A lanosterol derivative, and a preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, and provides a lanosterol derivative, a preparation method and application thereof.The structural formula of the lanosterol derivative provided by the present application is shown as formula I or formula II.The lanosterol derivative provided by the present application has both fat solubility and water solubility, can effectively penetrate into the pathological site in the body, and improves the drug utilization degree;the results of examples show that the solubility of the lanosterol derivative provided by the present application in water is obviously higher than that of lanosterol or 25-hydroxy lanosterol, and the lanosterol derivative shows obvious cataract treatment effect in animal body experiments, and has a broad prospect in the preparation of drugs for treating eye diseases or conditions.

Description

Technical Field

[0001] This invention 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, the present invention provides a lanosterol derivative, its preparation method, and its application. The lanosterol derivative provided by the present invention has both good water solubility and lipid solubility, can effectively penetrate into the diseased site in the body, and has high pharmaceutical utilization.

[0006] To achieve the above-mentioned objectives, the present invention 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]

[0009] 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.

[0010] In Formulas I and II, when X is oxygen, R and Y1, Y2 and Y3 are not simultaneously hydrogen.

[0011] 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.

[0012] Preferably, R in Formula I and Formula II is hydrogen or hydroxypyridinylformyl group.

[0013] Preferably, the lanosterol derivative is one or more of the following structures:

[0014]

[0015]

[0016]

[0017] This invention 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:

[0018] 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.

[0019]

[0020] 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:

[0021] 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.

[0022]

[0023] 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.

[0024]

[0025] 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.

[0026]

[0027] 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:

[0028] 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.

[0029]

[0030] 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:

[0031] 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.

[0032] 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:

[0033] 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.

[0034] 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:

[0035] 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.

[0036]

[0037] 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:

[0038] Lanosterol, pyridinium chlorochromate, sodium acetate, and solvent were mixed and oxidized to obtain a compound with the structure shown in formula D:

[0039]

[0040] 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.

[0041]

[0042] 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:

[0043] 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.

[0044] Preferably, the method for preparing the compound with the structure shown in Formula A-3 includes the following steps:

[0045] An esterification reaction was carried out by mixing a mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and catalyst. The resulting reaction solution was washed, dried and then rotary evaporated to obtain a crude product. The crude product was a mixture of compounds with the structure shown in Formula A-1 and compounds with the structure shown in Formula B-1.

[0046]

[0047] 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.

[0048]

[0049] 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.

[0050] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient being a lanosterol derivative or a pharmaceutically acceptable salt thereof as described in the above-described scheme.

[0051] Preferably, the pharmaceutically acceptable excipient is a form of excipient.

[0052] The present invention also provides the use of the lanosterol derivatives described above or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described above, in the preparation of medicaments for treating eye diseases or conditions.

[0053] The present invention 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.

[0054] The present invention provides a lanosterol derivative or a pharmaceutically acceptable salt thereof, wherein the structural formula of the lanosterol derivative is shown in Formula I or Formula II. This invention 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 pharmaceutical utilization. Furthermore, some of the compounds provided by this invention contain basic groups, enabling them to form salts with pharmaceutically acceptable acids, further enhancing their solubility in aqueous solutions. Under the action of various hydrolytic enzymes in the body, some of the compounds provided by this invention can release the active pharmaceutical ingredient 25-hydroxylanosterol or lanosterol. 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 by this invention 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 by this invention, 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.

[0055] The results of the examples show that the lanosterol derivative provided by the present invention 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

[0056] Figure 1 The lanosterol derivative with the structure shown in Formula I-1 prepared in Example 1 1 H-NMR spectrum;

[0057] Figure 2 The mass spectrum of the lanosterol derivative with the structure shown in Formula I-1 prepared in Example 1;

[0058] Figure 3 The lanosterol derivatives with the structures shown in Formulas I-8 prepared in Example 8 1 H-NMR spectrum;

[0059] Figure 4 Mass spectra of the lanosterol derivatives with structures of formula I-8 prepared in Example 8;

[0060] Figure 5 The HPLC peak area curve and corresponding regression equation for the lanosterol content in Example 12 are shown.

[0061] Figure 6 The content of 25-hydroxylanosterol in Example 12 is shown in the HPLC peak area curve and the corresponding regression equation.

[0062] Figure 7 The content of compound I-1 in Example 12 is shown as the HPLC peak area curve and the corresponding regression equation.

[0063] Figure 8 The image shows the lens transparency ratings for each group of animals in Example 14.

[0064] Figure 9 This is a screenshot of a Pantcam image taken before drug administration in the model control group of Example 15.

