WO2017186135A1 - 一种化合物及其在治疗白内障中的应用 - Google Patents

一种化合物及其在治疗白内障中的应用 Download PDF

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WO2017186135A1
WO2017186135A1 PCT/CN2017/082111 CN2017082111W WO2017186135A1 WO 2017186135 A1 WO2017186135 A1 WO 2017186135A1 CN 2017082111 W CN2017082111 W CN 2017082111W WO 2017186135 A1 WO2017186135 A1 WO 2017186135A1
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compound
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carbon atom
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饶燏
闫永彬
杨兴林
陈祥军
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清华大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring
    • C07J41/0033—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005
    • C07J41/0055—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of at least three carbon atoms which may or may not be branched, e.g. cholane or cholestane derivatives, optionally cyclised, e.g. 17-beta-phenyl or 17-beta-furyl derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/001—Oxiranes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
    • C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
    • C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
    • C07J73/005—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by nitrogen as hetero atom

Definitions

  • the invention relates to a compound and its application in treating cataract, and belongs to the field of biomedicine.
  • the lens is an important component of the refractive system of the eyeball and the only refractive interstitial with regulation ability.
  • the lens consists of a crystal capsule, a crystal epithelium, a crystal fiber and a suspensory ligament.
  • a cataract occurs if the lens is partially or completely turbid due to various reasons. Cataracts can cause vision loss in one or both eyes. Usually cataract development is slow, the symptoms mainly include blurred vision, dizziness, night vision ability, and serious blindness. The reduction of vision can seriously affect people's daily life, such as driving, reading, and reduced vision can also lead to mental illness. Cataracts occur frequently in people over the age of 40, and increase with age, and are associated with multiple factors, such as slow metabolism and degenerative diseases in the elderly.
  • ⁇ -, ⁇ - and ⁇ -crystallin are the most important soluble proteins in the lens.
  • ⁇ -crystallin is a dimer composed of two subunits belonging to the family of small heat shock proteins, which can effectively bind to damaged or failed proteins by blocking ATP without blocking ATP. The gathering.
  • Many different types of proteins can be isolated from the lens of cataract patients, many of which are in the form of high molecular weight protein aggregates. These protein aggregates result in turbid shading of the entire cataract lens. It is therefore desirable to provide a small molecule compound that reverses protein aggregation, which can reverse the aggregation of proteins in the cataract lens to become soluble proteins, which can be applied to the prevention or treatment of cataracts.
  • the present invention first provides a compound of formula I, a prodrug thereof, or a pharmaceutically acceptable salt thereof,
  • ring A is a six-membered ring or a seven-membered ring.
  • X is absent.
  • ring A is a seven-membered ring, X represents O or NH;
  • the carbon atom No. 3 is a single bond or a double bond with R 1 , and when it is a single bond, R 1 is selected from any of the following groups:
  • R 1 is selected from any of the following groups:
  • a carbon atom of No. 7, a carbon atom of No. 8, a carbon atom of No. 9 and a carbon atom of No. 11 are a single bond or a double bond;
  • R 2 is H or 2 R 2 forms a carbonyl group
  • R 3 is H or two R 3 forms a carbonyl group
  • R 4 is selected from any of the following groups:
  • R 5 and R 6 are each an alkyl group having 1 to 4 carbon atoms
  • X" and X"' are both selected from the group consisting of F, Cl, Br and I;
  • n 1 or 2.
  • R 1 , R 2 , R 3 , R 4 and n are as defined in Formula I.
  • R' 4 is selected from any of the following groups:
  • the compound of formula III is specifically of formula III-1, formula III-2, formula III-3, formula III-4, formula III-5, formula III-6, formula III-7, formula III-8 or formula III- 9 shows:
  • R' 1 is a single bond or a double bond with a carbon atom
  • R' 1 is selected from any of the following groups:
  • R' 1 is selected from any of the following groups:
  • the compound of formula IV is specifically of formula IV-1, formula IV-2, formula IV-3, formula IV-4, formula IV-5, formula IV-6, formula IV-7, formula IV-8, formula IV- 9.
  • R is a single bond or a double bond between a carbon atom
  • R" 1 is -OH
  • the compound of formula V is specifically as shown in formula V-1 or formula V-2:
  • X represents O or NH.
  • the compound of formula VII is specifically as shown in formula VII-1 or formula VII-2:
  • the compound of the present invention can be produced according to a conventional conventional method, for example, by a conventional reaction such as an oxidation reaction, a reduction reaction, and/or a condensation reaction.
  • a conventional reaction such as an oxidation reaction, a reduction reaction, and/or a condensation reaction.
  • the compounds provided by the present invention, prodrugs thereof or pharmaceutically acceptable salts thereof are useful for the treatment of cataracts.
  • the compound provided by the present invention, a prodrug thereof or a pharmaceutically acceptable salt thereof can be used for preventing, alleviating or reversing the aggregation of the lens protein in a cell;
  • CRY crystallin
  • the present invention will select ⁇ -CRY family mutants ⁇ A-Y118D, ⁇ B-R120G, ⁇ -CRY family mutant ⁇ B2-V187E, ⁇ -CRY family mutants ⁇ C-G129C and ⁇ D-W43R as cataract diseases. Models were used to test the effects of the compounds of the invention.
  • the active ingredient is a cataract-treating drug which is a compound provided by the present invention, a prodrug thereof or a pharmaceutically acceptable salt thereof, and is also within the scope of the present invention.
  • the small molecule having the novel structure provided by the invention has better activity in inhibiting protein aggregation caused by mutation of intracellular lens protein than the existing small molecule (such as C29, Science, 350, 674), and can improve the drug. It is absorbed by the body and has no toxic side effects on normal lens cells.
  • Figure 1 is a schematic representation of the lens protein mutant ⁇ B R120G misfolding in cells to form aggregate bodies.
  • Figure 2 is a graph showing the effect of a compound of the invention on the formation of aggregates of various lens protein mutants.
  • Figure 3 is a half effect concentration of the compound of Formula III-6 of the present invention on the aggregation of the lens protein mutant ⁇ B R120G.
  • Figure 4 is a graph showing the results of detection of the cytotoxicity of the compound of the formula III-6 of the present invention.
  • m-CPBA m-chloroperoxybenzoic acid
  • NaHCO 3 14 mg
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound of the formula IV-1 (500 mg) was dissolved in isopropyl alcohol, water (5 mL) and hypophosphorous acid (0.9 mL, 50% aqueous solution) were added to the above solution, and the reaction mixture was heated to reflux for 3 hours, diluted with water and filtered to give a white solid. The product was separated on a silica gel column (dichloromethane: ethyl acetate 2:1) to afford compound (450 mg).
  • the compound prepared was characterized to be structurally correct.
  • the lanosterol (100 mg), PCC (100 mg) and NaOAc (10 mg) were dissolved in dichloromethane and stirred at room temperature for 1 h. Dilute with water and extract with dichloromethane. The obtained organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness, and the product was obtained on silica gel column ( petroleum ether: ethyl acetate 20:1) to give the compound (90 mg).
  • the compound prepared was characterized to be structurally correct.
  • the compound of the formula IV-4 (100 mg), MeONH 2 ⁇ HCl (56 mg), NaOAc (150 mg) was dissolved in ethanol (10 ml), and the reaction mixture was stirred and stirred at 60 ° C for 3 h.
  • the reaction solution was cooled to room temperature, diluted with water and extracted with dichloromethane.
  • the obtained organic phase was dried over anhydrous sodium sulfate, and then the solvent was evaporated to dryness on a rotary evaporator.
  • the compound prepared was characterized to be structurally correct.
  • the compound of the formula IV-4 (100 mg), HONH 2 ⁇ HCl (48 mg), NaOAc (150 mg) was dissolved in ethanol (10 ml), and the reaction mixture was stirred at 60 ° C for 3 h.
  • the reaction solution was cooled to room temperature, diluted with water and extracted with dichloromethane.
  • the obtained organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness using a rotary evaporator.
  • the product obtained was purified by silica gel column ( petroleum ether: ethyl acetate 10:1) to give the compound of formula 1.
  • the compound of the formula (50 mg) was dissolved in anhydrous diethyl ether, and LiAlH 4 (50 mg) was added to the reaction mixture, and the mixture was reacted at room temperature for 3 h. Slowly add water to stop the reaction. Extract with dichloromethane. The obtained organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness on a rotary evaporator. The obtained product was isolated on silica gel column (dichloromethane: methanol 10:1) to give the compound (20 mg).
