Isocoumarin derivatives, processes for their preparation and use thereof

Abstract

The present application relates to a novel isocoumarin derivative, or a tautomer, stereoisomer, racemate, non-identical mixture of enantiomers, geometric isomer, solvate, pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutical composition comprising the compound. The present application also discloses a preparation method of the novel isocoumarin derivative and its application in pro-angiogenic drugs. It is found through a transgenic zebrafish model that the isocoumarin derivative provided by the present application has strong pro-angiogenic activity and low toxicity, and has the potential to be developed into a pro-angiogenic drug.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel isocoumarin derivative, a preparation method and application thereof, and in particular to the use of a novel isocoumarin derivative and a pharmaceutical composition thereof as a drug, especially as a pro-angiogenic drug. BACKGROUND

[0002] Angiogenesis refers to the growth of new capillaries and blood vessels from the existing blood vessel system, involving a series of complex biological processes such as endothelial cell proliferation, migration, basement membrane degradation, and blood vessel lumen formation. Imbalance of this process can lead to the pathogenesis of many diseases, such as cardiovascular and ischemic diseases, rheumatoid arthritis, ophthalmic and retinal disorders, etc. (Nature, 2005, 438: 932-936). The use of various pro-angiogenic or anti-angiogenic drugs or molecules to regulate angiogenesis can often effectively treat these pathological diseases clinically (Eur. J. Med. Res., 2022, 27:232). Insufficient angiogenesis leads to poor blood circulation and tissue death, and pro-angiogenic therapy has been considered as an attractive and promising approach to treat diseases related to blood vessel dysfunction, such as heart failure and ischemic stroke, which are still the leading causes of death worldwide (Curr. Opin. Pharmacol. 2018, 39: 60-67). So far, a variety of methods for inducing therapeutic angiogenesis have been identified, including cytokine therapy, gene therapy, small molecule therapy, and delivery of progenitor cells or stem cells (Curr. Med. Chem. 2017, 24: 3413-3432). However, all current treatment methods have the disadvantages of short-term efficacy, strong side effects, and low success rate (Coronary Artery Dis. 2017, 28: 605-613). Therefore, there is a great need to develop new therapeutic drugs to promote angiogenesis in ischemic tissues. SUMMARY

[0003] The present application aims to provide a strain of fungus Phomopsis prunorum The novel isocoumarin derivative derived from F4-3 and the preparation method and application thereof as a pro-angiogenic drug can meet the above needs of the prior art. Strain preservation information: preservation unit name: China Center for Type Culture Collection (CCTCC); preservation unit address: Wuhan University, Wuhan, Hubei, China; preservation date: May 24, 2022; preservation number: CCTCC No: M2023771; classification name: Phomopsis prunorumF4-3, see article (+)- / ()-Prunomarin A and (+)-pestalactone B, three new isocoumarin derivatives from the endophytic fungus Phomopsis prunorum, Tetrahedron Letters 75 (2021) 153205.

[0004] The present application specifically relates to a novel isocoumarin derivative of structure I, its tautomer, stereoisomer, non-equal mixture of racemate, enantiomer, geometric isomer, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof,

[0005] wherein R1 is H, -CH3 or -CHO; R2 is H, or -CH3.

[0006] In some embodiments, the compounds of the present application specifically include, but are not limited to, one of the following structures or a tautomer, stereoisomer, non-equal mixture of racemate, enantiomer, geometric isomer, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof of one of the following structures:

[0007] .

[0008] The present application provides a preparation method of the compound of formula I, characterized in that the activated Phomopsis prunorum The F4-3 strain is inoculated into a fungal fermentation medium and subjected to fermentation culture at room temperature 25-28 °C; after the fermentation is completed, the mycelium or fermentation broth is repeatedly soaked with an organic solvent, the organic phases are combined, concentrated, extracted, and concentrated under reduced pressure to obtain a crude extract, which is then subjected to normal phase silica gel column chromatography, Sephadex LH-20 gel column chromatography and HPLC high performance liquid column separation to obtain the novel isocoumarin compound.

