Adamantane modified camptothecin derivative

Abstract

The invention provides an adamantane modified camptothecin derivative, and particularly discloses a camptothecin analogue shown in the following formula (I) and pharmaceutically acceptable salt thereof, and the camptothecin analogue can be used as a prodrug of a topoisomerase I (TopoI) enzyme inhibitor and is applied to treatment of tumor diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an adamantane-modified camptothecin derivative and its preparation and application. Background Art

[0002] Camptothecin (CPT), a natural product isolated from the plant Camptotheca acuminata, is an excellent topoisomerase I (Topo 1) inhibitor. CPTs bind to the Topo 1-DNA complex, which is involved in key cellular processes such as replication, transcription, and recombination. While this complex is typically rapidly reversible in the absence of CPT, CPTs bind to it, causing the accumulation of DNA strand breaks during replication, ultimately leading to cell death during the S phase of the cell cycle.

[0003] Camptothecin's low solubility, poor stability, and significant adverse reactions have severely limited its clinical application. To address these issues, over the past few decades, structural modifications have been made to camptothecin through the design of prodrugs, the introduction of polar groups, and the use of targeting agents. SN38 is the most active CPT analog, and due to various chemical and pharmacological limitations, its clinical application is in the form of CPT-11 injection. Although CPT-11 is fully approved for the treatment of various cancers, only a small fraction of CPT-11 is converted into the active parent drug, SN38. Various side effects of CPT-11 have also been reported. Only a small fraction of patients benefit from camptothecin use, while the vast majority develop resistance. Protein degradation is an effective way to inhibit resistance.

[0004] Adamantane is a polycyclic cage molecule. Due to its simplicity and high symmetry, the adamantane group is a commonly used functional group in drug discovery to improve the pharmacological and pharmacokinetic properties of drugs. Adamantane has also been successfully used as a hydrophobic tag for protein degradation.

[0005] Therefore, our design strategy has found that camptothecin derivatives with degradation function are expected to effectively improve the current drug resistance and have important clinical application value. Modifying the chemical structure of camptothecin to obtain a more effective and safer therapeutic drug is very necessary. Summary of the Invention

[0006] The purpose of the present invention is to provide a more effective and safer camptothecin derivative.

[0007] The first aspect of the present invention provides a compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by the following formula (I):

[0008]

[0009] wherein Y is a Top I inhibitor module, which is a camptothecin analog;

[0010] A is selected from carbonate bonds NHC(O)NH, NHC(O), NH, OC(O), S, O, CRab, wherein Ra and Rb are each independently selected from the group consisting of H, deuterium, halogen, hydroxy, amino, nitro, C1-C4 alkyl, C1-C4 haloalkyl, and C1-C4 alkoxy;

[0011] L1 is a divalent linking group having -(L3) x -Shown structure, wherein x is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0012] Each of the L3 is independently selected from the group consisting of a chemical bond, CH2, CHD, CD2, C=O, O, S, NH, SO, SO2, P=O, NHCO, NHSO2, OCH2, and a 5-7 membered heterocyclic group; wherein the heterocyclic group comprises 1, 2 or 3 heteroatoms selected from N, S or O as the ring skeleton;

[0013] In addition to conventional substituents, the L1 may be optionally substituted by one -L2-Z substituent;

[0014] L2 is a chemical bond, a C2-C3 amide group, or a C2-C3 ester group;

[0015] L1 and L2 may each independently be substituted by one or more substituents selected from the group consisting of deuterium, halogen, hydroxy, amino, nitro, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 alkyl ester, C1-C4 alkanoyl, C1-C4 alkylamino, -C1-C4 alkyl-hydroxy;

[0016] Z is adamantyl group, preferably

[0017] In another preferred embodiment, the Y has a structure shown in the following formula (II):

[0018]

[0019] wherein R1 is selected from H, C1-C6 alkyl, NRaRb; Ra and Rb are each independently selected from H, C1-C4 alkyl;

[0020] m is selected from 0, 1 or 2;

[0021] R2 is selected from H, C1~C6 alkyl.

