Gyclicins and salts thereof

Abstract

The application relates to a cyclic icariin amino acid ester prodrug and a salt thereof, and belongs to the technical field of medicines, and particularly relates to a cyclic icariin amino acid ester prodrug formed between cyclic icariin and amino acids, a pharmaceutically acceptable salt thereof, and preparation and application thereof. Specifically, the application combines the structural characteristics of cyclic icariin and the inherent advantages of prodrugs, covalently combines amino acids with a 3-hydroxyl group in the structure of cyclic icariin to form a series of cyclic icariin amino acid ester prodrugs. The compound can significantly improve the water solubility, permeability and metabolic stability of cyclic icariin, improve the bioavailability of the medicine, and better exert an anti-tumor effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a cyclic icariin amino acid ester and a pharmaceutically acceptable salt thereof and a preparation method thereof. BACKGROUND

[0002] Cyclic icariin is an isomer of icariin, and its chemical name is 3,5-dihydroxy-2-(4-methoxy-phenyl)-8,8-dimethyl-9,10-dihydro-8H-pyrano[2,3-f]chromen-4-one, which is a flavonoid compound. Cyclic icariin can be separated from traditional Chinese medicinal material Icaria, or prepared by acid hydrolysis of Icaritin. Studies have found that cyclic icariin has good anti-tumor activity, can effectively inhibit various tumor cells and has no obvious toxic side effects. In addition, cyclic icariin also has multiple effects such as anti-osteoporosis, hypoglycemic, anti-inflammatory, anti-oxidation, improvement of cardiovascular and cerebrovascular, enhancement of immunity and improvement of estrogen level, and has good application prospect.

[0003] Cyclic icariin has poor solubility and permeability, and the hydroxyl group in the structure is easily metabolized in the intestine and liver in vivo, has a strong first-pass effect, has poor oral absorption, and thus has low bioavailability, which restricts the exertion of its efficacy. Therefore, it is necessary to improve the oral bioavailability of cyclic icariin. However, existing drug delivery systems such as liposomes, self-microemulsions, phospholipid complexes and polymer micelles have low drug loading capacity, poor preparation stability and no obvious improvement in oral absorption, and it is a great challenge to improve the bioavailability of cyclic icariin through preparation means.

[0004] A prodrug is a chemical modification of a drug to improve its bioavailability, and is an important means and strategy for designing and developing innovative drugs. Amino acids are diverse, inexpensive, safe and can form carbamate prodrugs with hydroxyl groups. The formation of a prodrug can increase the solubility of the parent drug in water. In order to improve the solubility, permeability and metabolism of cyclic icariin, the main metabolic sites of cyclic icariin are coupled with a series of amino acids through carbamate bonds to obtain a series of carbamate prodrugs, so as to improve the water solubility, membrane permeability and metabolic stability of cyclic icariin, and further improve its oral bioavailability and better exert its efficacy. SUMMARY

[0005] The present application aims to provide a cyclic icariin amino acid ester and a pharmaceutically acceptable salt thereof, and application of the prodrug in improving the water solubility and bioavailability of cyclic icariin.

[0006] The 3-hydroxyl group of the cyclic icariin is modified by an amino acid, wherein the amino acid is one of alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0007] Specifically, the cyclic icariin carbamate prodrug of the present application has the following general structure:

[0008]

[0009] wherein R is the residue of the selected amino acid, which is preferably glycine, L-isoleucine, L-phenylalanine, L-valine, L-leucine, L-alanine, L-glutamic acid, and further preferably L-leucine, which has the following structure:

[0010]

[0011] The synthesis of the cyclic icariin carbamate prodrug is carried out according to the following method:

[0012] Step 1: Compound I, a carbonylation reagent, is dissolved in a reaction solvent, and a basic catalyst is added dropwise to obtain compound II.

[0013] wherein the carbonylation reagent is selected from dimethyl carbonate, 4-nitrophenyl carbonate, phosgene, and solid phosgene;

[0014] the reaction solvent is selected from dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N,N-dimethylhexanamide; the basic catalyst is selected from triethylamine, potassium carbonate, sodium carbonate, strong potassium oxide, sodium hydroxide, and pyridine; and the dropwise temperature is -20℃-0℃.