[0009] In the above preparation method, the fungal fermentation medium contains rice 1.0%-80.0% (by weight, the same below), glucose 0.01%-5%, sodium chloride 0.01%-5%, sodium acetate 0.01%-5%, sodium propionate 0.01%-5%, methionine 0.01%-5%, shikimic acid 0.01%-5%, and the rest is water, and the culture time is preferably 20-100 days.

[0010] The stationary phase of the normal-phase silica gel column chromatography is preferably 200-300 mesh silica gel, and the mobile phase is preferably a mixture of ethyl acetate and petroleum ether in a volume ratio of 30%-100%; the mobile phase of the Sephadex LH-20 gel column chromatography is preferably a mixture of chloroform and methanol in a volume ratio of 1:1; the semi-preparative column used in the HPLC high-performance liquid chromatography is an ODS C18 column, preferably a Cosmosil 4.6x250 mm, 5 μm, the flow rate is preferably 1.0-5.0 mL / min, and the mobile phase is preferably a mixture of acetonitrile and water in a volume ratio of 30%-70%.

[0011] The present application relates to the use of the compounds of the present application and pharmaceutically acceptable salts thereof for the manufacture of a medicament for treating or alleviating a disease caused by insufficient vascularization in humans or animals.

[0012] The present application comprises a pharmaceutical composition comprising a therapeutically effective amount of the compounds of the present application in combination with at least one pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle.

[0013] In another aspect, the present application relates to the use of the compounds of the present application or a pharmaceutical composition thereof in a method for treating various diseases caused by insufficient vascularization, which comprises administering to a human or an animal a pharmaceutically acceptable therapeutically effective amount of the compounds of the present application or a pharmaceutical composition thereof.

[0014] The present application also comprises a method for treating or alleviating a disease caused by insufficient vascularization or the like, or a method for treating a condition susceptible to the disease, which comprises administering to a patient a therapeutically effective amount of the compounds of the present application.

[0015] The foregoing merely outlines some aspects of the present application, but is not limited to these aspects. These aspects and other aspects will be described in greater detail below. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Fluorescent image of the number of intersegmental vessels of zebrafish. DETAILED DESCRIPTION

[0017] Definitions and General Terminology

[0018] The present application will be put into context by listing the documents that the specific embodiments correspond to, and the examples are accompanied by structural formula and chemical formula explanations. The present application is intended to encompass all alternatives, variations, and equivalents that can be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments described herein. The present application is not intended to be limited to the methodologies and materials described. There are many documents and similar materials that are distinct from or contrary to the present application application, including but not limited to definitions of terms, usage of terms, described techniques, or the scope as controlled by the present application application.

[0019] The following definitions will apply unless otherwise indicated. For the purposes of the present application, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific gravity is as listed in the Handbook of Chemistry and Physics, 75thEd., 7-4. All th organic chemistry general principles are found in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, and all publications on the topic of organic synthesis, physical organic chemistry, and polymer science.

[0020] Unless otherwise indicated, the structural formulae described herein include all tautomeric forms (e.g., enantiomeric, diastereomeric, and geometric (or conformational) isomers): for example, where asymmetric centers exist, both the R , S R and S configurations, double bond ( Z ), ( E ) isomers, and ( Z ), ( E ) conformational isomers are included within the scope of the application. Thus, individual stereochemical isomers or mixtures of their enantiomers, diastereomers, or geometric (or conformational) isomers, of the compounds of the application are within the scope of the present application.

[0021] The term "prodrug" as used herein refers to a compound that is converted into a compound of the present invention in vivo. Such conversion is effected by hydrolysis of the prodrug in blood or by enzymatic conversion in blood or tissue to the parent structure. The prodrug class of compounds of the present invention can be esters, and among the esters that can serve as prodrugs in the present invention are benzoic acid esters, aliphatic (C1-C24) esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, a compound of the present invention containing a hydroxyl group can be acylated to provide a prodrug form of the compound. Other prodrug forms include phosphates, such as those compounds that are phosphorylated on a hydroxyl group of the parent. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series; Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; J. Rautio et al, Prodrugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270; and S. J. Hecker et al, Prodrugs of Phosphates and Phosphonates, J. Med. Chem., 2008, 51, 2328-2345.

[0022] Unless otherwise indicated, all tautomers of the compounds of the present invention are included within the scope of the present invention. In addition, unless otherwise indicated, the structural formulas of the compounds described herein include one or more isotopically enriched atoms.

