Icaritin methyl phosphate prodrug and preparation method and application of sodium salt of icaritin methyl phosphate prodrug

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of an icaritin methyl phosphate prodrug and sodium salt thereof. The preparation method comprises the following steps: reacting icaritin with di-tert-butyl chloromethyl phosphate under an alkaline condition to obtain di-tert-butyl icaritin methyl phosphate, then removing di-tert-butyl under an acidic condition to obtain an icaritin methyl phosphate prodrug, and further obtaining icaritin methyl sodium phosphate under the alkaline condition. The icaritin methyl phosphate prodrug provided by the invention has good water solubility and bioavailability, the preparation method is simple, reaction conditions are mild, and the icaritin methyl phosphate prodrug is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing icariin methyl phosphate prodrug and its sodium salt, and its application. Background Technology

[0002] Icaritin, chemically known as 3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methylbut-2-enyl)-1,2-benzopyran-4-one, is a hydrolysis product of icariin, the main active ingredient in Epimedium. It possesses various pharmacological effects, including anti-inflammatory, antioxidant, antitumor, neuroprotective, and osteoprotective properties. On January 10, 2022, icariin soft capsules (Icaritin, ICT) received conditional marketing approval for the treatment of unresectable hepatocellular carcinoma in patients who are unsuitable for or refuse standard therapy and have never received systemic therapy.

[0003] However, due to the poor water solubility of icariin (<10μg / mL), its dosage form diversity is greatly limited, thus losing the advantage of different routes of administration; at the same time, the extremely low water solubility also results in low bioavailability of icariin, which greatly limits its clinical application and efficacy.

[0004] Chinese patent application number 201510934944.0 discloses an icariin injection, which specifically comprises an icariin phospholipid complex, an injection oil, an emulsifier, a pH adjuster, and a co-emulsifier. Although this increases the lipid solubility of icariin and improves the drug loading of the icariin injection, it also poses safety risks such as hemolysin and phospholipid oxidation.

[0005] A self-microemulsion formulation of icariin is disclosed in Chinese patent application number 201680006886.8. After oral administration, it forms an O / W nanoemulsion with a particle size of 10-200 nm in vivo, but the improvement in bioavailability is not significant.

[0006] Patent application No. 201910477668.8 discloses a method for preparing icariin solid dispersion using hot melt extrusion technology, but it does not significantly improve the equilibrium solubility of icariin.

[0007] Patent application No. 202080044930.0 discloses a method for preparing icariin nanomicelle formulations. The method involves forming nanomicelles from icariin and polymer excipients, followed by concentration and the addition of flavoring agents, preservatives, and defoamers to produce a liquid formulation. Alternatively, the nanomicelles can be freeze-dried to prepare granules, capsules, or tablets. While this method improves the solubility of icariin to some extent and enriches the dosage forms, the preparation process is complex and the production cost is high, making it unsuitable for large-scale production and application.

[0008] In summary, the poor water solubility of icariin itself increases the difficulty of formulation development and introduces some safety concerns. Improving the water solubility of compounds through prodrug modification is a common compound structure optimization strategy, with amino acid esters, carbamates, and phosphate esters being common modification structures. Through in vivo carboxylesterases and phosphatases, the prodrug is hydrolyzed back to the original drug, thus exerting its original effect.

[0009] Patent application No. 202311340206.4 discloses a prodrug of icariin carbamate and its preparation method. Although the phase II metabolic stability of this type of prodrug is excellent, the improvement in water solubility is not significant, and it cannot effectively provide more dosage form options.

[0010] Therefore, there is an urgent need to develop a prodrug of icariin with good water solubility and excellent pharmaceutical properties, so as to enrich the route of administration, expand the application scope of icariin, and pave the way for its clinical application. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides a method for preparing icariin methyl phosphate prodrug and its sodium salt, as well as its applications.

