Light-controlled carbon monoxide and / or nitric oxide release compounds and their use in treatment of glaucoma

Abstract

The invention provides a light-controlled carbon monoxide and / or nitric oxide release compound and application thereof in glaucoma treatment, and particularly, the compound has a structure as shown in a formula I, and each group in the formula I is described in the specification. The compound releases carbon monoxide and / or nitric oxide under visible light irradiation. After being packaged, the compound permeates into an aqueous humor outflow channel through cornea, and is activated by visible light to release carbon monoxide and / or nitric oxide, so that the up-regulation of the content of soluble guanylate cyclase and the anti-oxidation effect are realized, the intraocular pressure of a glaucoma mouse model is obviously reduced, and the compound has important drug development potential. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a light-controlled carbon monoxide and / or nitric oxide releasing compound and its application in the treatment of glaucoma. Background Art

[0002] Glaucoma is an ocular neuropathy and the leading cause of blindness in adults. The pathogenesis and progression of glaucoma is a vicious cycle of oxidative and mechanical stress. Vascular dysregulation leads to persistent local inflammation and oxidative damage in the trabecular meshwork and Schlemm’s canal, which constitute the aqueous humor outflow pathway. Subsequently, damage to the outflow tissue leads to accumulation of aqueous humor and elevated intraocular pressure in the anterior chamber, which mechanically compresses the optic nerve head and further deteriorates vascular regulation. The biological consequence of this vicious cycle is optic nerve death and irreversible vision loss. Controlling further increases in intraocular pressure is the first priority of glaucoma interventions, including multiple glaucoma medications and laser or incisional surgery. However, if vascular regulation and redox homeostasis are not restored, further progression of glaucoma is inevitable. A molecular mechanism that can simultaneously control oxidative and mechanical stress is essential to stop the vicious cycle and may revolutionize the treatment of glaucoma. Summary of the invention

[0003] The object of the present invention is to provide a photo-controlled carbon monoxide and / or nitric oxide releasing compound and its application in the treatment of glaucoma.

[0004] In the first aspect of the present invention, there is provided a compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, or metabolite thereof:

[0005]

[0006] In Formula I,

[0007] X is O or S;

[0008] R1, R2, R3, and R4 are each independently selected from H, C1-C10 alkyl and nitroso; or R1, R2 and the nitrogen atom to which they are connected together form a 3-7-membered nitrogen-containing heterocyclic ring; or R3, R4 and the nitrogen atom to which they are connected together form a 3-7-membered nitrogen-containing heterocyclic ring;

[0009] R5 and R6 are each independently selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxyl and amino;

[0010] R7 is selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxy, amino, cyano and nitro;

[0011] p is 0, 1, 2 or 3;

[0012] q is 0, 1, 2, or 3;

[0013] t is 0, 1, 2, 3, or 4;

[0014] The C═O marked with “*” may optionally form a ketal or hemiketal structure.

[0015] The second aspect of the present invention provides a pharmaceutical composition, which comprises (i) the compound of formula I described in the first aspect of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite thereof, and (ii) a pharmaceutically acceptable carrier.

[0016] The third aspect of the present invention provides a compound of formula I described in the first aspect of the present invention, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite or the pharmaceutical composition described in the second aspect of the present invention for the preparation of a medicament for treating and / or preventing glaucoma.

[0017] The fourth aspect of the present invention provides a compound of formula I described in the first aspect of the present invention, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite or the pharmaceutical composition described in the second aspect of the present invention for use in lowering intraocular pressure.

[0018] The fifth aspect of the present invention provides a compound of formula I described in the first aspect of the present invention, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite or the pharmaceutical composition described in the second aspect of the present invention for use in up-regulating the content of soluble guanylate cyclase and / or anti-oxidation.

[0019] The sixth aspect of the present invention provides the use of a carbon monoxide-releasing compound in the preparation of a medicament for treating and / or preventing glaucoma.

[0020] In one or more embodiments, the present invention provides the compound of formula I described in the first aspect of the present invention, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite; or the pharmaceutical composition described in the second aspect of the present invention for use in regulating oxidative stress and / or mechanical stress in glaucoma.

[0021] In one or more embodiments, modulating oxidative stress manifests as restoring redox homeostasis in ocular blood vessels.

[0022] In one or more embodiments, restoring redox homeostasis comprises upregulating the level of soluble guanylate cyclase, anti-oxidation.

[0023] In one or more embodiments, modulation of mechanical stress manifests as downregulation of intraocular pressure in glaucoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the absorbance and fluorescence intensity spectrum of CND570 at different excitation wavelengths, as well as the changes of absorbance at 540nm and fluorescence intensity at 570nm with irradiation time (min).

[0025] Figure 2 This is the result of GC detecting the carbon monoxide produced by illumination CND570.

[0026] Figure 3 This is the result of EPR detection of nitric oxide produced by CND570 under illumination.

[0027] Figure 4 This is a confocal image of CND570 in trabecular meshwork cells (HTM).

[0028] Figure 5 It is the absorbance and fluorescence intensity spectrum of COD at different excitation wavelengths, as well as the changes of absorbance at 550nm and fluorescence intensity at 580nm with irradiation time (min).

[0029] Figure 6 This is the result of GC detecting the production of carbon monoxide by COD under light.

[0030] Figure 7 This is a confocal imaging image of COD in trabecular meshwork cells.

[0031] Figure 8 These are slice images of CND570 and COD in cornea and trabecular meshwork tissue.

[0032] Fig. 9 eNOS in CND570 and COD treatment - / - Results of the measurement of hydrogen peroxide levels in the trabecular meshwork and Schlemm's canal tissues of mice.

[0033] Fig.10 eNOS in CND570 and COD treatment - / - Imaging of soluble guanylate cyclase (sGC) in the trabecular meshwork and Schlemm's canal tissues of mice.

[0034] Fig.11 eNOS in CND570 and COD treatment - / - Changes in intraocular pressure in mice. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0036] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0037] Herein, “comprising”, “including”, “containing” and similar terms encompass the meanings of “consisting essentially of” and “consisting of”. For example, when “A comprises B and C” is disclosed herein, “A consists essentially of B and C” and “A consists of B and C” should be deemed to be disclosed herein.

[0038] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0039] In this document, unless otherwise specified, percentage refers to mass percentage and ratio refers to mass ratio.

