Lanosterol derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, and provides a lanosterol derivative as well as a preparation method and application thereof. The lanosterol derivative provided by the invention has a structural formula as shown in a formula I or a formula II. The lanosterol derivative provided by the invention has both fat solubility and water solubility, and can effectively permeate into a diseased region in a body, so that the medicinal availability is improved; results of the embodiment show that the solubility of the lanosterol derivative provided by the invention in water is obviously higher than that of lanosterol or 25-hydroxylanosterol, and the lanosterol derivative shows an obvious cataract treatment effect in animal in-vivo experiments and has a wide prospect in preparation of drugs for treating eye diseases or symptoms. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a lanosterol derivative and a preparation method and application thereof. Background Art

[0002] Cataract is the world's leading cause of blindness in ophthalmology, and the development of effective treatments for cataracts is of great significance. The onset of cataracts is affected by many factors, such as aging, heredity, local nutritional disorders, immune and metabolic abnormalities, prolonged exposure to strong light, smoking and alcohol abuse, malnutrition, long-term use of glucocorticoids, etc., which can cause opacity in the lens of the eyeball and visual impairment. Common types include senile cataracts, concurrent cataracts, traumatic cataracts, metabolic cataracts, etc., among which senile cataracts are the most common. The early symptoms of cataracts are not obvious, but as the degree of lens opacity deepens, symptoms such as blurred vision, diplopia, myopia, and glare will appear. Continuing development will lead to complete blindness of the patient.

[0003] At present, the main treatment for cataracts is surgery, which includes removing the cloudy lens and implanting an artificial lens. Although surgical treatment provides great help to patients, the cure rate of surgical treatment is much lower than the incidence rate, and there may be complications. Compared with surgical treatment, drug treatment of cataracts is an indispensable and effective method, with the advantages of good patient compliance, low treatment cost and few side effects. Protein aggregation plays an important role in the pathogenesis of cataracts. Studies in recent years have shown that gene mutations, protein amino acid residue isomerization, deamidation, ubiquitination, ion effects and protein interactions are all causes of protein aggregation. It is reported in related technologies that the content of protein aggregates can be reduced by lanosterol treatment, and lanosterol treatment can improve the transparency of isolated rabbit eye cataract lenses and dog cataract lenses, providing new ideas for the development of cataract treatment drugs.

[0004] However, lanosterol is a fat-soluble substance with poor solubility in water. When lanosterol is used 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 and a preparation method and application thereof. The lanosterol derivative provided by the present invention has good water solubility and fat solubility, can effectively penetrate into the diseased part of the body, and has high medicinal utilization.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0008]

[0009] In Formula I and Formula II: Y1, Y2 and Y3 are independently hydrogen, deuterium, C1-C4 alkyl or unsaturated hydrocarbon group; X is independently oxygen, sulfur or NR1 group, R1 in the NR1 group is hydrogen or alkyl; R is independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl;

[0010] In Formula I and Formula II, when X is oxygen, R and Y1, Y2 and Y3 are not hydrogen at the same time.

[0011] Preferably, the substituents in the substituted aryl, substituted heteroaryl, substituted arylformyl and substituted heteroarylformyl are independently one or more of halogen, deuterium, hydroxyl, thiol and methylthio.

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

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

[0014]

[0015]

[0016]

[0017] The present invention also provides a method for preparing the lanosterol derivatives described in the above scheme. When X in formula I is oxygen, Y1, Y2 and Y3 are hydrogen, and R is an arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl, the preparation method is method 1, and the method 1 comprises the following steps:

[0018] The compound with the structure shown in formula A-3, the compound with the structure shown in formula C, a condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative;

[0019]

[0020] When X in Formula I is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen, the preparation method is Method 2, which comprises the following steps:

[0021] A compound having a structure represented by formula A-3, pyridinium chlorochromate, sodium acetate and a solvent are mixed and subjected to an oxidation reaction to obtain a compound having a structure represented by formula A-4;

[0022]

[0023] The compound of the structure represented by the formula A-4, deuterated chloroform and a catalyst are mixed to carry out a deuteration reaction to obtain a compound of the structure represented by the formula A-5;

[0024]

[0025] The compound of the structure represented by the formula A-5, methanol and sodium deuterated borohydride are mixed for reduction reaction to obtain a lanosterol derivative of the structure represented by the formula I-6;

[0026]

[0027] When X in Formula I is oxygen, R, Y1 and Y2 are hydrogen, and Y3 is deuterium, the preparation method is Method 3, which comprises the following steps:

[0028] The compound having the structure shown in formula A-4, methanol and sodium deuterated borohydride are mixed and subjected to reduction reaction to obtain a lanosterol derivative having the structure shown in formula I-7;

[0029]

[0030] When X in Formula I is oxygen, Y1, Y2 and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl, or 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 4, which comprises the following steps:

[0031] 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 condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative;

[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 5, which comprises the following steps:

[0033] Mixing lanosterol, a compound having a structure shown in formula C, a condensation agent, a catalyst and a solvent for condensation reaction to obtain the lanosterol derivative;

[0034] When X in formula II is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen, the preparation method is method 6, which comprises the following steps:

[0035] The compound of the structure shown in formula A-3 is replaced by lanosterol, and the lanosterol derivative of the structure shown in formula B-2 is prepared according to the steps in method 2;

[0036]

[0037] When X in formula II is oxygen, R, Y1 and Y2 are hydrogen, and Y3 is deuterium, the preparation method is method 7, and the method 7 comprises the following steps:

[0038] Lanosterol, pyridinium chlorochromate, sodium acetate and a solvent are mixed and subjected to oxidation reaction to obtain a compound with a structure shown in formula D:

[0039]

[0040] The compound having the structure shown in formula D, methanol and sodium deuterated borohydride are mixed for reduction reaction to obtain a lanosterol derivative having 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 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 VIII, which comprises the following steps:

[0043] 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 condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative.

[0044] Preferably, the method for preparing the compound of the structure shown in formula A-3 comprises the following steps:

[0045] A mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and a catalyst are mixed to carry out an esterification reaction, and the obtained reaction solution is washed, dried and then rotary evaporated to obtain a crude product; the crude product is a mixture of a compound with a structure shown in formula A-1 and a compound with a structure shown in formula B-1;

[0046]

[0047] The crude product, N-bromosuccinimide and a solvent are mixed to carry out a bromination reaction, and 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 a structure shown in specific formula A-2;

[0048]

[0049] The compound with the structure shown in formula A-2, a reducing agent and a solvent are mixed to carry out a debromination-ester reduction reaction to obtain a compound with the structure shown in formula A-3.