[0012] The technical solution of the present invention is as follows:

[0013] The structure of the icariin methyl phosphate prodrug and its pharmaceutically acceptable salt, as shown in Formula I, is as follows:

[0014]

[0015] The structure of the icariin methyl phosphate prodrug and its pharmaceutically acceptable salt, as shown in Formula II, is as follows:

[0016]

[0017] The pharmaceutically acceptable salt is the sodium, potassium, or calcium salt of the icariin methyl phosphate prodrug, with sodium icariin methyl phosphate being more preferred.

[0018] This invention provides a method for preparing the icariin methyl phosphate prodrug, comprising the following steps:

[0019]

[0020] Step a): Icariin was dissolved in reaction solvent A, and di-tert-butylchloromethyl phosphate and an acid-binding agent were added. The mixture was stirred and homogenized at room temperature, and the reaction was heated. After the reaction was completed by HPLC monitoring, the reaction solution was cooled and poured into a separatory funnel. Solvent B was added, and the solution was washed successively with 0.1M HCl aqueous solution, water, and saturated saline solution. The organic phase was dried over anhydrous sodium sulfate and the organic solvent was removed under reduced pressure. The crude product was slurried at room temperature using solvent C, filtered, and washed to obtain crude di-tert-butylicariin methyl phosphate.

[0021] Step b): Dissolve crude di-tert-butylicariin methyl phosphate in reaction solvent D, stir at room temperature in the presence of acid, and remove the reaction solvent under reduced pressure to obtain crude icariin methyl phosphate.

[0022] Step c): Dissolve crude icariin methyl phosphate in reaction solvent E, adjust the pH with alkaline solution, stir at room temperature, and filter to obtain crude icariin methyl phosphate sodium.

[0023] Step d): Take crude icariin methyl phosphate sodium, add solvent F and dissolve it at a certain temperature. After the solution is clear, add an appropriate amount of activated carbon and stir. Filter, stir to precipitate crystals, filter, wash with anhydrous ethanol, and dry to obtain refined icariin methyl phosphate sodium.

[0024] In some embodiments, the reaction solvent A in step a) is selected from one or more of N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran, 1,4-dioxane, acetonitrile, and acetone, with DMF being preferred; the volume-to-mass ratio of icariin to the reaction solvent is 5–40 v / w (mL / g).

[0025] In some implementations, in step a), the molar ratio of di-tert-butylchloromethyl phosphate to icariin is 0.8 to 1.5.

[0026] In some embodiments, in step a), the acid-binding agent includes one or more of sodium hydroxide, potassium hydroxide, cesium carbonate, potassium carbonate, and sodium carbonate, preferably cesium carbonate. The molar ratio of the acid-binding agent to icariin is 1 to 5.

[0027] In some embodiments, in step a), solvent B is selected from one or more of ethyl acetate, dichloromethane, methyl tert-butyl ether, or trichloromethane.

[0028] In some embodiments, in step a), solvent C is selected from an 80% aqueous acetonitrile solution.

[0029] In some embodiments, in step b), the reaction solvent D is selected from one or more of dichloromethane, trichloromethane, or acetonitrile, preferably dichloromethane. The volume-to-mass ratio of crude di-tert-butylicin methyl phosphate to the reaction solvent is 20–100 v / w (mL / g).

[0030] In some embodiments, the acid in step b) is selected from hydrochloric acid (HCl), hydrobromic acid (HBr), or trifluoroacetic acid (TFA), preferably trifluoroacetic acid. The molar ratio of the added trifluoroacetic acid to di-tert-butylicariin methyl phosphate is 5–20.

[0031] In some embodiments, the reaction solvent E in step c) is selected from one or more of methanol, ethanol, acetone, acetonitrile, isopropanol, and propylene glycol, with ethanol being more preferred. The volume-to-mass ratio of crude icariin sodium methyl phosphate to the reaction solvent is 5–20 v / w (mL / g).

[0032] In some implementations, in step c), the pH is adjusted to 8-9 with a 4M sodium hydroxide solution.

