CN106866784B - Targeted mitochondrial antioxidant and preparation method and application thereof - Google Patents

Abstract

The invention discloses a compound which is shown as a formula I or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite and a pharmaceutically acceptable salt of the compound shown as the formula I, wherein Cbz represents benzyloxycarbonyl, Bn represents benzyl and Boc represents tert-butoxycarbonyl. The compound has stable physicochemical property, and compared with XJB-5-131, the compound has improved water solubility, stable physicochemical property, good bioactivity, small medicinal dose, improved in vivo stability, high bioavailability, small side effect, and outstanding targeted oxidation resistance, and can be effectively used for repairing mitochondrial oxidative damage and preventing or treating neurodegenerative diseases such as age-related macular degeneration.

Description

Targeted mitochondrial antioxidant and preparation method and application thereof

PRIORITY INFORMATION

This application claims priority and benefit to patent application No. 201510922167.8 filed on 11/12/2015 with the chinese national intellectual property office, and is incorporated herein by reference in its entirety.

Technical Field

The invention relates to the technical field of biochemistry, in particular to the field of compound preparation, and more particularly relates to a novel compound (formula I) and a preparation method and application thereof.

Background

Mitochondrial oxidative damage is associated with a number of neurodegenerative diseases including Dry senile maculopathy (Dry-AMD), Glaucoma (Glaucoma), Traumatic Brain Injury (TBI), Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD), and the like. Among them, Huntington's chorea is a genetic disease in which neurons in some regions of the brain are increasingly weakened. Disease symptoms, including loss of muscle coordination and decline in cognitive abilities, often manifest in middle age. The disease cannot be cured at present. Currently, physicians only administer patients with antidepressants or compounds that reduce the loss of motor coordination, both of which fail to slow the progression of the disease.

XJB-5-131 is a mitochondrial antioxidant with good targeting property, has good biological activity, and has been studied to show that XJB-5-131 has obvious curative effect in animal models of neurodegeneration of traumatic brain injury and animal models of Huntington's disease, and has good clinical application prospect (for example, see Nature Neuroscience 2012,15(10), 1407; Accounts of Chemical Research 2008,41(1), 87; J.Am.Chem.Soc.2005,127,12460, which is incorporated herein by reference in its entirety).

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a compound with stable physicochemical properties and better potential bioactivity and mitochondrial antioxidant property compared with XJB-5-131.

It should be noted that the present invention is completed based on the following work of the inventors: a plurality of analogues are obtained by carrying out structural optimization on XJB-5-131, and then a series of biological researches and animal model tests are carried out on the various analogues so as to screen out a novel mitochondrial antioxidant with good biological activity, outstanding targeted oxidation resistance and stable physicochemical property. As a result, the compound (namely the compound shown in the formula I) obtained by screening is stable in physicochemical property, higher in water solubility, more stable in crystal form, good in biological activity, small in medicinal dosage, high in-vivo bioavailability, small in side effect and prominent in targeted oxidation resistance compared with XJB-5-131. Furthermore, animal model experiments show that the compound I can effectively repair the injury of eye macula lutea of SD rats, prevent the apoptosis of retina layer cells of the rats and protect the retina cells, thereby relieving the eye symptoms of the rats.

Thus, according to one aspect of the invention, there is provided a compound. According to an embodiment of the invention, the compound is a compound of formula I or a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, a pharmaceutically acceptable salt of a compound of formula I,

wherein the content of the first and second substances,

cbz represents benzyloxycarbonyl, Bn represents benzyl, and Boc represents t-butoxycarbonyl.

The inventor finds that the compound has stable physicochemical properties, higher water solubility than XJB-5-131, easy crystallization, stable physicochemical properties, good biological activity, small medicinal dosage, high in-vivo bioavailability, small side effect and outstanding targeted antioxidation performance, can be effectively used for repairing mitochondrial oxidative damage and preventing or treating neurodegenerative diseases such as Dry senile maculopathy (Dry-AMD), Glaucoma (Glaucoma), Traumatic Brain Injury (TBI), Amyotrophic Lateral Sclerosis (ALS), Alzheimer Disease (AD), Parkinson Disease (PD), Huntington's Disease (HD) and the like.

It is to be noted that, unless otherwise indicated, all tautomeric forms of the compounds of the invention are included within the scope of the invention. In addition, unless otherwise indicated, the structural formulae of the compounds described herein include isotopically enriched concentrations of one or more different atoms.

The term "hydrate" as used herein refers to a compound provided by the present invention or a salt thereof, which further comprises water bound by non-covalent intermolecular forces in a chemical amount or in a non-chemical equivalent amount, and may be said to be an association of solvent molecules with water.

The term "solvate" refers to an association of one or more solvent molecules with a compound of the invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol.

The term "nitroxide" means that when a compound contains several amine functional groups, 1 or more than 1 nitrogen atom can be oxidized to form an N-oxide. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. The corresponding amines can be treated with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid) to form the N-oxide (see Advanced Organic Chemistry, Wiley Interscience, 4 th edition, Jerry March). In particular, the N-oxide may be prepared by the method of L.W.Deady (Syn.Comm.1977,7,509-514) in which an amine compound is reacted with m-chloroperoxybenzoic acid (mCPBA), for example, in an inert solvent such as methylene chloride.

The term "metabolite" refers to the product of the metabolism of a particular compound or salt thereof in vivo. Metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by assay methods as described herein. Such products may be obtained by administering the compound by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting a compound of the present invention with a mammal for a sufficient period of time.