[0023] "Metabolite" refers to a product produced through metabolism of a specified compound or salt thereof in the body. Metabolites of a compound can be identified using techniques known in the art, and may

[0024] The compounds of the present application can contain asymmetric or chiral centers, and thus exist in different stereoisomers. All stereoisomers of the compounds of the present application, including but not limited to, diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemates, form part of the present application. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D, L, or R, S are used to denote the absolute configuration of the molecule. The prefixes D and L or (+) and (-) are used to designate the sign of the rotation of plane-polarized light by the compound, that is, (+) if the compound is dextrorotatory and (-) if it is levorotatory. Many stereoisomers exhibit more or less pronounced differences in their chemical and / or physical properties. At a molecular level, these differences can result from a difference in the spatial orientation of the atoms of the molecule about one or more stereogenic centers. For example, the different stereoisomers of a given compound are not superimposable on one another.

[0025] The term "tautomer" or "tautomeric forms" refers to isomers of a molecule that differ only in the arrangement of atoms at a particular site or sites, such as the nitrogen in a keto-enol tautomerism. For example, proton tautomers (i.e., proton-shift tautomers) include tautomers that interconvert by a shift in a proton, such as keto-enol and imine-enamine isomerization. Atom valence (bonding) tautomers include tautomers that interconvert by a rearrangement of bonding electrons.

[0026] As used herein, "pharmaceutically acceptable salt" refers to organic and inorganic salts of the compounds of the present application. Pharmaceutically acceptable salts are well known in the art, for example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference. Pharmaceutically acceptable salts include those salts, which retain the desired pharmacological activity of the parent compound and do not impart undesired toxicological effects thereto. J. Pharmaceutical Sciencespharmaceutically acceptable, non-toxic salts of the compounds of this application include those derived from inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, nitric, and organic acids such as citric, maleic, fumaric, benzoic, palmoic, tartaric, salicylic, succinic, mandelic, methanesulfonic, ethanesulfonic, toluenesulfonic, sulfosalicylic, acetic, trifluoroacetic, and the like. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzenesulfonate, bisulfate, bisulfite, borate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, sulfocyanate, sulfamate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from an appropriate base include alkali metal, alkaline earth metal, ammonium, and N + (C1-C4 alkyl)4. Quaternary ammonium salts of any of the groups containing N in the compounds of this application are also contemplated. Water or oil-soluble or dispersible 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 appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed by the addition of inorganic acids to the free amines. Examples of pharmaceutically acceptable salts include those derived from inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, nitric, and organic acids such as citric, maleic, fumaric, benzoic, palmoic, tartaric, salicylic, succinic, mandelic, methanesulfonic, ethanesulfonic, toluenesulfonic, sulfosalicylic, acetic, trifluoroacetic, and the like. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzenesulfonate, bisulfate, bisulfite, borate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, sulfocyanate, sulfamate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from an appropriate base include alkali metal, alkaline earth metal, ammonium, and N

[0027] The salts of the compounds of this application can be prepared in a manner known per se, e.g., by means of ion exchange or by conversion of one salt into another salt.

[0028] The "solvate" of the present application refers to an association or complex of one or more solvent molecules and a compound of the present application. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol. The term "hydrate" refers to the complex that contains solely water as the solvent.

[0029] The solvates of the compounds of the present application, or the solvates of their salts, can be illustrated by the solvates of the specific compounds listed below, but are not limited to the present application:

[0030]

[0031] Salts of the compounds of the present application also include salts of intermediates used in preparing or purifying the compounds of the present application or salts of the separated enantiomers of the compounds of the present application, but are not necessarily pharmaceutically acceptable salts.

[0032] If the compounds of the present application are basic, salts can be prepared from any appropriate method available in the literature, for example, using inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid and the like. Or using organic acids, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid and salicylic acid; pyranosic acids, such as glucuronic acid and galacturonic acid; alpha-hydroxy acids, such as citric acid and tartaric acid; amino acids, such as aspartic acid and glutamic acid; aromatic acids, such as benzoic acid and cinnamic acid; sulfonic acids, such as p-toluenesulfonic acid, ethanesulfonic acid, and the like.