[0033] In some embodiments, in step d), solvent F is selected from one or more of methanol, ethanol, acetone, acetonitrile, isopropanol or water, preferably a 60% to 70% aqueous ethanol solution; the volume-to-mass ratio of crude icariin methyl phosphate sodium to solvent F is 20 to 40 v / w (mL / g).

[0034] The beneficial effects of this invention are:

[0035] 1. This invention discloses for the first time the preparation of the above-mentioned icariin methyl phosphate prodrug, which, after experimental verification, greatly improves the solubility of icariin in aqueous solution and its bioavailability in animals.

[0036] 2. The icariin methyl phosphate prodrug prepared by the method of the present invention has milder reaction conditions and simpler operation than the prior art. The present invention adopts a specific purification technique to obtain a high yield and high purity of the product, and does not require column chromatography purification, which is convenient for industrial production.

[0037] 3. By utilizing the advantages of good water solubility and high stability of icariin methyl phosphate prodrug, the possibility of diversifying the dosage forms of icariin has been made, further enriching the clinical application of icariin. Attached Figure Description

[0038] Figure 1 The pharmacokinetic curve of rats after oral administration of icariin sodium methyl phosphate solution.

[0039] Figure 2 The pharmacokinetic curve of icariin corn oil suspension in rats after oral administration. Detailed Implementation

[0040] The following embodiments are intended to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the invention to the scope of the embodiments described.

[0041] Example 1: Synthesis of di-tert-butylicariin methyl phosphate:

[0042] Add 100 mg of icariin (98%, 0.266 mM), 2 mL of DMF, di-tert-butylchloromethyl phosphate (86.93 mg, 95%, 0.319 mM), and Cs₂CO₃ (131.33 mg, 99%, 0.399 mM) to a 10 mL reaction flask. Stir at room temperature for 10 min, then heat to 60 °C. Monitor the reaction using high-performance liquid chromatography (HPLC). The reaction is complete after approximately 3 h. Pour the reaction solution into a separatory funnel, add 20 mL of ethyl acetate, and wash three times each with 20 mL of 0.1 M HCl, water, and saturated brine. Dry the organic phase with anhydrous sodium sulfate and remove the organic solvent under reduced pressure at 50 °C. Slurry the crude product with 80% acetonitrile for 30 min, filter, and wash three times with 80% acetonitrile to obtain 29.45 mg of crude di-tert-butylicariin methyl phosphate.

[0043] Example 2: Synthesis of di-tert-butylicariin methyl phosphate:

[0044] In a 10 mL reaction flask, add 100 mg of icariin (98%, 0.266 mM), 2.5 mL of acetonitrile, di-tert-butylchloromethyl phosphate (86.93 mg, 95%, 0.319 mM), and Cs₂CO₃ (131.33 mg, 99%, 0.399 mM). Stir at room temperature for 10 min, then heat to 60 °C. Monitor the reaction using high-performance liquid chromatography (HPLC). The reaction is complete after approximately 5 h. Pour the reaction solution into a separatory funnel, add 20 mL of ethyl acetate, and wash three times each with 20 mL of 0.1 M HCl, water, and saturated brine. Dry the organic phase with anhydrous sodium sulfate and remove the organic solvent under reduced pressure at 50 °C. Slurry the crude product with 80% acetonitrile for 30 min, filter, and wash three times with 80% acetonitrile to obtain 16.94 mg of crude di-tert-butylicariin methyl phosphate.

[0045] Example 3: Synthesis of di-tert-butylicariin methyl phosphate:

[0046] Add 100 mg of icariin (98%, 0.266 mM), 2 mL of DMF, di-tert-butylchloromethyl phosphate (86.93 mg, 95%, 0.319 mM), and K₂CO₃ (150.07 mg, 98%, 1.064 mM) to a 10 mL reaction flask. Stir at room temperature for 10 min, then heat to 50 °C. Monitor the reaction using high-performance liquid chromatography (HPLC). The reaction is complete after approximately 5 h. Pour the reaction solution into a separatory funnel, add 20 mL of ethyl acetate, and wash three times each with 20 mL of 0.1 M HCl, water, and saturated brine. Dry the organic phase with anhydrous sodium sulfate and remove the organic solvent under reduced pressure at 50 °C. Slurry the crude product with 80% acetonitrile for 30 min, filter, and wash three times with 80% acetonitrile to obtain 27.99 mg of crude di-tert-butylicariin methyl phosphate.