Various pharmaceutically acceptable salt forms of the compounds of the present invention are useful. The term "pharmaceutically acceptable salts" means those salt forms that are readily apparent to the pharmaceutical chemist as being substantially non-toxic and providing the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion. Other factors, more practical in nature, are also important for selection, these are: cost of raw materials, ease of crystallization, yield, stability, hygroscopicity and, as a result, flowability of the drug substance. Briefly, the pharmaceutical composition can be prepared by combining the active ingredient with a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable salts" as used herein refers to both organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as are: berge et al, description of the descriptive pharmaceutical acceptable salts in detail in J. pharmaceutical Sciences,66:1-19,1977. Pharmaceutically acceptable non-toxic acid salts include, but are not limited to, salts of inorganic acids formed by reaction with amino groups such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates, and salts of organic acids such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or those obtained by other methods described in the literature above, such as ion exchange. Other pharmaceutically acceptable salts include adipates, malates, 2-hydroxypropionates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, cyclopentylpropionates, digluconates, dodecylpropionatesSulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts obtained with appropriate bases include alkali metals, alkaline earth metals, ammonium and N⁺(C_1-4Alkyl radical)₄A salt. The present invention also contemplates quaternary ammonium salts formed from compounds containing groups of N. Water-soluble or oil-soluble or dispersion products can be obtained by quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations resistant to formation of counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C_1-8Sulfonates and aromatic sulfonates. Amine salts such as, but not limited to, N '-dibenzylethylenediamine, chloroprocaine, choline, ammonia, isopropylamine, benzathine (benzathine), choline (cholinate), lysine, meglumine (meglumine), piperazine, tromethamine, diethanolamine and other hydroxyalkylamine salts, ethylenediamine, N-methyl reduced glucamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidin-1' -ylmethyl-benzimidazolium and other alkylamine salts, piperazine and tris (hydroxymethyl) aminomethane; alkaline earth metal salts such as, but not limited to, barium, calcium and magnesium salts; transition metal salts such as, but not limited to, zinc salts.

The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are usually carried out in water or an organic solvent or a mixture of both. Generally, where appropriate, it is desirable to use a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. In, for example, "Remington's Pharmaceutical Sciences", 20 th edition, Mack Publishing Company, Easton, Pa., (1985); and "handbook of pharmaceutically acceptable salts: properties, Selection and application (Handbook of Pharmaceutical Salts: Properties, Selection, and Use) ", Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002) may find some additional lists of suitable Salts.

In addition, the compounds disclosed herein, including their salts, may also be obtained in the form of their hydrates or in the form of solvents containing them (e.g., ethanol, DMSO, etc.), for their crystallization. The compounds disclosed herein may form solvates with pharmaceutically acceptable solvents (including water), either inherently or by design; thus, the present invention is intended to include both solvated and unsolvated forms.

The term "treating" or "treatment" as used herein refers, in some embodiments, to ameliorating a disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof). In other embodiments, "treating" refers to moderating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a parameter of the body), or both. In other embodiments, "treating" or "treatment" refers to preventing or delaying the onset, occurrence, or worsening of a disease or disorder.

According to a further aspect of the invention there is provided a method for preparing a compound as hereinbefore described. According to an embodiment of the invention, the method comprises the steps of:

contacting 3-yne-1-butanol with imidazole and tert-butyldiphenylchlorosilane so as to obtain (3-yne-1-butyloxy) tert-butyldiphenylsilyl ether;

contacting 3-methylbutanal with (R) -tert-butylsulfenamide, magnesium sulfate and PPTS, so as to obtain (R) - ((E) -isoamylimine) -tert-butylsulfenamide;

contacting (3-yne-1-butyloxy) tert-butyldiphenylsilyl ether with (R) - ((E) -isoamylimine) -tert-butylsulfinamide, so as to obtain 8-tert-butyldiphenylsiloxy-2-methyl-5-octene- (S) -4-aza- (R) -tert-butylsulfinamide;

contacting 8-tert-butyldiphenylsiloxy-2-methyl-5-octene- (S) -4-aza- (R) -tert-butylsulfinamide with a solid sodium bicarbonate to obtain (S, E) -tert-butyl- (8-hydroxy-2-methyl-5-octene) carbamate;

contacting (S, E) -tert-butyl- (8-hydroxy-2-methyl-5-octene) carbamate with jones reagent to obtain (S, E) -5- ((tert-butylcarbonyl) amino) -7-methyl-3 octenoic acid;

contacting (S, E) -5- ((tert-butylcarbonyl) amino) -7-methyl-3 octenoic acid with (S) -4-benzyl-3-oxazolidinone to obtain tert-butyl ((S, E) -8- ((S) -4-benzyl-2-oxazolidinone) -2-methyl-8-carbonyl-5-enyl) -carbamate;

contacting tert-butyl ((S, E) -8- ((S) -4-benzyl-2-oxazolidinone) -2-methyl-8-carbonyl-5-enyl) -carbamate with tetrahydrofuran and benzyl bromide to obtain tert-butyl ((4S,7S, E) -7-benzyl-8- ((S) -4-benzyl-oxazolidinone) -2-methyl-8-carbonyl-5-enyl) -carbamate;

contacting tert-butyl ((4S,7S, E) -7-benzyl-8- ((S) -4-benzyl-oxazolidinone) -2-methyl-8-carbonyl-5-enyl) -carbamate with hydrogen peroxide, lithium hydroxide monohydrate, and sodium thiosulfate to obtain (2S,5S, E) -2-benzyl-5- ((tert-butoxycarbonyl) amino) -7-methyl-3-octenoic acid;

contacting (2S,5S, E) -2-benzyl-5- ((tert-butoxycarbonyl) amino) -7-methyl-3-octenoic acid with (S) -5- ((benzyloxycarbonyl) amino) -2- ((R) -3-methyl-2 ((S) -pyrrolidine-2-carboxamido) -butyrylamino) pentanoic acid methyl ester, so as to obtain (S) -methyl 2- ((R) -2- ((S) -1- (2S,5S, E) -2-benzyl-5- ((tert-butoxycarbonyl) amino) -7-methyl-3-octene) pyrrolidinyl-2-carboxamido) -3-methylbutanamido) -5- ((benzyloxycarbonyl) amino) pentanoate; and