[0015] Step 2: Compound II is added dropwise into cyclic icariin dissolved in an aprotic solvent, and the reaction is carried out at room temperature for 1-3h to obtain compound III;

[0016] the aprotic solvent is selected from dimethyl sulfoxide, N,N-dimethylformamide, and N-methyl pyrrolidone; and the reaction temperature is 10℃-40℃.

[0017] Step 3: Compound III is dissolved in an acid, and then ice-bath for 30 min, and then the reaction is carried out at room temperature for 1-3h, and the solvent is removed by rotary evaporation to prepare a liquid phase separation, and compound IV is obtained;

[0018] wherein the acid is selected from hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, and p-toluenesulfonic acid; and the reaction temperature is 0℃-40℃.

[0019] Step 4: Compound IV is dissolved in different bases to obtain its salt form.

[0020] The bases used include: potassium hydroxide, potassium bicarbonate, potassium carbonate, sodium hydroxide, sodium bicarbonate, sodium carbonate, ammonia, diethylamine, triethylamine.

[0021]

[0022] R represents the residue of an amino acid;

[0023] a: carbonylation reagent (dimethyl carbonate, 4-nitrophenyl carbonate, phosgene or solid phosgene), basic catalyst (triethylamine, potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, pyridine) and solvent (dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, N,N-dimethylhexanamide);

[0024] b: solvent (dimethyl sulfoxide, N,N-dimethylformamide and N-methyl pyrrolidone);

[0025] c: acid (hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, p-toluenesulfonic acid).

[0026] Advantages and beneficial effects of the present application:

[0027] The present application first synthesizes amino acid-modified icariin carbamate prodrugs and pharmaceutically acceptable salts thereof, and these compounds can significantly improve the water solubility, permeability and metabolic stability of icariin. After oral administration of icariin, the compound of Example 2 and the compound of Example 5, the in vivo exposure of icariin is significantly increased; the absolute bioavailability is 0.11%, 5.7% and 2.8%, respectively. Compared with oral icariin, the oral bioavailability of the compound of Example 2 and the compound of Example 5 is increased by 52 times and 25 times, respectively, the oral bioavailability is significantly improved, and the intended purpose is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 3 is the residual percentage-time curve of icariin and its prodrugs after phase II reaction in rat liver microsomes (n=3);

[0029] Figure 2 Figure 4 is the blood drug concentration-time curve of icariin, the compound of Example 2 and the compound of Example 5 (n=5);

[0030] a. Oral icariin (200 mg / kg);

[0031] b. Oral compound of Example 2 (200 mg / kg of icariin);

[0032] c. Orally administered compound of Example 5 (200 mg / kg, calculated as cycloicariin);

[0033] d. Intravenous injection of cycloicin (2 mg / kg);

[0034] Figure 3 (A) Tumor volume changes in 4T1 tumor-bearing mice after oral administration of cycloicin or the compound of Example 2; (B) Tumor images of 4T1 tumor-bearing mice 15 days after administration; (C) Tumor bearing rate of 4T1 tumor-bearing mice 15 days after administration; (D) Body weight changes in 4T1 tumor-bearing mice (n=6) after oral administration of cycloicin or the compound of Example 2. Detailed Implementation

[0035] The following specific examples are used to further illustrate the invention, but are by no means intended to limit its scope.

[0036] The following compounds were prepared according to the synthetic route of cycloicariin carbamate prodrug.

[0037] Example 1

[0038] Step 1: Dissolve glycine tert-butyl hydrochloride (1 g) and solid phosgene (1.2 g) in dichloromethane, purge with nitrogen, control the dropping temperature at -20℃ to 0℃, and add triethylamine dropwise to obtain compound II.

[0039] Step 2: Dissolve 1.47 g of cycloicariin in... N , N In dimethylformamide, a reaction solution containing compound II was added dropwise, nitrogen gas was introduced, and the reaction was carried out at room temperature for 2 h. The reaction solution was diluted with water, extracted three times with dichloromethane, washed successively with aqueous solution and saturated brine, filtered, and evaporated to dryness to remove the solvent, yielding a mixture containing compound III.

