Lanosterol derivative, preparation method therefor and use thereof

Abstract

The present application relates to the technical field of medicine and provides a lanosterol derivative, a preparation method therefor and a use thereof. The structural formula of the lanosterol derivative provided by the present application is as shown in formula I or II. The lanosterol derivative provided by the present application has both lipid solubility and water solubility, and can effectively penetrate into the pathological site in the body, thereby improving the drug utilization rate. The results of embodiments show that the lanosterol derivative provided by the present application has a significantly higher solubility in water than lanosterol or 25-hydroxylanosterol and shows a significant therapeutic effect on a cataract in animal in vivo experiments. Therefore, the derivative has wide prospects in the preparation of drugs for treating eye diseases or conditions.

Description

A lanosterol derivative, its preparation method and application Technical Field

[0001] This application relates to the field of pharmaceutical technology, and in particular to a lanosterol derivative, its preparation method, and its application. Background Technology

[0002] Cataracts are the leading cause of blindness worldwide, making the development of effective treatments crucial. The onset of cataracts is influenced by various factors, such as aging, genetics, local nutritional deficiencies, immune and metabolic abnormalities, prolonged exposure to strong light, smoking, excessive alcohol consumption, malnutrition, and long-term use of corticosteroids. All of these can lead to clouding of the lens in the eye, resulting in visual impairment. Common types include age-related cataracts, complicated cataracts, traumatic cataracts, and metabolic cataracts, with age-related cataracts being the most common. Early symptoms of cataracts are often subtle, but as the clouding deepens, blurred vision, double vision, myopia, and glare may occur, eventually leading to complete blindness.

[0003] Currently, the main treatment for cataracts is surgery, which involves removing the cloudy lens and implanting an artificial lens. While surgery greatly helps patients, its cure rate is far lower than the incidence rate, and complications are possible. Compared to surgery, drug therapy is an indispensable and effective method for treating cataracts, offering advantages such as better patient compliance, lower treatment costs, and fewer side effects. Protein aggregation plays a crucial role in the pathogenesis of cataracts. Recent studies have shown that gene mutations, protein amino acid residue isomerization, deamidation, ubiquitination, ionic interactions, and protein-protein interactions are all causes of protein aggregation. Related technologies report that treatment with lanosterol can reduce the content of protein aggregates, and lanosterol treatment can improve the transparency of isolated rabbit and canine cataract lenses, providing new insights for the development of cataract treatment drugs.

[0004] However, lanosterol is a fat-soluble substance and has poor solubility in water. When using lanosterol to treat cataracts, poor drug absorption is likely to occur after topical administration to the eye. Summary of the Invention

[0005] In view of this, this application provides a lanosterol derivative, its preparation method, and its application. The lanosterol derivative provided in this application has both good water solubility and lipid solubility, can effectively penetrate into the diseased site in the body, and has high pharmaceutical bioavailability.

[0006] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0007] A lanosterol derivative or a pharmaceutically acceptable salt thereof, the structural formula of said lanosterol derivative being shown in Formula I or Formula II:

[0008] In Formulas I and II: Y1, Y2, and Y3 are independently hydrogen, deuterium, C1-C4 alkyl, or unsaturated hydrocarbon groups; X is independently oxygen, sulfur, or an NR1 group, wherein R1 in the NR1 group is hydrogen or an alkyl group; R is independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl.

[0009] In Formulas I and II, when X is oxygen, R and Y1, Y2 and Y3 are not simultaneously hydrogen.

[0010] Preferably, the substituents in the substituted aryl, substituted heteroaryl, substituted arylformyl, and substituted heteroarylformyl groups are independently one or more of halogen, deuterium, hydroxyl, mercapto, and methylthio.

[0011] Preferably, R in Formula I and Formula II is hydrogen or hydroxypyridinylformyl group.

[0012] Preferably, the lanosterol derivative is one or more of the following structures:

[0013] This application also provides a method for preparing the lanosterol derivative described in the above scheme. When X in Formula I is oxygen, Y1, Y2 and Y3 are hydrogen, and R is arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl, the preparation method is Method One, which includes the following steps:

[0014] The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula A-3, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent and carrying out a condensation reaction.

[0015] R-OH form C;

[0016] When X is oxygen, Y1, Y2, and Y3 are deuterium, and R is hydrogen in Formula I, the preparation method is Method II, which includes the following steps:

[0017] The compound with the structure shown in Formula A-3, pyridinium chlorochromate, sodium acetate, and solvent were mixed and subjected to an oxidation reaction to obtain the compound with the structure shown in Formula A-4.

[0018] The compound with the structure shown in Formula A-4, deuterated chloroform, and a catalyst were mixed and subjected to a deuteration reaction to obtain the compound with the structure shown in Formula A-5.

[0019] The compound with the structure shown in Formula A-5, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula I-6.

[0020] When X is oxygen, R, Y1, and Y2 are hydrogen, and Y3 is deuterium in Formula I, the preparation method is Method Three, which includes the following steps:

[0021] The compound having the structure shown in A-4, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula I-7.

[0022] When X is oxygen, Y1, Y2, and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, in Formula I; or when X is oxygen, Y1 and Y2 are hydrogen, Y3 is deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Four, which includes the following steps:

[0023] The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula I-6 or the compound with the structure shown in Formula I-7, the compound with the structure shown in Formula C, a condensing agent, a catalyst and a solvent.

[0024] When X in Formula II is oxygen, Y1, Y2, and Y3 are hydrogen, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Five, which includes the following steps:

[0025] Lanosterol, a compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent are mixed and subjected to a condensation reaction to obtain the lanosterol derivative.

[0026] When X is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen in Formula II, the preparation method is Method Six, which includes the following steps:

[0027] The lanosterol derivative with the structure shown in Formula A-3 was prepared by replacing the compound with lanosterol according to the steps in Method 2, and the lanosterol derivative with the structure shown in Formula B-2 was obtained.

[0028] When X is oxygen, R, Y1, and Y2 are hydrogen, and Y3 is deuterium in Formula II, the preparation method is Method Seven, which includes the following steps:

[0029] Lanosterol, pyridinium chlorochromate, sodium acetate, and solvent were mixed and oxidized to obtain a compound with the structure shown in formula D:

[0030] The compound with the structure shown in Formula D, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula B-3.

[0031] When X in Formula II is oxygen, Y1, Y2, and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, or when X is oxygen, Y1 and Y2 are hydrogen, Y3 is deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Eight, which includes the following steps:

[0032] The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula B-2 or the compound with the structure shown in Formula B-3, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent.

[0033] Preferably, the method for preparing the compound with the structure shown in Formula A-3 includes the following steps:

[0034] An esterification reaction was carried out by mixing a mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and a catalyst to obtain a crude product; the crude product is a mixture of compounds with the structure shown in Formula A-1 and compounds with the structure shown in Formula B-1.

[0035] The crude product, N-bromosuccinimide, and solvent were mixed and subjected to a bromination reaction. The resulting reaction solution was then subjected to rotary evaporation, extraction, drying, rotary evaporation again, and silica gel column separation to obtain a compound with the structure shown in formula A-2.

[0036] The compound with the structure shown in Formula A-2, a reducing agent, and a solvent are mixed and subjected to a debromination-esterification reduction reaction to obtain the compound with the structure shown in Formula A-3.

[0037] This application also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient is a lanosterol derivative or a pharmaceutically acceptable salt thereof as described in the above-described scheme.

[0038] Preferably, the pharmaceutically acceptable excipient is a form of excipient.

[0039] This application also provides the use of the lanosterol derivatives or pharmaceutically acceptable salts thereof described in the above schemes, or the pharmaceutical compositions described in the above schemes, in the preparation of medicaments for treating eye diseases or conditions.