[0040] Herein, when describing embodiments or examples, it should be understood that they are not used to limit the present invention to these embodiments or examples. On the contrary, all substitutes, improvements and equivalents of the methods and materials described in the present invention can be included in the scope limited by the claims.

[0041] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0042] the term

[0043] Definitions of standard chemical terms can be found in references (including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4THED." Vols. A (2000) and B (2001), Plenum Press, New York). Unless otherwise stated, conventional methods within the technical scope of the art, such as mass spectrometry, NMR, IR and UV / VIS spectroscopy and pharmacological methods, are used. Unless specifically defined, the terms used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and drugs and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, the manufacturer's instructions for the use of the kit can be used, or the reaction and purification can be carried out in a manner known in the art or in accordance with the description of the present invention. The above-mentioned techniques and methods can usually be implemented according to the descriptions in the multiple summary and more specific documents cited and discussed in this specification, according to conventional methods well known in the art. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural parts and compounds.

[0044] Certain chemical groups defined herein are preceded by simplified symbols to indicate the total number of carbon atoms present in the group. For example, a C1-C6 alkyl group refers to an alkyl group as defined below having a total of 1 to 6 carbon atoms. The total number of carbon atoms in the simplified symbols does not include carbons that may be present in substituents of the group.

[0045] When a variable is mentioned in the present invention as "selected from: ...", it means that the variable is selected from any one of the options listed after the colon, or, if possible, it means that the variable is selected from one or more of the options listed after the colon.

[0046] All numerical ranges mentioned in the present invention are intended to include both endpoints of the range, all integers within the range, and sub-ranges formed by these integers.

[0047] In addition to the foregoing, when used in the specification and claims of the present invention, the following terms have the meanings indicated below unless otherwise specifically stated.

[0048] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0049] "Hydroxy" refers to an -OH group.

[0050] "Nitroso" refers to -N=O.

[0051] "Nitro" refers to -NO2.

[0052] "Cyano" refers to -CN.

[0053] "Amino" refers to -NH2.

[0054] In the present invention, the term "Ci-Cj" represents a range of carbon atoms, wherein i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and each integer point between the endpoints, wherein j is greater than i. For example, C1-C6 represents a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms and 6 carbon atoms. For another example, the term "C1-C10" represents 1 to 10, particularly 1 to 8, particularly 1 to 6, particularly 1 to 5, particularly 1 to 4, particularly 1 to 3 or particularly 1 to 2 carbon atoms.

[0055] In the present invention, as a group or part of other groups (for example, in groups such as alkyl substituted with halogens (such as fluorine, chlorine, bromine or iodine), the term "alkyl" refers to a fully saturated straight or branched hydrocarbon chain group consisting only of carbon atoms and hydrogen atoms connected to the rest of the molecule by a single bond, which has, for example, 1 to 10 (preferably 1 to 8, more preferably 1 to 6) carbon atoms. In some embodiments, the alkyl contains 1 to 9 carbon atoms, 1 to 8 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms or 1 to 2 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, n-octyl, n-nonyl, n-decyl, etc. Each alkyl group of each embodiment of the present invention is preferably a C1-C4 alkyl group. Unless otherwise specifically stated in the specification, the alkyl group may be optionally substituted.

[0056] In the present invention, the term "alkoxy" as a group or a part of other groups refers to a "-O-alkyl" group, wherein the alkyl group has the meaning described above.

[0057] As used herein, "ketal" refers to a product obtained by reacting a ketone through its carbonyl group with two molecules of an alcohol, or a ketone can be obtained by a continuous addition reaction with a halohydrin (eg, 2-bromoethanol) to obtain the same structure.

[0058] As used herein, "himeketal" refers to the product obtained by the reaction of a ketone via its carbonyl group with one molecule of alcohol.

[0059] As used herein, "subject" is a mammal, such as a primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc., especially a primate, more specifically a human. In some embodiments, the mammal is selected from: humans; domestic animals, such as laboratory animals, household pets or farm animals, such as cats, dogs, pigs, cows, sheep, goats, horses, rabbits; and non-domestic animals, such as wild animals, etc.

[0060] The term "pharmaceutically acceptable" as used herein refers to a substance (such as a carrier or diluent) that does not affect the biological activity or properties of the active pharmaceutical ingredient (i.e., the compound of the present invention, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite), and is relatively non-toxic, i.e., the substance can be administered to an individual without causing adverse biological reactions or interacting in an adverse manner with any component contained in the pharmaceutical composition. Pharmaceutically acceptable carriers or excipients include, but are not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that has been approved by, for example, the U.S. Food and Drug Administration (FDA) for use in humans or farmed animals.

[0061] The term "pharmaceutical composition" refers to a preparation containing a compound of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite thereof, and a medium generally accepted in the art for delivering biological (pharmacological) active compounds to mammals (e.g., humans). The medium includes a pharmaceutically acceptable carrier or excipient. The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and then exert biological (pharmacological) activity. In general, the pharmaceutical composition of the present invention contains 0.1% by weight to 99.5% by weight of a pharmaceutically active ingredient (i.e., a compound of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite thereof). In some embodiments, the pharmaceutical composition of the present invention contains 0.5%-90% by weight, for example, 1%, 1.5%, 2%, 5%, 10%, 15%, 20%, 25%, 30% or 50% by weight of the pharmaceutically active ingredient.

[0062] The compounds of the present invention may exist in a variety of different forms or derivatives, all of which are within the scope of the present invention. These forms or derivatives include, for example, tautomers, stereoisomers (e.g., enantiomers, diastereomers), racemic mixtures, geometric isomers, salts, prodrugs, solvates, isotope substitutions, different crystal forms or polymorphs, and metabolites (specifically active metabolites in this article).

[0063] In the present invention, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0064] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, and the like; organic acid salts include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, decanoates, undecylenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamate, pyroglutamate, aspartate, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginate, ascorbate, salicylates, 4-aminosalicylates, naphthalene disulfonates, and the like. These salts may be prepared by methods known in the art.

[0065] "Pharmaceutically acceptable base addition salt" refers to a salt formed with an inorganic base or an organic base that can maintain the biological effectiveness of the free acid without other side effects. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. The salt derived from organic base includes but is not limited to the following salt: primary amines, secondary amines and tertiary amines, substituted amines, including natural substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins etc. Preferred organic bases include isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. These salts can be prepared by methods known in the profession.