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound of the formula IV-7 (100 mg), HONH 2 ⁇ HCl (48 mg), NaOAc (150 mg) was dissolved in ethanol (10 ml), and the reaction mixture was stirred at 60 ° C for 3 h.
  • the reaction solution was cooled to room temperature, diluted with water and extracted with dichloromethane.
  • the obtained organic phase is dried over anhydrous sodium sulfate, and then the solvent is evaporated to dryness using a rotary evaporator.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound of the formula IV-7 (100 mg), MeONH 2 ⁇ HCl (56 mg), and NaOAc (150 mg) were dissolved in ethanol (10 ml), and the reaction mixture was stirred and stirred at 60 ° C for 3 h.
  • the reaction solution was cooled to room temperature, diluted with water and extracted with dichloromethane.
  • the obtained organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness on a rotary evaporator.
  • the obtained product was isolated on silica gel column ( petroleum ether: ethyl acetate 10:1) to give the compound of formula IV-12 (95 mg).
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • the compound of the formula IV-8 (50 mg) was dissolved in a mixed solvent of dichloromethane / trifluoroacetic acid (1 mL / 1 ml), and allowed to react at room temperature for 2 h. Dilute with water and extract with dichloromethane. Wash with saturated sodium bicarbonate. The obtained organic phase was dried over anhydrous sodium sulfate, and then the solvent was evaporated to dryness on a rotary evaporator. The obtained product was separated by silica gel column ( petroleum ether: ethyl acetate 30:1) to give compound 5F (20 mg) of formula VII-1. .
  • the compound prepared was characterized to be structurally correct.
  • the compound prepared was characterized to be structurally correct.
  • DAPI-Fluoromount-G fluorescent seal tablets (0100-20) were purchased from Southern Biotech (SBA).
  • Anti-p62 antibody (ab56416) was purchased from Abcam.
  • the crude lanosterol was purchased from TCI (C0427) with a purity of >50.0% (GC).
  • Cell counting kit-8 (CK04-500) was purchased from Japan Tongren Chemical.
  • the lens proteins ⁇ A-Y118D, ⁇ B-R120G, ⁇ B2-V187E, ⁇ C-G129C and ⁇ D-W43R mutants were constructed by the cloning of the research group. The specific process is as follows:
  • DNA molecule 1 was inserted between the XhoI and BamHI restriction sites of the pEGFP-N1 vector (Clontech) to obtain recombinant plasmid 1.
  • DNA molecule 1 A DNA molecule obtained by mutating the nucleotide 354 of the DNA molecule shown in SEQ ID NO: 1 from the T to G (the corresponding amino acid at position 118 is mutated from Y to D).
  • the DNA molecule shown in SEQ ID NO: 1 of the Sequence Listing is the open reading frame of CRY- ⁇ A.
  • DNA molecule 2 was inserted between the XhoI and HindIII cleavage sites of the pEGFP-N1 vector (Clontech) to obtain recombinant plasmid 2.
  • DNA molecule 2 A DNA molecule obtained by mutating the 360th nucleotide of the DNA molecule shown in SEQ ID NO: 2 of the sequence listing from A to G (the corresponding amino acid at position 120 is mutated from R to G).
  • the DNA molecule shown in SEQ ID NO: 2 of the Sequence Listing is the open reading frame of CRY- ⁇ B.
  • DNA molecule 3 was inserted between the XhoI and HindIII cleavage sites of the pEGFP-N1 vector (Clontech) to obtain recombinant plasmid 3.
  • DNA molecule 3 A DNA molecule obtained by mutating the 562th nucleotide of the DNA molecule shown in SEQ ID NO: 3 of the sequence listing from T (corresponding 187th amino acid from V to E). The DNA molecule shown in SEQ ID NO: 3 of the Sequence Listing is the open reading frame of CRY- ⁇ B2.
  • DNA molecule 4 was inserted between the XhoI and HindIII restriction sites of the pEGFP-N1 vector (Clontech) to obtain recombinant plasmid 4.
  • DNA molecule 4 A DNA molecule obtained by mutating the nucleotide 387 of the DNA molecule shown in SEQ ID NO: 4 of the sequence listing from G to T (corresponding 129th amino acid is mutated from G to C).
  • the DNA molecule shown in SEQ ID NO: 3 of the Sequence Listing is an open reading frame of CRY- ⁇ C.
  • DNA molecule 5 was inserted between the XhoI and HindIII restriction sites of the pEGFP-N1 vector (Clontech) to obtain a recombinant plasmid 5.
  • DNA molecule 5 A DNA molecule obtained by mutating the 129th nucleotide of the DNA molecule shown in SEQ ID NO: 5 of the sequence listing from T to C (the corresponding amino acid at position 43 is mutated from W to R).
  • the DNA molecule shown in SEQ ID NO: 3 of the Sequence Listing is an open reading frame for CRY- ⁇ D.
  • HeLa Human lens epithelial cells HLE-B-3 ( CRL-11421 (TM ) was purchased from the ATCC cell bank and culture conditions were performed as recommended by ATCC.
  • This example uses the following method to determine the effect of the compounds of the present invention on the aggregation of lens proteins ⁇ A-Y118D, ⁇ B-R120G, ⁇ B2-V187E, ⁇ C-G129C and ⁇ D-W43R mutants in HeLa cells.
  • the lens protein ⁇ B-R120G mutant misfolds in the cell and forms aggregated bodies visible under the microscope. As shown in Fig. 1, the morphology of the aggregates is uniform, and the aggregation ratio is stable, which is the study of chemical drugs to alleviate protein misfolding. The ideal model. Therefore, the present invention first uses the lens protein ⁇ B-R120G mutant as a research model to screen for effective compounds.
  • HeLa cells in good condition were inoculated into a 12-well plate in which the cell slides were pre-plated, and the cell density was 40% to 50%; after 24 hours of culture, the cell density was about 80%, that is, cell transfection can be performed, and the specific operation is according to Invitrogen.
  • the company's transfection reagent Lipofectamine TM 2000 recommended the protocol; after transfection for 4h, it was replaced with DMEM fresh medium for 16h. At this time, the lens protein ⁇ B-R120G mutant was expressed in a large amount in the cells, forming a certain proportion of small aggregation.
  • the cell culture medium was changed to opti-MEM, and the compound of the present invention was added to a final concentration of 4 uM; after 4 hours of culture, it was replaced with DMEM fresh medium and cultured for further 4 hours; preparation of immunofluorescent label: PBS washing cells Climb the tablets 3 times, fix the cells in 4% paraformaldehyde for 30 minutes at room temperature, wash the cells with PBS for 3 times, incubate the cells for 0.5 minutes at 0.4% Triton X-100, wash the cells for 3 times with PBS, 4% goat serum.
  • Block for 40 minutes at room temperature incubate the p62 antibody for 1 hour at room temperature, wash the cells with PBS for 3 times, incubate the corresponding source of fluorescent secondary antibody for 40 minutes at room temperature, wash the cells with PBS for 3 times, and seal with DAPI-Fluoromount-G fluorescent sealing tablets.
  • room temperature in the dark for about 1 hour At room temperature in the dark for about 1 hour; Application of Three-channel microscope Zeiss 710 was observed, taking the case of the single-blind aggregated cells statistical analysis of efficacy of the compounds of the present invention.
  • the compounds of the present invention were statistically analyzed for aggregation of the lens protein ⁇ B-R120G mutant in HeLa cells by the above method, and the results are shown in Table 1.
  • the data in Table 1 shows that the compound of the present invention has a mitigating effect on the aggregation phenomenon of the lens protein ⁇ B-R120G mutant in HeLa cells, especially the compound represented by the formula III-3, the compound of the formula III-6, and III.
  • a compound represented by -8; a compound of the formula IV-1, a compound of the formula IV-7 and a compound of the formula IV-12 have a significant mitigating effect on the aggregation of the lens protein ⁇ B-R120G mutant in HeLa cells. .
  • Proteins in the lens are misfolded after mutation, and most of them cause cataract diseases. Therefore, it has been important to verify whether the pharmacological effects of the compounds of the present invention have universal effects in a variety of lens protein mutants for the treatment of cataract diseases.
  • the present invention selects the lens protein ⁇ A-Y118D, ⁇ B2-V187E, ⁇ C-G129C and ⁇ D-W43R mutants for further verification of the drugs effective in preliminary screening.
  • the specific program is the same as the process of drug screening.
  • the results show that the compounds of the present invention, formula III-6 and formula IV-1, have significant effects in a cell model in which a plurality of lens protein mutants form aggregates, as shown in FIG.