[0022] In another preferred embodiment, the Y has a structure selected from the following group:

[0023]

[0024] In another preferred embodiment, L1 has -(L3) x -shown structure, wherein x is selected from the following group: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and each of the L3 is independently selected from the following group: chemical bond, CH2, C=O, O, S, NH, SO, SO2, NHCO, OCH2,

[0025]

[0026] In another preferred embodiment, L1 is selected from the following groups: -(CH2)n-,

[0027]

[0028] wherein n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0029] In another preferred embodiment, L2 is a chemical bond,

[0030] In another preferred embodiment, A is a carbonate bond NH, NHC(O)NH, O, S, CH2; preferably, a carbonate bond

[0031] In another preferred embodiment, the compound is selected from the following group:

[0032]

[0033]

[0034] The second aspect of the present invention provides the use of the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof for preparing a pharmaceutical composition for treating tumor diseases; the tumor is selected from the following group: gastric cancer, rectal cancer, lung cancer, cervical cancer and pancreatic cancer.

[0035] In another preferred embodiment, the compound of formula I is used to prepare a prodrug of a Topo I enzyme inhibitor.

[0036] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises: the compound according to the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0037] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 .A series of compound cytotoxicity results, taking HCT116 as an example.

[0039] Figure 2 Compound 4b induces apoptosis in MIA Paca2 cells. MIA Paca2 cells were treated with various concentrations of SN38 and 4b. (A) Apoptosis analysis of MIA Paca2 cells treated with 4b (10, 50, and 100 nM) for 24 hours. (B) Apoptosis analysis of MIA Paca2 cells treated with 4b (10, 50, and 100 nM) for 48 hours.

[0040] Figure 3 Western blot analysis of topoisomerase I in MIA Paca2 cells after treatment with compounds SN38 and 4b.

[0041] Figure 4 In vivo therapeutic efficacy of compound 4b in an HCT 116 mouse xenograft model. Nude mice bearing HCT 116 xenografts were treated with a control group, SN38 (10 and 20 mg / kg), or 4b (5 mg / kg) via tail vein injection. (A) Tumor volume was recorded, and relative tumor volume was plotted; (B) Mouse body weight was recorded, and relative body weight change was plotted; (C) H&E-stained images of tumors and major organs; scale bar = 100 μm.

[0042] Figure 5 Western blot analysis of topoisomerase I in MIA Paca2 cells treated with compounds 4a-f and 10a-c.

[0043] Figure 6 In vivo therapeutic efficacy of compound 4b in a MIA Paca2 mouse xenograft model. Nude mice bearing MIA Paca2 xenografts were treated with a control group, irinotecan (30 mg / kg), or 4b (10 mg / kg) by tail vein injection. (A) Tumor volume was recorded, and relative tumor volume was plotted; (B) Mouse body weight was recorded, and relative body weight change was plotted; (C) Photos of tumors in each group; (D) Mouse tumor weight was recorded. DETAILED DESCRIPTION

[0044] After extensive and in-depth research, extensive screening, and testing, the present inventors have provided a camptothecin analog, its preparation, and application. The camptothecin analog of the present invention exhibited significant cytotoxicity in four different tumor cell lines and can be used to treat tumor-related diseases. The present invention is also a typical Topo I inhibitor, exhibiting strong antiproliferative activity in Topo I-highly expressing cell lines and can be used as a Topo I inhibitor prodrug. Furthermore, the compounds of the present invention exhibited no significant toxic side effects in mouse models, demonstrating increased safety in clinical applications. In summary, the compounds of the present invention represent a novel class of Topo I anti-tumor drugs. This is the basis for the completion of the present invention.

[0045] the term

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0048] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0049] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0050] The experimental methods in the following examples of the present invention, where specific conditions are not specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0051] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0052] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0053] In this application, the term "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character "three" generally indicates that the related objects are in an "or" relationship.

[0054] In the compounds of the present invention, when any variable (such as R 10 、R 11 If a substituent (e.g., ) occurs more than once in any component, its definition at each occurrence is independent of its definition at every other occurrence. Likewise, combinations of substituents and variables are permissible so long as such combinations result in a stable compound. A line drawn from a substituent into the ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic, this means that such bonds may be attached only to any suitable carbon atom in an adjacent ring. It will be understood that one of ordinary skill in the art can select substituents and substitution patterns in the compounds of the present invention to provide compounds that are chemically stable and readily synthesized from readily available starting materials using techniques in the art and the methods set forth below. If a substituent is itself substituted with more than one group, it will be understood that these groups may be on the same carbon atom or on different carbon atoms so long as the structure is stable.

[0055] As used herein, the term "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, the definition of "C1-C8" in "C1-C8 alkyl" includes groups having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms in a straight or branched arrangement. The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms. For example, "cycloalkyl" includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and the like.