contacting methyl (S) -2- ((R) -2- ((S) -1- (2S,5S, E) -2-benzyl-5- ((tert-butoxycarbonyl) amino) -7-methyl-3-octene) pyrrolidinyl-2-carboxamido) -3-methylbutanamido) -5- ((benzyloxycarbonyl) amino) pentanoate with sodium hydroxide to give a crude product, and then contacting the crude product with 4-amino 2,2,6, 6-tetramethylpiperidine oxide, 1-hydroxybenzotriazole, carbodiimide, and 4-dimethylaminopyridine to obtain the compound described previously.

Therefore, the method can be used for efficiently preparing the compound shown in the formula I, and has the advantages of few steps, low cost, good stability of the obtained compound, and outstanding bioactivity and mitochondrion targeting oxidation resistance.

Wherein, when useful in therapy, a therapeutically effective amount of a compound of formula I and pharmaceutically acceptable salts thereof may be administered as a raw chemical or may be provided as an active ingredient in a pharmaceutical composition. Thus, according to a further aspect of the invention, there is also provided a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises a compound as described above. The inventor finds that the pharmaceutical composition can effectively repair mitochondrial oxidative damage, and thus can be effectively used for preventing or treating neurodegenerative diseases such as Dry age-related macular degeneration (Dry-AMD), Glaucoma (Glaucoma), Traumatic Brain Injury (TBI), Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD) and the like.

According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, or any combination thereof.

Suitable carriers, adjuvants and excipients are well known to those skilled in the art and are described in detail, for example, in Ansel h.c.et al, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (2004) Lippincott, Williams & Wilkins, philidelphia; gennaro a.r.et al, Remington: the Science and Practice of Pharmacy (2000) Lippincott, Williams & Wilkins, Philadelphia; and Rowe R.C., Handbook of Pharmaceutical Excipients (2005) Pharmaceutical Press, Chicago.

The term "therapeutically effective amount" as used herein refers to the total amount of each active ingredient sufficient to show meaningful patient benefit. When the active ingredient alone is used for separate administration, the term refers only to that ingredient. When used in combination, the term refers to the combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, sequentially or simultaneously. The compounds of formula I and their pharmaceutically acceptable salts are as described above. The carrier, diluent or excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. According to another aspect of the present disclosure there is also provided a process for the preparation of a pharmaceutical formulation, which process comprises admixing a compound of formula I, or a pharmaceutically acceptable salt thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients. The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, and which are effective for their intended use.

In general, the compounds of the present invention are administered in therapeutically effective amounts by any conventional means of administration for substances that exert similar effects. The appropriate dosage range will depend upon a variety of factors such as the severity of the disease being treated, the age and relative health of the subject being administered, the potency of the compound used, the route and form of administration, the indication for which administration is being effected and the preferences and experience of the relevant medical practitioner. One of ordinary skill in the art of treating such diseases will be able to determine, without undue experimentation and relying on personal knowledge and the disclosure of this application, a therapeutically effective amount of a compound of the present invention for a given disease.

Typically, the compounds of the invention are administered in the form of pharmaceutical formulations including those suitable for oral (including buccal and sublingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous and intravenous) administration or for administration by inhalation or insufflation. The preferred mode of administration is generally oral, and may be adjusted to the level of pain using a suitable daily dosage regimen.

One or more compounds of the present invention may be presented in pharmaceutical compositions and unit dosage forms together with one or more conventional adjuvants, carriers or diluents. The pharmaceutical compositions and unit dosage forms can contain conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and the unit dosage forms can contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. The pharmaceutical compositions may be applied in the form of solids such as tablets or filled capsules, semisolids, powders, sustained release formulations or liquids such as solutions, suspensions, emulsions, elixirs or filled capsules for oral use; or in the form of suppositories for rectal or vaginal administration; or in the form of a sterile injectable solution for parenteral use.

The compounds of the present invention may be formulated in a variety of orally administered dosage forms. Pharmaceutical compositions and dosage forms may comprise one or more compounds of the invention or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutically acceptable carrier may be a solid or a liquid. Formulations in solid form include: powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. In powders, the carrier is typically a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is usually mixed with a carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, a low melting wax, cocoa butter, and the like. The term "formulation" is intended to include preparations of the active compound that contain the encapsulating material as a carrier to provide a capsule in which the active ingredient, with or without a carrier, is surrounded by the carrier in association therewith. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges are solid forms suitable for oral administration.

Other forms suitable for oral administration include liquid form preparations (including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions) or solid form preparations which are intended to be converted into a liquid form preparation immediately before use. Emulsions may be prepared in solutions such as aqueous propylene glycol or may contain emulsifying agents such as lecithin, sorbitan monooleate or acacia. Aqueous solutions can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickening agents. Aqueous suspensions may be formulated by dispersing the finely divided active ingredient in water with viscous material, for example, natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents. Liquid form preparations include solutions, suspensions, and emulsions, which may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

The compounds of the invention may be formulated for parenteral administration (e.g., by injection, such as bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. Examples of oily or non-aqueous carriers, diluents, solvents or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g. olive oil) and injectable organic esters (e.g. ethyl oleate), and may contain formulatory agents such as preservatives, wetting agents, emulsifying or suspending agents, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic packaging of a sterile solid or by lyophilisation of a solution for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.