  • the present invention initially uses the lens protein ⁇ B-R120G mutant as a research model to calibrate the half-effect concentration EC50 value of the compound of the formula III-6 of the present invention, as shown in FIG. 3, and the result shows the compound represented by the formula III-6.
  • the efficacy of the substance is an order of magnitude higher than that of the natural compound lanosterol, and the drug treatment of cataract diseases has great potential.
  • a safe and effective drug is the research principle of the present invention, and the present invention will further test whether the compound of the formula III-6 of the present invention is cytotoxic to cells.
  • One control group was established to transfect HLE-B3 cells with empty vector; five experimental groups were transfected with HLE- of lens protein ⁇ A-Y118D, ⁇ B-R120G, ⁇ B2-V187E, ⁇ C-G129C and ⁇ D-W43R mutants.
  • B3 cells mimic cataract diseases caused by mutations in different lens proteins.
  • HLE-B3 cells in good condition were inoculated into 12-well plates, the cell density was 40%-50%; after 24 hours of culture, the cell density was about 80%, that is, cell transfection can be performed, and the lens proteins ⁇ A-Y118D and ⁇ B were transfected respectively.
  • the specific procedure was carried out according to the recommended protocol of Invitrogen's transfection reagent Lipofectamine TM 2000; after transfection for 4 h, it was replaced with DMEM fresh medium for 16 h. At this time, the lens protein mutant was abundantly expressed in the cells; 0.25% trypsin was used.
  • EDTA EDTA digested and dispersed, inoculated into 96-well plates, 2000 cells per well, continued to culture for 12 hours; cell culture medium was changed to opti-MEM, and compound 1G of the present invention was added to set two concentration gradients, final concentration Incubate for 12 hours at 5 uM or 50 nM; add 10 ⁇ L of CKK-8, incubate for 1 hour, and measure absorbance at 450 nm.
  • the small molecule having the novel structure provided by the invention has better activity in inhibiting protein aggregation caused by mutation of intracellular lens protein than the existing small molecule (such as C29, Science, 350, 674), and can improve the drug. It is absorbed by the body and has no toxic side effects on normal lens cells.

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Abstract

本发明公开了一种化合物及其在治疗白内障中的应用。所述化合物的结构式如式Ⅰ所示。式Ⅰ所示化合物、其前药或其药学上可接受的盐可用于阻止、缓解或者逆转晶状体蛋白在细胞内的聚集；在晶状体细胞中，90％以上的蛋白组分是晶状体蛋白(crystallin，CRY)，包括α-、β-和γ-CRY三个家族，而晶状体蛋白发生突变后，会引发细胞内的蛋白聚集，导致白内障疾病，本发明将选取α-CRY家族突变体αA-Y118D、αB-R120G、β-CRY家族突变体βB2-V187E、γ-CRY家族突变体γC-G129C和γD-W43R为白内障疾病的研究模型检测了本发明化合物的效果。本发明提供的具有新颖结构的小分子，与现有的小分子(如C29，Science,350,674)相比，在抑制细胞内晶状体蛋白突变导致的蛋白聚集具有更好的活性，且提高药物的可被机体吸收性，并且对正常晶状体细胞没有毒副作用。

Description

一种化合物及其在治疗白内障中的应用 技术领域
本发明涉及一种化合物及其在治疗白内障中的应用，属于生物医药领域。
背景技术
晶状体是眼球屈光系统的重要组成部分，也是唯一具有调节能力的屈光间质，晶状体由晶体囊、晶体上皮、晶体纤维和悬韧带组成。如果晶状体由于各种原因造成其部分或全部混浊，则发生白内障。白内障会导致单眼或者双眼的视力降低。通常白内障发展缓慢，症状主要包括视力模糊、晕光、夜视能力降低，严重会致盲。视力的降低会严重的影响人们的日常生活，比如开车、阅读，视力的降低也会导致心理疾病的产生。白内障多发于40岁以上的人群，随着且随年龄增长而增多，与多因素相关，如与老年人代谢缓慢，发生退行性病变有关，也有人认为与日光长期照射，内分泌紊乱，代谢障碍等因素有关。外伤、药物、放射性物质、并发症等也会引起后天性白内障，另外，有一些先天性的白内障患者，多在出生前后即已存在，有内生性与外生性两类，内生性者与胎儿发育障碍有关，外生性者是母体或胎儿的全身病变对晶状体造成损害所致。
国际公认的快速有效治疗白内障方法是手术治疗，通过手术将患者浑浊的晶状体取出，然后植入人工晶体。但总体而言手术治疗费用较高，对患者而言是很大的经济负担，随着人类平均寿命的延长、人口老龄化的出现，这一难题更为突出，因此寻找有效、安全、廉价的药物治疗白内障具有重要的现实意义。
晶状体内90％的蛋白是由晶状体蛋白组成。其中α-、β-和γ-晶状体蛋白是晶状体内最主要的可溶性蛋白。其中，α-晶状体蛋白是由两个亚基组成的二聚体，属于小热休克蛋白家族，它可以在不依赖ATP的情况下有效地结合损伤的或者未能正确折叠的蛋白质从而阻止这些蛋白的聚集。从白内障患者的晶状体中可以分离得到的很多不同种类的蛋白，其中有很多是以高分子量的蛋白聚集体形式存在。这些蛋白聚集体导致了整个白内障晶状体的浑浊遮光性。因此需要提供一种可以逆转蛋白聚集的小分子化合物，其可以逆转白内障晶状体内的蛋白聚集，使其变为可溶蛋白，该小分子可以应用到白内障的预防或者治疗中。
发明内容
本发明的目的是提供一种新具有新颖结构的小分子化合物，该小分子化合物在抑制晶状体细胞内蛋白聚集方面具有更好的活性，并且对正常晶状体细胞没有 毒副作用。
本发明首先提供式Ⅰ所示化合物、其前药或其药学上可接受的盐，
Figure PCTCN2017082111-appb-000001
式Ⅰ中，环A为六元环或七元环，当环A为六元环时，X不存在，当环A为七元环时，X表示O或NH；
3号碳原子与R1之间为单键或双键，当为单键时，R1选自如下基团中任一种：
Figure PCTCN2017082111-appb-000002
当为双键时，R1选自如下基团中任一种：
Figure PCTCN2017082111-appb-000003
7号碳原子、8号碳原子、9号碳原子和11号碳原子相邻碳原子之间为单键或双键；
当8号碳原子与9号碳原子之间为单键时，8号碳原子与9号碳原子之间存在环氧结构或7号碳原子与8号碳原子之间为双键或9号原子与11号碳原子之间为双键；
当7号碳原子与8号碳原子之间为单键时，R2为H或2个R2形成羰基；
当7号碳原子与8号碳原子之间为双键时，7号碳原子上连接一个R2，R2为H；
当9号碳原子与11号碳原子之间为单键时，R3为H或2个R3形成羰基；
当9号碳原子与11号碳原子之间为双键时，11号碳原子上连接一个R3，R3为H；
R4选自如下基团中任一种：
Figure PCTCN2017082111-appb-000004
其中，R5和R6均为碳原子数为1-4的烷基；
X”和X”’均选自F、Cl、Br和I；
n为1或2。
所述化合物的结构式进一步如式Ⅱ所示，
Figure PCTCN2017082111-appb-000005
式Ⅱ中，R1、R2、R3、R4和n的定义同式Ⅰ中。
所述化合物的结构式具体如式Ⅲ所示，
Figure PCTCN2017082111-appb-000006
式Ⅲ中，R’4选自如下基团中任一种：
Figure PCTCN2017082111-appb-000007
式Ⅲ所示化合物具体如式Ⅲ-1、式Ⅲ-2、式Ⅲ-3、式Ⅲ-4、式Ⅲ-5、式Ⅲ-6、式Ⅲ-7、式Ⅲ-8或式Ⅲ-9所示：
Figure PCTCN2017082111-appb-000008
