell suspension was 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 minutes, centrifuged at 1500rpm for 5 minutes, and then the supernatant was removed and resuspended in serum-free culture medium. The above steps were repeated twice to obtain the required labeled cell suspension, which was transplanted into the zebrafish yolk sac by microinjection, with about 100 cells transplanted per tail to establish a zebrafish-human small cell lung cancer sensitive strain transplant tumor model, and the zebrafish injected with ovarian cancer cells were placed at 35°C and cultured to 3dpf. Test the effect of cordycepin and prepared functional salt on the proliferation of anti-small cell lung cancer in zebrafish: When the tumor zebrafish model was inhibited to 3dpf, zebrafish with good transplant tumor consistency were selected under a microscope and randomly allocated to 6-well plates, with 30 tails per well. At 3 dpf, the above compounds were diluted according to the MTD dose, dissolved in DMSO and added to each well. A normal control group, a model control group and a solvent control group (DMSO) were set up at the same time. The fish water capacity of each well was 3 mL. After the zebrafish in each experimental group were continued to be cultured at 35°C until 5 dpf, 10 zebrafish were randomly selected from each experimental group for observation under a fluorescence microscope, photographed and saved. The image was analyzed using Nikon NIS-Elements l3.10 advanced image processing software, and the fluorescence intensity (S) of the zebrafish transplanted tumor was calculated. The inhibition results are shown in Table 3 below. The growth inhibition effect of Xiaoaiping injection and paclitaxel alone on zebrafish-sensitive human ovarian cancer strains was calculated using 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 Fig.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 functional salt 12 and immune checkpoint inhibitors PD-1 and PD-L1 are used simultaneously, the anti-tumor effect can be greatly improved (size 2504mm 3 Down to 602mm 3 ).

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A cordycepin salt, characterized in that: The salt is a nucleotide salt.

2. The cordycepin salt according to claim 1, characterized in that The nucleotide salt is selected from uridylate, cyclophosphate adenylate, cytidylate, adenylate, guanylate, inosinate, or xanthate.

3. A cordycepin salt, characterized in that: The salt is a natural product organic acid salt, and the natural product organic acid salt is selected from chlorogenic acid salt, caffeic acid salt, sialic acid salt, hibiscus acetate salt, glycyrrhizic acid salt, glycyrrhizic acid salt, oleanolic acid salt, ursolic acid salt, shikimic acid salt, salvianolic acid salt, quinic acid salt, deoxycholic acid salt, betulinic acid salt, gambogic acid salt, or rhein salt.

4. A cordycepin salt, characterized in that: The salt is selected from food organic acid salts, amino acid salts, vitamin salts, or inorganic acid salts; the salt does not include hydrochloride, sulfate, phosphate, maleate, tartrate, oxalate, and fumarate; Preferably, The food organic acid salt is selected from fructose-1,6-diphosphate, malate, acetate, malonate, butyrate, valerate, oleate, palmitate, stearate, laurate, maleate, citrate, tartrate, succinate, gluconate, lactobionate, lauryl sulfonate, or isethionate; The amino acid salt is selected from aspartate or glutamate; The vitamin salt is selected from vitamin B3 salt, vitamin B5 salt, vitamin C salt, or vitamin M salt; The inorganic acid salt is selected from hydrochloride, phosphate, phosphite, or borate.

5. The method for preparing the cordycepin salt according to any one of claims 1 to 4, characterized in that: The method comprises dissolving any one of nucleotides, natural organic acids, food organic acids, amino acids, vitamins and inorganic acids, and cordycepin in a solvent, filtering, and obtaining the cordycepin salt; or, The method comprises dissolving any one of nucleotides, natural organic acids, food organic acids, amino acids, vitamins and inorganic acids, and cordycepin in a solvent, crystallizing, filtering, and obtaining the cordycepin salt.

6. The preparation method according to claim 5, characterized in that: The molar ratio of any one of the nucleotides, natural product organic acids, food organic acids, amino acids, vitamins and inorganic acids to cordycepin is 0.1 to 10:1, preferably 1-2:0.6-3, 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.

7. A pharmaceutical composition, characterized in that The invention comprises a) the cordycepin salt according to any one of claims 1 to 4, 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; preferably, the immune checkpoint inhibitor is PD-1 and / or PD-L1; preferably, the mass ratio of the cordycepin salt to the other active substance is 2-4:1, preferably 3:1; preferably, the dosage form of the pharmaceutical composition is selected from tablets, pills, capsules, dripping pills, syrups, disintegrants, injections, sustained-release agents, or kits.

9. Use of the cordycepin salt according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a product for preventing and treating diseases related to cell functional damage and mutation in mammals; preferably, the disease related to cell functional damage and mutation is a tumor.

10. The use according to claim 9, 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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