[0033] If the compounds of the present application are acidic, salts can be prepared from any appropriate method available in the literature, for example, using inorganic or organic bases, such as ammonia (primary, secondary, tertiary amines), alkali metal hydroxides or alkaline earth metal hydroxides, and the like. Suitable salts include, but are not limited to, organic salts from amino acids such as glycine and arginine, ammonia, such as primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine, and piperazine, and inorganic salts from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0034] According to another aspect, pharmaceutical compositions of the present application feature a compound of the present application, or a compound of the examples, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of compound in the compositions of the present application is effective to detectably treat or reduce a disease in a patient caused by insufficient angiogenesis.

[0035] The compounds of the present application exist in free form or in suitable, pharmaceutically acceptable derivatives. According to the present application, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of esters, or any other adduct or derivative which upon administration to a patient is capable of providing (directly or indirectly) a compound of the present application, or a metabolite or residue thereof.

[0036] As described in this invention, pharmaceutically acceptable compositions of this invention further comprise pharmaceutically acceptable carriers, excipients, or excipients, such as those used in this invention, including any solvent, diluent, or other liquid excipient, dispersant or suspending agent, surfactant, isotonic agent, thickener, emulsifier, preservative, solid binder, or lubricant, etc., suitable for a particular target dosage form. As described in the following literature: In Remington: The Science and Practice of Pharmacy, 21st edition, 2005; ed. DB Troy, Lippincott Williams Wilkins, Phil Adelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York, the content of this literature, together with the present description, shows that different carriers can be used in the formulation of pharmaceutically acceptable compositions and their known methods of preparation. Except for any conventional carrier media that are incompatible with the compounds of the present invention, such as any adverse biological effects produced or interactions with any other component of a pharmaceutically acceptable composition that occur in a harmful manner, their use is also within the scope of this invention.

[0037] Substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, aluminum, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-blocking polymers, lanolin, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl cellulose. Sodium thiosulfate, ethyl cellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol, phosphate buffer solution, and other non-toxic and suitable lubricants such as sodium lauryl sulfate and magnesium stearate, colorants, release agents, coatings, sweeteners, flavorings and spices, preservatives and antioxidants.

[0038] The pharmaceutical compositions of the present application can be orally administered, injected, administered by spray inhalation, administered topically, administered rectally, administered nasally, administered buccally, administered vaginally or administered via an implanted reservoir. They can be in the form of capsules, tablets, pills, powders, granules, and aqueous or oily suspensions, or solutions. Oral administration can be in the form of tablets, pills, capsules, dispersible powders, granules, or suspensions, syrups, and elixirs, or in a form suitable for topical administration: ointments, gels, medicated gums and the like, or in the form of a sterile injectable solution or suspension for parenteral administration. The compounds of the present application can also be administered parenterally or intraperitoneally. Solutions or suspensions of these active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose, polyvinylpyrrolidone, and the like. Dispersions can also be prepared in glycerol, liquid, polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0039] The pharmaceutical forms suitable for injection include: sterile aqueous solutions or dispersions and sterile powders (for the extemporaneous preparation of sterile injectable solutions or dispersions). In all cases, these forms must be sterile and must be fluid to the extent that can be easily administered with a syringe. They must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, alcohols, such as glycerol, propylene glycol and liquid polyethylene glycols, suitable mixtures thereof and vegetable oils.

[0040] The compounds can be administered in a local rather than systemic fashion. For example, the compounds can be injected directly into an organ, often in a dilute form or in a sustained release form. In addition, pharmaceutical compositions containing a compound of the present application can be used in targeting drug delivery systems, such as liposomes. The liposomes will target the organ and be taken up selectively by that organ. In addition, compositions containing a compound of the present application can be provided in a rapid release, delayed release or sustained release form.

[0041] For inhalation administration, the compounds of the present application can be in the form of an aerosol, a gas mist or a powder. The pharmaceutical compositions of the compounds of the present application can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0042] The compounds of the present application can also be prepared as rectal compositions such as, for example, suppositories, rectal gels, rectal foams, rectal aerosols, suppositories, gel suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers, such as polyvinylpyrrolidone, PEG, and the like. In suppository form, low-melting wax such as, but not limited to, a mixture of fatty acid glycerides or cocoa butter is optionally first melted and then cooled to solidify with the active compound incorporated.