[0047] Example 4: Synthesis of di-tert-butylicariin methyl phosphate:

[0048] Add 100 mg of icariin (98%, 0.266 mM), 2 mL of DMF, di-tert-butylchloromethyl phosphate (57.95 mg, 95%, 0.213 mM), and Cs₂CO₃ (131.33 mg, 99%, 0.399 mM) to a 10 mL reaction flask. Stir at room temperature for 10 min, then heat to 50 °C. Monitor the reaction using high-performance liquid chromatography (HPLC). The reaction is complete after approximately 2 h. Pour the reaction solution into a separatory funnel, add 20 mL of ethyl acetate, and wash three times each with 20 mL of 0.1 M HCl, water, and saturated brine. Dry the organic phase with anhydrous sodium sulfate and remove the organic solvent under reduced pressure at 50 °C. Slurry the crude product with 80% acetonitrile for 30 min, filter, and wash three times with 80% acetonitrile to obtain 37.09 mg of crude di-tert-butylicariin methyl phosphate.

[0049] Example 5: Synthesis of Icariin Sodium Methylphosphate:

[0050] In a 10 mL reaction flask, crude di-tert-butylicariin methyl phosphate (40 mg, 83.66%, 0.057 mM), 3 mL of dichloromethane, and 84.23 μL of trifluoroacetic acid (99%, 1.134 mM) were added. The mixture was stirred at room temperature for 1 h, and the dichloromethane was removed by concentration under reduced pressure. 0.6 mL of isopropanol was added to the residue, and the pH was adjusted to 8.5 using 4 M sodium hydroxide solution. The mixture was stirred at room temperature for 1 h, and filtered to obtain 8.24 mg of crude icariin methyl phosphate sodium salt.

[0051] Example 6: Synthesis of Icariin Sodium Methylphosphate:

[0052] Add icariin (1000.39 mg, 98%, 2.661 mM), 10 mL DMF, di-tert-butylchloromethyl phosphate (869.60 mg, 95%, 3.194 mM), and Cs₂CO₃ (1313.81 mg, 99%, 3.992 mM) to a 25 mL reaction flask. Stir at room temperature for 10 min, then heat to 50 °C. Monitor the reaction using high-performance liquid chromatography (HPLC). The reaction is complete after approximately 2.5 h. Pour the reaction solution into a separatory funnel, add 100 mL of ethyl acetate, and wash three times each with 100 mL of 0.1 M HCl, water, and saturated brine. Dry the organic phase with anhydrous sodium sulfate and remove the organic solvent under reduced pressure at 50 °C. The crude product was slurried with 80% acetonitrile for 30 min, filtered, and washed three times with 80% acetonitrile to obtain 519.19 mg of crude di-tert-butylicariin methyl phosphate, with a yield of 23.17%.

[0053] The crude di-tert-butylicariin methyl phosphate (500 mg, 70.1072%, 0.594 mM), 25 mL of dichloromethane, and 445.62 μL of trifluoroacetic acid (99%, 5.939 mM) were added to a 50 mL reaction flask. The mixture was stirred at room temperature for 3 h, and the dichloromethane was removed by concentration under reduced pressure. 5 mL of ethanol was added to the residue, and the pH was adjusted to 8 using 4 M sodium hydroxide solution. The mixture was stirred at room temperature for 20 min, and filtered to obtain 408.47 mg of crude icariin methyl phosphate sodium salt.