[0066] As used herein, the term "prodrug" refers to compounds that are metabolized (i.e., converted in vivo) into pharmacologically active compounds of the present invention after administration. When the compounds of the present invention are difficult to absorb by themselves, their bioavailability can be improved by making them into prodrugs. Examples of prodrugs of the compounds of the present invention may include simple esters of compounds containing carboxyl groups (e.g., by reacting with C according to methods known in the art). 1-4 esters obtained by condensation of hydroxyl groups (for example, by condensation of hydroxyl groups with C 1-4 Monocarboxylic acid, C 3-6 esters obtained by condensation of dicarboxylic acids or their anhydrides, such as succinic anhydride or fumaric anhydride); imines of compounds containing amino groups (for example, by reacting with C 1-4 imines obtained by condensation of aldehydes or ketones); carbamates of compounds containing amino groups, such as those described by Leu et al. (J. Med. Chem., 42: 3623-3628 (1999)) and Greenwald et al. (J. Med. Chem., 42: 3657-3667 (1999)); acetals or ketal of compounds containing hydroxy groups (such as those obtained by condensation with chloromethyl methyl ether or chloromethyl ethyl ether according to methods known in the art).

[0067] As used herein, the term "solvate" refers to an aggregate comprising one or more molecules of the compounds of the invention and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the invention may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvated forms. The compounds of the invention may be true solvates, while in other cases, the compounds of the invention may only retain adventitious water or a mixture of water plus some adventitious solvent.

[0068] As used herein, "stereoisomers" refer to compounds composed of the same atoms, bonded by the same bonds, but having different three-dimensional structures. The present invention is intended to encompass various stereoisomers and mixtures thereof.

[0069] When the compounds of the present invention contain olefinic double bonds, and unless specified otherwise, it is intended that the compounds of the present invention include both E- and Z- geometric isomers.

[0070] "Tautomer" refers to an isomer formed when a proton is shifted from one atom of a molecule to another atom of the same molecule. All tautomeric forms of the compounds of the present invention are intended to be encompassed within the scope of the present invention.

[0071] The compounds of the present invention or their pharmaceutically acceptable salts may contain one or more chiral carbon atoms and may therefore give rise to enantiomers, diastereomers and other stereoisomeric forms. Each chiral carbon atom may be defined as (R)- or (S)- based on stereochemistry. The present invention is intended to include all possible isomers, as well as racemates and optically pure forms thereof. The compounds of the present invention may be prepared using racemates, diastereomers or enantiomers as starting materials or intermediates. Optically active isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as crystallization and chiral chromatography.

[0072] Conventional techniques for preparing / isolating individual isomers include chiral synthesis from appropriate optically pure precursors, or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high performance liquid chromatography, see, for example, Gerard Gübitz and Martin G. Schmid (Eds.), ChiRal Sepa Rations, Methods and Protocols, Methods in Molecular Biology, Vol. 243, 2004; A. M. Stalcup, ChiRal Sepa Rations, Annu. Rev. Anal. Chem. 3: 341-63, 2010; Fumiss et al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRaCTICAL ORGANIC CHEMISTRY. sup. TH ED., Longman Scientific and Technical Ltd., Essex, 1991, 809-816; Heller, Acc. Chem. Res. 1990, 23, 128, the disclosures of which are incorporated herein by reference in their entirety.

[0073] The present invention also includes all suitable isotopic substitutions (or isotopic variants) of the compounds of the present invention or their pharmaceutically acceptable salts. Isotopic variants of the compounds of the present invention or their pharmaceutically acceptable salts are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass commonly found in nature. Isotopes that can be incorporated into the compounds of the present invention and their pharmaceutically acceptable salts include, but are not limited to, isotopes of H, C, N and O, for example 2 H. 3 H. 11 C. 13 C. 14 C. 15 N. 17 O. 18 O.35 S. 18 F. 36 Cl and 125 I. Isotopic variations of the compounds of the present invention or pharmaceutically acceptable salts thereof can be prepared by conventional techniques using appropriate isotopic variations of suitable reagents.

[0074] Herein, unless otherwise specified, the wavy line on each structural formula or group generally indicates the position where the structural formula or group is connected to other parts in the compound.

[0075] As used herein, the terms "crystal form", "crystalline form" and "polymorph" are used interchangeably and refer to a crystal structure in which a compound (or its salt, solvate or other derivative such as a prodrug or metabolite) can be crystallized in different crystal packing arrangements (all with the same elemental composition). Different crystal forms typically have different X-ray diffraction patterns, infrared spectra, melting points, density hardness, crystal shape, optical and electrical properties, stability and solubility. Recrystallization solvents, crystallization rates, storage temperatures or other factors may cause one of the crystal forms to dominate. Polymorphs of a compound can be prepared by crystallization under different conditions.

[0076] As used herein, the terms "metabolite" and "active metabolite" are used interchangeably and refer to derivatives that are pharmacologically active and result from the metabolism of the parent pharmaceutical active ingredient in the subject. For example, such metabolites can result from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound or salt or prodrug.

[0077] As used herein, "prevention" refers to preventing the disease or condition from occurring in a mammal (preferably a human), especially when the mammal is susceptible to the condition but has not yet been diagnosed with the condition, or to preventing a disease or condition that has been cured from recurring in a mammal (preferably a human).

[0078] As used herein, "treatment" encompasses the treatment of a disease or condition of interest in a mammal, preferably a human, suffering from the disease or condition of interest, including one or more of the following: (i) inhibiting the disease or condition, i.e., arresting its development; (ii) alleviating the disease or condition, i.e., causing regression of the disease or condition; and (iii) alleviating the symptoms caused by the disease or condition, i.e., alleviating pain without resolving the underlying disease or condition.