[0040] This application also provides an eye drop comprising the following components in parts by weight: 2-4 parts of active ingredient, 5-7 parts of hydroxypropyl methylcellulose, 30-50 parts of polysorbate, 10-20 parts of boric acid, 1-2 parts of borax, 0.03-0.06 parts of benzalkonium chloride, and 1000 parts of water; wherein the active ingredient is the lanosterol derivative described in the above scheme or a pharmaceutically acceptable salt thereof.

[0041] This application provides a lanosterol derivative or a pharmaceutically acceptable salt thereof, the structural formula of which is shown in Formula I or Formula II. This application modifies the structure of lanosterol or 25-hydroxylanosterol, providing lanosterol derivatives with the structures shown in Formula I or Formula II. These lanosterol derivatives possess both lipid and water solubility, allowing them to effectively penetrate diseased sites in the body and improve drug utilization. Furthermore, some of the compounds provided in this application contain basic groups, enabling them to form salts with pharmaceutically acceptable acids, further enhancing their solubility in aqueous solutions. Some of the compounds provided in this application can release the active pharmaceutical ingredient 25-hydroxylanosterol or lanosterol under the action of various hydrolytic enzymes in the body. The hydroxynicotinic acid or hydroxyisonicotinic acid produced under the action of hydrolytic enzymes also possess certain antioxidant and anti-inflammatory effects. In addition, some of the compounds provided in this application contain deuterium atoms. Primary or secondary alcohol hydroxyl groups are oxidized in humans or animals under the catalysis of various dehydrogenases. The deuterium-containing lanosterol derivatives provided in this application, due to the significantly higher bond energy of the carbon-deuterium bond compared to the carbon-hydrogen bond, can mitigate this reaction and increase the metabolic stability of the compounds.

[0042] The results of the examples show that the lanosterol derivative provided in this application has a significantly higher solubility in water than lanosterol or 25-hydroxylanosterol, and exhibits a significant cataract treatment effect in animal experiments. Attached Figure Description

[0043] Figure 1 shows the lanosterol derivative with the structure shown in Formula I-1 prepared in Example 1. 1 H-NMR spectrum;

[0044] Figure 2 is a mass spectrum of the lanosterol derivative with the structure shown in Formula I-1 prepared in Example 1;

[0045] Figure 3 shows the lanosterol derivatives with structures of formula I-8 prepared in Example 8. 1 H-NMR spectrum;

[0046] Figure 4 shows the mass spectrum of the lanosterol derivatives with the structure shown in Formula I-8 prepared in Example 8;

[0047] Figure 5 shows the HPLC peak area curve and corresponding regression equation of lanosterol content in Example 12;

[0048] Figure 6 shows the HPLC peak area curve and corresponding regression equation of the 25-hydroxylanosterol content in Example 12.

[0049] Figure 7 shows the content of compound I-1 in Example 12 and the HPLC peak area curve and corresponding regression equation;

[0050] Figure 8 shows the lens transparency rating of each group of animals in Example 14;

[0051] Figure 9 shows a screenshot of a Pantcam image taken before drug administration in the model control group of Example 15.

[0052] Figure 10 shows a screenshot of a Pantcam image taken before administration of the 0.2% lanosterol derivative I-1 group in Example 15.

[0053] Figure 11 is a screenshot of Pantcam images taken before administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0054] Figure 12 is a screenshot of Pantcam image analysis taken 20 days after drug administration in the model control group of Example 15;

[0055] Figure 13 is a screenshot of Pantcam image analysis taken 20 days after administration of the 0.2% lanosterol derivative I-1 group in Example 15;

[0056] Figure 14 is a screenshot of Pantcam image analysis taken 20 days after administration of the 0.4% lanosterol derivative I-1 group in Example 15.

[0057] Figure 15 is a screenshot of Pantcam image analysis taken 40 days after drug administration in the model control group of Example 15;

[0058] Figure 16 is a screenshot of Pantcam image analysis taken 40 days after administration of the 0.2% lanosterol derivative I-1 group in Example 15;

[0059] Figure 17 is a screenshot of Pantcam image analysis taken 40 days after administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0060] Figure 18 is a screenshot of Pantcam image analysis taken 60 days after drug administration in the model control group of Example 15;

[0061] Figure 19 is a screenshot of Pantcam image analysis taken 60 days after administration of the 0.2% lanosterol derivative I-1 group in Example 15;

[0062] Figure 20 is a screenshot of Pantcam image analysis taken 60 days after administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0063] Figure 21 shows slit lamp photographs of the animals in each group in Example 15. Detailed Implementation

[0064] This application provides a lanosterol derivative or a pharmaceutically acceptable salt thereof, characterized in that the structural formula of the lanosterol derivative is shown in Formula I or Formula II:

[0065] In Formulas I and II: Y1, Y2, and Y3 are independently hydrogen, deuterium, C1-C4 alkyl, or unsaturated hydrocarbon groups; X is independently oxygen, sulfur, or an NR1 group, wherein R1 in the NR1 group is hydrogen or an alkyl group; R is independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl.

[0066] In Formulas I and II, when X is oxygen, R and Y1, Y2 and Y3 are not simultaneously hydrogen.

[0067] In this application, when R1 in the NR1 group is an alkyl group, the number of carbon atoms in the alkyl group is preferably 1 to 10, more preferably 1 to 5.

[0068] In this application, the substituents in the substituted aryl, substituted heteroaryl, substituted arylformyl, and substituted heteroarylformyl groups are preferably one or more of halogen, deuterium, hydroxyl, mercapto, and methylthio; the heteroaryl group in the heteroaryl or substituted heteroarylformyl group is preferably pyridyl.

[0069] In this application, R in Formula I and Formula II is preferably hydrogen or hydroxypyridinylformyl group, and the hydroxypyridinylformyl group can specifically be any one of the following structures:

[0070] In this application, in Formulas I and II, Y1, Y2, and Y3 are preferably hydrogen or deuterium.

[0071] In this application, the lanosterol derivative is preferably one or more of the structures shown in Formula I-1 to Formula I-9 and Formula II-1 to Formula II-2 (see above for specific structures).

[0072] In this application, the pharmaceutically acceptable salt of the lanosterol derivative can be an organic acid salt or an inorganic acid salt, wherein the organic acid salt preferably includes one or more of acetate, methanesulfonate and tartrate; and the inorganic acid salt preferably includes one or more of hydrochloride, sulfate and phosphate.

[0073] This application also provides a method for preparing the lanosterol derivative described in the above scheme. According to the structure of the lanosterol derivative, the preparation method is divided into method one to method eight, which are described below.

[0074] In this application, when X in Formula I is oxygen, Y1, Y2 and Y3 are hydrogen, and R is arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl, the preparation method is Method 1, which includes the following steps:

[0075] The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula A-3, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent and carrying out a condensation reaction.

[0076] R-OH type C.

[0077] In this application, the method for preparing the compound with the structure shown in Formula A-3 preferably includes the following steps:

[0078] An esterification reaction was carried out by mixing a mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and 4-dimethylaminopyridine, and the resulting reaction solution was washed, dried and then rotary evaporated to obtain a crude product; the crude product is a mixture of compounds with the structure shown in Formula A-1 and compounds with the structure shown in Formula B-1;

[0079] The crude product, N-bromosuccinimide, and solvent were mixed and subjected to a bromination reaction. The resulting reaction solution was then subjected to rotary evaporation, extraction, drying, rotary evaporation again, and silica gel column separation to obtain a compound with the structure shown in formula A-2.

[0080] The compound with the structure shown in Formula A-2, a reducing agent, and a solvent are mixed and subjected to a debromination-esterification reduction reaction to obtain the compound with the structure shown in Formula A-3.