The compounds of the present invention may be formulated for topical application to the epidermis in the form of an ointment, cream or lotion, or in the form of a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or coloring agents. Formulations suitable for topical administration in the mouth include: lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The compounds of the present invention may be formulated for administration in the form of suppositories. The low melting wax, such as a fatty acid glyceride mixture or cocoa butter, can be melted first and the active ingredient dispersed homogeneously, for example by stirring. The molten homogeneous mixture is then poured into a suitably sized mold, allowed to cool and solidify.

The compounds of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

The compounds of the present invention may be formulated for nasal administration. The solutions or suspensions can be applied directly to the nasal cavity by conventional means, for example, with a dropper, pipette or nebulizer. The formulations may be in single or multiple dose form. For a dropper or pipette multi-dose form, this may be achieved by the patient administering an appropriate, predetermined volume of solution or suspension. For a nebulizer, this can be achieved, for example, by a metered atomizing spray pump.

The compounds of the invention may be formulated for aerosol administration, particularly to the respiratory tract and including intranasal administration. The compounds typically have small particle sizes, for example, on the order of 5 microns or less. The particle size may be obtained by methods well known in the art, for example by micronization. The active ingredient is provided in pressurized packs containing a suitable propellant, such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane, or carbon dioxide or other suitable gas. The aerosol may also suitably contain a surfactant such as lecithin. The dosage of the medicament may be controlled by a metering valve. Alternatively, the active ingredient may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone. The powder carrier will form a gel in the nasal cavity. The powder compositions may be presented in unit dosage form, for example in gelatin capsules or cartridges or blister packs from which the powder may be administered by means of an inhaler.

If desired, the formulations may be prepared with enteric coatings suitable for sustained or controlled release administration of the active ingredient. For example, the compounds of the present invention may be formulated as transdermal or subcutaneous drug delivery devices. These delivery systems are advantageous when sustained release of the compound is necessary and when patient compliance with the treatment regimen is critical. The compounds in transdermal delivery systems are often attached to a skin-adherent solid carrier. The compounds of interest may also be used in combination with a penetration enhancer, such as laurocapram (1-dodecyl azepan-2-one). The sustained release delivery system may be inserted subcutaneously into the subcutaneous layer by surgery or injection. Subcutaneous implants encapsulate compounds in a liquid soluble film, such as silicone rubber, or a biodegradable polymer, such as polylactic acid.

The pharmaceutical preparation is preferably in unit dosage form. In this form, the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as tablets, capsules, and powders or ampoules in vials. In addition, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in a packaged form.

Other suitable pharmaceutical carriers and their formulations are described in Remington: the Science and Practice of Pharmacy1995Martin, edited by E.W, Mack Publishing Company, 19 th edition, Easton, Pennsylvania.

According to another aspect of the present invention, there is also provided the use of a compound or pharmaceutical composition as described above in the manufacture of a medicament for the prevention or treatment of a neurodegenerative disease.

According to an embodiment of the invention, the neurodegenerative disease is dry age-related macular, glaucoma, traumatic brain injury, amyotrophic lateral sclerosis, alzheimer's disease, parkinson's disease, huntington's disease.

It is noted that the compounds and pharmaceutical compositions of the present invention, in addition to being beneficial for human therapy, may also find application in veterinary therapy for pets, animals of the introduced species, and mammals in farm animals. Examples of other animals include horses, dogs, and cats. Herein, the compound of the present invention includes pharmaceutically acceptable derivatives thereof.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIGS. 1-2 show results of pH stability studies according to one embodiment of the present invention;

FIG. 3 shows the results of the test of the inhibitory effect of Compound I on Erastin-induced HT1080 cell iron death, according to one embodiment of the present invention;

FIG. 4 shows the results of a test for the recovery of damaged ARPE-19 cells from hydrogen peroxide by Compound I, according to an embodiment of the present invention;

FIG. 5 shows the results of visual electrophysiological examination of Compound I, according to one embodiment of the present invention.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of Compound I

A,Intermediate 13 synthesis:

first, intermediate 13 was synthesized according to the following synthetic route:

the method comprises the following steps: synthesis of Compound 5

Compound 4(10g, 37.6mmol) was dissolved in methanol (150mL), cooled to 0 deg.C, and thionyl chloride (22g, 0.19mmol) was added dropwise thereto.

The reaction solution was gradually warmed to room temperature with stirring, and stirring was continued for 12 hours. The reaction mixture was concentrated under reduced pressure to give compound 5(11.7g) in 98% yield.

Step two: synthesis of Compound 2

Compound 1(6.45g, 30mmol) and D-Valine methyl ester hydrochloride (5.53g, 33mmol) were dissolved in dichloromethane (200mL) and cooled to 0 ℃. To the above solution were added 1-hydroxybenzotriazole (4.46g, 33mmol), carbodiimide (6.3g, 33mmol) and DIEA (8.51g, 66 mmol).

The reaction was gradually warmed to room temperature, stirred for 12h, quenched with aqueous hydrochloric acid (100mL, 1M), and extracted with dichloromethane (200 mL. times.3). The organic phases were combined and washed with saturated aqueous sodium chloride (100 mL). Separating, collecting organic phase, drying, and concentrating. The residue was subjected to silica gel column chromatography to give compound 2(9.1g) in 93% yield.

Step three: synthesis of Compound 3

Compound 2(2.0g, 6.1mmol) was dissolved in tetrahydrofuran/methanol/water (8mL/4mL/2mL), cooled to 0 deg.C, and a lithium hydroxide solution (10mL, 1M aqueous solution) was added dropwise thereto.

The reaction was stirred at 0 ℃ for 2 h. Most of the tetrahydrofuran was removed under reduced pressure, and the residue was acidified with aqueous hydrochloric acid (20mL,1M) and then extracted with ethyl acetate (50 mL. times.2). The organic phases were combined and washed with saturated aqueous sodium chloride (30 mL). The organic phase was collected by liquid separation, dried and concentrated to obtain compound 3(1.8g) as a white solid with a yield of 95%.