[0056] As used herein, the term "alkenyl" includes straight or branched alkenyl groups. For example, C2-C6 alkenyl refers to a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0057] As used herein, the term "alkynyl" includes straight or branched chain alkynyl groups. For example, C2-C6 alkynyl refers to a straight or branched chain alkynyl group having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

[0058] As used herein, the term "cycloalkyl" refers to a cyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms. 10Alkenyl refers to a cyclic saturated aliphatic hydrocarbon group having 3 to 10 carbon atoms. It can be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It can also be a bicyclic ring, such as a bridged ring or a spiro ring.

[0059] As used herein, the term "heterocyclyl" or "heterocycloalkyl" refers to a saturated or partially saturated cyclic group having a specific number of ring atoms (e.g., 3-10 ring atoms), wherein 1-3 atoms are heteroatoms selected from N, S, and O. It can be a monocyclic, bicyclic, or polycyclic ring, such as a fused ring, a bridged ring, or a spirocyclic ring. Specific examples include oxetanyl, azetidinyl, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, and pyrrolidinyl.

[0060] As used herein, the term "alkylamino" refers to an amino group substituted by an alkyl group. For example, a "C1-C6 alkylamino" refers to an amino group substituted by a C1-C6 alkyl group, which may be monosubstituted or disubstituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, tert-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-tert-butylamino, etc.

[0061] As used herein, the term "alkoxy" refers to a group having an alkyl-oxy structure. For example, "C1-C6 alkoxy" refers to a linear or branched alkoxy group having 1 to 6 carbon atoms, including methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, and the like.

[0062] As used herein, the term "haloalkyl" represents an alkyl group wherein one or more hydrogen atoms are replaced by a halogen, wherein alkyl is as defined above.

[0063] As used herein, the term "haloalkoxy" represents an alkoxy group having one or more hydrogen atoms replaced by a halogen, wherein alkoxy is as defined above.

[0064] As will be understood by those skilled in the art, "halogen" as used herein is intended to include F, Cl, Br and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "Halogenated" means substituted with an atom selected from F, Cl, Br, and I.

[0065] Unless otherwise specified as "substituted or unsubstituted", the groups of the present invention may be substituted by substituents selected from the following groups: halogen, nitrile, nitro, hydroxyl, amino, C1-C6 alkyl-amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C2-C6 alkenyl, halogenated C2-C6 alkynyl, halogenated C1-C6 alkoxy, allyl, benzyl, C6-C12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, etc.

[0066] The present invention includes the free form of the compound of formula (I) (herein, formula (I) includes formula (II)), and also includes its pharmaceutically acceptable salts, its stereoisomers and its prodrug molecules. The term "free form" refers to the compound in a non-salt form. The pharmaceutically acceptable salts included include not only the exemplary salts of the specific compounds described herein, but also all typical pharmaceutically acceptable salts of the free form of the compound of formula (I) or formula (II). The free form of the specific salt of the compound can be isolated using techniques known in the art. For example, the free form can be regenerated by treating the salt with an appropriate dilute aqueous base solution, such as a dilute aqueous solution of NaOH, a dilute aqueous solution of potassium carbonate, a dilute aqueous ammonia and a dilute aqueous solution of sodium bicarbonate. The free form is somewhat different from its respective salt form in certain physical properties, such as solubility in polar solvents, but for the purposes of the invention, such acid salts and base salts are equivalent to their respective free forms in other pharmaceutical aspects.

[0067] Pharmaceutically acceptable salts of the present invention can be synthesized from compounds of the present invention containing a basic or acidic moiety by conventional chemical methods. Typically, salts of basic compounds are prepared by ion exchange chromatography or by reacting a free base with a stoichiometric amount or an excess of an inorganic or organic acid in the desired salt form in a suitable solvent or combination of solvents. Similarly, salts of acidic compounds are formed by reaction with a suitable inorganic or organic base.

[0068] Therefore, the pharmaceutically acceptable salts of the compounds of this invention include conventional non-toxic salts of the compounds of this invention formed by reacting an alkaline compound of this invention with an inorganic or organic acid. For example, conventional non-toxic salts include salts prepared from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, p-aminobenzenesulfonic acid, 2-acetoxy-benzoic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic ....

[0069] If the compound of the present invention is acidic, suitable "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, ferric salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, and the like. Ammonium salts, calcium salts, magnesium salts, potassium salts, and sodium salts are particularly preferred. Salts derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[0070] Berg et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977: 66: 1-19, describe in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.