Figure PCTCN2017082111-appb-000009
所述化合物的结构式具体如式Ⅳ所示，
Figure PCTCN2017082111-appb-000010
式Ⅳ中，R’1与碳原子之间为单键或双键；
当为单键时，R’1选自如下基团中任一种：
Figure PCTCN2017082111-appb-000011
当为双键时，R’1选自如下基团中任一种：
Figure PCTCN2017082111-appb-000012
R”4选自如下基团中任一种：
Figure PCTCN2017082111-appb-000013
式Ⅳ所示化合物具体如式Ⅳ-1、式Ⅳ-2、式Ⅳ-3、式Ⅳ-4、式Ⅳ-5、式Ⅳ-6、式Ⅳ-7、式Ⅳ-8、式Ⅳ-9、式Ⅳ-10、式Ⅳ-11、式Ⅳ-12或式Ⅳ-13所示：
Figure PCTCN2017082111-appb-000014
Figure PCTCN2017082111-appb-000015
所述化合物的结构式具体如式Ⅴ所示，
Figure PCTCN2017082111-appb-000016
Figure PCTCN2017082111-appb-000017
式Ⅴ中，R”1与碳原子之间为单键或双键；
当为单键时，R”1为-OH；
当为双键时，R”1为
Figure PCTCN2017082111-appb-000018
式Ⅴ所示化合物具体如式Ⅴ-1或式Ⅴ-2所示：
Figure PCTCN2017082111-appb-000019
所述化合物的结构式具体如式Ⅵ所示，
Figure PCTCN2017082111-appb-000020
所述化合物的结构式进一步如式Ⅶ所示，
Figure PCTCN2017082111-appb-000021
式Ⅶ中，X表示O或NH。
式Ⅶ所示化合物具体如式Ⅶ-1或式Ⅶ-2所示：
Figure PCTCN2017082111-appb-000022
本发明化合物可根据现有的常规方法进行制备，如采用氧化反应、还原反应和/或缩合反应等常规反应进行。
本发明所提供的化合物、其前药或其药学上可接受的盐可用于治疗白内障。
本发明提供的化合物、其前药或其药学上可接受的盐可用于阻止、缓解或者逆转晶状体蛋白在细胞内的聚集；
在晶状体细胞中，90％以上的蛋白组分是晶状体蛋白(crystallin，CRY)，包括α-、β-和γ-CRY三个家族，而晶状体蛋白发生突变后，会引发细胞内的蛋白聚集，导致白内障疾病，本发明将选取α-CRY家族突变体αA-Y118D、αB-R120G、β-CRY家族突变体βB2-V187E、γ-CRY家族突变体γC-G129C和γD-W43R为白内障疾病的研究模型来检测本发明化合物的效果。
活性成分为本发明提供的化合物、其前药或其药学上可接受的盐的治疗白内障的药物也属于本发明的保护范围。
本发明提供的具有新颖结构的小分子，与现有的小分子(如C29，Science,350,674)相比，在抑制细胞内晶状体蛋白突变导致的蛋白聚集具有更好的活性，且提高药物的可被机体吸收性，并且对正常晶状体细胞没有毒副作用。
附图说明
图1为晶状体蛋白突变体αB R120G在细胞内发生错误折叠形成聚集小体的示意图。
图2为本发明化合物对多种晶状体蛋白突变体形成聚集体的效应。
图3为本发明式Ⅲ-6所示化合物对晶状体蛋白突变体αB R120G聚集的半效应浓度。
图4为本发明式Ⅲ-6所示化合物细胞毒性的检测结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、式Ⅲ-1所示化合物的制备
羊毛甾醇(50％，购于TCI，100mg)溶于二氯甲烷(30mL)，在冰浴下，m-CPBA(间氯过氧苯甲酸)(85％，28mg)和NaHCO3(14mg)间隔3h分两批加入到上述溶液中，室温搅拌过夜。反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝10：1，体积比)，得式Ⅲ-1所示化合物(45mg)和式Ⅲ-1’所示化合物(40mg)。
Figure PCTCN2017082111-appb-000023
式Ⅲ-1’所示化合物：1H-NMR(400MHz,CDCl3)δ(ppm)3.23-3.21(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.5,79.1,50.7,50.6,49.9,44.6,39.7,39.0,37.2,36.6,35.7,31.1,31.0,29.9,28.4,28.1,28.0,26.7,24.4,24.3,23.0,22.7,21.2,19.3,18.9,18.4,15.9,15.6.
式Ⅲ-1所示化合物：1H-NMR(400MHz,CDCl3)δ(ppm)3.21(d,J＝8.0Hz,1H),2.69-2.67(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.6,134.4,79.0,77.5,77.2,76.8,65.0,64.9,58.5,58.2,50.51,50.48,50.4,49.9,44.6,39.0,37.1,36.4,36.3,35.7,32.9,32.7,31.10.31.08,30.9,28.4,28.3,28.1,28.0,26.6,26.0,25.7,25.06,25.05,24.4,21.1,19.3,18.9,18.79,18.76,18.7.
经表征，所制备的化合物结构正确。
实施例2、式Ⅲ-2所示化合物的制备
高碘酸钠(135mg)加入到35mL的乙醚中，搅拌的条件下，式Ⅲ-1所示化合物(283mg)加入到上述反应液中，室温下搅拌15min，水加入到反应液中，有机层采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶 柱分离(石油醚：乙酸乙酯＝15：1，体积比)，得式Ⅲ-2所示化合物(120mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)9.76(s,1H),3.24-3.21(m,1H),2.46-2.35(m,2H),0.6(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)203.4,134.6,134.4,79.1,77.5,77.2,76.9,53.6,50.5,50.4,50.0,44.7,41.3,39.0,37.2,36.2,35.7,31.1,30.9,28.4,28.3,28.1,28.0,26.6,24.4,21.1,19.3,18.5,18.4,15.9,15.6.
经表征，所制备的化合物结构正确。
实施例3、式Ⅲ-3所示化合物的制备
羊毛甾醇(50％，购于TCI，220mg)溶于THF/H2O(20mL/5mL)，NBS(54mg)加入到反应液中，反应液在室温下搅拌2h。反应液加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝5：1，体积比)，得式Ⅲ-3所示化合物(110mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)4.13-3.94(m,1H),3.25-3.21(m,1H),0.69(s,3H).
经表征，所制备的化合物结构正确。
实施例4、式Ⅲ-4所示化合物的制备
将40mg式Ⅲ-2所示化合物，25mg NaOAc，14mg NH2OH·HCl溶于5ml1,4-二氧六环-水(v/v，2：1)中，置于室温下搅拌12h。TLC检测其反应完全，将反应产物用饱和食盐水洗涤，乙酸乙酯萃取。分离有机相，用无水硫酸钠干燥后，置于旋转蒸发仪上减压蒸干。采用硅胶柱(v/v，乙酸乙酯：石油醚＝1：5)分离纯化，得到式Ⅲ-4所示化合物17mg。
1H-NMR(400MHz,CDCl3)δ(ppm)3.24(dd,J＝11.6Hz,J＝4.4Hz),0.88(s,3H),0.81(s,3H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)153.1,134.6,134.5,79.2,50.6,50.5,50.4,50.0,44.7,39.1,37.2,36.5,36.3,35.8,33.1,32.6,31.1,31.0,29.9,28.4,28.3,28.1,28.0,26.7,24.4,21.2,19.3,18.54,18.48,18.4,15.9,15.6.
经表征，所制备的化合物结构正确。
实施例5、式Ⅲ-5所示化合物的制备
将40mg式Ⅲ-2所示化合物，3ml二乙胺溶于二氯乙烷中搅拌1h后，于0℃下缓慢加入42mg NaBH(OAc)3。恢复至室温并继续搅拌12h。加入少许酸猝灭反应后，用饱和碳酸氢钠洗涤，乙酸乙酯萃取。分离有机相，用无水硫酸钠干燥后，置于旋转蒸发仪上减压蒸干。采用硅胶柱(v/v，二氯甲烷：甲醇＝20:1) 分离纯化，得到式Ⅲ-5所示化合物40mg。
1H-NMR(400MHz,CDCl3)δ(ppm)3.21(dd,J＝11.2Hz,J＝3.6Hz,1H),2.27-2.55(m,4H),0.79(s,3H),0.67(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.54.134.48,79.0,53.5,50.53,50.48,49.9,46.9,44.6,39.0,37.1,36.5,35.7,34.2,31.1,31.0,28.3,28.1,28.0,26.6,24.4,23.4,21.1,19.3,18.9,18.4,15.9,15.6,11.5.
经表征，所制备的化合物结构正确。
实施例6、式Ⅲ-6所示化合物的制备
式Ⅲ-3所示化合物(80mg)溶于无水THF(10mL)中，LiAlH4(38mg)加入到反应液中，反应液加热回流2h。冷却到室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝5：1，体积比)，得式Ⅲ-6所示化合物(40mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.25-3.22(m,1H),2.03-1.89(m,5H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.58,134.56,79.1,71.3,50.7,50.6,50.0,44.7,44.6,39.0,37.2,36.9,36.6,35.8,31.2,31.0,29.5,29.4,28.4,28.1,28.0,26.7,24.4,21.3,21.2,19.3,18.8,18.4,15.9,15.6.