[0054] The crude icariin methyl phosphate sodium salt was added to a 25 mL pear-shaped flask, 14 mL of 67% ethanol was added, the temperature was raised to 60 °C, and after the solution was clear, an appropriate amount of activated carbon was added and stirred. The mixture was filtered, allowed to cool naturally to room temperature, and then cooled in an ice bath for 1 h. The filtered product yielded 208.79 mg of icariin methyl phosphate sodium salt (yellowish-white solid, HPLC 97.99%, yield 65.98%).

[0055] The identification information for icariin, di-tert-butylicariin methyl phosphate, and icariin methyl phosphate is shown in Table 1:

[0056] Table 1. Information on icariin, di-tert-butylicariin methyl phosphate, and icariin methyl phosphate.

[0057]

[0058] Test Example 1: Stability of Icariin Sodium Methylphosphate in Aqueous Solution:

[0059] Since prodrugs are designed to be cleaved into the parent drug via chemical or enzyme-mediated pathways, most prodrug solutions cannot be heat-sterilized, and some prodrug solutions are chemically unstable at room temperature. Therefore, the stability of icariin sodium methyl phosphate in aqueous solution at room temperature was investigated.

[0060] Angiotensin sodium methyl phosphate was dissolved in water to prepare a 1 mg / mL solution, and left at room temperature for 24 h. The change in its content was then investigated, and the results are shown in Table 2.

[0061] Table 2. Aqueous stability of icariin methyl phosphate sodium at room temperature

[0062]

[0063] The results showed that icariin sodium methyl phosphate has good stability in aqueous solution at room temperature and has good potential for drug development.

[0064] Test Example 2: Solubility test of icariin sodium methyl phosphate at different pH values:

[0065] The poor water solubility of icariin is a major reason that limits its oral absorption and dosage form diversification. Therefore, examining the solubility of icariin sodium methyl phosphate under different pH conditions is beneficial for predicting the in vivo solubility and absorption of icariin sodium methyl phosphate, and also provides a reference for dosage form development.

[0066] Excessive sodium icariin methyl phosphate was added to aqueous phases with different pH values ​​and shaken at room temperature until equilibrium was reached. The equilibrium solubility of sodium icariin methyl phosphate in aqueous phases with different pH values ​​was obtained, and the results are shown in Table 3.

[0067] Table 3. Solubility of icariin sodium methyl phosphate in aqueous phases at different pH values.

[0068]

[0069] The results showed that the solubility of icariin sodium methyl phosphate in water was pH-dependent, with poor solubility under acidic conditions, greatly improved solubility under neutral and alkaline conditions, and the highest solubility at pH 7.4.

[0070] Test Example 3: Drug metabolism kinetics study in rats:

[0071] The prodrug of icariin methyl phosphate has good water solubility and stability in vitro. In order to investigate its transformation in vivo and improve its bioavailability, male Sprague-Dawley rats were selected for pharmacokinetic experiments.

[0072] The drug was divided into an icariin corn oil suspension group and an icariin sodium methyl phosphate aqueous solution group (the dosage was 10 mg / kg for both groups). Three SD rats were administered the drug by gavage. The rats were fasted overnight but allowed free access to water. Blood samples were collected from the fundus venous plexus at 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, and 8 h after administration. Plasma was collected after centrifugation at 4000 rpm for 10 min for the concentration determination of icariin (ICT), icariin diglucuronide metabolite (G3), and icariin methyl phosphate (3-P-ICT). The results are shown in Table 4. Figure 1 , Figure 2 .

[0073] Table 4. Pharmacokinetic parameters of rats after gavage administration of ICT or 7-P-ICT (n=3)

[0074]

[0075] The results showed that, compared with the group treated with icariin corn oil suspension by gavage, the C of ICT in the plasma of rats in the icariin sodium methyl phosphate prodrug group was significantly lower. max With AUC 0-t All were significantly improved, with bioavailability increasing 23.3 times compared to the ICT gavage group; the AUC of icariin diglucuronide metabolite was also significantly higher. 0-t This also indicates that 3-P-ICT has a high degree of hydrolysis in rats.