[0040] Step 3: The mixture of compounds III was dissolved in trifluoroacetic acid (TFA, 20 mL), under nitrogen protection, in an ice bath for 30 min, and then reacted at room temperature for 1 h. The solvent was removed by vacuum distillation, the mixture was washed three times with anhydrous dichloromethane, evaporated to dryness, and separated by preparative liquid chromatography to obtain 3-glycylcycloicariin.

[0041] The preparation method of Example 1 was used to implement Examples 2-7.

[0042] Example 2

[0043] Step 1: Dissolve L-leucine tert-butyl hydrochloride and solid phosgene in dichloromethane, purge with nitrogen, control the dropping temperature at -20℃ to 0℃, and add triethylamine dropwise to obtain compound II.

[0044] Second step: dissolve the compound II into N, N-dimethylformamide, add the reaction solution containing compound II dropwise, fill in nitrogen, react at room temperature for 2 hours, dilute the reaction solution with water, extract with dichloromethane for three times, wash with water solution and saturated brine solution in turn, filter, spin dry to remove the solvent, and obtain a mixture containing compound III.

[0045] Third step: dissolve the compound III mixture into trifluoroacetic acid (TFA), protect with nitrogen, ice bath for 30 minutes, then react at room temperature for 1 hour. Evaporate the solvent under reduced pressure, wash with anhydrous dichloromethane for three times, spin dry, separate by preparative liquid phase, and obtain 3-L-leucyl icariin.

[0046] Example 3

[0047] First step: dissolve L-phenylalanine tert-butyl ester hydrochloride and solid phosgene into dichloromethane, fill in nitrogen, control the dropwise temperature to be -20℃-0℃, and add triethylamine dropwise to obtain compound II.

[0048] Second step: dissolve the compound II into N, N-dimethylformamide, add the reaction solution containing compound II dropwise, fill in nitrogen, react at room temperature for 2 hours, dilute the reaction solution with water, extract with dichloromethane for three times, wash with water solution and saturated brine solution in turn, filter, spin dry to remove the solvent, and obtain a mixture containing compound III.

[0049] Third step: dissolve the compound III mixture into trifluoroacetic acid (TFA), protect with nitrogen, ice bath for 30 minutes, then react at room temperature for 1 hour. Evaporate the solvent under reduced pressure, wash with anhydrous dichloromethane for three times, spin dry, separate by preparative liquid phase, and obtain 3-L-phenylalaninyl icariin.

[0050] Example 4

[0051] First step: dissolve L-phenylalanine tert-butyl ester hydrochloride and solid phosgene into dichloromethane, fill in nitrogen, control the dropwise temperature to be -20℃-0℃, and add triethylamine dropwise to obtain compound II.

[0052] Second step: dissolve the compound II into N, N-dimethylformamide, add the reaction solution containing compound II dropwise, fill in nitrogen, react at room temperature for 2 hours, dilute the reaction solution with water, extract with dichloromethane for three times, wash with water solution and saturated brine solution in turn, filter, spin dry to remove the solvent, and obtain a mixture containing compound III.

[0053] Third step: dissolve the compound III mixture into trifluoroacetic acid (TFA), protect with nitrogen, ice bath for 30 minutes, then react at room temperature for 1 hour. Evaporate the solvent under reduced pressure, wash with anhydrous dichloromethane for three times, spin dry, separate by preparative liquid phase, and obtain 3-L-phenylalaninyl icariin.

[0054] Example 5

[0055] First step: L-valine tert-butyl ester hydrochloride, solid phosgene was dissolved in dichloromethane, filled with nitrogen, the temperature of dropwise addition was controlled at -20-0 ℃, triethylamine was added dropwise, compound II was obtained.

[0056] Second step: Icaritin was dissolved in N,N-dimethylformamide, the reaction solution containing compound II was added dropwise, filled with nitrogen, reacted at room temperature for 2 h, the reaction solution was diluted with water, extracted with dichloromethane for three times, washed with aqueous solution and saturated brine in turn, filtered, and the solvent was removed by rotary evaporation to obtain a mixture containing compound III.