[0079] "Effective amount" refers to a therapeutically effective amount or a preventive effective amount. "Therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result (alleviates one or more symptoms of the disease or condition being treated to some extent, such as reducing the intraocular pressure of glaucoma, restoring the redox homeostasis of glaucoma ocular blood vessels) at the required dose and for the required period of time. The therapeutically effective amount of a compound may vary according to the following factors: the disease state, age, sex and weight of the subject, and the ability of the compound to cause the desired response in the subject. The dosing regimen may be adjusted to provide the best therapeutic response. The therapeutically effective amount is also an amount in which any toxic or harmful effects of the compound are exceeded by the therapeutic beneficial effects. "Preventive effective amount" refers to an amount that effectively achieves the desired preventive result (such as preventing the occurrence of the disease in a subject at risk of suffering from the disease and the recurrence of the disease in a cured sick subject) at the required dose and for the required period of time. Typically, the preventive dose is used in a subject before or in the early stages of the disease, so that the preventive effective amount may be less than the therapeutically effective amount. Techniques such as dose escalation tests can be used to determine the effective amount suitable for any individual case.

[0080] As used herein, the terms "administer", "apply", "administer" and the like refer to methods that can deliver a compound or pharmaceutical composition to a desired site for biological action. Methods of administration known in the art can be used in the present invention. These methods include, but are not limited to, oral administration, intraduodenal administration, parenteral administration (including intrapulmonary, intranasal administration; intrathecal, intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion), topical administration and rectal administration. Those skilled in the art are familiar with administration techniques that can be used for the compounds and methods described herein, such as those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa. In a preferred embodiment, the compounds of the present invention, or pharmaceutically acceptable salts, enantiomers, diastereomers, solvates, isotope-substituted products, polymorphs, prodrugs, metabolites thereof, are administered by topical administration.

[0081] As used herein, "combination", "drug combination", "combination drug", or "combination therapy" and the like refer to a drug therapy obtained by mixing or combining more than one active ingredient, including fixed and non-fixed combinations of active ingredients, and also including combinations of two or more different treatments. The term "fixed combination" refers to the simultaneous administration of at least one compound described herein and at least one other active ingredient to a patient in the form of a single entity or a single dosage form. The term "non-fixed combination" refers to the simultaneous administration, combined administration, or sequential administration at variable intervals of at least one compound described herein and at least one other active ingredient to a patient in the form of separate entities. These also apply to cocktail therapy, for example the administration of three or more active ingredients.

[0082] Compounds of formula I

[0083] The compounds of the present invention can treat and / or prevent the occurrence and development of glaucoma by blocking the vicious cycle of oxidative stress and mechanical stress. The present invention provides a compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite thereof:

[0084]

[0085] In Formula I,

[0086] X is O or S;

[0087] R1, R2, R3, and R4 are each independently selected from H, C1-C10 alkyl and nitroso; or R1, R2 and the nitrogen atom to which they are connected together form a 3-7-membered nitrogen-containing heterocyclic ring; or R3, R4 and the nitrogen atom to which they are connected together form a 3-7-membered nitrogen-containing heterocyclic ring;

[0088] R5 and R6 are each independently selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxyl and amino;

[0089] R7 is selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxy, amino, cyano and nitro;

[0090] p is 0, 1, 2 or 3;

[0091] q is 0, 1, 2, or 3;

[0092] t is 0, 1, 2, 3, or 4;

[0093] The C═O marked with “*” may optionally form a ketal or hemiketal structure.

[0094] In some embodiments, in the compound of formula I, X is O; R1, R2, R3, R4 are each independently selected from H, C1-C10 alkyl and nitroso; R5 and R6 are each independently selected from C1-C10 alkyl and C1-C10 alkoxy; R7 is selected from C1-C10 alkyl and C1-C10 alkoxy.

[0095] Preferably, X is O.

[0096] Preferably, R1, R2, R3 and R4 are each independently selected from H, C1-C10 alkyl and nitroso. Preferably, at least one, at least two, at least three or all four of R1, R2, R3 and R4 are nitroso. In some embodiments, R1 is nitroso and R2 is C1-C10 alkyl. In some embodiments, R3 is nitroso and R4 is C1-C10 alkyl.

[0097] Preferably, R5, R6 and R7 are each independently selected from C1-C10 alkyl and C1-C10 alkoxy.

[0098] Preferably, p is 0.

[0099] Preferably, q is 0.

[0100] Preferably, t is 0.

[0101] In some embodiments, the compound of formula I is selected from:

[0102]

[0103] The compounds of formula I of the present invention are a class of photo-controlled carbon monoxide-releasing compounds, and may optionally photo-controlled release of nitric oxide.

[0104] Preferably, the present invention provides a light-controlled carbon monoxide and nitric oxide co-releasing compound having a structure shown in Formula I, wherein:

[0105] X is O or S;

[0106] R1 is a nitroso group, and R2, R3 and R4 are each independently selected from H, a C1-C10 alkyl group and a nitroso group; or R3 is a nitroso group, and R1, R2 and R4 are each independently selected from H, a C1-C10 alkyl group and a nitroso group; or R1 and R3 are a nitroso group, and R2 and R4 are each independently selected from H, a C1-C10 alkyl group and a nitroso group;

[0107] R5 and R6 are each independently selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxyl and amino;

[0108] R7 is selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxy, amino, cyano and nitro;

[0109] p is 0, 1, 2 or 3;

[0110] q is 0, 1, 2, or 3;

[0111] t is 0, 1, 2, 3, or 4;

[0112] The C═O marked with “*” may optionally form a ketal or hemiketal structure.

[0113] Pharmaceutical composition

[0114] The present invention provides a pharmaceutical composition comprising (i) a compound of formula I of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite thereof, and (ii) a pharmaceutically acceptable carrier.

[0115] For the purposes of the present invention, the pharmaceutical composition of the present invention is an aqueous topical formulation, such as an eye drop. A pharmaceutically acceptable carrier suitable for use in the pharmaceutical composition of the present invention is one that has no persistent adverse effects on the treated eye or its function, or on the general health of the treated individual. Temporary effects (such as mild irritation or a "stinging" sensation) are common when topical eye drops are administered, and the presence of such temporary effects is not inconsistent with pharmaceutical acceptability.

[0116] "Local administration" is understood to define all ocular routes, ie topical and injectable administration, as well as administration via implantable systems.