[0043] In addition, the compounds of the present application can also be used in combination with pro-angiogenic drugs. Specifically including, but not limited to, Danhong injection, ferulic acid, Danshen, etc.

[0044] Pharmaceutical compositions can be prepared according to conventional methods using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients can be used as suitable. Pharmaceutical compositions containing the compounds of the present application can be manufactured according to conventional methods e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0045] The pharmaceutical compositions will contain at least one pharmaceutically acceptable carrier, diluent or excipient and a compound of the present application as the active ingredient in free acid, free base or pharmaceutically acceptable salt form. In addition, the pharmaceutical compositions can also include other medically or pharmaceutically acceptable agents, carriers, adjuvants, such as preservatives, stabilizers, wetting agents or emulsifiers, solubility

[0046] Methods of preparing the compositions comprising the compounds described herein include bringing the compounds into association with one or more inert, pharmaceutically acceptable excipients or carriers in solid, semi-solid, or liquid form. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions, emulsions, gels, suspensions, and the like, containing the compounds dissolved or suspended therein. Semi-solid compositions include, but are not limited to, gels, suspensions, and creams. Compositions can be in the form of liquid solutions or suspensions, solid forms suitable for reconstitution into liquid or suspension form, or emulsions. These compositions also can contain minor amounts of nontoxic, nontherapeutic additives, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0047] The compounds of the present application are preferably prepared as dosage units to facilitate accurate dosing and uniformity of dosage. The term "dosage unit" as used herein refers to a physically discrete unit of the pharmaceutical agent appropriate for the patient to be treated. It will be appreciated, however, that the actual amount of the compound or composition of the present application required for use in treatment will vary depending on numerous factors, including the specific condition being treated, and the severity of the condition, the activity of the particular compound, the specific composition employed, the age, body weight, general health condition, sex and diet of the patient, the time of administration, the route of administration, and the rate of excretion of the particular compound being employed, the duration of the treatment, the use of other drugs or compounds in combination or in conjunction with the specific compound, and other factors well known in the medical arts.

[0048] The compounds of the present application can be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase the penetration in biological compartments (e.g., blood, lymphatic system, central nervous system), those which increase oral availability, those which increase solubility to allow administration by injection, those which alter metabolism and those which alter excretion. The compounds of the present application can be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase the penetration in biological compartments (e.g., blood, lymphatic system, central nervous system), those which increase oral availability, those which increase solubility to allow administration by injection, those which alter metabolism and those which alter excretion.

[0049] Generally, the compounds of the present application can be prepared by the methods as described herein, and the following examples will further illustrate the present application, unless otherwise specified.

[0050] For a further understanding of the present application, the following examples are provided. These examples are provided to better illustrate the present application and are not intended to limit the scope of the application or the manner in which it can be practiced.

[0051] Example 1

[0052] Fungus Phomopsis prunorum Strain fermentation, extraction and isolation of F4-3

[0053] The medium used for fermentation culture contains rice 60.0% (wt / wt, same below), glucose 2.4%, sodium chloride 2.5%, sodium acetate 1.3%, sodium propionate 2.0%, methionine 4.8%, shikimic acid 1.2%, 6-fluoroshikimic acid 3.0%, chorismic acid 1.6%, and the rest is water. The fungal strain is fermented at 25-28°C for 40 days.

[0054] The fermentation obtained bacterial body is extracted with ethyl acetate or chloroform-methanol mixture (1:1) 3 times, concentrated under reduced pressure, and then extracted with ethyl acetate 3 times to obtain a crude extract; the crude extract is concentrated, and then subjected to normal phase silica gel column chromatography (the mobile phase is 80% ethyl acetate-petroleum ether mixed solvent, by volume), Sephadex LH-20 gel column chromatography (the mobile phase is a mixed solvent of chloroform:methanol=1:1, by volume), and then HPLC high performance liquid preparation chromatography (ODS C18 column, the mobile phase is a mixed solvent of 60% acetonitrile-water, by volume), and the obtained eluate is concentrated to obtain novel isocoumarin compounds 1-4, respectively.