[0050] The present invention also provides a pharmaceutical composition, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient is the lanosterol derivative described in the above scheme or a pharmaceutically acceptable salt thereof.

[0051] Preferably, the pharmaceutically acceptable excipient is an excipient.

[0052] The present invention also provides the use of the lanosterol derivative or a pharmaceutically acceptable salt thereof or the pharmaceutical composition described in the above scheme in the preparation of a drug for treating eye diseases or disorders.

[0053] The present invention also provides an eye drop comprising the following components in parts by mass: 2 to 4 parts of an active ingredient, 5 to 7 parts of hydroxypropyl methylcellulose, 30 to 50 parts of polysorbate, 10 to 20 parts of boric acid, 1 to 2 parts of borax, 0.03 to 0.06 parts of benzalkonium chloride, and 1000 parts of water; the active ingredient is the lanosterol derivative or a pharmaceutically acceptable salt thereof described in the above scheme.

[0054] The present invention provides a lanosterol derivative or a pharmaceutically acceptable salt thereof. The structural formula of the lanosterol derivative is shown in Formula I or Formula II. The present invention improves the structure of lanosterol or 25-hydroxylanosterol, and provides lanosterol derivatives of the structure shown in Formula I or Formula II. The lanosterol derivatives provided by the present invention are both fat-soluble and water-soluble, can effectively penetrate into the diseased part in the body, and improve the medicinal utilization; further, some of the compounds provided by the present invention contain basic groups in the molecules, which can form salts with pharmaceutically acceptable acids, and further improve their solubility in aqueous solutions; some of the compounds provided by the present invention can release the active pharmaceutical ingredient 25-hydroxylanosterol or lanosterol under the action of various hydrolases in the body, and the hydroxynicotinic acid or hydroxyisonicotinic acid produced under the action of the hydrolases also has certain antioxidant and anti-inflammatory effects; in addition, some of the compounds provided by the present invention contain deuterium atoms in their structures, and the primary or secondary alcohol hydroxyl groups will be oxidized under the catalysis of various dehydrogenases in the human or animal body. The deuterium-containing lanosterol derivatives provided by the present invention can alleviate this reaction and increase the metabolic stability of the compound because the bond energy of the carbon-deuterium bond is much higher than that of the carbon-hydrogen bond.

[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 in vivo experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The lanosterol derivative of 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 derivative of the structure shown in formula I-8 prepared in Example 8 1 H-NMR spectrum;

[0059] Figure 4 The mass spectrum of the lanosterol derivative with the structure shown in formula I-8 prepared in Example 8;

[0060] Figure 5 is the lanosterol content and HPLC peak area curve and the corresponding regression equation in Example 12;

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

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

[0063] Figure 8 This is a lens transparency scoring diagram for each group of animals in Example 14;

[0064] Fig. 9 This is a screenshot of the Pantcam photography and analysis of the model control group in Example 15 before administration;

[0065] Fig.10 This is a screenshot of the Pantcam photography and analysis of the 0.2% lanosterol derivative I-1 group before administration in Example 15;

[0066] Fig.11 This is a screenshot of the Pantcam photography and analysis before administration of the 0.4% lanosterol derivative I-1 group in Example 15;

[0067] Fig.12 This is a screenshot of the Pantcam photography and analysis of the model control group in Example 15 after 20 days of administration;

[0068] Fig.13 This is a screenshot of the Pantcam photography and analysis of the 0.2% lanosterol derivative I-1 group in Example 15 after 20 days of administration;

[0069] Fig.14 This is a screenshot of the Pantcam photography and analysis of the 0.4% lanosterol derivative I-1 group in Example 15 after 20 days of administration;

[0070] Fig.15This is a screenshot of the Pantcam photography and analysis of the model control group in Example 15 after 40 days of drug administration;

[0071] Fig.16 This is a screenshot of the Pantcam photography and analysis of the 0.2% lanosterol derivative I-1 group in Example 15 after 40 days of administration;

[0072] Fig.17 This is a screenshot of the Pantcam photography and analysis of the 0.4% lanosterol derivative I-1 group in Example 15 after 40 days of administration;

[0073] Fig.18 This is a screenshot of the Pantcam photography and analysis of the model control group in Example 15 after 60 days of administration;

[0074] Fig.19 This is a screenshot of the Pantcam photography and analysis of the 0.2% lanosterol derivative I-1 group in Example 15 after 60 days of administration;

[0075] Fig. 20 This is a screenshot of the Pantcam photography and analysis of the 0.4% lanosterol derivative I-1 group in Example 15 after 60 days of administration;

[0076] Fig.21 These are slit lamp photos of each group of animals in Example 15. DETAILED DESCRIPTION

[0077] The present invention provides a lanosterol derivative or a pharmaceutically acceptable salt thereof, characterized in that the structural formula of the lanosterol derivative is as shown in Formula I or Formula II:

[0078]

[0079] In Formula I and Formula II: Y1, Y2 and Y3 are independently hydrogen, deuterium, C1-C4 alkyl or unsaturated hydrocarbon group; X is independently oxygen, sulfur or NR1 group, R1 in the NR1 group is hydrogen or alkyl; R is independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl;

[0080] In Formula I and Formula II, when X is oxygen, R and Y1, Y2 and Y3 are not hydrogen at the same time.

[0081] In the present invention, when R1 in the NR1 group is an alkyl group, the number of carbon atoms of the alkyl group is preferably 1-10, more preferably 1-5.

[0082] In the present invention, the substituents in the substituted aryl, substituted heteroaryl, substituted arylcarboxyl and substituted heteroarylcarboxyl are independently preferably one or more of halogen, deuterium, hydroxyl, thiol and methylthio; the heteroaryl in the heteroaryl or substituted heteroarylcarboxyl is preferably pyridyl.