[0071] Unless otherwise specified, the structural formulas described herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers, and geometric isomers (or conformers)): for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, etc. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformers) are all within the scope of the present invention.

[0072] As used herein, the term "tautomer" refers to structural isomers of different energies that can interconvert across a low energy barrier. For example, proton tautomers (i.e., prototropy) include interconversion via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion via reorganization of some of the bonding electrons.

[0073] As used herein, the term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

[0074] As used herein, the term "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

[0075] The compound of the present invention can also be in the form of a prodrug. As used herein, the term "prodrug" refers to a compound that produces an active compound when metabolized (e.g., in vivo or in vitro). In some embodiments, the prodrug can be inactive, or have an activity lower than that of the free drug, but can provide favorable processing, administration or metabolic characteristics. The exemplary prodrug moiety of the present invention can be connected to the free drug by the hydroxyl, amino, phosphate or thiophosphate backbone of nucleotides, and can include ester, carbamate, carbonyl, thioester, amide, isocyanate, urea, thiourea or other physiologically acceptable metabolically unstable parts. In some embodiments, the prodrug is activated by enzymatic hydrolysis.

[0076] The present disclosure also includes isotopically labeled compounds that are identical to compounds of formula (I) (including compounds of formula (II)), but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of suitable isotopes for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, such as, but not limited to, 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 31 P. 35 S. 18 F and 36 Cl. With heavier isotopes (such as deuterium, i.e. 2 H) substitution may offer certain therapeutic advantages due to greater metabolic stability, such as greater in vivo half-life or lower dosage requirements, and may therefore be preferred in certain circumstances. The compounds may be combined with positron emitting isotopes for use in medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron emitting isotopes that may be incorporated into compounds of formula (I) or (II) are 11 C. 13 N. 15 O and 18 F. Isotopically labeled compounds of formula (I) or (II) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically labeled reagent in place of a non-isotopically labeled reagent.

[0077] The compounds disclosed herein can exist in solvated and unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and the present invention is intended to encompass both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in crystalline form.

[0078] Pharmaceutical composition and administration method

[0079] Since the compounds of the present invention are CDK12 / 13 protein degraders, the compounds and pharmaceutically acceptable salts thereof, as well as other compound forms disclosed herein, can be included in pharmaceutical compositions useful for treating, preventing, and alleviating diseases associated with CDK12 / 13 activity.

[0080] The pharmaceutical composition of the present invention comprises an effective amount, such as a safe and effective amount of a compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. "Effective amount" refers to an amount sufficient to induce a desired biological response (e.g., to treat a condition). "Safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition comprises 1 to 3000 mg (active dose range of 3 to 30 mg / kg) of the compound of the present invention / dose, and more preferably comprises 10 to 2000 mg of the compound of the present invention / dose. Preferably, "one dose" is a capsule or tablet.

[0081] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances suitable for human use, which must have sufficient purity and sufficiently low toxicity. "Compatibility" in this article means that the components of the composition can be mixed with the compounds of the present invention and each other without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier parts include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose and cellulose acetate), gelatin, talc, solid lubricants (such as stearic acid and magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil), polyols (propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers ( etc.), wetting agents (sodium lauryl sulfate etc.), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives and pyrogen-free water.

[0082] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration routes include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and topical administration.

[0083] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or bulking agents, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, some complex silicates, and sodium carbonate; (e) retarding solvents, such as paraffin; (f) absorption promoters, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also include a buffer.

[0084] Solid dosage forms, such as tablets, dragees, capsules, pills, and granules, can be prepared from coating and shell materials, such as enteric coatings and other materials well known in the art. They may contain opacifiers, and the active compound or compounds in such compositions that are released in a delayed manner in a portion of the digestive tract. Examples of useful embedding components are polymeric substances and waxes. If desired, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0085] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs. In addition to the active compound, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0086] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0087] Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures thereof.

[0088] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0089] Topical dosage forms for the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants (if required).

[0090] The compounds of the present disclosure may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0091] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is suitable for use in a mammal (e.g., a human) in need of treatment, wherein the dosage is a dosage that is considered pharmaceutically effective at the time of administration. For a 60 kg human, the daily dosage is generally 1-2000 mg, preferably 6-600 mg. Of course, the specific dosage should also be determined by taking into account factors such as the route of administration and the patient's health status, all of which are within the skill of a skilled physician.