[0057] Third step: The mixture of compound III was dissolved in trifluoroacetic acid (TFA), protected by nitrogen, ice-bath for 30 min, then reacted at room temperature for 1 h. The solvent was evaporated under reduced pressure, washed with anhydrous dichloromethane for three times, rotary evaporation, separated by preparative liquid phase to obtain 3-L-valine icaritin.

[0058] Example 6

[0059] First step: L-valine tert-butyl ester hydrochloride, solid phosgene was dissolved in dichloromethane, filled with nitrogen, the temperature of dropwise addition was controlled at -20-0 ℃, triethylamine was added dropwise, compound II was obtained.

[0060] Second step: Icaritin was dissolved in N,N-dimethylformamide, the reaction solution containing compound II was added dropwise, filled with nitrogen, reacted at room temperature for 2 h, the reaction solution was diluted with water, extracted with dichloromethane for three times, washed with aqueous solution and saturated brine in turn, filtered, and the solvent was removed by rotary evaporation to obtain a mixture containing compound III.

[0061] Third step: The mixture of compound III was dissolved in trifluoroacetic acid (TFA), protected by nitrogen, ice-bath for 30 min, then reacted at room temperature for 1 h. The solvent was evaporated under reduced pressure, washed with anhydrous dichloromethane for three times, rotary evaporation, separated by preparative liquid phase to obtain 3-L-valine icaritin.

[0062] Example 7

[0063] First step: L-valine tert-butyl ester hydrochloride, solid phosgene was dissolved in dichloromethane, filled with nitrogen, the temperature of dropwise addition was controlled at -20-0 ℃, triethylamine was added dropwise, compound II was obtained.

[0064] Second step: Icaritin was dissolved in N,N-dimethylformamide, the reaction solution containing compound II was added dropwise, filled with nitrogen, reacted at room temperature for 2 h, the reaction solution was diluted with water, extracted with dichloromethane for three times, washed with aqueous solution and saturated brine in turn, filtered, and the solvent was removed by rotary evaporation to obtain a mixture containing compound III.

[0065] Step 3: Compound III mixture was dissolved in trifluoroacetic acid (TFA) under nitrogen protection, and ice bath for 30 min, then room temperature reaction for 1 h. The solvent was evaporated under reduced pressure, washed with anhydrous dichloromethane for 3 times, spin dry, separated by preparative liquid phase, and 3-L-glutamylcycloartemisinin was obtained.

[0066] The identification information of cycloartemisinin and cycloartemisinin carbamate prodrug compounds modified by amino acids is shown in Table 1:

[0067] Table 1 Related information of cycloartemisinin and its prodrugs

[0068]

[0069] Example 8

[0070] Water equilibrium solubility test of cycloartemisinin carbamate prodrug

[0071] The poor water solubility of cycloartemisinin is an important reason to limit its bioavailability, so the equilibrium solubility of the series of prodrugs in water is beneficial to predict the dissolution and absorption of cycloartemisinin carbamate prodrug in vivo. Excess cycloartemisinin and amino acid modified cycloartemisinin carbamate prodrug were added to an appropriate amount of water, and shaken to equilibrium in a constant temperature oscillator at 37 ℃, to obtain the water equilibrium solubility of cycloartemisinin and amino acid modified cycloartemisinin carbamate prodrug. The results are shown in Table 2.

[0072] Table 2 Water equilibrium solubility of cycloartemisinin and its prodrugs

[0073]

[0074] The results show that each prodrug significantly improves the water solubility of cycloartemisinin, among which the water solubility of the compounds of Example 2 and Example 5 is better, which increases the water solubility by about 16.3 and 14 times, respectively.