[0083] In the present invention, R in Formula I and Formula II is preferably hydrogen or hydroxypyridylcarbonyl, and the hydroxypyridylcarbonyl may be any one of the following structures:

[0084]

[0085] In the present invention, in Formula I and Formula II, Y1, Y2 and Y3 are independently preferably hydrogen or deuterium.

[0086] In the present 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 the present invention, the pharmaceutically acceptable salt of the lanosterol derivative may 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 derivatives described in the above scheme. According to the structure of the lanosterol derivatives, the preparation methods are divided into method one to method eight, which are described below respectively.

[0089] In the present 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 1, which comprises the following steps:

[0090] The compound with the structure shown in formula A-3, the compound with the structure shown in formula C, a condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative;

[0091]

[0092] In the present invention, the method for preparing the compound of the structure represented by formula A-3 preferably comprises the following steps:

[0093] A mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and 4-dimethylaminopyridine are mixed for esterification reaction, and the obtained reaction solution is washed, dried and then rotary evaporated to obtain a crude product; the crude product is a mixture of a compound with a structure shown in formula A-1 and a compound with a structure shown in formula B-1;

[0094]

[0095] The crude product, N-bromosuccinimide and a solvent are mixed to carry out a bromination reaction, and 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 a structure shown in specific formula A-2;

[0096]

[0097] The compound with the structure shown in formula A-2, a reducing agent and a solvent are mixed to carry out a debromination-ester reduction reaction to obtain a compound with the structure shown in formula A-3.

[0098] The invention comprises the following steps: mixing a mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and a catalyst for esterification reaction; washing and drying the obtained reaction solution, and then removing the solvent by rotary evaporation to obtain a crude product. In the present invention, the solvent of the mixed solution of lanosterol and dihydrolanosterol is preferably petroleum ether; in a specific embodiment of the present invention, commercial lanosterol is used, wherein the mass fraction of lanosterol is 50-60%, and the fraction of dihydrolanosterol is 40-50%; the volume ratio of the total molar amount of lanosterol and dihydrolanosterol to acetic anhydride is preferably 23-24mmol:3-4mL, more preferably 23.47mmol:3.85mL; the catalyst used in 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 3-5h, more preferably 4h; in a specific embodiment of the present invention, TLC detection is preferably used until the raw material disappears. After the esterification reaction is completed, the present invention preferably cools the obtained reaction solution to room temperature and then washes it; the washing is preferably carried out in sequence using hydrochloric acid solution, sodium bicarbonate solution and water; the drying agent used in the drying is preferably anhydrous sodium sulfate. In the present invention, the crude product is a mixture of a compound of the structure shown in formula A-1 and a compound of the structure shown in formula B-1. The two have similar structures and are difficult to separate by conventional separation and purification methods. Therefore, the crude product is directly subjected to the next step of reaction.

[0099] After obtaining the crude product, the present invention mixes the crude product, N-bromosuccinimide (NBS) and a solvent to carry out a bromination reaction, and the obtained reaction solution is successively subjected to rotary evaporation, extraction, drying, rotary evaporation again and silica gel column separation to obtain a compound with a structure shown in specific formula A-2. In the present invention, the solvent is preferably a mixed solvent of tetrahydrofuran (THF) and water, and the volume ratio of tetrahydrofuran to water in the mixed solvent is preferably 3 to 5:1, more preferably 4:1; the molar ratio of the total molar amount of the compound of the structure shown in formula A-1 and the compound of the structure shown in formula B-1 in the crude product to NBS is preferably 10:5 to 6, more preferably 10:5.5; in the present invention, there is no unsaturated bond on the side chain of the compound of the structure shown in formula B-1, and the present invention controls the amount of NBS so that the compound of the structure shown in formula B-1 does not react with NBS. After the bromination reaction is completed, the compound of the structure shown in formula B-1 can be separated by conventional methods; the temperature of the bromination reaction is room temperature, and the reaction time is preferably 1 to 3 hours, more preferably 2 hours; after the bromination reaction is completed, the present invention removes most of the THF by rotary evaporation, and then dilutes the residue with water, extracts with dichloromethane to obtain an organic phase, and then dries the organic phase with anhydrous sodium sulfate, and then rotary evaporation is performed again to remove the solvent, and the residue is separated by silica gel column.

[0100] After obtaining the compound of the structure shown in formula A-2, the present invention mixes the compound of the structure shown in formula A-2, a reducing agent and a solvent to perform a debromination-ester reduction reaction to obtain a compound of 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 of the structure shown in formula A-2 and the reducing agent is preferably 1:1.5-2.5, more preferably 1:2; the temperature of the debromination-ester reduction reaction is preferably 60-65°C, more preferably 63°C, and the reaction time is preferably 3-5h, more preferably 4h. After the debromination-ester reduction reaction is completed, it is preferably quenched with water, the obtained reaction solution is extracted with dichloromethane, the obtained organic phase is dried with anhydrous sodium sulfate, and then the solvent is removed by rotary evaporation, and the residue is separated by silica gel column chromatography to obtain a compound of the structure shown in formula A-3.

[0101] After obtaining the compound of formula A-3, the present invention mixes the compound of formula A-3, the compound of formula C, a condensing agent, a catalyst and a solvent for 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 of 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 of formula A-3 to the catalyst is preferably 2:1; the solvent is preferably dichloromethane.

[0102] In the present invention, when R in Formula I is hydroxypyridylcarbonyl, the compound of the structure represented by 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 of the structure represented by Formula A-3 to the compound of the structure represented by 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 a silica gel column to obtain the lanosterol derivative.

[0103] In the present invention, when X in Formula I is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen, the preparation method is Method 2, which comprises the following steps:

[0104] A compound having a structure represented by formula A-3, pyridinium chlorochromate, sodium acetate and a solvent are mixed and subjected to an oxidation reaction to obtain a compound having a structure represented by formula A-4;

[0105] The compound of the structure represented by the formula A-4, deuterated chloroform and a catalyst are mixed to carry out a deuteration reaction to obtain a compound of the structure represented by the formula A-5;

[0106] The compound of the structure represented by the formula A-5, methanol and sodium deuterated borohydride are mixed for reduction reaction to obtain a lanosterol derivative of the structure represented by the formula I-6;

[0107]

[0108] The present invention mixes a compound of the structure shown in formula A-3, pyridinium chlorochromate (PCC), sodium acetate and a solvent for oxidation reaction to obtain a compound of the structure shown in formula A-4. In the present invention, the solvent is preferably dichloromethane; the molar ratio of the compound of the structure shown in formula A-3 to PCC is preferably 1:1; the molar ratio of the compound of the structure shown in formula A-3 to sodium acetate is preferably 1:0.5-0.6; the temperature of the oxidation reaction is preferably room temperature, and TLC detection is used to react until the raw material disappears; after the oxidation reaction is completed, the present invention preferably mixes the obtained reaction solution with water and separates them to obtain an organic phase and an aqueous phase, extracts the aqueous phase with dichloromethane, combines the obtained extracted organic phase and the separated organic phase, and then dries them with anhydrous sodium sulfate, then rotary evaporation removes the solvent, and the obtained crude product is separated by silica gel column chromatography to obtain a compound having the structure shown in A-4.