[0117] The form of "topical administration" may be, for example, without limitation, eye drops, eye washes or ocular instillations, sprays, creams, ointments, gels, hydrogels, oleogels, hydrophilic lenses, inserts or implants. The dosage form may be, for example, without limitation, solutions, suspensions, colloidal systems (e.g., liposomes, emulsions, microemulsions, nanoemulsions, microparticles, nanoparticles, microspheres, vesicles (niosomes), dendrimers (dendrimers)), micelles, mixed micelles, coordination systems (e.g., cyclodextrin solutions), and non-implantable inserts (e.g., in the form of, for example, discs, films or strips, without limitation).

[0118] "Injection administration" may include, without limitation: intraocular (intravite, IVT) administration; periocular administration, including subconjunctival, subtenon's, retrobulbar and intrascleral administration.

[0119] "Intravitreal administration" can be performed as an injectable system or an implantable system. The dosage form can be, for example, without limitation, solutions, suspensions, colloidal systems (e.g., liposomes, emulsions, microemulsions, nanoemulsions, microparticles, nanoparticles, microspheres, vesicles, dendrimers), micelles, mixed micelles, and biodegradable or non-biodegradable implants (e.g., without limitation, in the form of rods, spikes, or pellets).

[0120] For both routes of administration (topical and injectable), depending on the compound being delivered, most of the above dosage forms can potentially prolong the residence time of the active ingredient on the ocular surface or in the vitreous, provide slow and sustained release of the encapsulated compound, and / or avoid toxicity and enhance ocular tolerance.

[0121] For topical or injectable administration, the excipient must be pharmaceutically acceptable and suitable for such ocular administration.

[0122] The aqueous medium used in the pharmaceutical composition of the present invention consists of water that does not contain physiologically and ophthalmologically unfavorable agents. The pharmaceutical composition of the present invention is in the form of an aqueous preparation having a pH that is physiologically compatible with the ocular route. "pH that is physiologically compatible with the ocular route" refers to a pH range of about 5.5 to about 8, preferably about 6.0 to about 7.5. The pH value of the preparation is adjusted by the following substances: an acid, such as acetic acid, boric acid, lactic acid, hydrochloric acid; a base, such as sodium hydroxide, sodium borate, sodium citrate, sodium acetate; or a pharmaceutically acceptable buffer solution, such as sodium phosphate buffer solution, potassium phosphate buffer solution, sodium citrate buffer solution. The aqueous preparation of the present invention is isotonic and physiologically adapted for ocular, topical and intraocular administration. The osmotic pressure of the preparation is close to the physiological pressure and is generally about 200mOSm to about 400mOsm, preferably about 260 to about 340mOsm. If necessary, the osmotic pressure can be adjusted with an appropriate amount of a physiologically and ophthalmologically acceptable excipient. Sodium chloride is usually used as a tonicity agent, and its concentration (expressed as m / V) does not exceed 0.9%. Equivalent amounts of one or more salts composed of cations and anions can also be used. Depending on the therapeutic indications of the present invention, the osmotic pressure can be optionally corrected by adding sugars or polyols alone or as a combined mixture. The formulations of the present invention have a viscosity of 0 to about 2000 centipoise, preferably less than about 100 centipoise, and more preferably less than about 30 centipoise.

[0123] The compositions of the present invention may include agents that increase viscosity, thereby extending the residence time of the active ingredient in front of the cornea after instillation. These viscosity enhancers may also have mucoadhesive properties. The present invention may use mucoadhesive polymers that can produce non-covalent bonds with glycoproteins that are significantly present in the conjunctiva, thereby confining the preparation to the eye, locally optimizing the residence time of the preparation and potentially enhancing the ocular bioavailability of the active ingredient, as well as reducing the frequency of administration to improve treatment compliance. These polymers are generally high molecular weight hydrocolloids. The polymers can be used alone or in combination in the present invention, and are, for example: cellulose derivatives, such as methylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose; acrylic acid derivatives, such as polyacrylates and functionalized derivatives thereof (or polycarbophils); carbomers; natural products, such as alginates, chitosan, pectin, hyaluronic acid and derivatives thereof; polysaccharide derivatives, such as gellan gum and derivatives thereof, xanthangum, carrageenans; copolymers, such as poloxamer. A polymer having in situ gelling ability can be introduced into the formulation of the pharmaceutical composition of the present invention. The so-called phase change system is a liquid, and allows ion activation to occur according to pH and temperature to form a gel compatible with ocular function. For example, the following polymers can be cited, such as polyacrylic acid derivatives, cellulose derivatives, methylcellulose, copolymers and poloxamers.

[0124] The pharmaceutical composition of the present invention may further comprise excipients known to those skilled in the art, such as surfactants, co-surfactants, co-solvents, penetrants, gelling agents, emulsifiers, antioxidants, preservatives, and polymers for sustained release.

[0125] The pharmaceutical composition can also be in the form of an insert or solid implant, which can be administered to the eye and release the active ingredient slowly. For example, the insert can be prepared using a water-soluble solid polymer. The inert polymer used to prepare the insert suitable for the ocular route and the ocular biocompatibility can be synthetic, semi-synthetic or natural products. The composition of the solid implant can also be composed of synthetic, semi-synthetic or natural polymers, and the polymer is preferably a biodegradable polymer, such as polyvinyl alcohol, polylactic acid-co-glycolic acid, poly-ε-caprolactone, hyaluronate. These biodegradable polymers can also encapsulate the active ingredient into microspheres, nanospheres or nanocapsules dispersed in the aqueous solution, thereby providing a sustained directional release of the active ingredient.

[0126] Other matrices, such as water-soluble granules (lentilles) impregnated or containing the active ingredient, can prolong the residence time of the active ingredient on the ocular surface.

[0127] The principles of manufacture and sterilization of these formulations are conventionally known in the art of dosage form technology.

[0128] In some embodiments, the pharmaceutically acceptable carrier is selected from DSPE-mPEG (distearoylphosphatidylethanolamine-polyethylene glycol) and water. Available DSPE-mPEG includes DSPE-mPEG2000, wherein "2000" indicates that the molecular weight of PEG is 2000.

[0129] application

[0130] The present invention provides use of a compound of formula I of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite thereof; or a pharmaceutical composition of the present invention in the preparation of a drug for treating and / or preventing glaucoma.

[0131] The present invention provides a compound of formula I of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite thereof; or the use of the pharmaceutical composition of the present invention in lowering intraocular pressure. In some embodiments, the intraocular pressure is glaucoma intraocular pressure.