[0055] Example 2

[0056] The compound in Example 1 is subjected to structural analysis test, and the following physicochemical property data is obtained:

[0057] Compound 1: white amorphous powder; [ α ] 25 D +20.0 ( c 0.05, MeCN); UV (MeCN) λ max (logε) 248 (5.16), 280 (0.48), 334 (1.81) nm; CD (MeCN) λ max (Δ ε ) 209 (‒5.71), 239(+24.41), 286 (−1.53), 330 (‒1.00) nm; HR-ESI-MS m / z 445.1130 [M + H] + (calcdfor C 23 H 23 O 10 , 445.1129); 1 H NMR (DMSO- d 6, 400 MHz) δ : 6.49 (s, 1H), 6.45 (t, J =2.3 Hz, 1H), 4.38 (br s, 1H), 4.30 (dd, J = 10.8, 8.0 Hz, 1H), 4.19 (td, J =10.0, 6.2 Hz, 1H), 3.08 (d, J = 17.9 Hz, 1H), 2.97 (d, J= 17.9 Hz, 1H), 2.65(dd, J = 17.5, 6.0 Hz, 1H), 2.56 (m, 2H), 2.25 (m, 1H), 2.14 (m, 1H), 2.10 (m,1H), 2.01 (s, 3H); 13 C NMR (100 MHz) δ : 170.7 (C), 167.6 (C), 165.6 (C), 164.5(C), 160.4 (C), 147.3 (C), 137.6 (CH), 135.8 (C), 128.8 (C), 109.7 (C), 108.7(C), 99.0 (CH), 97.4 (C), 84.5 (CH), 76.0 (C), 68.9 (CH), 66.0 (CH), 33.9(CH2), 31.0 (CH2), 29.0 (CH2), 18.6 (CH2), 8.0 (CH3).

[0058] Compound 2: white amorphous powder; [ α ] 25 D ‒38.0 ( c 0.1, MeCN); UV (MeCN) λ max (log ε)248 (5.16), 280 (0.48), 334 (1.81) nm; CD (MeCN) λ max (Δ ε ) 215 (4.11), 241 (−25.2), 286 (1.62), 331 (1.37) nm; HR-ESI-MS m / z 459.1277 [M + H] + (calcd forC 23 H 23 O 10 , 459.1286); 1 H NMR (DMSO- d 6, 400 MHz) δ : 6.55 (t, J = 2.3 Hz, 1H), 6.47(s, 1H), 4.45 (br s, 1H), 4.34 (dd, J= 10.8, 8.0 Hz, 1H), 4.16 (td, J = 10.0,6.2 Hz, 1H), 3.67 (s, 3H), 3.18 (d, J = 17.9 Hz, 1H), 2.80 (d, J = 17.9 Hz, 1H),2.75 (dd, J = 17.5, 6.0 Hz, 1H), 2.60 (m, 1H), 2.50 (m, 2H), 2.15 (m, 1H), 1.93(m, 1H), 2.02 (s, 3H); 13 C NMR (100 MHz) δ : 170.2 (C), 165.5 (C), 165.5 (C),163.9 (C), 160.5 (C), 147.1 (C), 139.7 (CH), 135.8 (C), 126.5 (C), 109.7 (C),108.8 (C), 98.9 (CH), 97.6 (C), 84.0 (CH), 76.0 (C), 68.4 (CH), 65.1 (CH),52.0 (CH3), 36.6 (CH2), 29.3 (CH2), 26.7 (CH2), 18.3 (CH2), 7.9 (CH3).