[0109] After obtaining the compound of the structure shown in formula A-4, the present invention mixes the compound of the structure shown in formula A-4, deuterated chloroform and a catalyst for deuteration reaction to obtain a compound of 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]decene-5-ene (TBD); the molar ratio of the compound of 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 12h; 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 of the structure shown in formula A-5.

[0110] After obtaining the compound of the structure shown in formula A-5, the present invention mixes the compound of the structure shown in formula A-5, methanol and sodium deuterated borohydride for reduction reaction to obtain the lanosterol derivative of the structure shown in formula I-6. In the present invention, the molar ratio of the compound of the structure shown in formula A-5 and sodium deuterated borohydride is preferably 1.0:0.5~1.0; the temperature of the reduction reaction is preferably 0°C~room temperature, and the reaction time is preferably 1~3 hours; after the reduction reaction is completed, the present invention preferably removes the methanol in the obtained reaction solution, and then mixes the obtained residue, water and ethyl acetate for phase separation, and the obtained organic phase is rotary evaporated to remove the solvent, and then the obtained crude product is separated by silica gel column chromatography to obtain the lanosterol derivative of the structure shown in formula I-6.

[0111] In the present invention, when X in Formula I is oxygen, R, Y1 and Y2 are hydrogen, and Y3 is deuterium, the preparation method is Method 3, which comprises the following steps:

[0112] The compound having the structure shown in formula A-4, methanol and sodium deuterated borohydride are mixed for reduction reaction to obtain a lanosterol derivative having the structure shown in formula I-7;

[0113]

[0114] In the present invention, the molar ratio of the compound of the structure shown in formula A-4 to sodium deuterated borohydride is preferably 1.0:0.5~1.0; the temperature of the reduction reaction is preferably 0°C~room temperature, and the reaction time is preferably 1~3 hours; the post-treatment method after the reduction reaction is the same as the post-treatment method of the reduction reaction in method 2, and will not be repeated here.

[0115] In the present 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 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 4, which comprises the following steps:

[0116] 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 condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative.

[0117] In the present invention, the types and amounts of the condensing agent, catalyst and solvent in the method 4, as well as the specific conditions of the condensation reaction are the same as those of the condensation reaction described in the method 1, and only the compound of the structure shown in formula I-6 or the compound of the structure shown in formula I-7 is used to replace the compound of the structure shown in formula A-3, which will not be repeated here.

[0118] In the present 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 5, which comprises the following steps:

[0119] Lanosterol, a compound with a structure shown in formula C, a condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative.

[0120] In the present invention, the types and amounts of the condensation agent, catalyst and solvent in the method five, as well as the specific conditions of the condensation reaction are the same as those of the condensation reaction described in the method one, and only lanosterol is used to replace the compound of the structure shown in formula A-3, which will not be repeated here.

[0121] In the present invention, when X in formula II is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen, the preparation method is method 6, and the method 6 comprises the following steps:

[0122] The compound of the structure shown in formula A-3 is replaced by lanosterol, and the lanosterol derivative of the structure shown in formula B-2 is prepared according to the steps in method 2;

[0123]

[0124] In the present invention, the specific operating conditions of the method six are the same as those of the method two, except that lanosterol is used to replace the compound with the structure shown in formula A-3 for oxidation reaction, the product obtained by the oxidation reaction is used to replace the compound with the structure shown in formula A-4 for deuteration reaction, and the product obtained by the reduction reaction is used to replace the compound with the structure shown in formula A-5 for reduction reaction. The specific conditions of the oxidation reaction, deuteration reaction and reduction reaction are not repeated here.

[0125] In the present invention, when X in formula II is oxygen, R, Y1 and Y2 are hydrogen, and Y3 is deuterium, the preparation method is method 7, and the method 7 comprises the following steps:

[0126] Lanosterol, pyridinium chlorochromate, sodium acetate and a solvent are mixed and subjected to oxidation reaction to obtain a compound with a structure shown in formula D:

[0127] The compound having the structure shown in formula D, methanol and sodium deuterated borohydride are mixed for reduction reaction to obtain a lanosterol derivative having the structure shown in formula B-3;

[0128]

[0129] In the present invention, the oxidation reaction conditions in the method seven are the same as those in the method two, and only lanosterol is used to replace the compound with the structure shown in formula A-3; the reduction reaction conditions in the method seven are the same as those in the method three, and only 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 the present 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 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 VIII, and the Method VIII comprises the following steps:

[0131] 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 condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative.

[0132] In the present invention, the conditions for the condensation reaction in the method eight are the same as those in the method one, and only the compound of the structure shown in formula B-2 or the compound of the structure shown in formula B-3 is used to replace the compound of the structure shown in formula A-3, which will not be repeated here.

[0133] The present invention also provides a pharmaceutical composition, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient is the lanosterol derivative described in the above scheme or a pharmaceutically acceptable salt thereof.

[0134] In the present invention, the pharmaceutically acceptable excipient is preferably an excipient; the present invention has no special requirements on the type of the excipient, and those familiar to those skilled in the art can be used.

[0135] The present invention also provides the use of the lanosterol derivative or its pharmaceutically acceptable salt described in the above scheme or the pharmaceutical composition described in the above scheme in the preparation of a drug for treating eye diseases or conditions; the eye diseases include cataracts or retinal degeneration.