Claims

1. The icariin methyl phosphate prodrug of Formula I and its pharmaceutically acceptable salt, the structure of which is shown below:

2. The icariin methyl phosphate prodrug of Formula II and its pharmaceutically acceptable salt, the structure of which is shown below: The pharmaceutically acceptable salt is the sodium, potassium, or calcium salt of the icariin methyl phosphate prodrug, with sodium icariin methyl phosphate being more preferred.

3. The method for preparing the icariin methyl phosphate prodrug according to claim 2 comprises the following steps: Step a): Icariin was dissolved in reaction solvent A, and di-tert-butylchloromethyl phosphate and an acid-binding agent were added. The mixture was stirred and homogenized at room temperature, and the reaction was heated. After the reaction was completed by HPLC monitoring, the reaction solution was cooled and poured into a separatory funnel. Solvent B was added, and the solution was washed successively with 0.1M HCl aqueous solution, water, and saturated saline solution. The organic phase was dried over anhydrous sodium sulfate and the organic solvent was removed under reduced pressure. The crude product was slurried at room temperature using solvent C, filtered, and washed to obtain crude di-tert-butylicariin methyl phosphate. Step b): Dissolve crude di-tert-butylicariin methyl phosphate in reaction solvent D, stir at room temperature in the presence of acid, and remove the reaction solvent under reduced pressure to obtain crude icariin methyl phosphate. Step c): Dissolve crude icariin methyl phosphate in reaction solvent E, adjust the pH with alkaline solution, stir at room temperature, and filter to obtain crude icariin methyl phosphate sodium. Step d): Take crude icariin methyl phosphate sodium, add solvent F and dissolve it at a certain temperature. After the solution is clear, add an appropriate amount of activated carbon and stir. Filter, stir to precipitate crystals, filter, wash with anhydrous ethanol, and dry to obtain refined icariin methyl phosphate sodium.

4. The preparation method according to claim 3, characterized in that, In step a), the reaction solvent A is selected from one or more of DMF, DMSO, tetrahydrofuran, 1,4-dioxane, acetonitrile, and acetone, with DMF being preferred; the volume-to-mass ratio of reaction solvent A to icariin is 5-40:1; and the molar ratio of di-tert-butylchloromethyl phosphate to icariin is 0.8-1.5:

1.

5. The preparation method according to claim 3, characterized in that, In step a), the acid-binding agent includes one or more of sodium hydroxide, potassium hydroxide, cesium carbonate, potassium carbonate, and sodium carbonate, preferably cesium carbonate; the molar ratio of the acid-binding agent to icariin is 1 to 5:1; in step a), solvent B is selected from one or more of ethyl acetate, dichloromethane, methyl tert-butyl ether, or trichloromethane; in step a), solvent C is selected from an 80% aqueous solution of acetonitrile.

6. The preparation method according to claim 3, characterized in that, In step b), the reaction solvent D is selected from one or more of dichloromethane, trichloromethane, or acetonitrile, preferably dichloromethane. The volume-to-mass ratio of reaction solvent D to crude di-tert-butylicariin methyl phosphate is 20–100:1; the acid in step b) is selected from HCl, HBr, or TFA, preferably TFA; the molar ratio of added TFA to di-tert-butylicariin methyl phosphate is 5–20:

1.

7. The preparation method according to claim 3, characterized in that, In step c), the reaction solvent E is selected from one or more of methanol, ethanol, acetone, acetonitrile, isopropanol, and propylene glycol, with ethanol being more preferred. The volume-to-mass ratio of reaction solvent E to crude icariin methyl phosphate is 5–20:1; in step c), the pH is adjusted to 8–9 with 4M sodium hydroxide solution.

8. The preparation method according to claim 3, characterized in that, In step d), solvent F is selected from one or more of methanol, ethanol, acetone, acetonitrile, isopropanol or water, preferably a 60% to 70% aqueous ethanol solution; the volume-to-mass ratio of solvent F to crude icariin methyl phosphate sodium is 20 to 40:

1.

9. The use of the icariin methyl phosphate prodrug according to claim 1 or 2 in the preparation of antitumor drugs.

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