[0092] The present invention will be further described below in conjunction with specific implementations. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0093] General formula for the synthesis of compounds 4a-f

[0094] (General formula for the synthesis of intermediate compounds)

[0095]

[0096] 4a-f Synthetic formula

[0097]

[0098]

[0099] General formula for the synthesis of compounds 10a-c

[0100]

[0101] Example 1 Synthesis of Compound 4a

[0102]

[0103] After compound 1 (1 eq) and compound 2 (1.2 eq) were stirred in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0104] Compound 3 (1 eq) and compound 4 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0105] 1 H NMR (400MHz, DMSO) δ8.23(d,J=9.1Hz,1H),8.17(d,J=2.6Hz,1H),7.79(dd,J=9.2,2.6Hz,1H),7.33(s,1H),5.44(s,2H),5.35(s,2H),3.89( s,2H),3.21(q,J=7.6Hz,2H),1.94–1.82(m,4H),1.68(d,J=16.8Hz,8H),1.58(d,J=2.9Hz,6H),1.43(d,J=2.9Hz,3H),0.88(t,J=7.4Hz,3H).

[0106] 13 C NMR (101MHz, DMSO) δ172.62,162.30,153.43,151.54,150.63,149.96,147.81,144.37,143.68,127.33,127.05,126.65 ,124.73,116.73,113.76,100.45,73.94,72.73,65.33,50.69,39.09,38.24,34.55,30.53,29.29,22.93,15.01,7.71.

[0107] ESI-HRMS calculation for C 57 H 71 N3O 13 m / z[M+H] + 585.2597, found [M+H] + 585.2595.

[0108] Example 2 Synthesis of Compound 4b

[0109]

[0110] After compound 1 (1 eq) and compound 2 (1.2 eq) were stirred in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0111] Compound 3 (1 eq) and compound 4 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0112] 1 H NMR (400MHz, DMSO) δ8.22(d,J=9.1Hz,1H),8.15(d,J=2.6Hz,1H),7.77(dd,J=9. 1,2.5Hz,1H),7.33(s,1H),5.46–5.32(m,4H),4.33(t,J=7.3Hz,2H),3.20(q,J= 7.5Hz,2H),1.95–1.81(m,5H),1.65(q,J=12.5Hz,6H),1.54–1.52(m,6H),1.51( d,J=7.3Hz,1H),1.30(d,J=5.5Hz,1H),1.28–1.23(m,3H),0.88(t,J=7.3Hz,3H).

[0113] 13 C NMR (101MHz, DMSO) δ172.93,157.27,153.38,152.61,150.49,149.81,147.04,146.31,146.00,131.90,129.12,127.5 0,125.52,119.55,115.52,97.16,72.84,65.78,50.03,42.27,42.22,36.90,31.78,30.77,28.40,22.71,14.33,8.24.

[0114] ESI-HRMS calculation for C 35 H 38 N2O7m / z[M+H]+ 495.2402, found [M+H] + 495.2396

[0115] Example 3 Synthesis of Compound 4c

[0116]

[0117] After compound 1 (1 eq) and compound 2 (1.0 eq) were stirred in DCM, Et3N (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0118] After compound 3 (1 eq) and compound 4 (1.2 eq) were stirred in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 5. The crude compound was then purified by flash column chromatography to obtain the desired product 5.

[0119] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0120] 1 H NMR (400MHz, DMSO) δ8.21(d,J=9.2Hz,1H),8.15(d,J=2.6Hz,1H),7.77(dd,J=9.2,2.5Hz,1H),7.33 (s,1H),5.44(s,2H),5.32(s,2H),4.39(d,J=13.5Hz,2H),4.15(d,J=6.1Hz,2H),3.24–3.15(m,2H) ,2.78(t,J=12.8Hz,2H),2.01(d,J=4.0Hz,1H),1.96(t,J=3.2Hz,3H),1.87(d,J=2.9Hz,7H),1.78– 1.69(m,3H),1.66(q,J=3.2Hz,6H),1.29(t,J=7.6Hz,3H),1.18–1.07(m,2H),0.88(t,J=7.3Hz,3H).

[0121] 13C NMR(101MHz,DMSO)δ174.12,172.47,156.80,152.85,152.16,150.01,149.32,146.57,145.82,145.55,131.44,128.66,127.02,125.00, 119.08,115.04,96.71,72.52,72.37,65.26,49.56,44.44,40.95,38.53,36.11,36.08,35.05,30.29,28.60,27.95,22.24,13.85,7.75.