[0136] The present invention also provides an eye drop, comprising the following components in parts by weight: 2 to 4 parts of an active ingredient, 5 to 7 parts of hydroxypropyl methylcellulose, 30 to 50 parts of polysorbate, 10 to 20 parts of boric acid, 1 to 2 parts of borax, 0.03 to 0.06 parts of benzalkonium chloride, and 1000 parts of water, wherein the part by weight of the active ingredient is preferably 2 or 4 parts, the part by weight of hydroxypropyl methylcellulose is preferably 6 parts, the part by weight of polysorbate is preferably 40 parts, the part by weight of boric acid is preferably 16 parts, the part by weight of borax is preferably 1.6 parts, and the part by weight of benzalkonium chloride is preferably 0.05 parts; the active ingredient is the lanosterol derivative or a pharmaceutically acceptable salt thereof described in the above scheme; the polysorbate is preferably polysorbate 80; and the water is preferably sterile water for injection.

[0137] In the present invention, the preparation method of the eye drops preferably comprises: dissolving hydroxypropyl methylcellulose with part of the water to obtain solution I; dissolving boric acid, borax and benzalkonium chloride with part of the water, mixing the obtained 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 prescription amount to obtain solution II; adding the active ingredient to solution II for high shear emulsification to obtain solution III; high-pressure homogenizing the solution III to obtain solution IV; finally adding the remaining water to fix the volume of solution IV; the rotation speed of the high shear emulsification is preferably 10000r / min, and the time is preferably 5-10 minutes; the pressure of the high-pressure homogenization is preferably 240bar-2600bar, and the number of high-pressure homogenization is preferably 1 time; after high-pressure homogenization, it is preferred to rinse the high-pressure homogenizer pipeline with an appropriate amount of water, collect the rinse liquid, and mix it with the drug solution.

[0138] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0139] Example 1

[0140] This example synthesizes the lanosterol derivative with the structure shown in formula I-1, and the specific reaction route is as follows:

[0141]

[0142] The specific steps are:

[0143] Step 1, preparation of intermediate A-1: ​​Add DMAP (2.35mmol) and 3.85mL of acetic anhydride to 100mL of petroleum ether solution of lanosterol (mixture of lanosterol and dihydrolanosterol 10g, 23.47mmol, containing 50% lanosterol). Reflux the reaction for 4h, detect by TLC until the raw material disappears, and cool to room temperature. Wash with 2×10mL of 5wt% hydrochloric acid, 2×10mL of 10wt% sodium bicarbonate, and 2×20mL of water in sequence, and dry over anhydrous sodium sulfate. After rotary evaporation to remove the solvent, the crude product is directly subjected to the next step of reaction.

[0144] Step 2, preparation of intermediate A-2: The above crude product - a mixture of A1 and B1 (50%, 10 mmol) was dissolved in THF / H2O (240 mL / 60 mL), NBS (5.5 mmol) was added to the reaction solution, and 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, the solvent was removed by rotary evaporation, and the crude product was separated on a silica gel column (petroleum ether: ethyl acetate = 5:1, volume ratio) 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 THF solution of LiAlH4 (20 mmol) was added at 0°C, and the mixture was heated at 63°C for 4 h under nitrogen protection. After cooling to room temperature, 1.6 mL of water was added to quench the reaction, 150 mL of dichloromethane was added, and 50 mL of water was added to separate the organic phase. 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, volume ratio) to obtain intermediate A-3.

[0146] Step 4, preparation of target compound formula I-1: intermediate A-3 (3mmol), DMAP (1.5mmol), 6-hydroxynicotinic acid (6mmol), DCC (8mmol) were added to a round-bottom flask, nitrogen protection, anhydrous dichloromethane 40mL was added at 0°C, and the reaction was carried out at room temperature for 20h. The solvent was removed. The residue was directly separated on a silica gel column (petroleum ether: ethyl acetate = 4:1, volume ratio) to obtain a lanosterol derivative of 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 having the structure shown in formula I-1 1 H-NMR spectrum, Figure 2 It is the mass spectrum of the lanosterol derivative with the structure shown in formula I-1.

[0147] Example 2

[0148]

[0149] The structure of the target compound in Example 2 is shown in Formula I-2. The synthesis method is the same as that in Example 1, except that 2-hydroxynicotinic acid is 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 a structure as shown in Formula I-3. The synthesis method is the same as in Example 1, using 5-hydroxynicotinic acid to react 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 structure of the target compound in Example 4 is shown in Formula I-4. The synthesis method is the same as that in Example 1, and 2-hydroxyisonicotinic acid is 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 structure of the target compound in Example 5 is shown in Formula I-5. The synthesis method is the same as that in Example 1, and 3-hydroxynicotinic acid is 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 structure of the target compound in Example 6 is shown in Formula I-6, and the synthesis route is as follows:

[0162]

[0163] The specific steps are:

[0164] Step 1: Dissolve compound A-3 (1 mmol) in 20 mL of dichloromethane, add 215 mg of PCC and 45 mg of NaOAc. Stir at room temperature and react until the starting material disappears by TLC. 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 with a rotary evaporator, and separate the crude product with a silica gel column 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 the solvent by rotary evaporation, and purify the crude product by column chromatography to obtain intermediate A-5.

[0166] Step 3: Dissolve the intermediate A-5 (1 mmol) in methanol, add sodium deuterated borohydride (1 mmol) in an ice bath, and react for 1 hour. After the reaction is completed, remove the methanol, add water and ethyl acetate to the residue and stir, separate the organic phase, remove the solvent by rotary evaporation, and separate the deuterated 25-hydroxylanosterol I-6 by silica gel column chromatography; the structural identification data are as follows: Ms: 413.7 (M + -34(2OH)).

[0167] Example 7

[0168]

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

[0170] Referring to the synthesis steps in Example 6, after preparing intermediate A-4, intermediate A-4 (1 mmol) was dissolved in 15 mL of methanol, sodium deuterated borohydride (1 mmol) was added under ice bath and reacted 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 structure in Example 8 is shown in Formula I-9. The synthesis method refers to the synthesis steps in Example 1, and the compound I-6 replaces the intermediate A-3 and reacts with 6-hydroxynicotinic acid in the presence of a dehydrating agent DCC and a catalyst DMAP to obtain the product. The product structure identification data is 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 having the structure shown in formula I-8 1 H-NMR spectrum, Figure 4 It is the mass spectrum of the lanosterol derivative with the structure shown in formula I-8.