[0081] In this application, a mixed solution of lanosterol and dihydrolanosterol, acetic anhydride, and a catalyst are mixed and subjected to an esterification reaction to obtain a crude product. In this application, the solvent for the mixed solution of lanosterol and dihydrolanosterol is preferably petroleum ether; in a specific embodiment of this application, commercially available lanosterol is used, wherein the mass fraction of lanosterol is 50-60% and the mass fraction of dihydrolanosterol is 40-50%; the ratio of the total molar amount of lanosterol and dihydrolanosterol to the volume of acetic anhydride is preferably 23-24 mmol:3-4 mL, more preferably 23.47 mmol:3.85 mL; the catalyst used for the esterification reaction is preferably 4-(N,N-dimethyl)aminopyridine (DMAP); the molar ratio of the total molar amount of lanosterol and dihydrolanosterol to 4-(N,N-dimethyl)aminopyridine is preferably 9.5-10.5:1; the esterification reaction is preferably carried out under reflux conditions, and the esterification reaction time is preferably 3-5 h, more preferably 4 h; in a specific embodiment of this application, TLC detection is preferably used until the raw material disappears. After the esterification reaction is completed, this application preferably washes and dries the resulting reaction solution, then removes the solvent by rotary evaporation. Specifically, this application preferably cools the resulting reaction solution to room temperature before washing; the washing is preferably performed sequentially using hydrochloric acid solution, sodium bicarbonate solution, and water; the drying agent is preferably anhydrous sodium sulfate. In this application, the crude product is a mixture of compounds with the structure shown in Formula A-1 and compounds with the structure shown in Formula B-1. Since their structures are similar and conventional separation and purification methods are difficult to use, this crude product is directly used in the next reaction step.

[0082] After obtaining the crude product, this application mixes the crude product, N-bromosuccinimide (NBS), and solvent to carry out a bromination reaction. The resulting reaction solution is then subjected to rotary evaporation, extraction, drying, further rotary evaporation, and silica gel column separation to obtain a compound with the structure shown in formula A-2. In this application, the solvent is preferably a mixture of tetrahydrofuran (THF) and water, with a volume ratio of tetrahydrofuran to water of 3-5:1, more preferably 4:1. The total molar amount of the compound with the structure shown in formula A-1 and the compound with the structure shown in formula B-1 in the crude product, and the molar ratio of NBS, are preferably 10:5-6, more preferably 10:5.5. In this application, the compound with the structure shown in formula B-1 has no unsaturated bonds on its side chains. This application controls the amount of NBS to prevent the compound with the structure shown in formula B-1 from reacting with NBS. After the bromination reaction is completed, The compound with the structure shown in Formula B-1 can be separated by conventional methods; the bromination reaction is carried out at room temperature, and the reaction time is preferably 1 to 3 hours, more preferably 2 hours; after the bromination reaction is completed, most of the THF is removed by rotary evaporation, and then the residue is diluted with water and extracted with dichloromethane to obtain an organic phase. The organic phase is then dried with anhydrous sodium sulfate, and the solvent is removed by rotary evaporation again. The residue is then separated by silica gel column chromatography. The eluent used for silica gel column chromatography is a mixed solvent of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate in the mixed solvent is preferably 5:1.

[0083] After obtaining the compound with the structure shown in Formula A-2, this application mixes the compound with the structure shown in Formula A-2, a reducing agent, and a solvent to carry out a debromination-esterification reduction reaction to obtain the compound with the structure shown in Formula A-3. In this application, the reducing agent is preferably LiAlH4, and the solvent is preferably THF; the molar ratio of the compound with the structure shown in Formula A-2 to the reducing agent is preferably 1:1.5 to 2.5, more preferably 1:2; the temperature of the debromination-esterification reduction reaction is preferably 60 to 65°C, more preferably 63°C, and the reaction time is preferably 3 to 5 h, more preferably 4 h. After the debromination-esterification reduction reaction is completed, the reaction is preferably quenched with water, the resulting reaction solution is extracted with dichloromethane, the resulting organic phase is dried with anhydrous sodium sulfate, the solvent is removed by rotary evaporation, and the residue is separated by silica gel column chromatography to obtain the compound with the structure shown in Formula A-3.

[0084] After obtaining the compound with the structure shown in Formula A-3, this application mixes the compound with the structure shown in Formula A-3, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent to carry out a condensation reaction to obtain the lanosterol derivative. In this application, the condensing agent is preferably dicyclohexylcarbodiimide (DCC), and the molar ratio of the compound with the structure shown in Formula A-3 to the condensing agent is preferably 1:2 to 3, more preferably 3:8; the catalyst used in the condensation reaction is preferably DMAP, and the molar ratio of the compound with the structure shown in Formula A-3 to the catalyst is preferably 2:1; the solvent is preferably dichloromethane.

[0085] In this application, when R in Formula I is hydroxypyridinylformyl, the compound with the structure shown in Formula C is preferably hydroxynicotinic acid, specifically 6-hydroxynicotinic acid, 2-hydroxynicotinic acid, 2-hydroxyisonicotinic acid, 5-hydroxynicotinic acid, or 3-hydroxynicotinic acid; the molar ratio of the compound with the structure shown in Formula A-3 to the compound with the structure shown in Formula C is preferably 1:2 to 2.5, more preferably 1:2; the temperature of the condensation reaction is preferably room temperature, and the reaction time is preferably 20 to 24 hours, more preferably 20 hours; after the condensation reaction is completed, the obtained reaction solution is preferably desolventized, and the residue is separated by silica gel column chromatography to obtain the lanosterol derivative.

[0086] In this application, when X is oxygen, Y1, Y2, and Y3 are deuterium, and R is hydrogen in Formula I, the preparation method is Method II, which includes the following steps:

[0087] The compound with the structure shown in Formula A-3, pyridinium chlorochromate, sodium acetate, and solvent were mixed and subjected to an oxidation reaction to obtain the compound with the structure shown in Formula A-4.

[0088] The compound with the structure shown in Formula A-4, deuterated chloroform, and a catalyst were mixed and subjected to a deuteration reaction to obtain the compound with the structure shown in Formula A-5.

[0089] The compound with the structure shown in Formula A-5, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula I-6.

[0090] This application involves mixing a compound with the structure shown in Formula A-3, pyridinium chlorochromate (PCC), sodium acetate, and a solvent to undergo an oxidation reaction to obtain a compound with the structure shown in Formula A-4. In this application, the solvent is preferably dichloromethane; the molar ratio of the compound with the structure shown in Formula A-3 to PCC is preferably 1:1; the molar ratio of the compound with the structure shown in Formula A-3 to sodium acetate is preferably 1:0.5–0.6; the oxidation reaction is preferably carried out at room temperature, monitored by TLC, and continues until the reactants disappear; after the oxidation reaction is complete, this application preferably mixes the resulting reaction solution with water and separates them to obtain an organic phase and an aqueous phase. The aqueous phase is extracted with dichloromethane, and the extracted organic phase and the separated organic phase are combined and dried with anhydrous sodium sulfate. The solvent is then removed by rotary evaporation, and the crude product is separated by silica gel column chromatography to obtain a compound with the structure shown in Formula A-4.

[0091] After obtaining the compound with the structure shown in Formula A-4, this application mixes the compound with the structure shown in Formula A-4, deuterated chloroform, and a catalyst to carry out a deuteration reaction to obtain the compound with the structure shown in Formula A-5. In this application, the catalyst used for the deuteration reaction is preferably 1,5,7-triazabicyclo[4.4.0]decen-5-ene (TBD); the molar ratio of the compound with the structure shown in Formula A-4 to the catalyst is preferably 1:0.1; the temperature of the deuteration reaction is preferably room temperature, and the reaction time is preferably 12 h; after the deuteration reaction is completed, this application preferably removes the solvent by rotary evaporation of the obtained reaction solution, and purifies the obtained crude product by column chromatography to obtain the compound with the structure shown in Formula A-5.