Step three: synthesis of Compound 6

Compound 3(5g, 15.9mmol) and compound 5(6g, 19mmol) were dissolved in dichloromethane (100mL) and cooled to 0 ℃. To the above solution were added 1-hydroxybenzotriazole (2.36g, 17.5mmol), carbodiimide (3.34g, 17.5mmol) and triethylamine (3.21g, 31.8 mmol).

The reaction was stirred at 0-15 ℃ for 6h, then quenched with aqueous hydrochloric acid (20mL,1M) and extracted with dichloromethane (100 mL. times.2). The organic phases were combined and washed with saturated aqueous sodium chloride (50 mL). Separating, collecting organic phase, drying, and concentrating. The residue was subjected to silica gel column chromatography to give compound 6(8.5g) in 93% yield.

Step four: synthesis of Compound 13

Compound 6(1.2g, 2.08mmol) was dissolved in dichloromethane (10mL), cooled to 0 deg.C, and TFA (4mL) was added dropwise thereto. The reaction solution was gradually warmed to room temperature and stirred for 2 h. The solvent and a large amount of trifluoroacetic acid were removed by concentration under reduced pressure, and then ethyl acetate and saturated aqueous sodium carbonate solution were added to Ph 8-9. Extracted with ethyl acetate (50 mL. times.2). The organic phases were combined and washed with saturated aqueous sodium chloride (50 mL). The organic phase was collected by liquid separation, dried and concentrated to obtain compound 13(1.27g) as a white foamy solid with a yield of 98%.

II, synthesizing the compound I

Compound I is prepared according to the process of the present invention, combining the following synthetic routes, in particular as follows:

1. synthesis of Compound 8:

compound 7(40g, 0.57mol) and imidazole (43g, 0.63mol) were dissolved in dichloromethane (1L) and stirred for 10 minutes.

Tert-butyldiphenylchlorosilane (150g, 0.55mol) was dissolved in dichloromethane (500L) and then added dropwise to the above solution. The reaction was stirred at ambient temperature for 20 h.

The reaction mixture was concentrated, and separated by silica gel column chromatography to give compound 8(162g) in 96% yield.

2. Synthesis of Compound 11:

compound 9(32.1mL, 0.29mol) was dissolved in dichloromethane (1.5L). To this was added successively compound 10(30g,0.25mol), magnesium sulfate (146g, 1.21mol) and PPTS (6.2g, 0.025 mol).

The mixture was stirred at room temperature for 20h, filtered through celite, and concentrated. The residue was subjected to silica gel column chromatography to give compound 11(42g) in 91% yield.

3. Synthesis of Compound 12:

compound 8(9.8g, 27.9mmol) was dissolved in dry dichloromethane (162mL), to which Cp was added₂Zr hydrochloride (7.7g, 29.8mmol), was stirred at room temperature for 10 min.

Cooling the yellow reaction solution to 0 ℃, and slowly adding AlMe into the yellow reaction solution₃(14.9mL, 2.0M in toluene, 29.8 mmol). Stirring was then continued for 10 min.

Compound 11 was dissolved in dry dichloromethane (27mL), added to the above solution and reacted for 12h with stirring.

Ice water (10mL) was slowly added to the reaction mixture in ice bath, and an aqueous hydrochloric acid solution (20mL,1M) was added thereto. Stirring and separating the liquid. The aqueous phase was extracted with dichloromethane (100mL) and the organic phases were combined and washed with saturated sodium chloride solution (150 mL). The organic phase was collected, dried and concentrated. The residue was subjected to silica gel column chromatography to give compound 12(8.6g) in 92% yield.

4. Compound 14 synthesis:

compound 12(20g, 40mmol) was dissolved in 1, 4-dioxane (120mL), cooled to 0 deg.C, and concentrated hydrochloric acid (40mL) was added dropwise with stirring.

The reaction solution was stirred at room temperature for 16 hours, and extracted with ethyl acetateTake (200 mL. times.3). The aqueous phase was retained and 1, 4-dioxane (200mL) was added thereto, stirred for 5min, the reaction cooled to 0 deg.C and sodium bicarbonate solid (81g, 0.96mol) was added slowly followed by dropwise addition (Boc)₂O(10.5g，48mmol)。

The reaction mixture was stirred at room temperature for 8 hours, extracted with ethyl acetate (200 mL. times.3), the organic phases were combined, washed with saturated sodium chloride (200mL), dried and concentrated. The residue was subjected to silica gel column chromatography to give compound 14(9g) in 87% yield.

5. Synthesis of Compound 15:

compound 14(3g, 11.8mmol) was dissolved in acetone (150mL), cooled to 0 deg.C, and stirred.

To the above solution was added Jones reagent (11.8mL, 2.5M), and the reaction was stirred under ice-bath conditions for 2 h.

To the reaction mixture was added isopropanol (5mL), stirred for 5min, and filtered. To the resulting filtrate were added water (50mL) and ethyl acetate (300 mL). Stirring and separating the liquid. The organic phase was washed with saturated sodium chloride (100 mL). Separating, collecting organic phase, drying, and concentrating. The residue was subjected to silica gel column chromatography to give compound 15(3g) in 94% yield.

6. Synthesis of Compound 17:

compound 15(2.6g, 9.6mmol) was dissolved in dry tetrahydrofuran (50mL) and cooled to-78 ℃. To the above solution was added triethylamine (1.46mL, 10.4mmol) and PivCl (1.25mL, 10 mmol). The reaction solution was stirred at-78 deg.C for 20min, warmed to room temperature and stirred for 30 min. The reaction was then cooled to-78 ℃ again.