经表征，所制备的化合物结构正确。
实施例7、式Ⅲ-7所示化合物的制备
将50mg式Ⅲ-2所示化合物溶于无水5ml THF中。加入4ml 4M异丙基溴化镁的四氢呋喃溶液。TLC检测反应完成后，加水终止反应，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝5：1，体积比)，得式Ⅲ-7所示化合物17mg。
1H-NMR(400MHz,CDCl3)δ(ppm)3.33-3.32(m,1H),3.23(dd,J＝11.6Hz,J＝4.8Hz,1H),0.81(s,3H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.54,134.51,79.1,50.6,50.5,44.6,39.0,37.2,35.7,31.1,31.0,28.1,28.0,27.0,26.6,24.4,22.8,21.1,19.3,19.2,19.0,18.4,16.8,15.9,15.6,14.3.
经表征，所制备的化合物结构正确。
实施例8、式Ⅲ-8所示化合物的制备
式Ⅲ-6所示化合物(50mg)于二氯甲烷(30mL)，在冰浴下，m-CPBA(85％，28mg)和NaHCO3(14mg)间隔3h分两批加入到上述溶液中，室温搅拌过夜。反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝5：1，体积比)，得式Ⅴ-1所示化合物(45mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.21-3.19(m,1H),0.76-0.75(m,6H)13C-NMR(100MHz,CDCl3)δ(ppm)78.6,71.2,70.8,68.3,49.0,48.5,44.5,43.7,41.9,38.6,38.0,36.7,36.4,33.0,29.42,29.36,28.6,28.4,27.3,27.0,23.6,21.6,21.2,20.1,19.1,17.1,16.6,16.4,15.2.
将30mg式Ⅴ-1所示化合物溶于3ml THF中，加入100μl 40％的氢氟酸溶液，室温下搅拌4天。TLC检测反应完成后用饱和碳酸氢钠溶液洗，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱(乙酸乙酯：石油醚＝1：3)分离得到27mg式Ⅲ-8所示化合物。
1H-NMR(400MHz,CDCl3)δ(ppm)5.47(s,br,1H),5.32(s,br,1H),3.24(dd,J＝11.2Hz,J＝4.4Hz,1H),0.56(s,3H)13C-NMR(100MHz,CDCl3)δ(ppm)146.0,142.8,120.3,116.5,79.1,71.2,51.2,50.5,49.2,44.5,43.9,38.8,38.0,37.5,36.8,36.4,35.8,31.6,29.5,29.4,28.3,28.1,27.9,25.7,23.1,22.9,21.3,18.6,15.9,15.8.
经表征，所制备的化合物结构正确。
实施例9、式Ⅲ-9所示化合物的制备
式Ⅲ-6所示化合物(44mg)溶于无水二氯甲烷中，冰浴条件下下，20μl DAST加入到反应体系中，反应1h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝10：1)，得式Ⅲ-9所示化合物(10mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.23(dd,J＝11.6Hz,J＝4.4Hz,1H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)150.5,140.8,122.1,116.4,96.9,95.3,59.1,50.4,50.35,45.5,44.8,42.1,41.9,37.2,36.7,36.6,36.3,30.5,29.8,29.6,28.2,27.0,26.9,26.8,26.6,24.5,24.0,23.3,23.2,22.9,20.9,18.9,16.0,15.9.
经表征，所制备的化合物结构正确。
实施例10、式Ⅳ-1所示化合物的制备
式Ⅲ-1所示化合物(80mg)，醋酸酐(300uL)，DMAP(2mg)，吡啶(1ml)和二氯甲烷(10ml)加入到反应瓶中。室温搅拌6h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得式Ⅳ-1所示化合物(80mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)4.51-4.47(m,1H),2.68(t,J＝6.4Hz,1H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)171.2,134.5,134.4,81.0,77.5,77.2,76.8,65.1,64.9,58.5,58.3,50.6,50.5,50.4,49.9,44.6,37.9,37.0,36.5,36.3,35.4,32.9,32.7,31.07,31.05,30.9,28.4,28.3,28.0,26.5,26.1,25.7,25.09,25.07, 24.4,24.3,21.5,21.1,19.3,18.9,18.8,18.7,18.2,16.7.
经表征，所制备的化合物结构正确。
实施例11、式Ⅳ-2所示化合物的制备
羊毛甾醇(80mg)、醋酸酐(300uL)、DMAP(2mg)、吡啶(1ml)和二氯甲烷(10ml)加入到反应瓶中。室温搅拌6h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得式Ⅳ-2所示化合物(80mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)5.12-5.08(m,1H),4.52-4.48(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)171.0,134.7,134.5,131.0,125.4,81.1,77.5,77.2,76.9,50.7,50.6,50.0,44.7,38.0,37.1,36.5,36.4,35.5,31.2,31.0,28.4,28.1,26.6,25.8,25.1,24.4,24.37,21.4,21.2,19.3,18.8,18.3,17.8,16.7,15.9.
经表征，所制备的化合物结构正确。
实施例12、式Ⅳ-3所示化合物的制备
式IV-1(500mg)溶解到异丙醇中，水(5mL)和次磷酸(0.9mL，50％水溶液)加入到上述溶液中，反应液加热回流3小时，用水稀释，过滤得白色固体。产品用硅胶柱分离(二氯甲烷：乙酸乙酯2：1)，得式Ⅳ-3所示化合物(450mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)4.51-4.47(m,1H),3.36-3.27(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)171.2,134.6,134.4,81.1,79.8,78.9,73.4,73.3,50.7,50.6,49.9,44.6,37.9,37.0,36.9,36.4,35.4,33.7,33.2,31.1,30.9,28.8,28.5,28.4,28.3,28.0,26.7,26.5,24.4,24.3,23.4,23.3,21.5,21.1,19.3,19.0,18.7,18.2,16.7,15.9.
经表征，所制备的化合物结构正确。
实施例13、式Ⅳ-4所示化合物的制备
羊毛甾醇(100mg)，PCC(100mg)和NaOAc(10mg)溶解到二氯甲烷中，室温搅拌1h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得式Ⅳ-4所示化合物(90mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)5.11-5.08(m,1H),2.62-2.54(m,1H),2.43-2.37(m,1H),0.71(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)218.0,135.5,133.2,131.1,125.3,51.3,50.5,50.0,47.5,44.6,37.0,36.44,36.37,36.2,34.7,31.1,31.0,28.3,26.5,26.3,25.9,25.0,24.4,21.4,21.2,19.6,18.8,18.75,17.8,16.0.
经表征，所制备的化合物结构正确。
实施例14、式Ⅳ-5所示化合物的制备
式Ⅳ-4所示化合物(100mg)，MeONH2·HCl(56mg)，NaOAc(150mg)溶于乙醇(10ml)，反应液60摄氏度加热搅拌3h。反应液冷却到室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝10：1，体积比)，得式Ⅳ-5所示化合物(95mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)9.11(s,br,1H),3.13(d,J＝3.6Hz,1H),0.90(d,J＝6Hz,3H),0.85(s,3H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)167.2,134.9,133.9,71.3,51.5,50.6,50.0,44.6,44.5,40.5,37.3,36.8,36.6,35.8,31.1,31.0,29.5,29.3,28.3,27.0,26.5,24.4,23.1,21.3,21.2,19.1,18.9,18.8,17.7,16.0.
经表征，所制备的化合物结构正确。
实施例15、式Ⅳ-6所示化合物的制备
式Ⅳ-4所示化合物(100mg)，HONH2·HCl(48mg)，NaOAc(150mg)溶于乙醇(10ml)，反应液60摄氏度加热搅拌3h。反应液冷却到室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯10：1)，得式1所示化合物。
Figure PCTCN2017082111-appb-000024
1H-NMR(400MHz,CDCl3)δ(ppm)9.14(s,1H),5.10(s,1H),3.14-3.10(m,1H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)167.2,135.0,134.0,131.0,125.4,51.5,50.5,50.0,44.6,40.5,37.3,36.5,36.4,35.8,31.1,31.0,28.3,27.1,26.5,25.9,25.1,24.4,23.2,21.2,19.1,18.9,18.8,17.8,17.7,16.0
式1所示化合物(50mg)溶于无水乙醚中，LiAlH4(50mg)加入到反应液中，室温反应3h。慢慢加入水来终止反应。用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(二氯甲烷：甲醇10：1)，得式Ⅳ-6所示化合物(20mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)8.25(s,2H),5.10(m,1H),2.90(d,J＝ 10.0Hz,1H),0.68(s,3H).