[0092] After obtaining the compound with the structure shown in Formula A-5, this application mixes the compound with the structure shown in Formula A-5, methanol, and sodium deuterated borohydride for a reduction reaction to obtain the lanosterol derivative with the structure shown in Formula I-6. In this application, the molar ratio of the compound with the structure shown in Formula A-5 to sodium deuterated borohydride is preferably 1:0.5 to 1; the temperature of the reduction reaction is preferably 0°C to room temperature, and the reaction time is preferably 1 to 3 hours; after the reduction reaction is completed, this application preferably removes the methanol from the resulting reaction solution, then mixes the obtained residue, water, and ethyl acetate for phase separation, removes the solvent from the obtained organic phase by rotary evaporation, and then separates the obtained crude product by silica gel column chromatography to obtain the lanosterol derivative with the structure shown in Formula I-6.

[0093] In this application, when X is oxygen, R, Y1, and Y2 are hydrogen, and Y3 is deuterium in Formula I, the preparation method is Method Three, which includes the following steps:

[0094] The compound with the structure shown in A-4, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula I-7.

[0095] In this application, the molar ratio of the compound with the structure shown in Formula A-4 to sodium deuterated borohydride is preferably 1.0:0.5 to 1; the temperature of the reduction reaction is preferably 0°C to room temperature, and the reaction time is preferably 1 to 3 hours; the post-treatment method after the reduction reaction is completed is the same as the post-treatment method of the reduction reaction in Method 2, and will not be described again here.

[0096] In this application, when X in Formula I is oxygen, Y1, Y2, and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, or when X is oxygen, Y1 and Y2 are hydrogen, Y3 is deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Four, which includes the following steps:

[0097] The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula I-6 or the compound with the structure shown in Formula I-7, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent.

[0098] In this application, the types and amounts of condensing agent, catalyst and solvent in method four, as well as the specific conditions of the condensation reaction, are the same as those in method one. Only the compound with the structure shown in formula I-6 or the compound with the structure shown in formula I-7 can be used instead of the compound with the structure shown in formula A-3. This will not be repeated here.

[0099] In this application, when X in Formula II is oxygen, Y1, Y2, and Y3 are hydrogen, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Five, which includes the following steps:

[0100] The lanosterol, a compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent are mixed and subjected to a condensation reaction to obtain the lanosterol derivative.

[0101] In this application, the types and amounts of condensing agent, catalyst and solvent in method five, as well as the specific conditions of the condensation reaction, are the same as those in method one. Only lanosterol is used to replace the compound with the structure shown in formula A-3, which will not be repeated here.

[0102] In this application, when X in Formula II is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen, the preparation method is Method Six, which includes the following steps:

[0103] The lanosterol derivative with the structure shown in Formula A-3 was prepared by replacing the compound with lanosterol according to the steps in Method 2, and the lanosterol derivative with the structure shown in Formula B-2 was obtained.

[0104] In this application, the specific operating conditions of method six are the same as those of method two, except that the compound with the structure shown in formula A-3 is replaced by lanosterol is subjected to an oxidation reaction, the product obtained from the oxidation reaction is replaced by a deuteration reaction, and the product obtained from the reduction reaction is replaced by a reduction reaction. The specific conditions of the oxidation reaction, deuteration reaction and reduction reaction are not described in detail.

[0105] In this application, when X in Formula II is oxygen, R, Y1, and Y2 are hydrogen, and Y3 is deuterium, the preparation method is Method Seven, which includes the following steps:

[0106] Lanosterol, pyridinium chlorochromate, sodium acetate, and solvent were mixed and oxidized to obtain a compound with the structure shown in formula D:

[0107] The compound with the structure shown in Formula D, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula B-3.

[0108] In this application, the oxidation reaction in method seven is under the same conditions as the oxidation reaction in method two, except that lanosterol is used to replace the compound with the structure shown in formula A-3; the reduction reaction in method seven is under the same conditions as the reduction reaction in method three, except that the compound with the structure shown in formula D is used to replace the compound with the structure shown in formula A-4.

[0109] In this application, when X in Formula II is oxygen, Y1, Y2, and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, or when X is oxygen, Y1 and Y2 are hydrogen, Y3 is deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Eight, which includes the following steps:

[0110] The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula B-2 or the compound with the structure shown in Formula B-3, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent.

[0111] In this application, the conditions for the condensation reaction in method eight are the same as those in method one, except that the compound with the structure shown in formula B-2 or the compound with the structure shown in formula B-3 is used instead of the compound with the structure shown in formula A-3, which will not be described in detail here.

[0112] This application also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient is a lanosterol derivative or a pharmaceutically acceptable salt thereof as described in the above-described scheme.

[0113] In this application, the pharmaceutically acceptable excipient is preferably a formulation; this application does not have any special requirements on the type of formulation, and any formulation well known to those skilled in the art can be used.

[0114] This application also provides the use of the lanosterol derivatives or pharmaceutically acceptable salts thereof described in the above schemes, or the pharmaceutical compositions described in the above schemes, in the preparation of medicaments for treating eye diseases or conditions, including cataracts or retinal degeneration.

[0115] This application also provides an eye drop comprising the following components in parts by weight: 2-4 parts of active ingredient, 5-7 parts of hydroxypropyl methylcellulose, 30-50 parts of polysorbate, 10-20 parts of boric acid, 1-2 parts of borax, 0.03-0.06 parts of benzalkonium chloride, and 1000 parts of water. Preferably, the active ingredient comprises 2 or 4 parts by weight, the hydroxypropyl methylcellulose comprises 6 parts by weight, the polysorbate comprises 40 parts by weight, the boric acid comprises 16 parts by weight, the borax comprises 1.6 parts by weight, and the benzalkonium chloride comprises 0.05 parts by weight. The active ingredient is a lanosterol derivative or a pharmaceutically acceptable salt thereof as described above. The polysorbate is preferably polysorbate 80. The water is preferably sterile water for injection.

[0116] In this application, the preferred method for preparing the eye drops includes: dissolving hydroxypropyl methylcellulose in a portion of water to obtain solution I; dissolving boric acid, borax, and benzalkonium chloride in a portion of water, mixing the resulting solution with solution I, then adding polysorbate, and then adding water until the weight of water in the system is 85-90% of the total water content in the prescription to obtain solution II; adding the active ingredient to solution II for high-shear emulsification to obtain solution III; homogenizing solution III under high pressure to obtain solution IV; and finally adding the remaining water to bring solution IV to a final volume. The rotation speed of the high-shear emulsification is preferably 10000 r / min, and the time is preferably 5-10 minutes. The pressure of the high-pressure homogenization is preferably 240 bar to 2600 bar, and the number of high-pressure homogenizations is preferably once. After high-pressure homogenization, the high-pressure homogenizer pipeline is preferably rinsed with an appropriate amount of water, the rinsing liquid is collected, and mixed with the drug solution.

[0117] The technical solutions of this application will be clearly and completely described below with reference to the embodiments therein. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0118] Example 1

[0119] This embodiment synthesizes the lanosterol derivative with the structure shown in Formula I-1 via the following specific reaction route:

[0120] The specific steps are as follows:

[0121] Step 1, Preparation of Intermediate A-1: ​​In 100 mL of a petroleum ether solution of lanosterol (10 g of a mixture of lanosterol and dihydrolanosterol, 23.47 mmol, containing 50% lanosterol), DMAP (2.35 mmol) and acetic anhydride (3.85 mL) were added. The reaction was refluxed for 4 h, and TLC was performed until the starting material disappeared. The mixture was then cooled to room temperature. The product was washed successively with 2 × 10 mL of 5 wt% hydrochloric acid, 2 × 10 mL of 10 wt% sodium bicarbonate, and 2 × 20 mL of water, and dried over anhydrous sodium sulfate. After removing the solvent by rotary evaporation, the crude product was directly used in the next reaction step.

[0122] Step 2, Preparation of Intermediate A-2: The mixture of the above crude products—A1 and B1 (50%, 10 mmol)—was dissolved in THF / H2O (240 mL / 60 mL), and NBS (5.5 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 2 h. Most of the THF was removed by rotary evaporation, diluted with water, and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 5:1, v / v) to obtain intermediate A-2.