[0122] ESI-HRMS calculation for C 40 H 45 N3O8m / z[M+H] + 696.3273, found [M+H] + 696.3279.

[0123] Example 4 Synthesis of Compound 4d

[0124]

[0125] After compound 1 (1 equiv) and compound 2 (1.0 equiv) were stirred in DCM, DMAP (1.2 equiv) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0126] After compound 3 (1 equiv) and compound 4 (1.2 equiv) were stirred in DCM, Et3N (1.5 equiv) was slowly added at 0°C and stirred at room temperature for 5 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 5. The crude compound was then purified by flash column chromatography to obtain the desired product 5.

[0127] Compound 5 (1 equiv) and compound 6 (1.1 equiv) were stirred in DMF, then DIPEA (1.5 equiv) was slowly added at 0°C and stirred at 50°C for 8 hours. After completion of the reaction, as monitored by TLC, saturated NaHCO₃ solution was added to quench the reaction, and the mixture was extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO₄, and evaporated to yield the crude compound. The crude compound was then purified by flash chromatography to yield the desired product.

[0128] 1H NMR (400MHz, DMSO) δ8.22(d,J=9.2Hz,1H),8.14(d,J=2.6Hz,1H),7.77(dd,J=9.2,2.5Hz,1H),7.33( s,1H),5.44(s,2H),5.33(s,2H),4.40–4.36(m,2H),4.14–4.10(m,2H),3.76–3.72(m,2H),3.61(dd, J=3.7,2.4Hz,2H),3.59(q,J=1.5Hz,4H),3.19(q,J=7.5Hz,2H),1.94(q,J=3.5Hz,3H),1.87(p,J=7. 0Hz, 2H), 1.80 (d, J = 3.0Hz, 7H), 1.64 (d, J = 3.4Hz, 6H), 1.29 (t, J = 7.6Hz, 3H), 0.88 (t, J = 7.3Hz, 3H).

[0129] 13 C NMR (101MHz, DMSO) δ176.91,172.94,157.28,153.45,152.65,150.49,149.76,147.05,146.30,146.00,131.95,129.16,127.50,125. 40,119.56,115.46,97.18,72.84,70.34,70.25,68.85,68.52,65.74,63.50,50.03,40.46,38.82,30.75,27.75,22.72,14.31,8.23.

[0130] ESI-HRMS calculation for C 40 H 46 N2O 11 m / z[M+H] + 731.3171, found [M+H] + 731.3174.

[0131] Example 5 Synthesis of Compound 4e

[0132]

[0133] After compound 1 (1 eq) and compound 2 (1.0 eq) were stirred in DCM, DMAP (1.2 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0134] After compound 3 (1 eq) and compound 4 (1.2 eq) were stirred in DCM, Et3N (1.5 eq) was slowly added at 0°C and stirred at room temperature for 5 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 5. The crude compound was then purified by flash column chromatography to obtain the desired product 5.

[0135] Compound 5 (1 equiv) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by the addition of saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0136] 1 H NMR (400MHz, DMSO) δ8.21(d,J=9.2Hz,1H),8.15(d,J=2.6Hz,1H),7.77(dd,J=9.2,2.5Hz,1H),7.33 (s,1H),5.44(s,2H),5.33(s,2H),4.41–4.37(m,2H),4.11–4.07(m,2H),3.75–3.71(m,2H),3.61–3 .55(m,6H),3.53(d,J=4.8Hz,8H),3.19(q,J=7.6Hz,2H),1.94(p,J=2.9Hz,4H),1.87(p,J=7.0Hz,2 H), 1.78 (d, J = 2.9Hz, 7H), 1.64 (dt, J = 6.3, 2.9Hz, 6H), 1.29 (t, J = 7.6Hz, 3H), 0.88 (t, J = 7.3Hz, 3H).

[0137] 13 C NMR (101MHz, DMSO) δ176.42,172.49,156.81,153.00,152.18,150.03,149.29,146.59,145.84,145.54,131.48,128.69,127.04,124.96, 119.10,115.04,96.72,72.39,69.87,69.83,69.78,68.34,68.07,68.05,63.05,49.57,39.98,38.34,30.28,27.29,22.26,13.86,7.77.

[0138] ESI-HRMS calculation for C 44H 54 N2O 13 m / z[M+H] + 819.3626, found [M+H] + 819.3627.