经表征，所制备的化合物结构正确。
实施例16、式Ⅳ-7所示化合物的制备
式Ⅲ-6所示化合物(100mg)，PCC(100mg)和NaOAc(10mg)溶解到二氯甲烷中，室温搅拌1h。加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯20：1)，得式Ⅳ-7所示化合物(90mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)2.56-2.40(m,2H),0.88(s,6H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)217.9,135.47,133.28,71.17,51.4,50.6,50.0,47.5,44.6,44.5,37.0,36.9,36.6,36.2,34.7,31.1,31.0,29.5,29.4,28.3,26.5,26.3,24.4,21.4,21.2,19.6,18.8,16.0.
经表征，所制备的化合物结构正确。
实施例17、式Ⅳ-8所示化合物的制备
式Ⅳ-7所示化合物(100mg)，HONH2·HCl(48mg)，NaOAc(150mg)溶于乙醇(10ml)，反应液60摄氏度加热搅拌3h。反应液冷却到室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝10：1，体积比)，得式Ⅳ-8所示化合物(95mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)9.11(s,br,1H),3.14-3.10(m,1H),0.91-0.89(m,6H),0.85(s,3H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)167.2,134.9,133.9,71.3,51.5,50.6,50.0,44.6,44.5,40.5,37.3,36.8,36.6,35.8,31.1,31.0,29.5,29.3,28.3,27.0,26.5,24.4,23.2,21.3,21.2,19.1,18.9,18.8,17.7,15.9.
经表征，所制备的化合物结构正确。
实施例18、式Ⅳ-9所示化合物的制备
将40mg式Ⅲ-6所示化合物溶于4ml吡啶中，加入12mg丁二酸酐和11mg DMAP。置于80℃下搅拌3h。TLC检测反应完成后用10％的盐酸洗去吡啶，并用乙酸乙酯萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱(二氯甲烷：甲醇＝20：1，体积比)分离得到31mg式Ⅳ-9所示化合物。
1H-NMR(400MHz,CDCl3)δ(ppm)4.52(dd,J＝11.6Hz,J＝4.8Hz,1H),2.69-2.63(m,4H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)172.0,170.7,134.6,134.3,81.6,71.4,60.6,50.6,49.9,44.6,44.5,38.0,37.0,36.8,36.6,35.3,31.1,30.9,29.5,29.4,29.3,29.1,28.5,28.4,28.0,26.5,24.4,24.2,21.3.21.2,21.1, 19.3,18.8,18.2,16.7.
经表征，所制备的化合物结构正确。
实施例19、式Ⅳ-10所示化合物的制备
式1所示化合物(50mg)溶于无水乙醚中，LiAlH4(50mg)加入到反应液中，室温反应3h。慢慢加入水来终止反应。用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(二氯甲烷：甲醇：三乙胺＝10：1：0.1，体积比)，得式Ⅳ-11所示化合物(20mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)2.68(s,1H),2.46-2.43(m,1H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.6,134.2,70.1,68.4,59.7,51.1,50.9,50.5,49.7,44.3,43.9,38.3,37.04,36.97,36.9,36.7,36.4,35.8,35.6,31.0,30.9,30.5,27.9,27.7,27.0,26.3,25.4,25.2,23.3,20.69,20.65,18.4,18.13,18.07.
经表征，所制备的化合物结构正确。
实施例20、式Ⅳ-11所示化合物的制备
式Ⅳ-7所示化合物(100mg)、MeONH2·HCl(56mg)、NaOAc(150mg)溶于乙醇(10ml)，反应液60摄氏度加热搅拌3h。反应液冷却到室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯10：1)，得式Ⅳ-12所示化合物(95mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.81(s,3H),3.00(d,J＝14.4Hz,1H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)166.2,134.9,134.0,77.5,77.2,76.8,71.3,61.2,51.6,50.7,50.0,44.62,44.56,40.2,37.2,36.9,36.6,35.8,31.1,31.0,29.5,29.4,28.4,27.2,26.5,24.4,23.4,21.3,21.2,19.1,18.8,18.3,16.0.
经表征，所制备的化合物结构正确。
实施例21、式Ⅳ-12和式Ⅳ-13所示化合物的制备
式Ⅲ-6所示化合物(90mg)溶于无水THF(10mL)中，NaH(5个当量)和碘甲烷(5个当量)加入到反应液中。室温搅拌12h。反应完毕后，逐滴加入水来终止反应。二氯甲烷加入到反应液中，有机相用无水硫酸钠干燥，然后旋转蒸发仪蒸干。所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝30：1)得式Ⅳ-13所示化合物(30mg)和式Ⅳ-14所示化合物(30mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.36(s,3H),3.69-3.65(m,1H),0.69(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.7,134.5,88.8,77.5,77.2,76.8,74.8,57.7,51.1,50.7,50.0,49.2,44.6,40.5,39.0,37.2,37.0,36.6,35.6,31.2,31.0,28.4, 28.1,26.6,25.2,24.4,22.8,21.2,20.7,19.3,18.9,18.3,16.3,15.9.
1H-NMR(400MHz,CDCl3)δ(ppm)3.37(s,3H),3.17(s,3H),2.69-2.65(m,1H),0.68(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)134.7,134.5,88.8,71.3,57.7,51.1,50.7,50.0,44.7,44.6,39.0,37.2,36.9,36.6,35.7,31.2,31.0,29.5,29.4,28.4,28.1,26.6,24.4,22.8,21.3,21.2,19.3,18.8,18.3,16.3,15.9.
经表征，所制备的化合物结构正确。
实施例22、式Ⅴ-1所示化合物的制备
式Ⅲ-6所示化合物(50mg)溶于二氯甲烷(30mL)，在冰浴下，m-CPBA(85％，28mg)和NaHCO3(14mg)间隔3h分两批加入到上述溶液中，室温搅拌过夜。反应液用饱和碳酸氢钠溶液洗涤，无水碳酸钠干燥，然后再旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝5：1，体积比)，得式Ⅴ-1所示化合物(45mg)
1H-NMR(400MHz,CDCl3)δ(ppm)3.21-3.19(m,1H),0.76-0.75(m,6H)13C-NMR(100MHz,CDCl3)δ(ppm)78.6,71.2,70.8,68.3,49.0,48.5,44.5,43.7,41.9,38.6,38.0,36.7,36.4,33.0,29.42,29.36,28.6,28.4,27.3,27.0,23.6,21.6,21.2,20.1,19.1,17.1,16.6,16.4,15.2.
经表征，所制备的化合物结构正确。
实施例23、式Ⅴ-2所示化合物的制备
将37mg式Ⅴ-1所示化合物溶于3ml二氯甲烷，加入37mg PCC和4mg NaOAc，室温下搅拌2h。TLC检测反应完成后水洗，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离。将分离产物溶于乙醇，加入12mg盐酸羟胺和20mg醋酸钠。60℃搅拌2h。TLC检测反应完成后水洗，先用旋转蒸发仪蒸发除去大部分溶剂后，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱(乙酸乙酯：石油醚＝5：1，体积比)分离得到21mg式Ⅴ-2所示化合物。
1H-NMR(400MHz,CDCl3)δ(ppm)3.14-3.10(m,1H),1.00(d,J＝6Hz,3H),0.85(s,3H),0.70(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)166.4,71.3,70.7,68.3,49.0,48.5,44.5,43.7,43.1,40.0,38.1,36.7,36.4,32.9,32.1,29.44,29.38,28.6,27.6,26.9,23.9,22.8,21.7,21.2,20.1,19.1,17.4,17.2,17.0,16.4.
经表征，所制备的化合物结构正确。
实施例24、式Ⅵ所示化合物的制备
5H(30mg)、RuCl3·3H2O(1mg)和TBHP(indecane，5-6M)加入到2mL的 环己烷中。室温反应6h。旋转蒸发仪旋干，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝5：1，体积比)，得到的化合物溶于10％KOH的乙醇溶液中，回流3h，冷却至室温，加水稀释，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯＝3：1，体积比，得式Ⅵ所示化合物C(10mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)3.29-3.25(m,1H),2.89(d,J＝14.0Hz,1H),2.75(d,J＝14.0Hz,1H),2.60(d,J＝14.0Hz,1H),2.50-2.44(m,2H),0.80(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)202.6,202.4,151.9,150.8,77.8,71.2,51.8,50.3,49.3,49.1,47.5,44.4,39.9,39.0,36.6,36.3,34.2,32.3,29.5,29.4,28.0,27.8,27.5,26.0,21.1,18.7,17.7,17.0,15.6.