[0123] Step 3, Preparation of Intermediate A-3: Intermediate A-2 (10 mmol) was added to a round-bottom flask, and a LiAlH4 THF solution (20 mmol) was added at 0 °C. Under nitrogen protection, the mixture was heated to 63 °C for 4 h. After cooling to room temperature, the reaction was quenched with 1.6 mL of water, followed by the addition of 150 mL of dichloromethane and 50 mL of water. The organic phase was separated, and the aqueous phase was extracted twice with 50 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The crude product was separated by silica gel column chromatography (ethyl acetate: petroleum ether = 5:1, v / v) to obtain intermediate A-3.

[0124] Step 4: Preparation of the target compound, formula I-1: Intermediate A-3 (3 mmol), DMAP (1.5 mmol), 6-hydroxynicotinic acid (6 mmol), and DCC (8 mmol) were added to a round-bottom flask under nitrogen protection. 40 mL of anhydrous dichloromethane was added at 0 °C, and the reaction was carried out at room temperature for 20 h. The solvent was removed. The residue was directly separated by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1, v / v) to obtain the lanosterol derivative with the structure shown in formula I-1. The structural identification data of the product are as follows: 1H-NMR (400MHz, CDCl3) δ8.15(d,J=2.5Hz,1H),8.00(dd,J=9.6,2.5Hz,1H),6.58(d,J=9.6Hz,1H ),4.70(dd,J=11.5,4.3Hz,1H),2.04(d,J=14.7Hz,3H),1.94(d,J=12.4Hz,2H),1.81-1.76(m,2 H),1.73-1.66(m,4H),1.53-1.33(m,9H),1.28(d,J=19.4Hz,2H),1.22(s,7H),1.11(d,J=10.2H z,2H),1.04(s,3H),0.98(s,3H),0.92(d,J=1.9Hz,5H),0.89(s,3H),0.70(s,3H).Ms:566.28(M + +1). Figure 1 shows the lanosterol derivative with the structure shown in Formula I-1. 1 Figure 2 shows the H-NMR spectrum of the lanosterol derivative with the structure shown in Formula I-1.

[0125] Example 2

[0126] The target compound in Example 2 has the structure shown in Formula I-2. The synthesis method was the same as in Example 1, except that 2-hydroxynicotinic acid was reacted with A-3. The structural identification data of the obtained product are as follows: 1 H NMR (400MHz, Chloroform-d) δ = 8.19 (d, J = 9.62Hz, 1H), 6.85 (m, 1H), 7.57 (d, J = 8.65Hz, 1H), 4.52-4.32 (m, 1H), 4.13 (s , 1H), 3.43(s, 1H), 2.01-1.56(m, 13H), 1.60-1.51(m, 19H), 1.02(s, 3H), 1.07(s, 3H), 0.87-0.83(m, 8H), 0.79(s, 3H). Ms:566.26(M + +1).

[0127] Example 3

[0128] The target compound in Example 3 has the structure shown in Formula I-3. The synthesis method is the same as in Example 1, involving the reaction of 5-hydroxynicotinic acid with A-3. The structural identification data of the product are as follows: 1H NMR (400MHz, Chloroform-d) δ = 7.96 (s 1H), 8.03 (s, 1H), 8.17 (s, 1H), 4.52-4.32 (m, 1H), 4.25 (s, 1H), 1.91-1.59 (m, 13H ), 1.58-1.48(m, 19H), 1.05(s, 3H), 1.03(s, 3H), 0.88-0.82(m, 8H), 0.81(s, 3H). Ms:566.26(M + +1).

[0129] Example 4

[0130] The target compound in Example 4 has the structure shown in Formula I-4. The synthesis method is the same as in Example 1, involving the reaction of 2-hydroxyisonicotinic acid with A-3. The structural identification data of the product are as follows: 1 H NMR (400MHz, Chloroform-d) δ = 10.69 (s 1H), 7.93 (d, J=8.39Hz, 1H), 7.19 (d, J=7.35Hz, 1H), 4.41-4.30 (m, 1H), 4.39 (s, 1H), 1.98-1 .63 (m, 13H), 1.61-1.52 (m, 19H), 1.05 (s, 3H), 1.05 (s, 3H), 0.90-0.84 (m, 8H), 0.83 (s, 3H). Ms:566.26(M + +1).

[0131] Example 5

[0132] The target compound in Example 5 has the structure shown in Formula I-5. The synthesis method is the same as in Example 1, using 3-hydroxynicotinic acid reacted with A-3. The structural identification data of the product are as follows: 1 H NMR (400MHz, Chloroform-d) δ = 12.53 (s 1H), 8.35 (s,), 7.89 (d, J = 8.69Hz, 1H), 7.97 (d, J = 9.01Hz, 1H) 4.42-4.33 (m, 1H), 4.38 (s, 1H), 1. 99-1.65 (m, 13H), 1.61-1.55 (m, 19H), 1.08 (s, 3H), 1.07 (s, 3H), 0.91-0.85 (m, 8H), 0.85 (s, 3H). Ms:566.26(M + +1).

[0133] Example 6

[0134] The target compound in Example 6 has the structure shown in Formula I-6, and the synthetic route is as follows:

[0135] The specific steps are as follows:

[0136] Step 1: Dissolve compound A-3 (1 mmol) in 20 mL of dichloromethane, add 215 mg PCC and 45 mg NaOAc. Stir at room temperature and monitor the reaction by TLC until the starting material disappears. After the reaction is complete, add 10 mL of water, separate the organic phase, and extract the aqueous phase with dichloromethane. Dry the obtained organic phase with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and separate the crude product by silica gel column chromatography to obtain intermediate A-4.

[0137] Step 2: Dissolve intermediate A-4 (1 mmol) in 15 mL of deuterated chloroform, add catalyst TBD (1 mmol), stir at room temperature for 12 h, remove solvent by rotary evaporation, and purify the crude product by column chromatography to obtain intermediate A-5.

[0138] Step 3: Intermediate A-5 (1 mmol) was dissolved in methanol, and sodium deuterated borohydride (1 mmol) was added under ice bath conditions. The reaction was carried out for 1 hour. After the reaction was complete, methanol was removed, and the residue was added to water and ethyl acetate with stirring. The organic phase was separated, the solvent was removed by rotary evaporation, and silica gel column chromatography was used to separate deuterated 25-hydroxylanosterol I-6. The structural identification data are as follows: Ms: 413.7 (M + -34(2OH)).

[0139] Example 7

[0140] The target compound in Example 7 has the structure shown in Formula I-7. The synthesis method is as follows:

[0141] Following the synthesis steps in Example 6, intermediate A-4 was prepared. Intermediate A-4 (1 mmol) was then dissolved in 15 mL of methanol, and sodium deuterated borohydride (1 mmol) was added under ice bath conditions, followed by a reaction for 1 hour. The structural identification data of the product are as follows: Ms: 411.7 (M + -34(2OH)).

[0142] Example 8

[0143] The target compound in Example 8 has the structure shown in Formula I-9. The synthesis method follows the steps in Example 1, except that compound I-6 replaces intermediate A-3, and the compound is reacted with 6-hydroxynicotinic acid in the presence of dehydrating agent DCC and catalyst DMAP. The product structure identification data are as follows: 1H-NMR (400MHz, CDCl3) δ8.17(d,J=2.5Hz,1H),8.01(dd,J=9.6,2.5Hz,1H),6.59(d,J=9.6Hz,1H),2.10-2.00(m,4H),1.75(dd,J=17. 9,11.1Hz,3H),1.58-1.34(m,10H),1.26(s,6H),1.22(s,6H),1.04(s,3H),0.98(s,3H),0.93-0.88(m,9H),0.70(s,3H).Ms: 569.3(M + +1). Figure 3 shows the lanosterol derivatives with the structure shown in Formula I-8. 1 Figure 4 shows the H-NMR spectrum of the lanosterol derivative with the structure shown in Formula I-8.