In another reaction flask, compound 16(1.99g, 11mmol) was dissolved in dry tetrahydrofuran (50mL), cooled to-78 deg.C, and n-butyllithium (4.2mL, 2.5M in tetrahydrofuran) was added dropwise and stirred for 20 min. The lithium salt solution of compound 16 was added dropwise to the above reaction solution containing compound 15, gradually warmed to room temperature, and stirred for 18 hours.

To the reaction mixture were added saturated ammonium chloride (25mL), water (30mL) and ethyl acetate (100 mL). Stirring and separating the liquid. The organic phase was collected and the aqueous phase was extracted with ethyl acetate (50 mL). The organic phases were combined and washed with saturated sodium chloride (50 mL). Separating, collecting organic phase, drying, and concentrating. The residue was subjected to silica gel column chromatography to give compound 17(2.6g) in 63% yield.

7. Synthesis of Compound 18:

compound 17(3.3g, 7.67mmol) was dissolved in dry tetrahydrofuran (67mL) and cooled to-78 ℃. NaHMDS (4.22mL, 2M in tetrahydrofuran) was diluted with dry tetrahydrofuran (5mL) and added dropwise to the solution.

After the reaction mixture was stirred at-78 ℃ for 30min, benzyl bromide (13.1g, 76.7mmol) was added thereto. The reaction solution was kept at-45 ℃ and stirred for 12 hours, and the reaction was quenched with saturated ammonium chloride (50mL), followed by extraction with ethyl acetate (100 mL. times.3), and the organic phase was collected and washed with saturated sodium chloride (100 mL). Separating, collecting organic phase, drying, and concentrating. The residue was subjected to silica gel column chromatography to give compound 18(3.3g) in 83% yield.

8. Compound 19 synthesis:

compound 18(1.5g, 2.9mmol) was dissolved in tetrahydrofuran/water (25mL, 4:1 vol), cooled to 0 deg.C, to which was added hydrogen peroxide (1.2mL, 30%), followed by lithium hydroxide monohydrate (480mg, 11.5mmol) and stirred for 1 h.

A saturated solution of sodium thiosulfate (10mL) was added to the reaction mixture, and the pH was adjusted to 5 with saturated sodium dihydrogen phosphate. Extract with ethyl acetate (50mL), collect the organic phase, dry, and concentrate. The residue was subjected to silica gel column chromatography to give compound 19(1g) in 94% yield.

9. Synthesis of Compound 20:

compound 19(0.2g, 0.55mmol) and compound 13(314.0mg, 0.66mmol) were dissolved in dry dichloromethane (5mL) and cooled to 0 ℃. To the above solution were added HATU (274.0mg, 0.72mmol) and DIEA (142.0mg, 1.1 mmol).

The reaction solution was gradually warmed to room temperature, stirred for 3 hours, concentrated to remove the solvent, added with ethyl acetate (30mL) and dilute hydrochloric acid (20mL,1M), stirred and extracted. The organic phase was collected by separation, dried and concentrated to give compound 20(465mg) which was used in the next reaction without further purification.

10. Synthesis of Compound I:

compound 20(465mg, 0.55mmol) was dissolved in tetrahydrofuran/methanol/water (2mL/0.5mL/0.5mL), cooled to 0 deg.C, and a lithium hydroxide solution (1.1mL, 1M aqueous solution) was added dropwise thereto.

The reaction solution was gradually warmed to room temperature with stirring, and stirring was continued for 2 h. The solvent was removed by concentration, the pH was adjusted to 2-3 with aqueous hydrochloric acid (1M), followed by extraction with ethyl acetate (30mL × 2), and the organic phases were combined and washed with saturated aqueous sodium chloride (50 mL). Separating, collecting organic phase, drying, concentrating to obtain crude acid (380mg), and directly using in next condensation reaction.

The crude acid (380mg, 0.55mmol) and 4-amino 2,2,6, 6-tetramethylpiperidine oxide (97mg, 0.57mmol) were dissolved in dry dichloromethane (5mL) and cooled to 0 ℃. To the above solution were added HATU (232mg, 0.61mmol) and DIEA (121mg, 0.94 mmol).

The reaction solution was gradually warmed to room temperature, stirred for 3 hours, concentrated to remove the organic solvent, added with dilute hydrochloric acid (20Ml,1M), extracted with ethyl acetate (30Ml × 2), and the organic phases were combined and washed with saturated aqueous sodium chloride (50 Ml). Separating, collecting organic phase, drying, and concentrating. The residue was subjected to preliminary purification by silica gel column chromatography and then to HPLC preparation to give Compound I (83mg) in 18% yield in three steps.

The target molecular structure contains nitroxide free radicals, and cannot be characterized by conventional nuclear magnetism due to the paramagnetic property of the target molecular structure. The method is characterized in that the ascorbic acid is used for reducing the nitroxide free radicals into the nitroxide free radicals, so that nuclear magnetism characterization is carried out. Mass spectrometry was performed for both molecules simultaneously. The results are as follows:

¹H NMR(400MHz,CDCl₃)ppm 7.33-7.29(m,6H),7.21-7.15(m,4H),5.63-5.49(m,4H),5.27-5.09(m,4H),4.55-4.46(m,3H),4.15-4.12(m,3H),3.48-3.09(m,8H),2.78-2.75(m,2H),2.30-2.20(m,4H),1.89-1.73(m,5H),1.56-1.49(m,4H),1.42-1.33(m,10H),1.26-1.13(m,12H),0.99-0.95(m,6H),0.87(d,J＝6.4Hz,6H)

compound I MS (ESI): 959.6[ M +1 ]]⁺(ii) a Nitrogen-hydroxyl compound: 960.8[ M +1 ]]⁺