经表征，所制备的化合物结构正确。
实施例25、式Ⅶ-1所示化合物的制备
式Ⅳ-8所示化合物(50mg)溶于二氯甲烷/三氟乙酸(1mL/1ml)的混合溶剂中，室温反应2h。加水稀释，用二氯甲烷萃取。饱和碳酸氢钠洗涤。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱分离(石油醚：乙酸乙酯30：1)，得式Ⅶ-1所示化合物5F(20mg)。
1H-NMR(400MHz,CDCl3)δ(ppm)8.72(s,1H),3.12(d,J＝15.2Hz,1H),2.19-2.10(m,1H),0.72(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)167.3,135.0,134.0,89.5,51.5,50.6,50.0,44.6,40.9,40.5,37.3,36.43,36.38,35.8,31.1,31.0,28.3,27.1,26.5,25.8,25.7,24.4,23.2,21.2,20.6,19.1,18.9,18.7,17.7,16.0.
经表征，所制备的化合物结构正确。
实施例26、式Ⅶ-2所示化合物的制备
将66mg式Ⅳ-7所示化合物溶于5ml二氯甲烷中，加入80mg m-CPBA(纯度大于80％)和24mg NaHCO3。室温下搅拌12h。TLC检测反应完成后水洗，用二氯甲烷萃取。所得有机相采用无水硫酸钠干燥，然后再用旋转蒸发仪蒸干溶剂，所得产品采用硅胶柱(乙酸乙酯：石油醚＝1：3，体积比)分离得到53mg式Ⅶ-2所示化合物。
1H-NMR(400MHz,CDCl3)δ(ppm)2.27(dd,J＝12.2Hz,J＝3.4Hz,1H),0.87(s,6H),0.74(s,3H)；13C-NMR(100MHz,CDCl3)δ(ppm)175.0,85.9,71.5,71.2,69.8,49.3,48.4,44.4,44.1,43.4,40.5,36.6,36.3,33.1,32.4,32.3,31.8,29.4,29.3,28.5,26.9,25.6,24.9,22.0,21.6,21.2,20.1,19.1,19.0,16.5.
经表征，所制备的化合物结构正确。
下述实施例27-28所用的部分试验材料的来源如下：
本测试例用到的部分实验材料来源：
高纯度羊毛甾醇(L5768)、胆固醇(C3045)、二甲基亚砜(D8418)、多聚甲醛、Triton X-100和NP-40(Nonidet P40)购买自Sigma公司。
DAPI-Fluoromount-G荧光封片剂(0100-20)购买自SouthernBiotech(SBA)。
Anti-p62抗体(ab56416)购买自Abcam公司。
羊毛甾醇粗品购买自TCI(C0427)，纯度>50.0％(GC)。
细胞培养基、抗生素、转染试剂LipofectamineTM 2000均购买自Invitrogen公司。
Cell counting kit-8(CK04-500)购买自日本同仁化学。
其他未注明具体来源的化学试剂均为分析纯。
晶状体蛋白αA-Y118D、αB-R120G、βB2-V187E、γC-G129C和γD-W43R突变体为本研究组克隆构建，具体过程如下：
将DNA分子1插入pEGFP-N1载体(Clontech)的XhoⅠ和BamHⅠ酶切位点之间，得到重组质粒1。DNA分子1：将序列表的序列1所示的DNA分子的第354位核苷酸由T突变为G(相应的第118位氨基酸由Y突变为D)得到的DNA分子。序列表的序列1所示的DNA分子为CRY-αA的开放阅读框。
将DNA分子2插入pEGFP-N1载体(Clontech)的XhoⅠ和HindⅢ酶切位点之间，得到重组质粒2。DNA分子2：将序列表的序列2所示的DNA分子的第360位核苷酸由A突变为G(相应的第120位氨基酸由R突变为G)得到的DNA分子。序列表的序列2所示的DNA分子为CRY-αB的开放阅读框。
将DNA分子3插入pEGFP-N1载体(Clontech)的XhoⅠ和HindⅢ酶切位点之间，得到重组质粒3。DNA分子3：将序列表的序列3所示的DNA分子的第562位核苷酸由T突变为A(相应的第187位氨基酸由V突变为E)得到的DNA分子。序列表的序列3所示的DNA分子为CRY-βB2的开放阅读框。
将DNA分子4插入pEGFP-N1载体(Clontech)的XhoⅠ和HindⅢ酶切位点之间，得到重组质粒4。DNA分子4：将序列表的序列4所示的DNA分子的第387位核苷酸由G突变为T(相应的第129位氨基酸由G突变为C)得到的DNA分子。序列表的序列3所示的DNA分子为CRY-γC的开放阅读框。
将DNA分子5插入pEGFP-N1载体(Clontech)的XhoⅠ和HindⅢ酶切位点之间，得到重组质粒5。DNA分子5：将序列表的序列5所示的DNA分子的第129位核苷酸由T突变为C(相应的第43位氨基酸由W突变为R)得到的DNA分子。序列表的序列3所示的DNA分子为CRY-γD的开放阅读框。
HeLa
Figure PCTCN2017082111-appb-000025
人类晶状体上皮细胞HLE-B-3(
Figure PCTCN2017082111-appb-000026
CRL-11421TM)均购买自ATCC细胞库，培养条件按ATCC公司所建议的条件进行。
本发明测试例中未注明具体条件的实验方法，通常按照常规条件。
实施例27、本发明化合物对晶状体蛋白突变体在细胞内聚集的影响
本实施例用以下方法来测定本发明化合物对晶状体蛋白αA-Y118D、αB-R120G、βB2-V187E、γC-G129C和γD-W43R突变体在HeLa细胞内聚集情况的影响。
测试中以溶剂DMSO为空白对照，Lanosterol(羊毛甾醇)为阳性对照，Cholesterol(胆固醇)为阴性对照，并测试了现有化合物C29，作为本发明化合物的对照。具体方法如下：
Figure PCTCN2017082111-appb-000027
(1)以晶状体蛋白αB-R120G突变体为研究模型研究本发明化合物的性能
晶状体蛋白αB-R120G突变体在细胞内发生错误折叠，形成显微镜下可见的聚集小体，如图1所示，其形成聚集小体的形态均一，聚集比例稳定，是研究化学药物缓解蛋白质错误折叠的理想模型。因此，本发明首先采用晶状体蛋白αB-R120G突变体为研究模型筛选有效的化合物。
具体实验方案如下：
将状态良好的HeLa细胞接种到预先已铺好细胞爬片的12孔板中，细胞密度40％～50％；培养24h左右，细胞密度80％左右，即可以进行细胞转染，具体操作按照Invitrogen公司的转染试剂LipofectamineTM 2000所建议方案进行；转染4h后，换成DMEM新鲜培养基继续培养16h，这时晶状体蛋白αB-R120G突变体在细胞内已大量表达，形成一定比例的聚集小体；将细胞培养基换做opti-MEM，并加入本发明化合物，至终浓度为4uM；培养4h后，换成DMEM新鲜培养基，再培养4h；进行免疫荧光标片的制备：PBS洗细胞爬片3遍，4％多聚甲醛室温固定细胞30分钟，PBS洗细胞爬片3遍，0.4％Triton X-100室温孵育细胞爬片15分钟，PBS洗细胞爬片3遍，4％山羊血清室温封闭40分钟，p62抗体室温孵育1小时，PBS洗细胞爬片3遍，相应来源的荧光二抗室温孵育40分钟，PBS洗细胞爬片3遍，用DAPI-Fluoromount-G荧光封片剂封片，室温避光 放置1小时左右；应用Zeiss 710三通道显微镜进行观察，采取单盲统计细胞内的聚集情况，分析本发明化合物的药效。
所有样品设三组平行实验，并对实验进行了三次重复，通过上述方法统计分析本发明化合物对晶状体蛋白αB-R120G突变体在HeLa细胞内聚集情况，结果如表1中所示。
表1中数据显示，本发明化合物对对晶状体蛋白αB-R120G突变体在HeLa细胞内形成的聚集现象具有缓解作用，尤其是式Ⅲ-3所示化合物、式Ⅲ-6所示化合物式、Ⅲ-8所示化合物；式Ⅳ-1所示化合物、式Ⅳ-7所示化合物和式Ⅳ-12所示化合物对晶状体蛋白αB-R120G突变体在HeLa细胞内形成的聚集现象具有明显的缓解作用。
表1本发明化合物对晶状体蛋白αB-R120G突变体在细胞内聚集影响的统计
化合物 聚集比例 化合物 聚集比例 化合物 聚集比例