[0144] Example 9

[0145] The target compound in Example 9 has the structure shown in Formula I-9. The synthesis method followed the steps in Example 1, using compound I-7 instead of intermediate A-3, and reacted with 6-hydroxynicotinic acid in the presence of dehydrating agent DCC and catalyst DMAP. The structural identification data of the product are as follows: 1 H NMR (400MHz, CDCl3) δ8.17(d,J=2.6Hz,1H),8.01(dd,J=9.6,2.5Hz,1H),6.58(d,J=9.6Hz,1H),2.04(d,J=7.7Hz,4H),1.97-1.90(m,3H),1 .81-1.75(m,2H),1.54-1.30(m,12H),1.26(dd,J=5.7,3.1Hz,3H),1.22(s,7H),1.04(s,3H),0.98(s,3H),0.94-0.86(m,9H),0.70(s,3H). Ms:567.3(M + +1).

[0146] Example 10

[0147] The target compound in Example 10 has the structure shown in Formula II-1. The synthesis method followed the steps in Example 1, using lanosterol as the starting material instead of intermediate A-3, and reacting it directly with 6-hydroxynicotinic acid in the presence of the dehydrating agent DCC. The structural identification data of the product are as follows: 1H-NMR (400MHz, CDCl3) δ8.15(s,1H),7.99(dd,J=9.6,2.5Hz,1H),6.57(d,J=9.6Hz,1H),4.6 9(dd,J=11.5,4.4Hz,1H),3.47(d,J=10.5Hz,1H),2.04(s,3H),1.93(d,J=13.0Hz,2H),1.83 -1.66(m,6H),1.59-1.43(m,4H),1.43-1.35(m,4H),1.33(s,2H),1.21(s,7H),1.16-1.06(m ,2H),1.03(s,3H),0.97(s,3H),0.91(d,J=7.6Hz,5H),0.89(s,3H),0.70(s,3H).Ms:548.3(M + +1).

[0148] Example 11

[0149] The target compound in Example 11 has the structure shown in Formula II-2. The synthesis method followed some steps from Example 6, using lanosterol as a starting material, which was oxidized by PCC, reduced with sodium deuterated borohydride, and then reacted with 6-hydroxynicotinic acid in the presence of the dehydrating agent DCC. The structural identification data of the product are as follows: Ms: 549.3 (M + +1).

[0150] Example 12

[0151] The solubility of lanosterol, 25-hydroxylanosterol, and some of the compounds provided in this application in water was determined by HPLC, and the specific steps are as follows:

[0152] Step 1: Plot the relationship curve between the content of the compound to be tested and the HPLC peak area (taking lanosterol as an example).

[0153] Analytical conditions: Column, Agilent 959990-902 (4.6×250mm, 5μm), column temperature, 35℃; mobile phase, methanol; flow rate, 1.0mL / min; detection wavelength, 210nm; injection volume, 20μL; elution mode, isocratic elution.

[0154] Weigh approximately 10.00 mg of lanosterol and place it in a 100 mL volumetric flask. Add an appropriate amount of methanol, sonicate to dissolve, and dilute to the mark with methanol. Shake well to obtain a stock solution for plotting the content of each compound versus HPLC peak area curves. Pipette an appropriate amount of the above stock solution into a 25 mL volumetric flask, dilute to volume with methanol, and shake well to obtain lanosterol solutions of different concentrations. Inject the solutions into the chromatogram according to the chromatographic conditions under "Analytical Conditions" and record the peak area. Plot the peak area (y) on the ordinate and the concentration (x) on the abscissa to establish a regression equation: y = 11639032.6320x - 8583.6303, with a correlation coefficient R. 2 =0.9997; the experimental results show that the linear relationship is good in the concentration range of 0.1000 mg / mL to 0.0025 mg / mL.

[0155] Using the same method, the content and HPLC peak area curves and regression equations of 25-hydroxylanosterol and some compounds provided in this application were obtained. The content and HPLC peak area curve of lanosterol and the corresponding regression equation are shown in Figure 5, where the vertical axis represents the measured HPLC peak area, and the horizontal axis represents the corresponding lanosterol content in methanol solution. The content and HPLC peak area curve of 25-hydroxylanosterol and the corresponding regression equation are shown in Figure 6. The content and HPLC peak area curve of compound I-1 and the corresponding regression equation are shown in Figure 7.

[0156] Step 2: Compound Solubility Test. Excess lanosterol, 25-hydroxylanosterol, and a portion of the compounds provided in this application were placed in 5 mL of purified water. The solutions were placed in a constant temperature water bath at 37 ± 0.5 °C and magnetically stirred (500 rpm), maintaining a solid excess throughout the experiment. After 6 h, the solutions were removed, centrifuged at 10000 rpm for 5 min, and the supernatant was collected. After passing through a 0.22 μm polyethersulfone filter membrane, each sample was analyzed by HPLC according to the methods described above. The experimental results are shown in Table 1.

[0157] Table 1. Solubility of lanosterol and its derivatives in pure water

[0158] As can be seen from the results in Table 1, the lanosterol derivative provided in this application has significantly higher solubility in water than lanosterol or 25-hydroxylanosterol due to the addition of polar groups in the molecule, which improves its water solubility.

[0159] Example 13

[0160] Hepatic microsomal metabolic stability testing of 25-hydroxylanosterol, compounds I-6 and I-7. Since carbon-deuterium bonds are more stable than carbon-hydrogen bonds, replacing hydrogen atoms with deuterium atoms at readily metabolizable sites in drug moleculehoods may alter the drug's metabolic rate, resulting in a longer half-life, lower clearance, and better bioavailability. Considering that the hydrogen atom on the carbon containing the secondary hydroxyl group in 25-hydroxylanosterol is a potential metabolic site, and since drug metabolism primarily occurs in the liver, this application selected compounds I-6, I-7, and 25-hydroxylanosterol for hepatic microsomal metabolic stability testing. Specifically:

[0161] Step 1: Determination of the standard curve. Accurately weigh appropriate amounts of the analyte samples 25-hydroxylanosterol and deuterated 25-hydroxylanosterol, and dissolve them in calculated-purity acetonitrile to achieve a concentration of 1 mg / mL. The standard curve range is selected as 0, 2, 4, 6, 8, and 10 μM / L. Plot the standard curve based on the peak areas obtained from HPLC analysis.

[0162] Step 2: Prepare the test system (using a 0.5 mg / mL liver microsomal protein system) for HPLC analysis. Thaw liver microsomes stored at -80℃ in an ice bath. Add the analyte (1 mg / mL), NADPHA solution (10 μL), and solution B (2 μL) together and mix well. Incubate at 37℃ for 10 minutes, then place on ice. Mix liver microsomes (5 μL) with 0.1M PBS buffer (181 μL) and the two analyte solutions (2 μL) and place on ice. Finally, add to the NADPH mixture and incubate at 37℃. Set sampling time points at 0 min, 5 min, 10 min, 15 min, 20 min, 30 min, 60 min, 90 min, 120 min, and 240 min (two replicates for each time point). At the set incubation time points, add an equal volume of pre-cooled acetonitrile to the incubation system to terminate the reaction. After mixing, the samples were centrifuged at 12500 rpm for 10 minutes at 4℃. The supernatant was filtered through a microporous membrane and analyzed by HPLC. The half-life and scavenging rate of each analyte were calculated based on the peak area and the standard curve. A negative control group was used without NADPHA solution and solution B. A blank control group was used containing only the analytes and 0.1 M PBS buffer. The HPLC conditions were: column temperature: 25℃; mobile phase: 100% methanol isocratic elution; flow rate: 0.80 mL / min; detection wavelength: 210 nm; injection volume: 20 μL. The test results of 25-hydroxylanosterol and deuterated compounds I-6 and I-7 are shown in Table 2.