[0139] Example 6 Synthesis of Compound 4f

[0140]

[0141] After compound 1 (1 eq) and compound 2 (1.0 eq) were stirred in DCM, Et3N (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0142] After stirring compound 3 (1 eq) and compound 4 (1.2 eq) in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to give crude compound 5. The crude compound was then purified by flash column chromatography to give the desired product 5.

[0143] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0144] 1 H NMR (400MHz, DMSO) δ8.19(d,J=9.2Hz,1H),8.12(d,J=2.6Hz,1H),7.75(dd,J=9.1,2.5Hz,1H),7 .32(s,1H),5.43(s,2H),5.30(s,2H),4.25(t,J=6.5Hz,2H),3.17(q,J=7.5Hz,2H),3.02(q,J=6. 6Hz,2H),1.96–1.92(m,3H),1.90–1.83(m,2H),1.74(d,J=3.0Hz,6H),1.71–1.66(m,2H),1.63(d t,J=6.0,3.0Hz,6H),1.37(dt,J=12.7,7.0Hz,4H),1.28(t,J=7.6Hz,5H),0.88(t,J=7.3Hz,3H).

[0145] 13 C NMR (101MHz, DMSO) δ176.69,172.48,156.79,152.98,152.11,150.02,149.32,146.54,145.82,145.50,131.41,128.63,127.01,125.01,119 .08,115.02,96.71,72.38,68.83,65.27,49.55,39.73,38.79,38.32, 36.18,30.29,29.03,27.98,27.70,25.90,24.94,22.25,13.86,7.78.

[0146] ESI-HRMS calculation for C 40 H 47 N3O8m / z[M+H] + 698.3363, found [M+H] + 698.3363.

[0147] Example 7 Synthesis of Compound 4g

[0148]

[0149] After compound 1 (1 eq) and compound 2 (1.0 eq) were stirred in DCM, Et3N (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0150] After stirring compound 3 (1 eq) and compound 4 (1.2 eq) in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to give crude compound 5. The crude compound was then purified by flash column chromatography to give the desired product 5.

[0151] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0152] 1H NMR (400MHz, DMSO) δ8.25–8.21(m,1H),7.96(d,J=8.6Hz,1H),7.49(t,J=2.2Hz,1H),7.41(dd,J=8.4,2.4 Hz,1H),7.29(d,J=0.9Hz,1H),6.40(t,J=5.2Hz,1H),5.42–5.32(m,2H),5.01(d,J=1.0Hz,2H),4.64(s,1 H),4.22(t,J=6.2Hz,2H),3.15(q,J=4.8Hz,2H),2.13(dq,J=13.7,8.1Hz,1H),2.06–1.96(m,3H),1.94–1 .82(m,1H),1.87(s,3H),1.76–1.67(m,8H),1.55–1.47(m,2H),1.46–1.30(m,4H),0.90(t,J=8.0Hz,3H).

[0153] 13 C NMR (101MHz, DMSO) δ179.63,172.62,157.50,153.42,150.32,150.05,149.29,146.70,142.66,131.42,131.15,130.61,126.72,124. 66,118.92,115.33,98.49,72.73,66.80,65.33,50.47,42.47,41.16,38.74,36.41,30.53,29.62,28.71,28.33,26.63,25.75,7.71.

[0154] ESI-HRMS calculation for C 38 H 48 N3O8m / z[M+H] + 698.3363, found [M+H] + 670.3084.

[0155] Example 8 Synthesis of Compound 10a

[0156]

[0157] After compound 1 (1 eq) and compound 2 (2 eq) were stirred in DCM, Et3N (2.2 eq) was slowly added at 0°C and stirred at room temperature for 5 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0158] After compound 3 (1 eq) and compound 4 (1.2 eq) were stirred in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 5. The crude compound was then purified by flash column chromatography to obtain the desired product 5.

[0159] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0160] 1 H NMR (400MHz, DMSO) δ8.24(d,J=9.2Hz,1H),8.14(d,J=2.6Hz,1H),7.78(dd,J=9.2,2.5 Hz,1H),7.33(s,1H),5.44(s,2H),5.35(s,2H),4.24(s,2H),4.01(s,4H),3.19(q,J=7. 5Hz,2H),2.01(d,J=7.5Hz,1H),1.96(d,J=5.1Hz,6H),1.88(d,J=7.8Hz,1H),1.84(d,J =2.9Hz,12H),1.66(t,J=2.9Hz,12H),1.29(s,4H),1.04(s,3H),0.89(d,J=7.3Hz,3H).