[0075] Example 9

[0076] Phase II metabolism of cycloartemisinin and its carbamate prodrugs

[0077] Serious phase II metabolism is a major factor limiting the oral bioavailability of epimedium. To determine whether the designed prodrugs can improve the metabolic stability of epimedium, we selected the compound of Example 2 and the compound of Example 5, which have higher water solubility, and used in vitro liver microsomal incubation method to simulate the metabolism of prodrugs in vivo. The specific incubation process is as follows: the rat liver microsomes, magnesium chloride, D-glucaric acid 1,4-lactone, triton and the test drug were prepared into appropriate concentrations with buffer, added to the stoppered glass test tube, pre-incubated in the 37 ℃ constant temperature water bath shaking box, 5'-uridine diphosphate glucuronide trisodium solution was added to start the glucuronidation reaction, incubated in the 37 ℃ constant temperature water bath shaking box, three parallel samples were taken at different time points for determination. The percentage of remaining prodrug and parent drug was used as an index to evaluate the metabolism of epimedium and prodrugs in rat liver microsomes. The results are shown in Table 1. Figure 1

[0078] The results show that the glucuronidation reaction of epimedium and its carbamate prodrugs is time-dependent. With the extension of incubation time, epimedium is rapidly metabolized, while the compound of Example 2 and the compound of Example 5 are slowly metabolized, which can significantly improve the phase II metabolic stability of epimedium.

[0079] Example 10

[0080] In vivo intestinal perfusion study of epimedium and its carbamate prodrugs

[0081] In order to study whether the prodrugs can improve the permeability of the parent drug, we conducted in vivo intestinal perfusion test of epimedium, the compound of Example 2 and the compound of Example 5 in rats. About 10 cm long rat jejunum was taken, both ends were cannulated, epimedium and the compound of Example 2 and the compound of Example 5 were dissolved in Kreb-Ringers nutrient solution (pH 6.0) respectively, the concentration was 5 μM, the perfusion solution containing the drug was passed through the rat jejunum at a flow rate of 0.2 mL / min, and the membrane permeability of epimedium and the compound of Example 2 and the compound of Example 5 in the jejunum was obtained.

[0082] Table 3 Membrane permeability of epimedium and its prodrugs

[0083]

[0084] The results show that compared with epimedium, the effective permeability coefficient of the compound of Example 2 and the compound of Example 5 is increased by 2.2 times and 1.8 times respectively, and the permeability is significantly improved.

[0085] Example 11

[0086] Pharmacokinetic study in rats

[0087] ​The water solubility of the compounds of Examples 2-7 is improved to different degrees after the amino acid modification of the icariin to form the carbamates, compared with the icariin, wherein the water solubility of the compound of Example 2 and the compound of Example 5 is increased by 16.3 and 14 times, respectively, and the water solubility is increased the most. After the hydroxyl group is protected, the biophase metabolism of the compound of Example 2 and the compound of Example 5 is obviously reduced. In the in-vivo intestinal perfusion test, the effective permeability coefficient of the compound of Example 2 and the compound of Example 5 is found to be increased by 2.2 times and 1.8 times, respectively, and the permeability is significantly improved, which preliminarily proves the expected purpose in-vitro. In order to further study the improvement of the bioavailability of the design, the compound of Example 2 and the compound of Example 5 with high water solubility, good permeability and not prone to phase II metabolism are selected for the in-vivo pharmacokinetic study.

[0088] The experimental group and the control group of Sprague-Dawley rats are respectively given the icariin, the compound of Example 2 and the compound of Example 5 (200 mg / kg of icariin) by gavage, and at the same time, the icariin solution (2 mg / kg) is injected intravenously, and the concentration of icariin in the plasma is determined, and the results are shown in Table 4 and Figure 2 .

[0089] Table 4 Pharmacokinetic parameters of icariin in rats after oral administration of icariin, the compound of Example 2 and the compound of Example 5 (200 mg / kg of icariin)

[0090]

[0091] From Table 4 and Figure 2 , it can be seen that after the icariin, the compound of Example 2 and the compound of Example 5 are given by gavage, the area under the curve (AUC 0-t ) of the compound of Example 2 and the compound of Example 5 is obviously higher than that of icariin, and the in-vivo exposure of icariin is significantly increased; according to the data of the intravenous injection of icariin, the absolute bioavailability of icariin and its prodrugs is 0.11%, 5.7% and 2.8%, respectively. Compared with the oral icariin, the oral bioavailability of the compound of Example 2 and the compound of Example 5 is increased by 52 times and 25 times, respectively, and the oral bioavailability is significantly improved.