[0163] Table 2. Hepatic microsomal metabolic stability of 25-hydroxylanosterol and deuterated compounds I-6 and I-7

[0164] The results of the liver microsomal metabolic stability test in the table show that deuteration of the hydrogen atom on the carbon containing the secondary hydroxyl group of 25-hydroxylanosterol significantly improves its metabolic stability. Among the compounds, compound I-6, with both the hydrogen atom on the hydroxyl group and the adjacent carbon deuterated, exhibits the longest half-life (19.85 min) and the lowest in vitro clearance rate, while the undeuterated 25-hydroxylanosterol has the shortest half-life (10.53 min) and the highest in vitro clearance rate. These results demonstrate that deuteration at appropriate sites in compounds can indeed alter their metabolic stability and further improve their bioavailability.

[0165] Example 14 Experimental Study on the Effects of Lanosterol Derivatives on Sodium Selenite-Induced Cataract Model in New Zealand Rabbits

[0166] 1. Laboratory animals

[0167] New Zealand rabbits, 2.0–3.0 months old, 1.5–2.5 kg, standard grade, both male and female, 32 rabbits available.

[0168] 2. Formulation and preparation process of lanosterol derivatives

[0169] 2.1 Prescription:

[0170] Table 3 Eye Drop Prescriptions

[0171] 2.2 Preparation process:

[0172] (1) First, dissolve hydroxypropyl methylcellulose in about 10% boiling water for injection, add an appropriate amount of cold water for injection, stir until completely dissolved, and obtain solution I.

[0173] (2) Dissolve the prescribed amounts of boric acid, borax and benzalkonium chloride in an appropriate amount of water for injection and add them to solution I. While stirring, slowly add the prescribed amount of polysorbate 80 and add water for injection to about 90% of the prescribed amount. Stir until homogeneous to obtain solution II.

[0174] (3) Weigh out the prescribed amount of lanosterol, compound I-1 or II-1 and add it to solution II. Use a high-shear emulsifier at a speed of 10000r / min to disperse for 5-10 minutes until the dispersion is uniform, and obtain solution III.

[0175] (4) Place solution III in a high-pressure homogenizer and homogenize it once at a pressure of 250 bar ± 10 bar. Collect the drug solution and rinse the high-pressure homogenizer pipeline with an appropriate amount of sterile water for injection. Collect the rinsing solution and mix it with the drug solution to obtain solution IV.

[0176] (5) Dilute solution IV to 1L with sterile water for injection.

[0177] (6) Samples were taken to test pH, osmotic pressure and content. After passing the test, the samples were dispensed in a sterile environment using low-density polyethylene pharmaceutical eye drop bottles, with 5 ml of medicine in each bottle.

[0178] 3. Modeling, grouping, and drug administration

[0179] 3.1 Modeling: 32 New Zealand rabbits with clear and normal lenses were selected for modeling. After anesthetizing the animals, 0.1 mL of 10 mM sodium selenite solution was slowly injected into the anterior chamber. The day of injection was recorded as D0.

[0180] 3.2 Grouping: On day 3, animals with successful modeling were randomly and evenly grouped according to the degree of lens opacity grade and sex. They were divided into a model control group, a lanosterol eye drop group (0.4wt%), a lanosterol derivative I-1 eye drop group (0.4wt%), and a lanosterol derivative II-1 (0.4wt%) eye drop group, with 8 animals in each group, including both males and females.

[0181] 3.3 Drug administration: On day 4, the right eye was treated with eye drops according to the set requirements, 3 times a day, with an interval of about 3 hours each time, for 21 consecutive days. The left eye was not treated and served as the normal control.

[0182] 4. Indicator Testing

[0183] Slit-lamp photography was used to grade and score the ophthalmic images before administration and on days 13 and 20 (D3, D16, and D23). On day 24, about 2 hours after the second ophthalmic drop, the animal's eyeball was dissected, and the lens, including the capsule, was completely separated. The lens was placed on graph paper and photographed to show the clarity of the squares photographed through the lens, and the clarity of the lens was scored.

[0184] 5. Experimental Results

[0185] (1) Effect on lens opacity grading in cataract model animals

[0186] Table 4. Results of lens opacity grading in a New Zealand rabbit cataract model induced by sodium selenite ( n=8) Note: Compared with A: * indicates P<0.05, ** indicates P<0.01; compared with D3, ▲P<0.05, ▲▲P<0.01.

[0187] The lens opacity grading results in Table 4 show that, compared with the model control group and before drug administration (D3), the lens opacity grading conversion scores of animals in the lanosterol eye drops group, lanosterol derivative I-1 eye drops group, and lanosterol derivative II-1 eye drops group were significantly reduced at D16 and D23 (P < 0.05 or P < 0.01); compared with the lanosterol eye drops group, the lens opacity grading conversion scores of animals in the lanosterol derivative I-1 eye drops group and lanosterol derivative II-1 eye drops group showed a significant decreasing trend at D16 and D23.

[0188] (2) Effect on lens transparency score in cataract model animals

[0189] Table 5. Scoring results of lens transparency in the New Zealand rabbit cataract model induced by sodium selenite ( n=8) Note: Compared with A: * indicates P<0.05, ** indicates P<0.01.

[0190] Figure 8 shows the lens transparency rating for each group of animals.

[0191] The lens transparency scores in Table 5 show that, compared with the model control group, the lens transparency scores of the lanosterol eye drops group, the lanosterol derivative I-1 eye drops group, and the lanosterol derivative II-1 eye drops group were all significantly lower (P < 0.05 or P < 0.01); compared with the lanosterol eye drops group, the lens transparency score of the lanosterol derivative I-1 eye drops group showed a significant decreasing trend.

[0192] 6. Conclusion

[0193] In summary, lanosterol eye drops, lanosterol derivative I-1 eye drops, and lanosterol derivative II-1 eye drops significantly improved the cataract model in New Zealand rabbits induced by sodium selenite, as evidenced by a reduction in lens opacity and lens transparency scores. At the same concentration, lanosterol derivative I-1 and lanosterol derivative II-1 eye drops showed superior improvement compared to lanosterol eye drops in the New Zealand rabbit cataract model.

[0194] Example 15: Experimental Study on the Effects of Lanosterol Derivatives on Spontaneous Cataracts in Cynomolgus Monkeys

[0195] 1. Laboratory animals

[0196] Crab-eating macaques weigh 4.0–8.0 kg, common grade, both male and female, 12 individuals.

[0197] 2. Screening, grouping, and administration

[0198] Screening: The opacity of the lenses in both eyes of the cynomolgus monkeys was examined by slit-lamp photography. Twelve cynomolgus monkeys with spontaneous cataracts were screened and randomly divided into three groups: a model control group, a low-concentration group (0.2 wt%) of lanosterol derivative I-1 eye drops, and a high-concentration group (0.4 wt%) of lanosterol derivative I-1 eye drops. Each group contained four animals and eight eyes. The day of animal enrollment was recorded as D0. The formulation and preparation method of the eye drops were the same as in Example 14.

[0199] Administration: After grouping, administer eye drops to both eyes as set, 4 times a day, with an interval of about 3 hours between each administration, for 60 consecutive days.

[0200] 3. Indicator Testing

[0201] The average density (Avg) and maximum density (Max) of the lens of each group of cynomolgus monkeys were measured using a Pentacam HR anterior segment analyzer before administration (D0), on D20, D40, and D60.

[0202] Slit-lamp photography was used to collect lens images of each group of animals before administration (D0), D20, D40, and D60. Finally, the lens images of cynomolgus monkeys taken by slit lamp were scored according to the Clinical Lens Opacity Grading System III (LOCS III).

[0203] 4. Experimental Results

[0204] (1) Effects on lens density in cynomolgus monkeys with spontaneous cataracts

[0205] Table 6. Results of the average lens density (Avg) test in cynomolgus monkeys with spontaneous cataracts. n=8) Note: Compared with before administration, ▲P<0.05, ▲▲P<0.01.

[0206] Table 7. Results of the test on the maximum lens density (Max) in cynomolgus monkeys with spontaneous cataracts. n=8) Note: Compared with before administration, ▲P<0.05, ▲▲P<0.01.