[0132] The present invention provides a compound of formula I of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite thereof; or the use of the pharmaceutical composition of the present invention in up-regulating the content of soluble guanylate cyclase and / or anti-oxidation. In some embodiments, the content of the soluble guanylate cyclase is the content of soluble guanylate cyclase in the trabecular meshwork and Schlemm's canal tissue of glaucoma. In some embodiments, the anti-oxidation is to control the content of peroxide, for example, to lower the content of hydrogen peroxide. Preferably, the content of hydrogen peroxide is the content of hydrogen peroxide in the trabecular meshwork and Schlemm's canal tissue of glaucoma.

[0133] The present invention also provides the use of a compound that releases carbon monoxide in the preparation of a medicament for treating and / or preventing glaucoma. In some embodiments, the compound that releases carbon monoxide is a compound of formula I of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite thereof. The compound that releases carbon monoxide may also release nitric oxide.

[0134] The present invention also provides a compound of formula I of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope substitution, polymorph, prodrug, metabolite thereof; or the use of the pharmaceutical composition of the present invention in regulating oxidative stress and / or mechanical stress in glaucoma. Regulating oxidative stress is manifested in restoring the redox homeostasis of ocular blood vessels, such as upregulating the content of soluble guanylate cyclase, anti-oxidation (such as downregulating the content of hydrogen peroxide). In some embodiments, the content of the soluble guanylate cyclase is the content of soluble guanylate cyclase in the trabecular meshwork and Schlemm's canal tissue of glaucoma. In some embodiments, the content of hydrogen peroxide is the content of hydrogen peroxide in the trabecular meshwork and Schlemm's canal tissue of glaucoma. Regulating mechanical stress is manifested in downregulating the intraocular pressure of glaucoma.

[0135] Treatment

[0136] For the purposes of the present invention, those skilled in the art will understand that the methods of treatment and uses described herein are intended to include administering to a subject in need thereof a therapeutically effective amount of a compound of formula I of the present invention, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite thereof; and / or a pharmaceutical composition of the present invention, and exciting under visible light irradiation. In some embodiments, the administration is to the eye of a human or animal. The visible light is green light, preferably with a wavelength between 500-550 nm, for example 510-530 nm, such as 520 nm. The intensity of the visible light is 5-20 mw / cm 2 , preferably 10-15mw / cm 2 The irradiation is to irradiate the ocular surface for 5-20 minutes, for example, 10-15 minutes.

[0137] The amount of active ingredient administered to the subject is generally described as being effective for treatment (even if temporarily or symptomatically), and thus the method comprises administering one or more drops in the subject's eye one or more times daily. Of course, a clinician with ordinary skills can determine the ideal dosage based on an assessment of the clinical condition and the concentration of the ingredients contained in the drug.

[0138] The present invention has the following beneficial effects:

[0139] The present invention provides a class of small molecule compounds of formula I, which release carbon monoxide and / or nitric oxide and rhodamine dye under visible light irradiation. After being encapsulated by DSPE-mPEG, the compounds penetrate through the cornea to reach the aqueous humor outflow channel, and are activated by visible light (such as green light) to release carbon monoxide and / or nitric oxide, thereby achieving the upregulation of soluble guanylate cyclase content and anti-oxidation effects, resulting in a significant reduction in intraocular pressure in a glaucoma mouse model, and have important drug development potential.

[0140] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0141] In Examples 1-11, local treatment, local application, local therapy or local spot treatment refers to dripping the sample to be tested onto the surface of the eyeball.

[0142] eNOS - / - The mice were glaucoma mice purchased commercially.

[0143] Preparation Example 1: Synthesis of CND570 and COD

[0144] 1. The present invention provides a synthetic route of compound CND570:

[0145]

[0146] (1) Synthesis of compound 3: Compound 1 (400 mg, 1.30 mmol, 1.00 equivalent) and ninhydrin (compound 2, 230 mg, 1.30 mmol, 1.00 equivalent) were weighed and dissolved in 50 mL of acetonitrile in a 100 mL round-bottom flask. Then 5 ml of acetic acid was added and stirred for 0.5 h, and then 0.5 ml of sulfuric acid was added. Then the reaction was carried out at 70°C for 12 hours. The solvent was evaporated under reduced pressure. Then the residue was neutralized with saturated NaHCO3. The reaction solution was extracted three times with water and dichloromethane, and the organic layer was collected and dried over anhydrous sodium sulfate. The inorganic salt was filtered and the filtrate was dried under reduced pressure. The crude product was separated by silica gel column chromatography (solvent system: petroleum ether: ethyl acetate = 10:1, V / V) to obtain compound 3 (250 mg, brown solid, yield 43%).

[0147] (2) Synthesis of compound CND570: Compound 3 (100 mg, 1.00 equivalent, 0.22 mmol) and tert-butyl nitrosoate (68 mg, 3.00 equivalent, 0.66 mmol) were refluxed in 20 mL THF for 3 h, and the solvent was evaporated under reduced pressure. The crude product was separated by silica gel column chromatography (solvent system: petroleum ether: ethyl acetate = 10:1, V / V) to obtain compound CND570 (35 mg, dark green solid, yield 39%).

[0148] 1H NMR (400MHz, CDCl3) δ7.99(d,J=7.8Hz,1H),7.81(t,J=7.6Hz,1H),7.59(t,J=7.6Hz,1H),7.37(d,J=7.9Hz,1H),7.33(d,J=2.3Hz,1H),7.10(dd,J=8 .4,2.3Hz,1H),6.52(d,J=8.5Hz,1H),6.42(d,J=2.5Hz,1H),6.22–6.10(m ,2H),3.97(q,J=7.2Hz,2H),3.26(q,J=7.1Hz,4H),1.08(t,J=5.8Hz,9H). 13 C NMR (101MHz, CDCl3) δ196.24,189.57,151.28,151.14,150.57,147.98,141.15,138.49,136.72,128.66,128.23,127.68,1 27.33,122.47,119.77,112.48,107.57,106.83,106.34,97.76,49.75,43.43,37.72,11.50,10.69.ESI-HRMS,m / z,[M+Na] + ,calcd.for C 27 H 25 N3O4Na + ,478.1743; found 478.1746.