[0059] Compound 3: white amorphous powder; [ α ] 25 D ‒156.0 ( c 0.1, MeCN); UV (MeCN) λ max (logε) 226 (4.08), 272 (5.42), 328 (1.78) nm; CD (MeCN) λ max (Δ ε ) 215 (+8.33), 230(−14.4), 265 (−30.8), 332 (−8.0) nm; HR-ESI-MS m / z 509.1067 [M + Na] + (calcd forC 24 H 22 O 11 Na, 509.1060); 1 H NMR (DMSO-d 6, 400 MHz) δ : 10.42 (s, 1H), 6.58 (t, J = 2.3Hz, 1H), 4.49 (br s, 1H), 4.38 (dd, J = 10.8, 8.0 Hz, 1H), 4.18 (td, J = 10.0,6.2 Hz, 1H), 3.70 (s, 3H), 3.27 (d, J = 16.8 Hz, 1H), 3.07 (m, 1H), 2.94 (d, J =16.8 Hz, 1H), 2.99 (dd, J = 17.2, 6.0 Hz, 1H), 2.61 (m, 1H), 2.54 (m, 1H), 2.18(m, 1H), 2.02 (s, 3H), 1.74 (m, 1H); 13 C NMR (100 MHz) δ : 195.6 (C), 170.0 (C),168.9 (C), 165.6 (C), 165.1 (C), 164.4 (C), 152.0 (C), 142.0 (C), 139.9 (CH),126.6 (C), 110.5 (C), 110.2 (C), 109.3 (CH), 99.7 (C), 84.2 (CH), 75.4 (C),68.4 (CH), 65.1 (CH), 52.0 (CH3), 37.6 (CH2), 29.2 (CH2), 28.2 (CH2), 24.5(CH2), 7.3 (CH3).

[0060] Compound 4: white amorphous powder; [ α ] 25 D +148.0 ( c 0.1, MeCN); UV (MeCN) λ max (logε) 226 (4.08), 272 (5.42), 328 (1.78) nm; CD (MeCN) λ max (Δ ε) 212 (‒7.63), 229(+16.1), 265 (+17.8), 333 (+7.09) nm; HR-ESI-MS m / z 473.1074 [M + H] + (calcd forC 23 H 21 O 11 , 473.1078); 1 H NMR (DMSO- d 6, 400 MHz) δ : 10.31 (s, 1H), 6.56 (t, J = 2.3Hz, 1H), 4.42 (br s, 1H), 4.32 (dd, J = 10.8, 8.0 Hz, 1H), 4.32 (td, J = 10.0,6.2 Hz, 1H), 3.22 (d, J = 16.8 Hz, 1H), 3.03 (m, 1H), 2.91 (d, J = 16.8 Hz, 1H),2.78 (dd, J = 16.7, 6.2 Hz, 1H), 2.68 (m, 1H), 2.27 (m, 1H), 2.25 (m, 1H), 1.98(s, 3H), 1.94 (m, 1H); 13 C NMR (100 MHz) δ : 195.1 (C), 170.3 (C), 168.6 (C),165.5 (C), 165.1 (C), 164.4 (C), 152.4 (C), 141.7 (C), 137.6 (CH), 128.5 (C),110.5 (C), 110.1 (C), 109.1 (CH), 99.6 (C), 83.7 (CH), 75.3 (C), 68.5 (CH),65.5 (CH), 35.3 (CH2), 32.2 (CH2), 29.5 (CH2), 25.0 (CH2), 7.2 (CH3).

[0061] Example 3

[0062] Pro-angiogenic activity test based on zebrafish model

[0063] Transgenic TG (VEGFR2: GFP) zebrafish and AB wild-type zebrafish were provided by the Drug Screening Research Lab of the Shandong Academy of Sciences. Zebrafish were bred in a zebrafish breeding system under standard conditions of 14 h light / 10 h dark, 28 °C.

[0064] Zebrafish embryo culture: Zebrafish were bred under standard conditions of 14 h light / 10 h dark, water temperature 28 °C, pH 7.2-7.5, and were fed with artificial granular bait and newly hatched Artemia nauplii at regular intervals. When collecting eggs, healthy mature zebrafish were placed in a mating tank at a ratio of 1 / 1 or 1 / 2 for males and females, and fertilized eggs were obtained the next day at 9-10 o'clock. After disinfection and cleaning of the fertilized eggs, they were transferred to zebrafish embryo culture water and incubated at 28 °C under controlled light.