That is, the compound prepared in this example comprises the following structure:

wherein, the names of the compounds are respectively as follows:

compound 1: (S) -1- (tert-butyloxycarbonyl) pyrolidine-2-carboxylic acid, N-tert-butyloxycarbonyl-L-proline;

compound 2: (S) -tert-butyl2- (((R) -1-methoxy-3-methyl-1-oxobutan-2-yl) carbomoyl) pyrollidine-1-carboxylate, (S) -tert-butyl2- (((R) -1-methoxy-3-methyl-1-oxo-2-butyl) carbamoyl) pyrrolidine-1-carboxylic acid;

compound 3: (R) -2- ((S) -1- (tert-butyloxycarbonyl) pyrolidine-2-carboxamid) -3-methylbutanic acid, (R) -2- ((S) -1- (tert-butyloxycarbonyl) pyrrolidine-2-carboxamido) -3-methylbutanoic acid;

compound 4: (S) -2-amino-5- (((phenyloxy) carbonyl) amino) pentanic acid, (S) -2-amino-5- ((benzyloxycarbonyl) amino) pentanoic acid;

compound 5: (S) -methyl 2-amino-5- (((benzyloxy) carbonyl) amino) pentanoate hydrochloride, (S) -2-amino-5- ((benzyloxycarbonyl) amino) pentanoic acid methyl ester hydrochloride;

compound 6: (S) -tert-butyl2- (((R) -1- (((S) -5- (((benzyloxy) carbonyl) amino) -1-methoxy-1-oxopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbomoyl) pyrollidine-1-carboxylate, (S) -tert-butyl2- (((R) -1- (((S) -5- (((benzyloxy) carbonyl) amino) -1-methoxy-1-oxo-2-pentyl) amino) -3-methyl-1-oxo-2-butyl) carbamoyl) pyrrolidine-1-carboxylic acid;

compound 7: but-3-yn-1-ol, 3-alkyne-1-butanol;

compound 8: (but-3-yn-1-yloxy) (tert-butyl) diphenylsilane, (3-yne-1-butyloxy) tert-butyldiphenylsilyl ether;

compound 9: 3-methylbutanal, 3-methylbutanal;

compound 10: (R) -2-methylpropionamide-2-sulfinamide, (R) -tert-butyl sulfenamide;

compound 11: (R, E) -2-methyl-N- (3-methybutylidene) propane-2-sulfinamide, (R) - ((E) -isoamylimine) -tert-butyl sulfenamide;

compound 12: (R) -N- ((S, E) -8- ((tert-butyldiphenylsilylyl) oxy) -2-methyl-5-en-4-yl) -2-methylpropane-2-sulfenamide, 8-tert-butyldiphenyloxy-2-methyl-5-octene- (S) -4-aza- (R) -tert-butylsulfinamide;

compound 13: (S) -methyl 5- (((benzylioxy) carbonyl) amino) -2- ((R) -3-methyl-2- ((S) -pyrolidine-2-carboxamido) butanamido) pentanoate, (S) -5- ((benzyloxycarbonyl) amino) -2- ((R) -3-methyl-2 ((S) -pyrrolidine-2-carboxamido) -butyrylamino) pentanoic acid methyl ester;

compound 14: (S, E) -tert-butyl (8-hydroxy-2-methyl-5-en-4-yl) carbamate, (S, E) -tert-butyl- (8-hydroxy-2-methyl-5-octene) carbamate;

compound 15: (S, E) -5- ((tert-butylcarbonyl) amino) -7-methyl-3-enoic acid, (S, E) -5- ((tert-butylcarbonyl) amino) -7-methyl-3-octenoic acid;

compound 16: (S) -4-benzyloxyzolidin-2-one, (S) -4-benzyl-3-oxazolidinone;

compound 17: tert-butyl ((S, E) -8- ((S) -4-benzyl-2-oxozolidin-3-yl) -2-methyl-8-oxo-5-en-4-yl) carbamate, tert-butyl ((S, E) -8- ((S) -4-benzyl-2-oxazolidinone) -2-methyl-8-carbonyl-5-alkenyl) -carbamate;

compound 18: tert-butyl ((4S,7S, E) -7-benzyl-8- ((S) -4-benzyl-2-oxoazolidin-3-yl) -2-methyl-8-oxooct-5-en-4-yl) carbamate, tert-butyl ((4S,7S, E) -7-benzyl-8- ((S) -4-benzyl-oxazolidinone) -2-methyl-8-carbonyl-5-enyl) -carbamate;

compound 19: (2S,5S, E) -2-benzyl-5- ((tert-butoxycarbonyl) amino) -7-methyl-3-enoic acid, (2S,5S, E) -2-benzyl-5- ((tert-butoxycarbonyl) amino) -7-methyl-3-octenoic acid;

compound 20: (S) -methyl 2- ((R) -2- ((S) -1- ((2S,5S, E) -2-benzyl-5- ((tert-butylcarbonyl) amino) -7-methyl-3-enyl) pyrolidine-2-carboxamido) -3-methylbutanamido) -5- (((phenyloxy) carbonyl) amino) pentanate, (S) -2- ((R) -2- ((S) -1- (2S,5S, E) -methyl 2-benzyl-5- ((tert-butoxycarbonyl) amino) -7-methyl-3-octene) pyrrolidinyl-2-carboxamido) -3-methylbutanamido) -5- ((benzyloxycarbonyl) amino) pentanoate.