[0149] 2. The present invention provides a synthetic route of compound COD:

[0150]

[0151] Compound 3 (100 mg, 1.00 equivalent, 0.22 mmol), 2-bromoethanol (33 mg, 1.20 equivalent, 0.27 mmol) and K2CO3 (243 mg, 8.00 equivalent, 1.76 mmol) were added to 20 mL THF and refluxed for 24 h. The solid in the mixture was filtered out and the filtrate was concentrated under reduced pressure to obtain a viscous residue. The reaction solution was extracted three times with water and dichloromethane, and the collected organic layer was dried over anhydrous sodium sulfate. The inorganic salts were filtered and the filtrate was decompressed and dried. The crude product was separated by silica gel column chromatography (solvent system: petroleum ether: ethyl acetate = 10: 1, V / V) to obtain compound COD (77 mg, yellow solid, yield 70%).

[0152] 1H NMR (400MHz, CDCl3) δ7.93(d,J=7.7Hz,1H),7.72(td,J=7.6,1.3Hz,1H),7.54(t,J=7.5Hz,1H),7.38(d,J=7.8Hz,1H),6.44(d,J=2.6Hz,2H), 6.40(d,J=8.7Hz,2H),6.30(dd,J=8.8,2.6Hz,2H),4.09–4.00(m,2H),3.65–3.55(m,2H),3.37(qd,J=7.3,2.0Hz,8H),1.20(t,J=7.0Hz,12H). 13 C NMR (101MHz, CDCl3) δ202.37,154.91,153.45,148.13,136.70,135.10,130.34,128.50,127.4 7,122.81,110.15,109.86,106.73,98.14,66.29,51.37,44.34,12.72.ESI-HRMS,m / z,[M+Na] + ,Calcd for C 31 H 34 N2O4Na + ,521.2416;found,521.2418.

[0153] Preparation Example 2: DSPE-mPEG2000 encapsulation of CND570 or COD

[0154] CND570 or COD was dissolved in 10 mL of dichloromethane in a 25 mL round-bottom culture bottle. Then, 8 mg of DSPE-mPEG2000 was added to each culture bottle. The mixture was ultrasonically treated for 1 hour to form a dispersed system. The dichloromethane was then removed under reduced pressure by a rotary evaporator. Then, 1 mL of deionized pure water was added to the culture bottle, and then high-frequency ultrasonic treatment was used for 30 minutes to convert large particles into small-sized micelles. After that, the solution was filtered through a 0.22 μm microporous membrane 5 times to obtain a 2 mM polymer micelle stock solution of CND570 or COD.

[0155] Example 1: Photoactivated UV absorption and fluorescence emission spectra of CND570

[0156] Test method: CND570 (2 μM) was dissolved in PBS (10 mM, pH=7.4) with 5% DMSO as a co-solvent and illuminated at 365-650 nm to test its UV absorption and fluorescence emission spectra.

[0157] The test results are as follows Figure 1As shown in Figure a, 365nm (185mW / cm 2 ), Figure b is 395nm (190mW / cm 2 ), Figure c is 450nm (195mW / cm 2 ), Figure d is 520nm (130mW / cm 2 ), Figure e is 600nm (130mW / cm 2 ), Figure f is 650nm (220mW / cm 2 ).

[0158] Example 2: Gas chromatography (GC) detection of carbon monoxide release by photoactivation of CND570

[0159] Test method: CND570 (0.1 mM) was dissolved in PBS (10 mM, pH = 7.4) with 5% DMSO as co-solvent, using 520 nm, 130 mW / cm 2 The cells were exposed to light for 10 min.

[0160] The GC detection light CND570 produces a carbon monoxide result graph. The test results are as follows Figure 2 shown.

[0161] Example 3: Electron paramagnetic (EPR) detection of CND570 light-activated nitric oxide release

[0162] Test method: CND570 (20 μM) was dissolved in PBS (10 mM, pH = 7.4) with 10% DMSO as co-solvent and the wavelength was 520 nm and 130 mW / cm 2 The cells were exposed to light for 10 min.

[0163] The results of using EPR to detect the nitric oxide produced by CND570. Figure 3 shown.

[0164] Example 4: Confocal imaging of CND570 in trabecular meshwork cells (HTM)

[0165] Test method: Photoactivated imaging was performed using the laser built into the Nikon confocal for 15 minutes. The staining method was to incubate HTM cells with CND570 (2μM) for 15 minutes. Then, the cells were incubated with lysosomal stain (100nM) for 15 minutes and then incubated with COP490 (2μM) and PdCl2 (4μM) or DAF-FM-DA (5μM) with or without PTIO (10μM) for 30 minutes. Wash three times with PBS to remove excess dye. Cells were irradiated with the built-in 532nm laser to collect fluorescence images on a Nikon confocal scanning microscope. The blue channel of COP490 or DAF-FM-DA was excited at 488nm (em: 500-550nm), the green channel of CND570 was excited at 561nm (em: 570-620nm), and the lysosomal stain channel was excited at 647nm (em: 663-738nm). For CO detection, cells need to be incubated for 15 minutes after illumination before imaging.

[0166] Test results such as Figure 4 Figure a is the photoactivated imaging of CND570 (2 μM) in HTM cells; Figure b is the fluorescence analysis of Figure a; Figure c is the co-localization imaging of CND570 and lysosome commercial dye after illumination; Figure d is the detection of nitric oxide release by CND570 photoactivated in cells; Figure e is the detection of carbon monoxide release by CND570 photoactivated in cells.

[0167] Example 5: UV absorption and fluorescence emission spectra of COD photoactivation

[0168] Test method: COD (2 μM) was dissolved in PBS (10 mM, pH=7.4) with 5% DMSO as a co-solvent and illuminated at 520 nm to test its UV absorption and fluorescence emission spectra.

[0169] Test results such as Figure 5 As shown. Figure a is the UV absorption spectrum, and Figure b is the fluorescence emission spectrum.

[0170] Example 6: Gas Chromatography (GC) Detection of COD Photo-activated Release of Carbon Monoxide

[0171] Test method: COD (0.1 mM) was dissolved in PBS (10 mM, pH = 7.4) with 5% DMSO as co-solvent, using 520 nm, 130 mW / cm 2 The cells were exposed to light for 10 min.

[0172] The results of using GC to detect the carbon monoxide produced by light COD. Figure 6 shown.