[0065] Figure 10 Screenshots taken with Pantcam before administration of the 0.2% lanosterol derivative I-1 group in Example 15;

[0066] Figure 11 Screenshots taken with Pantcam before administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0067] Figure 12 This is a screenshot taken by Pantcam and analyzed 20 days after drug administration to the model control group in Example 15.

[0068] Figure 13 Screenshots taken with Pantcam after 20 days of administration of the 0.2% lanosterol derivative I-1 group in Example 15;

[0069] Figure 14 Screenshots taken with Pantcam after 20 days of administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0070] Figure 15This is a screenshot taken by Pantcam and analyzed 40 days after drug administration in the model control group of Example 15.

[0071] Figure 16 Screenshots taken with Pantcam after 40 days of administration of the 0.2% lanosterol derivative I-1 group in Example 15;

[0072] Figure 17 Screenshots taken with Pantcam after 40 days of administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0073] Figure 18 This is a screenshot taken by Pantcam and analyzed in Example 15, showing the model control group 60 days after drug administration.

[0074] Figure 19 Screenshots taken with Pantcam after 60 days of administration of the 0.2% lanosterol derivative I-1 group in Example 15;

[0075] Figure 20 Screenshots taken with Pantcam after 60 days of administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0076] Figure 21 These are slit lamp photographs of the animals in each group in Example 15. Detailed Implementation

[0077] This invention provides a lanosterol derivative or a pharmaceutically acceptable salt thereof, characterized in that the lanosterol derivative has the structural formula shown in Formula I or Formula II:

[0078]

[0079] 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.

[0080] In Formulas I and II, when X is oxygen, R and Y1, Y2 and Y3 are not simultaneously hydrogen.

[0081] In this invention, 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.

[0082] In this invention, 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 in the heteroaryl or substituted heteroarylformyl group is preferably pyridyl.

[0083] In this invention, 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:

[0084]

[0085] In this invention, in Formulas I and II, Y1, Y2 and Y3 are preferably hydrogen or deuterium.

[0086] In this invention, 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 (see above for specific structures).

[0087] In this invention, 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.

[0088] The present invention also provides a method for preparing the lanosterol derivative described above. According to the structure of the lanosterol derivative, the preparation method is divided into method one to method eight, which are described below.

[0089] In this invention, 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:

[0090] 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.

[0091]

[0092] In this invention, the method for preparing the compound with the structure shown in Formula A-3 preferably includes the following steps:

[0093] 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;

[0094]

[0095] 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.

[0096]

[0097] 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.

[0098] In this invention, a mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and catalyst are mixed and subjected to esterification reaction. The resulting reaction solution is washed, dried and then the solvent is removed by rotary evaporation to obtain crude product. In this invention, the solvent for the mixed solution of lanosterol and dihydrolanosterol is preferably petroleum ether; in a specific embodiment of this invention, 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 invention, TLC detection is preferably used until the raw material disappears. After the esterification reaction is completed, the present invention preferably cools the resulting reaction solution to room temperature and then washes it; the washing is preferably performed sequentially using hydrochloric acid solution, sodium bicarbonate solution and water; the drying agent is preferably anhydrous sodium sulfate. In the present invention, 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 two have similar structures and are difficult to separate using conventional separation and purification methods. Therefore, the crude product is directly carried out in the next reaction step.

[0099] After obtaining the crude product, the present invention mixes the crude product, N-bromosuccinimide (NBS) and solvent to carry out a bromination reaction. The resulting reaction solution is subjected to rotary evaporation, extraction, drying, rotary evaporation again and silica gel column separation in sequence to obtain a compound with the structure shown in formula A-2. In this invention, the solvent is preferably a mixture of tetrahydrofuran (THF) and water, and the volume ratio of tetrahydrofuran to water in the mixed solvent is preferably 3-5:1, more preferably 4:1; the total molar amount of the compound with structure A-1 and the compound with structure B-1 in the crude product and the molar ratio of NBS are preferably 10:5-6, more preferably 10:5.5; in this invention, the compound with structure B-1 has no unsaturated bonds on its side chain, and the amount of NBS is controlled so that the compound with structure B-1 does not react with NBS. After the bromination reaction is completed, the compound with structure B-1 can be separated by conventional methods; the temperature of the bromination reaction is room temperature, and the reaction time is preferably 1-3 hours, more preferably 2 hours; after the bromination reaction is completed, most of the THF is removed by rotary evaporation, then the residue is diluted with water, extracted with dichloromethane to obtain an organic phase, the organic phase is dried with anhydrous sodium sulfate, and then the solvent is removed by rotary evaporation again. The residue is separated by silica gel column chromatography.