[0065] Measurement of intersegmental blood vessel number in zebrafish: At 24 h of fertilized egg development, the egg membrane was removed using a 1.0 mg / mL solution of chain protease E. Normal zebrafish embryos were selected under a stereomicroscope and transferred to a 24-well culture plate, 10 embryos per well. Normal control, PTK787 model, positive control, and drug treatment groups (using ferulic acid FA as the positive control, with a sample concentration of 80 µM) were set up and placed in an illuminated incubator (28 °C) to allow the embryos to continue to develop. After 48 h of drug action, the growth of fluorescent blood vessels was observed under a fluorescence microscope and the number of intersegmental blood vessels (ISVs) with blood flow in zebrafish was recorded. As shown in FIG. 1, the results show that the growth of intersegmental blood vessels in the blank control group was normal, and the growth of intersegmental blood vessels in the model group was significantly inhibited. Figure 1 ## p <0.01), indicating successful modeling; compared with the model group, the test compounds at a concentration of 80 µM could significantly increase the number of zebrafish intersegmental blood vessels with blood flow ( ** p <0.01), indicating that the test compounds had significant pro-angiogenic activity; at the same time, compound 1 at a concentration of 40 µM could also significantly increase the number of zebrafish intersegmental blood vessels with blood flow ( ** p <0.01), which was comparable to the positive drug ferulic acid. In addition, the test compounds at a concentration of 80 µM did not exhibit significant toxicity to zebrafish, had low toxicity, and had the potential to be developed as pro-angiogenic drugs.​

Claims

1. An isocoumarin derivative of the structure of formula I, characterized in that, The compound of formula I has the following structure: wherein R1is H or -CHO; R2is H, or -CH3; or a pharmaceutically acceptable salt of the isocoumarin derivative of formula I.

2. The isocoumarin derivative according to claim 1, characterized in that, having one of the following structures: 。 3. The process for the preparation of isocoumarin derivatives according to claim 1 or 2, characterized in that, comprising the following steps: (1) inoculating the activated Phomopsis prunorum F4-3 strain into a fungal fermentation medium containing 1.0 wt % - 80.0 wt % of rice, 0.01 wt % - 5 wt % of glucose, 0.01 wt % - 5 wt % of sodium chloride, 0.01 wt % - 5 wt % of sodium acetate, 0.01 wt % - 5 wt % of sodium propionate, 0.01 wt % - 5 wt % of methionine, 0.01 wt % - 5 wt % of shikimic acid, and the rest being water, and carrying out fermentation culture at room temperature for 20 - 100 days; (2) after the fermentation is completed, repeatedly soaking the mycelium or fermentation broth with an organic solvent, combining the organic phases, concentrating, extracting, and concentrating under reduced pressure to obtain a crude extract, and then subjecting the crude extract to normal-phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and HPLC high-performance liquid chromatography to obtain the isocoumarin derivative; The fungus Phomopsis prunorum The preservation unit of F4-3 is China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC No: M2023771. The preservation date is May 24, 2022.

4. The production method according to claim 3, wherein The fermentation medium used in the fermentation culture contains 60.0 wt % of rice, 2.4 wt % of glucose, 2.5 wt % of sodium chloride, 1.3 wt % of sodium acetate, 2.0 wt % of sodium propionate, 4.8 wt % of methionine, 1.2 wt % of shikimic acid, 3.0 wt % of 6-fluorosikimic acid, 1.6 wt % of chorismic acid, and the rest being water, and the fungal strain is fermented and cultured at 25 - 28 °C for 40 days.

5. The production method according to claim 3, wherein The stationary phase used in the normal-phase silica gel column chromatography is 200 - 300 mesh silica gel, and the mobile phase is a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 30% - 100%; the mobile phase used in the Sephadex LH-20 gel column chromatography is a mixed solvent of chloroform and methanol in a volume ratio of 1:1; and the chromatographic column used in the HPLC high-performance liquid chromatography is an ODS C18 column, the flow rate is 1.0 - 5.0 mL / min, and the mobile phase is a mixed solvent of acetonitrile and water in a volume ratio of 20% - 70%.

6. An angiogenic agent, characterized by comprising a protein having the amino acid sequence of SEQ ID NO:

1. The isocoumarin derivative of claim 1 or 2.

7. A pharmaceutical composition comprising one or more isocoumarin derivatives of claim 1 or 2; the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient.

8. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition further comprises at least one other pro-angiogenic drug; the pharmaceutical composition is any one of an injection, an oral preparation, a lyophilized powder injection, or a suspension.

9. Use of a solvate of the isocoumarin derivative of claim 1 or 2 as a pro-angiogenic drug lead compound for non-disease diagnosis and treatment purposes.

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