Example 2 measurement of physical and chemical Properties

1. Investigation of pH stability

Buffers with pH 1.2 and pH 8.0 were prepared, as follows:

pH 1.2 hydrochloric acid solution: 765ul concentrated hydrochloric acid is taken in a volumetric flask and diluted to 100ml, thus obtaining the product.

pH 8.0 phosphate buffer solution: 0.2mol/L potassium dihydrogen phosphate solution (solution A): 2.722g of monopotassium phosphate is taken, dissolved in water and diluted to 100 ml; 0.2mol/L sodium hydroxide solution (solution B): taking 800mg of sodium hydroxide, adding water for dissolving, and diluting to 100 ml; and taking 25ml of the solution A and 23.05ml of the solution B, and adding water to dilute to 100ml to obtain the compound.

The incubation was designed and performed as follows:

preparation of compound I stock solution: 1g of the compound I obtained in example 1 are dissolved in a volumetric flask and acetonitrile is added to 10ml to give a standard solution having a concentration of 100 mg/ml.

Then, 10.0. mu.L of compound I stock solution was added to 10mL of the buffer solution at 37 ℃ for 3min of pre-incubation to give a final concentration of 100. mu.g/mL. After reactions t ═ 0, 1,4 and 20 hours, 300. mu.L were sampled and tested on the machine. The content is detected by adopting an LC-MS standard curve method.

Relevant receipts are collected at different time points, and the result shows that the chromatographic peak area and the retention time of the compound I have no obvious change. The results show that compound I prepared in example 1 has good stability in buffers of different pH (see fig. 1-2).

As shown in FIGS. 1-2, the stability of Compound I at pH 8.0 and 1.2 was relatively stable under both acidic and basic conditions for 4 hours; after 20 hours, however, there was partial decomposition of compound I under acidic conditions.

2. Solubility test in buffer

10mg of the solid of Compound I (obtained as described in example 1) are added to 10ml of buffer solution, supersaturated and screwed on, and stirred at 37 ℃ for 24 h. Standing, taking supernatant, filtering, and testing on a machine. The content was measured by LC-MS standard curve method and compared with the standard curve to calculate the solubility in each pH buffer. At pH 7.4, the solubility of compound I was 0.6 mg/mL.

The result shows that compared with the compound XJB-5-131, the solubility of the compound I is greatly improved, and the compound I is beneficial to drug absorption.

EXAMPLE 3 Compound I has inhibitory effect on Erastin-induced apoptosis of HT1080 cells (Ferroptosis)

After the diluted cells are added into a 96-well plate for adherent growth, 10 mu of MERASTIN is added to induce the iron apoptosis (Ferroptosis) state of HT1080 cells, then compound I under different concentrations is added, after incubation is carried out for 24h in 6-well of each concentration, the cell viability is detected by using a CTG (CellTiter-Glo) method, and the half maximum effective concentration of the compound I is obtained and is EC 50-275.2 nM.

The experimental results show that: the inhibition effect of the compound I on Erastin induced HT1080 cell induced iron death shows that the compound I targets mitochondria and can eliminate free radicals generated in cells (see figure 3).

Example 4 Hydrogen peroxide Induction of ARPE cell death

The diluted cells were added to 96-well plates and after adherent growth, different concentrations of H2O2 (300. mu. mol/L) were added to normal groups and 6 wells per concentration. After adherent growth, drugs (1 × 10-1 × 10-5mol/L) with different concentrations are added, each concentration is 6 holes, and the control group is added with DMSO and culture solution mixed liquor (without drugs) with the same volume. Adding H2O2 (concentration of about 30% and 50% proliferation inhibition rate), incubating at 37 deg.C for a certain time, adding MTT solution, incubating for 4H/37 deg.C, pouring out most culture solution, adding 100mL DMSO to dissolve purple crystal, and reading with enzyme-labeling instrument to obtain OD value.

The experimental results show that: the compound I has a recovery effect on ARPE-19 cells damaged by hydrogen peroxide (see figure 4). As shown in FIG. 4, under the induction of 300. mu. mol/L hydrogen peroxide, the compound I has the effect of restoring ARPE-19 cells.

Example 5 Dry age-related macular degeneration (Dry-AMD) detection

After the quarantine of 24 SD rats is finished, visual electrophysiological detection is carried out and the FERG is recorded. Animals were randomized into three groups (n ═ 8), normal group, model group, compound I treatment group (group I, administered with compound I prepared in example 1) according to the FERG b wave amplitude. Model group and treatment group, wherein the model is made by illuminating for 8h in a 10000Lux white cold light source illumination box, and after the illumination is finished, the animal is transferred to an experimental animal room for normal feeding. After successful modeling, the normal group and the model group were each administered intravitreally with a blank physiological saline, and the compound I treatment group (group I, administered with compound I prepared in example 1) was each administered intravitreally with 0.1 mg/kg. The time was continuously observed for two cycles by the FERG wave amplitude, and the a-wave and b-wave amplitudes were recorded. Two weeks later, the animals were sacrificed and then the animals were sampled and HE stained sections were taken to observe retinal thickness changes and the like.

The results show that: at a concentration of 0.05mM, the b-wave amplitude change value of compound I was significantly different from that of the model group (see fig. 5). As shown in fig. 5, the SD rats had significant difference in b-wave at 0.05mM under white light illumination for 8h, indicating that compound I can protect retinal cell apoptosis.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims (5)

1. A compound of formula I or a pharmaceutically acceptable salt of a compound of formula I,

wherein the content of the first and second substances,

cbz represents benzyloxycarbonyl, Bn represents benzyl, and Boc represents t-butoxycarbonyl.

2. A pharmaceutical composition comprising a compound of claim 1.

3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, or any combination thereof.

4. Use of the compound of claim 1 or the pharmaceutical composition of claim 2 or 3 for the preparation of a medicament for the prevention or treatment of a neurodegenerative disease.

5. The use according to claim 4, wherein said neurodegenerative disease comprises dry senile maculopathy, glaucoma, traumatic brain injury, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, Huntington's disease.

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