[0092] Example 12

[0093] Pharmacodynamic evaluation

[0094] The compound of Example 2 with high oral absolute bioavailability is selected for the pharmacodynamic study to investigate its anti-tumor activity. Eighteen BALB / c mice (female, body weight of 18-22 g) are subcutaneously injected with the density of 1×10 7cells / mL of murine breast cancer cells (4T1). When the tumor volume on the tumor-bearing mice reached 100 mm 3 mm3, the mice were randomly divided into 6 groups, with 6 animals in each group. The tumor-bearing mice were orally administered with olive oil, Icaritin and the compound of Example 2 at an equivalent dose of 70 mg / kg, respectively, for 15 consecutive days. The tumor volume and body weight of the tumor-bearing mice were measured and recorded every other day. After the end of the efficacy experiment, all the tumor-bearing mice were sacrificed, and the tumor tissues were isolated and obtained. The tumor-bearing rate was calculated. The results are shown in Table 1. Figure 3

[0095] The results show that, compared with Icaritin, the compound of Example 2 exhibits good anti-tumor effect, which can effectively inhibit tumor growth, and has little effect on body weight and good safety. The compound designed in the present application significantly improves the bioavailability of Icaritin, and is safe and effective, and has good development prospects.​

Claims

1. A cyclic icariin carbamate prodrug or a pharmaceutically acceptable salt thereof, characterized in that, The prodrug is prepared by coupling the hydroxyl group at position 3 of the icariin with the amino group of an amino acid through a carbamate bond, the amino acid being selected from glycine, L-alanine, L-valine, L-isoleucine, L-leucine, L-phenylalanine, L-glutamic acid; The structural general formula of the icariin carbamate prodrug is: wherein R is the residue of an amino acid selected from the following structures: 。 2. A process for the preparation of the Epimedium-derived aminocarbamate prodrug of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The following steps are followed: Step 1: Compound I, a carbonylation reagent is dissolved in a reaction solvent, and a basic catalyst is added dropwise to obtain compound II; Step 2: Compound II is added dropwise into icariin dissolved in an aprotic solvent, and the reaction is carried out at room temperature for 1-3 hours to obtain compound III; Step 3: Compound III is dissolved in an acid, and then ice-bath for 30 min, and then the reaction is carried out at room temperature for 1-3 hours, and the solvent is removed by rotary evaporation to prepare a liquid phase separation to obtain compound IV; Step 4: Compound IV is dissolved in different bases to obtain its salt form; R represents the residue of the amino acid as claimed in claim 1.

3. The production method according to claim 2, wherein In the first step, the carbonylation reagent is selected from dimethyl carbonate, 4-nitrophenyl carbonate, phosgene, and solid phosgene; the basic catalyst is selected from triethylamine, potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, and pyridine; and the solvent is selected from dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dioxane, and N,N-dimethylhexanamide.

4. The production method according to claim 2, wherein In the second step, the aprotic solvent is selected from dimethyl sulfoxide, N,N-dimethylformamide, and N-methylpyrrolidone.

5. The production method according to claim 2, wherein In the third step, the acid is selected from hydrochloric acid, acetic acid, formic acid, maleic acid, lactic acid, carbonic acid, trifluoroacetic acid, phosphoric acid, and p-toluenesulfonic acid.

6. The production method according to claim 2, wherein In the fourth step, the base is selected from potassium hydroxide, potassium bicarbonate, potassium carbonate, sodium hydroxide, sodium bicarbonate, sodium carbonate, ammonia, diethylamine, and triethylamine.

7. A pharmaceutical composition, characterized by, The icariin carbamate prodrug as claimed in claim 1 or a pharmaceutically acceptable salt thereof.

8. Use of the icariin carbamate prodrug as claimed in claim 1 or the pharmaceutical composition as claimed in claim 7 in the preparation of a medicament for improving the solubility, permeability, metabolic stability, and bioavailability of icariin.

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