[0174] Example 9

[0175]

[0176] The target compound in Example 9 has a structure as shown in Formula I-9. The synthesis method is similar to the synthesis steps in Example 1, whereby Compound I-7 replaces Intermediate A-3 and reacts with 6-hydroxynicotinic acid in the presence of a dehydrating agent, DCC, and a 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 a structure as shown in Formula II-1. The synthesis method is similar to the steps in Example 1, with lanosterol as the raw material instead of intermediate A-3, and directly reacting with 6-hydroxynicotinic acid under the action of a dehydrating agent DCC to obtain the product. 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] Embodiment 11

[0181]

[0182] The target compound in Example 11 has a structure as shown in Formula II-2. The synthesis method refers to some steps in Example 6, using lanosterol as a raw material, oxidizing it with PCC, reducing it with sodium deuterated borohydride, and then reacting it with 6-hydroxynicotinic acid under the action of a dehydrating agent DCC to obtain it. The structural identification data of the product are as follows: Ms: 549.3 (M + +1).

[0183] Example 12

[0184] The solubility test of lanosterol, 25-hydroxylanosterol and some compounds provided by the present invention in water was carried out by HPLC method, and the specific steps are as follows:

[0185] Step 1: Draw a curve of the relationship between the content of the compound to be tested and the HPLC peak area (taking lanosterol as an example).

[0186] Analytical conditions: chromatographic column, Agilent 959990-902 (4.6×250mm, 5μm), column temperature, 35°C; mobile phase, methanol; flow rate, 1.0mL / min; detection wavelength, 210nm; injection volume, 20μL; elution mode, isocratic elution.

[0187] Weigh about 10.00 mg of lanosterol, place it in a 100 mL volumetric flask, add an appropriate amount of methanol, ultrasonically dissolve, dilute to the mark with methanol, and shake well as a reserve solution for drawing the curve of the content of each compound and the HPLC peak area. Use a pipette to transfer an appropriate amount of the above reserve solution to a 25 mL volumetric flask, make up to volume with methanol, and shake well to obtain lanosterol solutions of different concentrations. Inject into the chromatographic instrument for measurement according to the chromatographic conditions under the "Analysis Conditions" item, and record the peak area; use the peak area (y) as the ordinate and the concentration (x) as the abscissa to establish the regression equation of y=11639032.6320x-8583.6303, with a correlation coefficient R 2 =0.9997; The test results show that the linear relationship is good in the concentration range of 0.1000mg / mL to 0.0025mg / mL.

[0188] The same method was used to obtain the content and HPLC peak area curves and regression equations of 25-hydroxylanosterol and some compounds provided by the present invention. The content and HPLC peak area curves of lanosterol and the corresponding regression equations are shown in Figure 2. Figure 5 As shown, Figure 5 The ordinate is the measured HPLC peak area, and the abscissa is the corresponding lanosterol content in methanol solution. The curve of 25-hydroxylanosterol content and HPLC peak area and the corresponding regression equation are as follows: Figure 6 The content of compound I-1 and the HPLC peak area curve and the corresponding regression equation are shown in Figure 7 shown.

[0189] Step 2, compound solubility test. Take excess lanosterol, 25-hydroxylanosterol and some compounds provided by the present invention, and place them in 5 mL of pure water respectively. Place the solution in a constant temperature water bath at 37±0.5°C with magnetic stirring (500rpm), and maintain excess solid during the test. Take out the solution after 6 hours, centrifuge at 10000rpm for 5 minutes, take the supernatant, pass it through a 0.22μm polyethersulfone filter membrane, and perform HPLC analysis of each sample according to the method in the above-mentioned analysis conditions. The experimental results are shown in Table 1.

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

[0191]

[0192]

[0193] It can be seen from the results in Table 1 that the lanosterol derivative provided by the present invention has a significantly higher solubility in water than lanosterol or 25-hydroxylanosterol because polar groups are added to the molecule to improve its water solubility.

[0194] Example 13

[0195] 25-hydroxylanosterol, compound I-6 and I-7 liver microsomal metabolic stability test. Since the stability of carbon-deuterium bonds is higher than that of carbon-hydrogen bonds, replacing hydrogen with deuterium atoms at the easily metabolized sites of drug molecules may change the metabolic rate of the drug, so that the drug molecule has a longer half-life, a lower clearance rate, and better bioavailability. Considering that the hydrogen on the carbon where the secondary hydroxyl group in 25-hydroxylanosterol is located is a potential metabolic site, and the metabolism of the drug is mainly carried out in the liver, the present invention selects compounds I-6, I-7 and 25-hydroxylanosterol to test the liver microsomal metabolic stability. Specifically:

[0196] Step 1: Determination of standard curve. Accurately weigh appropriate amounts of the test samples 25-hydroxylanosterol and deuterated 25-hydroxylanosterol, and dissolve them in a calculated amount of pure acetonitrile to a concentration of 1 mg / mL. Select 0, 2, 4, 6, 8, 10 μM / L as the standard curve range. Draw a standard curve based on the peak area of ​​HPLC analysis.

[0197] Step 2, prepare the test system (using 0.5mg / mL liver microsomal protein system) for HPLC analysis. Melt the liver microsomes stored at -80℃ in an ice bath for standby use, and add the substrate to be tested (1mg / mL). Add NADPHA solution (10μL) and solution B (2μL) and mix them together, incubate at 37℃ for 10 minutes and then place on ice. Mix liver microsomes (5μL) with 0.1M PBS buffer (181μL) and two substrates to be tested (2μL) and place on ice. Finally, add to the NADPH mixed solution and incubate at 37℃. Set the sampling time points to 0 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes and 240 minutes (set two replicates for each time point). At the set incubation time point, add an equal volume of pre-cooled acetonitrile to the incubation system to terminate the reaction. After the sample is mixed, it is centrifuged at 4℃12500rpm for 10 minutes. The supernatant is filtered through a microporous membrane and detected by HPLC. The half-life and clearance rate of each test compound are calculated by comparing the peak area with the standard curve. In the experiment, the negative control group was used without NADPHA solution and B solution. The blank control group only contained the test compound and 0.1M PBS buffer. The HPLC conditions are: column temperature: 25℃; mobile phase: 100% methanol isocratic elution; flow rate: 0.80mL / min; detection wavelength: 210nm; injection volume: 20μL. The test results of 25-hydroxylanosterol and deuterated compounds I-6 and I-7 are shown in Table 2.