[0207] Figures 9-11 are screenshots of Pantcam images taken by animals in each group before drug administration; Figures 12-14 are screenshots of Pantcam images taken by animals in each group 20 days after drug administration; Figures 15-17 are screenshots of Pantcam images taken by animals in each group 40 days after drug administration; Figures 18-20 are screenshots of Pantcam images taken by animals in each group 60 days after drug administration.

[0208] The lens density results in Tables 6 and 7 show that, compared with before drug administration (D0), there were no significant differences in the average lens density (Avg) and maximum lens density (Max) at D20, D40, and D60 in the model control group (P > 0.05). However, the average lens density (Avg) and maximum lens density (Max) at D20, D40, and D60 were significantly reduced in the low (0.2%) and high (0.4%) concentration groups of lanosterol derivative I-1 eye drops (P < 0.05 or P < 0.01).

[0209] (2) Effect on the scoring of lens opacity in cynomolgus monkeys with spontaneous cataracts

[0210] Table 8. Scoring results of lens opacity in cynomolgus monkeys with spontaneous cataracts ( n=8) Note: Compared with before administration, ▲P<0.05, ▲▲P<0.01.

[0211] Figure 21 shows slit lamp photographs of each group of animals.

[0212] The results of the lens opacity scoring in Table 8 show that, compared with before drug administration (D0), there were no significant differences in the lens opacity scores of the model control group animals at D20, D40, and D60 (P>0.05). However, the lens opacity scores of the animals in the low (0.2wt%) and high (0.4wt%) concentration groups of lanosterol derivative I-1 eye drops were significantly reduced at D20, D40, and D60 (P<0.05 or P<0.01).

[0213] 5. Conclusion

[0214] In summary, low (0.2wt%) and high (0.4wt%) concentrations of lanosterol derivative I-1 eye drops have significant therapeutic effects on spontaneous cataracts in cynomolgus monkeys, manifested by reducing lens density and turbidity scores in the model animals.

[0215] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A lanosterol derivative or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the lanosterol derivative is shown in Formula I or Formula II: In Formulas I and II: Y1, Y2, and Y3 are independently hydrogen, deuterium, C1-C4 alkyl, or unsaturated hydrocarbon groups; X is independently oxygen, sulfur, or an NR1 group, wherein R1 in the NR1 group is hydrogen or an alkyl group; R is hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl. In Formulas I and II, when X is oxygen, R, Y1, Y2, and Y3 are not all hydrogen at the same time.

2. The lanosterol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The substituents in the substituted aryl, substituted heteroaryl, substituted arylformyl, and substituted heteroarylformyl groups are independently one or more of halogen, deuterium, hydroxyl, mercapto, and methylthio.

3. The lanosterol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, In Formulas I and II, R is hydrogen or hydroxypyridinylformyl group.

4. The lanosterol derivative or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, The hydroxypyridinyl formyl group is any one of the following structures:

5. The lanosterol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The lanosterol derivative is any one of the following structures:

6. The lanosterol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The pharmaceutically acceptable salts of the lanosterol derivatives are organic or inorganic acid salts, wherein the organic acid salts include one or more of acetate, methanesulfonate, and tartrate; and the inorganic acid salts include one or more of hydrochloride, sulfate, and phosphate.

7. The method for preparing the lanosterol derivative according to any one of claims 1 to 6, characterized in that, When X in Formula I is oxygen, Y1, Y2, and Y3 are hydrogen, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method One, which includes the following steps: The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula A-3, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent and carrying out a condensation reaction. R-OH form C; When X is oxygen, Y1, Y2, and Y3 are deuterium, and R is hydrogen in Formula I, the preparation method is Method II, which includes the following steps: The compound with the structure shown in Formula A-3, pyridinium chlorochromate, sodium acetate, and solvent were mixed and subjected to an oxidation reaction to obtain the compound with the structure shown in Formula A-4. The compound with the structure shown in Formula A-4, deuterated chloroform, and a catalyst were mixed and subjected to a deuteration reaction to obtain the compound with the structure shown in Formula A-5. The compound with the structure shown in Formula A-5, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula I-6. When X is oxygen, R, Y1, and Y2 are hydrogen, and Y3 is deuterium in Formula I, the preparation method is Method Three, which includes the following steps: The compound having the structure shown in A-4, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula I-7. When X is oxygen, Y1, Y2, and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, in Formula I; or when X is oxygen, Y1 and Y2 are hydrogen, Y3 is deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Four, which includes the following steps: The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula I-6 or the compound with the structure shown in Formula I-7, the compound with the structure shown in Formula C, a condensing agent, a catalyst and a solvent. When X in Formula II is oxygen, Y1, Y2, and Y3 are hydrogen, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Five, which includes the following steps: Lanosterol, a compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent are mixed and subjected to a condensation reaction to obtain the lanosterol derivative. When X is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen in Formula II, the preparation method is Method Six, which includes the following steps: The lanosterol derivative with the structure shown in Formula A-3 was prepared by replacing the compound with lanosterol according to the steps in Method 2, and the lanosterol derivative with the structure shown in Formula B-2 was obtained. When X is oxygen, R, Y1, and Y2 are hydrogen, and Y3 is deuterium in Formula II, the preparation method is Method Seven, which includes the following steps: Lanosterol, pyridinium chlorochromate, sodium acetate, and solvent were mixed and oxidized to obtain a compound with the structure shown in formula D: The compound with the structure shown in Formula D, methanol, and sodium deuterated borohydride were mixed and reduced to obtain the lanosterol derivative with the structure shown in Formula B-3. When X in Formula II is oxygen, Y1, Y2, and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, or when X is oxygen, Y1 and Y2 are hydrogen, Y3 is deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl, or substituted heteroarylformyl, the preparation method is Method Eight, which includes the following steps: The lanosterol derivative is obtained by mixing the compound with the structure shown in Formula B-2 or the compound with the structure shown in Formula B-3, the compound with the structure shown in Formula C, a condensing agent, a catalyst, and a solvent.

8. The preparation method according to claim 7, characterized in that, The preparation method of the compound with the structure shown in Formula A-3 includes the following steps: An esterification reaction was carried out by mixing a mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and a catalyst to obtain a crude product; the crude product is a mixture of compounds with the structure shown in Formula A-1 and compounds with the structure shown in Formula B-1. The crude product, N-bromosuccinimide, and solvent were mixed and subjected to a bromination reaction. The resulting reaction solution was then subjected to rotary evaporation, extraction, drying, rotary evaporation again, and silica gel column separation to obtain a compound with the structure shown in Formula A-2. The compound with the structure shown in Formula A-2, a reducing agent, and a solvent are mixed and subjected to a debromination-esterification reduction reaction to obtain the compound with the structure shown in Formula A-3.

9. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient is a lanosterol derivative as described in any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutically acceptable excipients are excipients.

11. The use of the lanosterol derivative of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 or 8 in the preparation of a medicament for treating an eye disease or condition.

12. The application according to claim 11, characterized in that, The eye diseases mentioned include cataracts or retinal degeneration.

13. An eye drop, characterized in that, The product comprises the following components in parts by weight: 2-4 parts of active ingredient, 5-7 parts of hydroxypropyl methylcellulose, 30-50 parts of polysorbate, 10-20 parts of boric acid, 1-2 parts of borax, 0.03-0.06 parts of benzalkonium chloride, and 1000 parts of water; wherein the active ingredient is a lanosterol derivative as described in any one of claims 1-6 or a pharmaceutically acceptable salt thereof.

14. The eye drops according to claim 13, characterized in that, The polysorbate is polysorbate 80.

15. A method for treating an eye disease, characterized in that, Treatment is performed via ocular administration; the ocular administration uses a lanosterol derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 7, a pharmaceutical composition as described in claim 9 or 10, or an eye drop as described in claim 13 or 14.

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