DMSO 73.8±4.0 式Ⅳ-1 34.2±4.0 式Ⅴ-1 44.7±16.1
Lanosterol 47.7±4.9 式Ⅳ-2 47.6±10.3 式Ⅴ-2 72.2±7.7
Cholesterol 73.7±5.0 式Ⅳ-3 61.0±8.9 式Ⅵ 55.5±10.2
C29 35.9±6.1 式Ⅳ-4 67.7±5.7 式Ⅶ-1 56.0±7.5
式Ⅲ‐1 53.8±7.6 式Ⅳ-5 47.3±9.8 式Ⅶ-2 43.6±5.9
式Ⅲ‐2 46.2±7.7 式Ⅳ-6 44.2±8.3    
式Ⅲ-3 40.2±7.7 式Ⅳ-7 41.6±13.4    
式Ⅲ-4 46.3±6.5 式Ⅳ-8 56.6±10.5    
式Ⅲ-5 65.9±9.0 式Ⅳ-9 51.8±9.3    
式Ⅲ-6 32.2±5.9 式Ⅳ-11 71.6±4.0    
式Ⅲ-7 48.4±9.3 式Ⅳ-11 40.9±8.2    
式Ⅲ-8 32.6±3.4 式Ⅳ-12 67.6±3.2    
式Ⅲ-9 59.0±5.8 式Ⅳ-13 60.6±4.1    
(2)多种晶状体蛋白突变体中验证本发明化合物的有效性。
晶状体内的蛋白质突变后发生错误折叠，大多都会导致白内障疾病。因此，在多种晶状体蛋白突变体中验证本发明化合物的药效是否具有普遍效应对白内障疾病的治疗具有重要的意义。
除αB R120G突变体外，本发明选用了晶状体蛋白αA-Y118D、βB2-V187E、γC-G129C和γD-W43R突变体对初步筛选有效的药物做进一步验证。具体方案同药物筛选的过程。通过进一步的验证，结果表明，本发明化合物式Ⅲ-6和式Ⅳ-1在多种晶状体蛋白突变体形成聚集的细胞模型中都具有明显的效果，如图2所示。
(3)初步标定本发明物的药效
本本发明初步以晶状体蛋白αB-R120G突变体为研究模型，标定本发明式Ⅲ-6所示化合物的半效应浓度EC50值，如图3所示，结果显示式Ⅲ-6所示化合 物的药效要比天然化合物羊毛甾醇有着数量级上的提升，白内障疾病的药物治疗具有巨大的潜力。
实施例29、本发明化合物细胞毒性的检测
安全有效的药物是本发明的研究原则，本发明将进一步检测本发明式Ⅲ-6所示化合物对细胞是否具有细胞毒性。测试设立1个对照组，转染空载体的HLE-B3细胞；5个实验组，转染晶状体蛋白αA-Y118D、αB-R120G、βB2-V187E、γC-G129C和γD-W43R突变体的HLE-B3细胞，模拟不同晶状体蛋白突变导致的白内障疾病。
用以下方法检测本发明化合物对不同细胞株的细胞毒性：
将状态良好的HLE-B3细胞接种到12孔板中，细胞密度40％～50％；培养24h左右，细胞密度80％左右，即可以进行细胞转染，分别转染晶状体蛋白αA-Y118D、αB-R120G、βB2-V187E、γC-G129C和γD-W43R突变体，及一个空载对照组。具体操作按照Invitrogen公司的转染试剂LipofectamineTM 2000所建议方案进行；转染4h后，换成DMEM新鲜培养基继续培养16h，这时晶状体蛋白突变体在细胞内已大量表达；用0.25％胰酶(EDTA)消化吹散后，接种到96孔板，每孔2000细胞，继续培养12小时；将细胞培养基换做opti-MEM，并加入本发明化合物1G，设定两个浓度梯度，终浓度为5uM或50nM，孵育12小时；加入10μL CKK-8，孵育1小时，450nm测吸收值。
所有样品设三组平行实验，并对实验进行了三次重复，通过上述方法统计分析本发明式Ⅲ-6所示化合物对正常晶状体上皮细胞以及转染各种晶状体蛋白突变体的细胞株均不存在细胞毒性，反而在一定程度上提高了细胞活力，结果如图4所示。
工业应用
本发明具有如下有益效果：
本发明提供的具有新颖结构的小分子，与现有的小分子(如C29，Science,350,674)相比，在抑制细胞内晶状体蛋白突变导致的蛋白聚集具有更好的活性，且提高药物的可被机体吸收性，并且对正常晶状体细胞没有毒副作用。

Claims (10)

  1. 式Ⅰ所示化合物、其前药或其药学上可接受的盐，
    Figure PCTCN2017082111-appb-100001
    式Ⅰ中，环A为六元环或七元环，当环A为六元环时，X不存在，当环A为七元环时，X表示O或NH；
    3号碳原子与R1之间为单键或双键，当为单键时，R1选自如下基团中任一种：
    Figure PCTCN2017082111-appb-100002
    当为双键时，R1选自如下基团中任一种：
    Figure PCTCN2017082111-appb-100003
    7号碳原子、8号碳原子、9号碳原子和11号碳原子相邻碳原子之间为单键或双键；
    当8号碳原子与9号碳原子之间为单键时，8号碳原子与9号碳原子之间存在环氧结构或7号碳原子与8号碳原子之间为双键或9号原子与11号碳原子之间为双键；
    当7号碳原子与8号碳原子之间为单键时，R2为H或2个R2形成羰基；
    当7号碳原子与8号碳原子之间为双键时，7号碳原子上连接一个R2，R2为H；
    当9号碳原子与11号碳原子之间为单键时，R3为H或2个R3形成羰基；
    当9号碳原子与11号碳原子之间为双键时，11号碳原子上连接一个R3，R3为H；
    R4选自如下基团中任一种：
    Figure PCTCN2017082111-appb-100004
    其中，R5和R6均为碳原子数为1-4的烷基；
    X”和X”’均选自F、Cl、Br和I；
    n为1或2。
  2. 根据权利要求1所述的化合物、其前药或其药学上可接受的盐，其特征在于：所述化合物的结构式如式Ⅱ所示，
    Figure PCTCN2017082111-appb-100005
    式Ⅱ中，R1、R2、R3、R4和n的定义同式Ⅰ中。
  3. 根据权利要求2所述的化合物、其前药或其药学上可接受的盐，其特征在于：所述化合物的结构式如式Ⅲ所示，
    Figure PCTCN2017082111-appb-100006
    式Ⅲ中，R’4选自如下基团中任一种：
    Figure PCTCN2017082111-appb-100007
  4. 根据权利要求2所述的化合物、其前药或其药学上可接受的盐，其特征在于：所述化合物的结构式如式Ⅳ所示，
    Figure PCTCN2017082111-appb-100008
    式Ⅳ中，R’1与碳原子之间为单键或双键；
    当为单键时，R’1选自如下基团中任一种：
    Figure PCTCN2017082111-appb-100009
    当为双键时，R’1选自如下基团中任一种：
    Figure PCTCN2017082111-appb-100010
    R”4选自如下基团中任一种：
    Figure PCTCN2017082111-appb-100011
  5. 根据权利要求2所述的化合物、其前药或其药学上可接受的盐，其特征在于：所述化合物的结构式如式Ⅴ所示，
    Figure PCTCN2017082111-appb-100012
    式Ⅴ中，R”1与碳原子之间为单键或双键；
    当为单键时，R”1为-OH；
    当为双键时，R”1为
    Figure PCTCN2017082111-appb-100013
  6. 根据权利要求2所述的化合物、其前药或其药学上可接受的盐，其特征在于：所述化合物的结构式如式Ⅵ所示，
    Figure PCTCN2017082111-appb-100014
  7. 根据权利要求1所述的化合物、其前药或其药学上可接受的盐，其特征在于：所述化合物的结构式如式Ⅶ所示，
    Figure PCTCN2017082111-appb-100015
    式Ⅶ中，X表示O或NH。
  8. 权利要求1-7中任一项所述化合物、其前药或其药学上可接受的盐在制 备治疗白内障的药物中的应用。
  9. 权利要求1-7中任一项所述化合物、其前药或其药学上可接受的盐在制备阻止、缓解或逆转晶状体蛋白在细胞内聚集的产品中的应用。
  10. 一种治疗白内障的药物，其活性成分为权利要求1-7中任一项所述化合物、其前药或其药学上可接受的盐。
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