[0198]

[0199] Table 2 Metabolic stability of 25-hydroxylanosterol and deuterated compounds I-6 and I-7 in liver microsomes

[0200] Sample name K (rate constant) T1 / 2 (half-life, minutes) CLint (min / mL / μg, clearance) 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 microsome metabolic stability test in the table show that after the hydrogen on the carbon where the secondary hydroxyl group in 25-hydroxylanosterol is deuterated, its metabolic stability is indeed significantly improved. Among them, the half-life of compound I-6 after the hydrogen on the carbon where the hydroxyl group is located and the adjacent carbon are deuterated is the longest (19.85min), and it has the lowest in vitro clearance rate, while the half-life of non-deuterated 25-hydroxylanosterol is the shortest (10.53min), and it has the highest in vitro clearance rate. The above results show that deuteration at appropriate sites in the compound does change its metabolic stability and further improve the bioavailability.

[0202] Example 14 Experimental study on the effect of lanosterol derivatives on sodium selenite-induced cataract model in New Zealand rabbits

[0203] 1. Experimental Animals

[0204] New Zealand rabbits, 2.0-3.0 months old, 1.5-2.5kg, ordinary grade, both male and female, 32 pieces.

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

[0206] 2.1 Prescription:

[0207] Table 3 Eye drops prescription

[0208]

[0209]

[0210] 2.2 Preparation process:

[0211] (1) First dissolve hydroxypropyl methylcellulose in about 10% boiled water for injection, add an appropriate amount of cold water for injection, and stir until completely dissolved to obtain Solution I.

[0212] (2) Use an appropriate amount of water for injection to dissolve the prescribed amount of boric acid, borax and benzalkonium chloride by stirring, add them to solution I, slowly add the prescribed amount of polysorbate 80 while stirring, add water for injection to about 90% of the prescribed amount, stir evenly, and obtain solution II.

[0213] (3) Weigh the prescribed amount of lanosterol and compound I-1 or II-1 and add them to solution II. Use a high shear emulsifier to disperse them at a speed of 10,000 r / min for 5-10 minutes until they are evenly dispersed to obtain solution III.

[0214] (4) Place solution III in a high-pressure homogenizer and homogenize once at a pressure of 250 bar ± 10 bar, collect the drug solution, and flush the high-pressure homogenizer pipeline with an appropriate amount of sterile water for injection, collect the flushing liquid, and mix it with the drug solution to obtain solution IV.

[0215] (5) Make up the volume of Solution IV to 1 L with sterile water for injection.

[0216] (6) Take samples for testing of pH, osmotic pressure, and content. If qualified, package them in a sterile environment using low-density polyethylene medicinal eye drop bottles, with 5 ml of liquid in each bottle.

[0217] 3. Modeling, grouping and drug administration

[0218] 3.1 Modeling: 32 New Zealand rabbits with transparent lenses and no abnormalities were selected for modeling. After the modeling animals were anesthetized, 0.1 mL of 10 mM sodium selenite solution was slowly injected into the anterior chamber. The day of modeling injection was recorded as D0.

[0219] 3.2 Grouping: On D3, the animals were randomly and balancedly divided into model control group, lanosterol eye drops group (0.4%), lanosterol derivative I-1 eye drops group (0.4%), and lanosterol derivative II-1 (0.4%) eye drops group according to the lens opacity grading score and gender of the successfully modeled animals, with 8 animals in each group, both male and female.

[0220] 3.3 Drug administration: On D4, the right eye of the model was administered eye drops according to the set requirements, 3 times a day, with an interval of about 3 hours between each time, for 21 consecutive days. The left eye was not modeled and not medicated as a normal control.

[0221] 4.Indicator detection

[0222] Slit lamp photography was used for grading and scoring before administration, and on days 13 and 20 (D3, D16, and D23) after administration. The eyeballs of the animals were removed approximately 2 hours after the second eye drop administration on D24, and the lens including the capsule was completely separated. The lens was placed on graph paper and photographed to show the clarity of the grid photographed through the lens, and the lens clarity was scored.

[0223] 5. Experimental Results

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

[0225] Table 4 Results of lens opacity grading in 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 scores in Table 4 showed that compared with the model control group and before 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 at D16 and D23 were significantly reduced (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 at D16 and D23 showed a significant downward trend, but no statistical difference was observed.

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

[0231] Table 5 Scoring results of lens transparency in 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 This is a lens transparency score chart for each group of animals.

[0237] The lens transparency score results in Table 5 show that: compared with the model control group, the lens transparency scores of animals in the lanosterol eye drops group, the lanosterol derivative I-1 eye drops group, and the lanosterol derivative II-1 eye drops group were significantly reduced (P < 0.05 or P < 0.01); compared with the lanosterol eye drops group, the lens transparency score of animals in the lanosterol derivative I-1 eye drops group showed a significant downward 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 have a significant improvement effect on the cataract model New Zealand rabbits induced by sodium selenite, which is manifested by reducing the lens opacity grade score and lens transparency score of the model animals. The same concentration of lanosterol derivative I-1 eye drops and lanosterol derivative II-1 eye drops have a slightly better improvement effect on the cataract model New Zealand rabbits than lanosterol eye drops.

[0240] Example 15 Experimental study on the effects of lanosterol derivatives on spontaneous cataract in cynomolgus monkeys

[0241] 1. Experimental Animals

[0242] Cynomolgus monkeys, 4.0-8.0kg, ordinary grade, both male and female, 12 pieces.

[0243] 2. Screening, grouping and drug administration

[0244] Screening: The lens opacity of both eyes of cynomolgus monkeys was examined by slit lamp photography, and 12 cynomolgus monkeys with spontaneous cataract were screened and randomly divided into 3 groups, namely, model control group, lanosterol derivative I-1 eye drops low concentration group (0.2%), and lanosterol derivative I-1 eye drops high concentration group (0.4%), with 4 animals and 8 eyes in each group. The day of animal enrollment was recorded as D0. The formula and preparation method of the eye drops are the same as those in Example 14.