[0100] After obtaining the compound with the structure shown in Formula A-2, the present invention 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 the present invention, 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.

[0101] After obtaining the compound with the structure shown in Formula A-3, the present invention 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 the present invention, 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.

[0102] In this invention, when R in Formula I is a hydroxypyridinylformyl group, 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.

[0103] In this invention, 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:

[0104] 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.

[0105] 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.

[0106] 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.

[0107]

[0108] This invention involves oxidizing a compound with the structure shown in Formula A-3, pyridinium chlorochromate (PCC), sodium acetate, and a solvent to obtain a compound with the structure shown in Formula A-4. In this invention, 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, this invention preferably separates the resulting reaction solution with water 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.

[0109] After obtaining the compound with the structure shown in Formula A-4, the present invention 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 the present invention, 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, the present invention 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.

[0110] After obtaining the compound with the structure shown in Formula A-5, the present invention performs a reduction reaction by mixing the compound with the structure shown in Formula A-5, methanol, and sodium deuterated borohydride to obtain the lanosterol derivative with the structure shown in Formula I-6. In the present invention, the molar ratio of the compound with the structure shown in Formula A-5 to sodium deuterated borohydride is preferably 1.0:0.5 to 1.0; 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, the present invention preferably removes methanol from the obtained reaction solution, then mixes the obtained residue, water, and ethyl acetate for phase separation, removes the solvent by rotary evaporation of the obtained organic phase, 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.

[0111] In this invention, 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:

[0112] 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.

[0113]

[0114] In this invention, 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 1.0; 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.

[0115] In this invention, 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:

[0116] 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.

[0117] In this invention, the types and amounts of condensing agent, catalyst and solvent, as well as the specific conditions of the condensation reaction in method four 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 to replace the compound with the structure shown in formula A-3. This will not be repeated here.

[0118] In this invention, 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:

[0119] 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.

[0120] In this invention, 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.

[0121] In this invention, 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:

[0122] 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.

[0123]

[0124] In this invention, 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 will not be repeated.

[0125] In this invention, 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:

[0126] Lanosterol, pyridinium chlorochromate, sodium acetate, and solvent were mixed and oxidized to obtain a compound with the structure shown in formula D:

[0127] 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.

[0128]

[0129] In this invention, 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.

[0130] In this invention, 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:

[0131] 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.

[0132] In this invention, 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. This will not be elaborated further here.

[0133] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient being a lanosterol derivative or a pharmaceutically acceptable salt thereof as described in the above-described scheme.

[0134] In this invention, the pharmaceutically acceptable excipient is preferably a formulation; this invention does not have any special requirements on the type of excipient, and any excipient well known to those skilled in the art can be used.

[0135] The present invention also provides the use of the lanosterol derivatives described above or pharmaceutically acceptable salts thereof or the pharmaceutical compositions described above in the preparation of medicaments for treating eye diseases or conditions, including cataracts or retinal degeneration.

[0136] The present invention 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.

[0137] In this invention, 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 preferred rotation speed for high-shear emulsification is 10000 r / min, and the preferred time is 5-10 minutes. The preferred pressure for high-pressure homogenization is 240 bar to 2600 bar, and the preferred number of high-pressure homogenization cycles is 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.

[0138] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0139] Example 1

[0140] This embodiment synthesizes the lanosterol derivative with the structure shown in Formula I-1 via the following specific reaction route:

[0141]

[0142] The specific steps are as follows:

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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 Lanosterol derivatives with the structure shown in Formula I-1 1 H-NMR spectrum, Figure 2 The mass spectrum of the lanosterol derivative with the structure shown in Formula I-1 is shown.

[0147] Example 2

[0148]

[0149] 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 A3. 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).

[0150] Example 3

[0151]

[0152] 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 A3. The structural identification data of the product are as follows: 1H NMR (400MHz, Chloroform-d) δ = 7.96 (s1H), 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).

[0153] Example 4

[0154]

[0155] The target compound in Example 4 has the structure shown in Formula I-4. The synthesis method is the same as in Example 1, using 2-hydroxyisonicotinic acid reacted with A3. The structural identification data of the product are as follows: 1 H NMR (400MHz, Chloroform-d) δ = 10.69 (s1H), 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).

[0156] Example 5

[0157]

[0158] 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 A3. The structural identification data of the product are as follows: 1 H NMR (400MHz, Chloroform-d) δ = 12.53 (s1H), 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).