[0173] Example 7: Confocal imaging of COD in trabecular meshwork cells

[0174] Test method: The same test method as in Example 4 was used to collect confocal imaging of COD in trabecular meshwork cells.

[0175] Test results such as Figure 7 Figure a is the photoactivated imaging of COD (2 μM) in HTM cells; Figure b is the fluorescence analysis of Figure a; Figure c is the co-localized imaging of COD and lysosome commercial staining after illumination; Figure d is the detection of COD releasing carbon monoxide after photoactivation in cells.

[0176] Example 8: Imaging of CND570 and COD in slices of cornea and trabecular meshwork tissue

[0177] Test method: eNOS - / - After topical application of DSPE-mPEG2000-encapsulated CND570, COD (3 drops, 2 μL / drop, 2 mM) to mice, tissue sections were removed 1 hour after administration, and the control group was water. 2 ) The sections were illuminated with light for 15 minutes and then imaged.

[0178] Test results such as Figure 8 As shown in the figure, Epi represents corneal epithelium; End represents corneal endothelium; CB represents ciliary body; TM represents trabecular meshwork.

[0179] Example 9: CND570 and eNOS in COD Treatment - / - Determination of hydrogen peroxide levels in the trabecular meshwork and Schlemm's canal tissues of rats

[0180] Test method: The mice (eNOS - / - ), the control group was water. 520nm light (15mW / cm 2 ) were irradiated on the ocular surface for 15 min. The trabecular meshwork and Schlemm's canal tissues were extracted for hydrogen peroxide measurement. ** indicates p<0.01, n=6.

[0181] Test results such as Fig. 9 shown.

[0182] Example 10: CND570 and eNOS in COD Treatment - / - Imaging of soluble guanylate cyclase (sGC) in the trabecular meshwork and Schlemm's canal of mice

[0183] Test method: The cell immunofluorescence staining method is: HTM cells were incubated with CND570 and COD for half an hour, and then illuminated for 15 minutes (all 5μM, 15mW / cm 2), and 2 hours later the cells were fixed with 4% formaldehyde for 30 minutes and then washed three times with PBS. The nonspecific binding sites of the cells were blocked with goat serum for 1 hour at room temperature, and then permeabilized with 0.2% TritonX-100 for 5 minutes. Then, the cells were incubated with guanylate cyclase (1:1000; Abcam) overnight at 4°C. After washing three times with PBS, the cells were incubated in secondary antibodies (Alexa Fluor 488, goat anti-rabbit) and Hoechst33258 solution (0.5 mg / mL; Sigma). Then they were observed by fluorescence microscopy. The immunofluorescence staining method of eye sections was as follows: CND570 encapsulated with DSPE-mPEG2000, COD solution (3 drops, 2 μL each, 2 mM), 520 nm (15 mW / cm 2 ) for 15 minutes, and the mouse eyes (eNOS / - / ) were treated topically. Two hours later, the eyes were removed and stored in 4% formaldehyde, then embedded in paraffin, sectioned, and immunofluorescence stained. The sections were observed under a fluorescence microscope.

[0184] The test results are as follows Fig.10 Figure a is the immunofluorescence analysis of sGC expression in HTM cells treated with CND570 or COD (5 μM), and b is the immunofluorescence analysis of eNOS in CND570 or COD treated with DSPE-mPEG2000. - / - Immunofluorescence analysis of sGC expression in rat trabecular meshwork / Schlemm's canal tissue sections, with water as the control group.

[0185] Example 11: CND570 and eNOS in COD Treatment - / - Changes in intraocular pressure (IOP) in mice

[0186] Test method: CND570 and COD (3 drops, 2 μL / drop, 2 mM) encapsulated with DSPE-mPEG200 were applied topically for treatment, respectively, at 520 nm (15 mw / cm 2 ) light was irradiated on the ocular surface for 15 min, and H2O was used as the control. The intraocular pressure was measured using a rebound tonometer (TonoLab; ICare Espoo, Finland). The IOP of each eye was measured three times and the results were averaged. * indicates p<0.05, *** indicates p<0.001 ****0.0001, n=12.

[0187] The test results are as follows Fig.11 shown.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, or metabolite thereof: In Formula I, X is O or S; R1, R2, R3, and R4 are each independently selected from H, C1-C10 alkyl and nitroso; or R1, R2 and the nitrogen atom to which they are connected together form a 3-7-membered nitrogen-containing heterocyclic ring; or R3, R4 and the nitrogen atom to which they are connected together form a 3-7-membered nitrogen-containing heterocyclic ring; R5 and R6 are each independently selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxyl and amino; R7 is selected from C1-C10 alkyl, C1-C10 alkoxy, halogen, hydroxy, amino, cyano and nitro; p is 0, 1, 2 or 3; q is 0, 1, 2, or 3; t is 0, 1, 2, 3, or 4; The "*" marked C=O may optionally form a ketal or hemiketal structure.

2. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, or metabolite thereof, characterized in that: X is O; R1, R2, R3, R4 are each independently selected from H, C1-C10 alkyl and nitroso; R5 and R6 are each independently selected from C1-C10 alkyl and C1-C10 alkoxy; R7 is selected from C1-C10 alkyl and C1-C10 alkoxy.

3. The compound of formula I according to claim 1, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, or metabolite thereof, characterized in that: The compound of formula I is selected from:

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises (i) a compound of formula I according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite thereof, and (ii) a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutically acceptable carrier is selected from DSPE-mPEG and water.

6. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is an aqueous topical preparation.

7. Use of the compound of formula I according to any one of claims 1 to 3, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite or the pharmaceutical composition according to any one of claims 4 to 6 in the preparation of a medicament for treating and / or preventing glaucoma.

8. Use of the compound of formula I as described in any one of claims 1 to 3, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite or the pharmaceutical composition as described in any one of claims 4 to 6 in lowering intraocular pressure.

9. Use of the compound of formula I according to any one of claims 1 to 3, or its pharmaceutically acceptable salt, enantiomer, diastereomer, solvate, isotope-substituted product, polymorph, prodrug, metabolite or the pharmaceutical composition according to any one of claims 4 to 6 in up-regulating the content of soluble guanylate cyclase and / or anti-oxidation.

10. Use of a carbon monoxide releasing compound in the preparation of a medicament for treating and / or preventing glaucoma.

Images