[0245] Drug administration: After grouping, eye drops were administered to both eyes according to the set requirements, 4 times a day, with an interval of about 3 hours between each time, for 60 consecutive days.

[0246] 3. Index detection

[0247] The average density (Avg) and maximum density (Max) of the lens of each group of cynomolgus monkeys were measured using Pentacam HR anterior segment analyzer before administration (D0), D20, D40, and D60.

[0248] Slit lamp photography was used to collect lens photographs of animals in each group before administration (D0), D20, D40, and D60. Finally, the slit lamp lens photographs of cynomolgus monkeys 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 cataract

[0251] Table 6 Test results of average lens density (Avg) of cynomolgus monkeys with spontaneous cataract

[0252]

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

[0254] Table 7 Test results of maximum lens density (Max) in cynomolgus monkeys with spontaneous cataract

[0255]

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

[0257] Figures 9 to 11 Screenshots of Pantcam photography and analysis of each group of animals before drug administration; Figure 12 to Figure 14 Screenshots of Pantcam photography and analysis of animals in each group 20 days after drug administration; Figure 15 to Figure 17 Screenshots of Pantcam photography and analysis of animals in each group 40 days after drug administration; Figures 18 to 20 These are screenshots of the Pantcam photography and analysis of each group of animals 60 days after drug administration.

[0258] The lens density results in Tables 6 and 7 show that compared with before administration (D0), there were no significant differences in the average lens density (Avg) and maximum lens density (Max) of the animals in the model control group at D20, D40, and D60 (P>0.05), while the average lens density (Avg) and maximum lens density (Max) of the animals in the low (0.2%) and high (0.4%) concentration groups of lanosterol derivative I-1 eye drops at D20, D40, and D60 were significantly reduced (P<0.05 or P<0.01).

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

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

[0261]

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

[0263] Fig.21 Slit lamp photographs were taken for each group of animals.

[0264] The lens opacity scoring results in Table 8 show that: compared with before administration (D0), there was no significant difference in the lens opacity scores of the model control group animals at D20, D40, and D60 (P>0.05), and 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 at D20, D40, and D60 were significantly reduced (P<0.05 or P<0.01).

[0265] 5. Conclusion

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

[0267] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

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 Formula I and Formula II: Y1, Y2 and Y3 are independently hydrogen, deuterium, C1-C4 alkyl or unsaturated hydrocarbon group; X is independently oxygen, sulfur or NR1 group, R1 in the NR1 group is hydrogen or alkyl; R is independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl; In Formula I and Formula II, when X is oxygen, R and Y1, Y2 and Y3 are not 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 are independently one or more of halogen, deuterium, hydroxyl, thiol and methylthio.

3. The lanosterol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: In the formula I and formula II, R is hydrogen or hydroxypicolinylcarbonyl.

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

5. The method for preparing the lanosterol derivative according to any one of claims 1 to 4, 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 1, which comprises the following steps: The compound with the structure shown in formula A-3, the compound with the structure shown in formula C, a condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative; R-OH formula C; When X in Formula I is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen, the preparation method is Method 2, which comprises the following steps: A compound having a structure represented by formula A-3, pyridinium chlorochromate, sodium acetate and a solvent are mixed and subjected to an oxidation reaction to obtain a compound having a structure represented by formula A-4; The compound of the structure represented by the formula A-4, deuterated chloroform and a catalyst are mixed to carry out a deuteration reaction to obtain a compound of the structure represented by the formula A-5; The compound of the structure represented by the formula A-5, methanol and sodium deuterated borohydride are mixed for reduction reaction to obtain a lanosterol derivative of the structure represented by the formula I-6; When X in Formula I is oxygen, R, Y1 and Y2 are hydrogen, and Y3 is deuterium, the preparation method is Method 3, which comprises the following steps: The compound having the structure shown in formula A-4, methanol and sodium deuterated borohydride are mixed and subjected to reduction reaction to obtain a lanosterol derivative having the structure shown in formula I-7; When X in Formula I is oxygen, Y1, Y2 and Y3 are deuterium, and R is arylformyl, substituted arylformyl, heteroarylformyl or substituted heteroarylformyl, or 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 4, which comprises the following steps: 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 condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative; 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 5, which comprises the following steps: Mixing lanosterol, a compound having a structure shown in formula C, a condensation agent, a catalyst and a solvent for condensation reaction to obtain the lanosterol derivative; When X in formula II is oxygen, Y1, Y2 and Y3 are deuterium, and R is hydrogen, the preparation method is method 6, which comprises the following steps: The compound of the structure shown in formula A-3 is replaced by lanosterol, and the lanosterol derivative of the structure shown in formula B-2 is prepared according to the steps in method 2; When X in formula II is oxygen, R, Y1 and Y2 are hydrogen, and Y3 is deuterium, the preparation method is method 7, and the method 7 comprises the following steps: Lanosterol, pyridinium chlorochromate, sodium acetate and a solvent are mixed and subjected to oxidation reaction to obtain a compound with a structure shown in formula D: The compound having the structure shown in formula D, methanol and sodium deuterated borohydride are mixed for reduction reaction to obtain a lanosterol derivative having 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 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 VIII, which comprises the following steps: 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 condensation agent, a catalyst and a solvent are mixed to carry out a condensation reaction to obtain the lanosterol derivative.

6. The preparation method according to claim 5, characterized in that: The preparation method of the compound of the structure shown in formula A-3 comprises the following steps: A mixed solution of lanosterol and dihydrolanosterol, acetic anhydride and a catalyst are mixed to carry out an esterification reaction, and the obtained reaction solution is washed, dried and then rotary evaporated to obtain a crude product; the crude product is a mixture of a compound with a structure shown in formula A-1 and a compound with a structure shown in formula B-1; The crude product, N-bromosuccinimide and a solvent are mixed to carry out a bromination reaction, and 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 a structure shown in specific formula A-2; The compound with the structure shown in formula A-2, a reducing agent and a solvent are mixed to carry out a debromination-ester reduction reaction to obtain a compound with the structure shown in formula A-3.

7. A pharmaceutical composition, characterized in that The invention comprises an active ingredient and a pharmaceutically acceptable auxiliary material; the active ingredient is the lanosterol derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutically acceptable auxiliary material is an excipient.

9. Use of the lanosterol derivative or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a medicament for treating eye diseases or disorders.

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

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