[0159] Example 6

[0160]

[0161] The target compound in Example 6 has the structure shown in Formula I-6, and the synthetic route is as follows:

[0162]

[0163] The specific steps are as follows:

[0164] 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.

[0165] 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.

[0166] 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)).

[0167] Example 7

[0168]

[0169] The target compound in Example 7 has the structure shown in Formula I-7. The synthesis method is as follows:

[0170] 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)).

[0171] Example 8

[0172]

[0173] 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: 1 H-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 Lanosterol derivatives with the structure shown in Formula I-8 1 H-NMR spectrum, Figure 4 The mass spectrum of the lanosterol derivative with the structure shown in Formula I-8 is shown.

[0174] Example 9

[0175]

[0176] The target compound in Example 9 has the structure shown in Formula I-9. The synthesis method followed the steps in Example 1, except that compound I-7 replaced intermediate A-3, and the compound was 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).

[0177] Example 10

[0178]

[0179] 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: 1 H-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).

[0180] Example 11

[0181]

[0182] 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).

[0183] Example 12

[0184] The solubility of lanosterol, 25-hydroxylanosterol, and some of the compounds provided in this invention in water was tested using HPLC, and the specific steps are as follows:

[0185] Step 1: Plot the relationship curve between the content of the analyte and the HPLC peak area (taking lanosterol as an example).

[0186] 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.

[0187] 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.

[0188] Using the same method, the content versus HPLC peak area curves and regression equations of 25-hydroxylanosterol and some compounds provided in this invention were obtained. The content versus HPLC peak area curves and corresponding regression equations of lanosterol are shown below. Figure 5 As shown, Figure 5 The vertical axis represents the measured HPLC peak area, and the horizontal axis represents the corresponding lanosterol content in methanol solution. The HPLC peak area curve and corresponding regression equation for 25-hydroxylanosterol content are shown below. Figure 6 As shown. The content of compound I-1 versus HPLC peak area curve and the corresponding regression equation are shown below. Figure 7 As shown.

[0189] Step 2: Compound Solubility Test. Excess lanosterol, 25-hydroxylanosterol, and a portion of the compounds provided in this invention were placed in 5 mL of purified water. The solutions were placed in a constant temperature water bath at 37±0.5℃ 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.

[0190] Table 1. Solubility of lanosterol and its derivatives in pure water

[0191]

[0192]

[0193] As can be seen from the results in Table 1, the lanosterol derivative provided by the present invention 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.

[0194] Example 13

[0195] 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 invention selected compounds I-6, I-7, and 25-hydroxylanosterol for hepatic microsomal metabolic stability testing. Specifically:

[0196] 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.

[0197] 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.

[0198]

[0199] Table 2. Hepatic microsomal metabolic stability of 25-hydroxylanosterol and deuterated compounds I-6 and I-7

[0200] Sample Name K (speed constant) T1 / 2 (half-life, minutes) CLint (min / mL / μg, clearance rate) 25-Hydroxylanosterol 0.06581 10.53 0.01316 Compound I-7 0.04936 14.11 0.00988 Compound I-6 0.03510 19.85 0.00695

[0201] 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, has 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 indicate that deuteration at appropriate sites in compounds can indeed alter their metabolic stability and further improve their bioavailability.

[0202] Example 14 Experimental Study on the Effects of Lanosterol Derivatives on Sodium Selenite-Induced Cataract Model in New Zealand Rabbits

[0203] 1. Laboratory animals

[0204] New Zealand rabbits, 2.0–3.0 months old, 1.5–2.5 kg, standard grade, both male and female, 32 rabbits available.

[0205] 2. Formulation and preparation process of lanosterol derivatives

[0206] 2.1 Prescription:

[0207] Table 3 Eye Drop Prescriptions

[0208]

[0209]

[0210] 2.2 Preparation process:

[0211] (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.

[0212] (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.

[0213] (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.

[0214] (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.

[0215] (5) Dilute solution IV to 1L with sterile water for injection.

[0216] (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.

[0217] 3. Modeling, grouping, and drug administration

[0218] 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.

[0219] 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 (0.4%), a lanosterol derivative I-1 eye drop group (0.4%), and a lanosterol derivative II-1 (0.4%) eye drop group, with 8 animals in each group, including both males and females.

[0220] 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.

[0221] 4. Indicator Testing

[0222] 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.

[0223] 5. Experimental Results

[0224] (1) Effect on lens opacity grading in cataract model animals

[0225] Table 4. Results of lens opacity grading in a New Zealand rabbit cataract model induced by sodium selenite.

[0226]

[0227]

[0228] Note: Compared with A: * indicates P<0.05, ** indicates P<0.01; compared with D3, ▲P<0.05, ▲▲P<0.01.

[0229] 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 drop group, lanosterol derivative I-1 eye drop group, and lanosterol derivative II-1 eye drop group were significantly reduced at D16 and D23 (P < 0.05 or P < 0.01). Compared with the lanosterol eye drop group, the lens opacity grading conversion scores of animals in the lanosterol derivative I-1 eye drop group and lanosterol derivative II-1 eye drop group showed a significant decreasing trend at D16 and D23, but no statistically significant difference was observed.

[0230] (2) Effect on lens transparency score in cataract model animals

[0231] Table 5. Scoring results of lens transparency in the New Zealand rabbit cataract model induced by sodium selenite.

[0232]

[0233]

[0234]

[0235] Note: Compared with A: * indicates P<0.05, ** indicates P<0.01.

[0236] Figure 8 Transparency rating charts for the lenses of each group of animals.

[0237] 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, but no statistical difference was observed.

[0238] 6. Conclusion

[0239] 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 slightly better improvement than lanosterol eye drops in the New Zealand rabbit cataract model.

[0240] Example 15: Experimental Study on the Effects of Lanosterol Derivatives on Spontaneous Cataracts in Cynomolgus Monkeys

[0241] 1. Laboratory animals

[0242] Crab-eating macaques weigh 4.0–8.0 kg, common grade, both male and female, 12 individuals.

[0243] 2. Screening, grouping, and administration

[0244] 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%) of lanosterol derivative I-1 eye drops, and a high-concentration group (0.4%) 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.

[0245] 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.

[0246] 3. Indicator Testing

[0247] The mean 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), D20, D40, and D60.

[0248] 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).

[0249] 4. Experimental Results

[0250] (1) Effects on lens density in cynomolgus monkeys with spontaneous cataracts

[0251] Table 6. Results of average lens density (Avg) test in cynomolgus monkeys with spontaneous cataracts.

[0252]

[0253] Note: Compared with before administration, ▲P<0.05, ▲▲P<0.01.

[0254] Table 7. Results of Maximum Lens Density (Max) Test in Cynomolgus Monkeys with Spontaneous Cataracts

[0255]

[0256] Note: Compared with before administration, ▲P<0.05, ▲▲P<0.01.

[0257] Figures 9-11 Screenshots of Pantcam images taken of each group of animals before drug administration; Figures 12-14 Screenshots of Pantcam images taken from each group of animals 20 days after drug administration; Figures 15-17 Screenshots of Pantcam images taken from each group of animals 40 days after drug administration; Figures 18-20 Screenshots of Pantcam images taken from each group of animals 60 days after drug administration.

[0258] 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).

[0259] (2) Effect on the scoring of lens opacity in cynomolgus monkeys with spontaneous cataracts

[0260] Table 8. Scoring results of lens opacity in cynomolgus monkeys with spontaneous cataracts.

[0261]

[0262] Note: Compared with before administration, ▲P<0.05, ▲▲P<0.01.

[0263] Figure 21 Take photos of each group of animals under slit lamps.

[0264] 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 (0.2%) and high (0.4%) concentration groups of lanosterol derivative I-1 eye drops were significantly reduced at D20, D40, and D60 (P<0.05 or P<0.01).

[0265] 5. Conclusion

[0266] In summary, both low (0.2%) and high (0.4%) concentrations of lanosterol derivative I-1 eye drops showed significant therapeutic effects on spontaneous cataracts in cynomolgus monkeys, manifested by a reduction in lens density and turbidity scores in the model animals.

[0267] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lanosterol derivative or a pharmaceutically acceptable salt thereof, characterized by, The lanosterol derivative has a structural formula as shown in formula I: Formula I; In formula I, Y1, Y2 and Y3 are independently hydrogen or deuterium; X is oxygen; and R is hydroxypyridylcarbonyl.

2. The lanosterol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, The lanosterol derivative is one or more of the following structures: 。 3. A pharmaceutical composition, characterized by, The active ingredient is the lanosterol derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1-2.

4. The pharmaceutical composition of claim 3, wherein, The pharmaceutically acceptable excipient is an excipient.

5. Use of the lanosterol derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1-2 or the pharmaceutical composition according to claim 3 or 4 in the preparation of a medicament for treating an eye disease or disorder.

6. An eye drop solution, characterized by, The active ingredient is the lanosterol derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1-2.

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