[0161] 13 C NMR (101MHz, DMSO) δ177.20,172.62,162.30,153.56,151.51,150.63,149.96,147.81,144.37,143.68,127.33,127.05,126.65,124.73,116 .73,113.76,100.45,72.73,69.73,69.72,65.33,50.69,41.52,41.33 ,39.59,37.67,36.58,30.53,29.29,28.44,22.93,16.44,15.01,7.71.

[0162] ESI-HRMS calculation for C 50 H 58 N2O 11 m / z[M+H]+ 863.4188, found [M+H] + 863.4113.

[0163] Example 9 Synthesis of Compound 10b

[0164]

[0165] After compound 1 (1 eq) and compound 2 (1.0 eq) were stirred in DCM, Et3N (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0166] After stirring compound 3 (1 eq) and compound 4 (1.2 eq) in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to give crude compound 5. The crude compound was then purified by flash column chromatography to give the desired product 5.

[0167] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0168] 1 H NMR (400MHz, DMSO) δ8.22(d,J=9.2Hz,1H),8.15(d,J=2.6Hz,1H),7.77(dd,J=9.2,2.5Hz,1H),7.33(s,1H),5 .44(s,2H),5.34(s,2H),4.26(t,J=6.6Hz,2H),3.90(d,J=2.7Hz,6H),3.19(q,J=7.5Hz,2H),2.97(q,J=6.5H z,2H),2.20–2.15(m,1H),2.03–1.98(m,1H),1.97–1.94(m,6H),1.91–1.85(m,2H),1.80(d,J=2.9Hz,12H),1 .65(dd,J=6.3,2.7Hz,12H),1.41(q,J=7.0Hz,4H),1.29(t,J=7.6Hz,6H),0.94(s,3H),0.89(d,J=7.3Hz,3H).

[0169] 13 C NMR (101MHz, DMSO) δ176.06,156.81,156.03,149.34,146.58,145.85,131.44,115.05,96.71,72.38,68.80,65.27,65 .01,64.96,49.58,40.24,40.15,38.81,38.35,35.88,31.30,29.03,27.97,27.27,25.79,24.90,16.69,13.86,7.76.

[0170] ESI-HRMS calculation for C 57 H 71 N3O 13 m / z[M+H] + 1006.5059, found [M+H] + 1006.5060.

[0171] Example 10 Synthesis of Compound 10c

[0172]

[0173] After compound 1 (1 eq) and compound 2 (1.0 eq) were stirred in DCM, Et3N (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0174] After stirring compound 3 (1 eq) and compound 4 (1.2 eq) in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to give crude compound 5. The crude compound was then purified by flash column chromatography to give the desired product 5.

[0175] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0176] 1H NMR (400MHz, DMSO) δ7.95(d,J=8.3Hz,1H),7.59(d,J=2.6Hz,1H),7.46(dd,J=8.4,2.7Hz,1H),7.26(d,J=0.9Hz,1H),5.37(dd,J=3 .8,1.1Hz,2H),5.35–5.29(m,1H),5.10(s,2H),4.64(s,1H),4.38(t,J=5.2Hz,2H),4.23(d,J=11.5Hz,2H),4.13(s,2H),3.98(d,J =11.5Hz,2H),3.74(t,J=5.2Hz,2H),3.70–3.62(m,4H),3.57(t,J=4.3Hz,2H),3.32(dt,J=5.7,4.3Hz,2H),3.23(q,J=7.6Hz,2H), 2.06(qd,J=5.7,4.6Hz,6H),2.00(d,J=5.0Hz,13H),1.79–1.67(m,7H),1.37(t,J=7.6Hz,3H),1.08(s,2H),0.90(t,J=8.0Hz,3H).

[0177] 13 C NMR (101MHz, DMSO) δ177.20,172.62,162.30,157.21,153.28,151.57,150.63,149. 96,147.81,144.37,143.68,127.33,127.05,126.65,124.73,116.73,113.76,100. 45,72.73,70.61,69.73,69.70,69.54,68.73,68.58,65.33,65.30,50.69,41.52,41.33,39.59,39.49,37.74,36.58,30.53,29.29,28.44,22.93,16.46,15.01,7.71.

[0178] ESI-HRMS calculation for C 57 H 71 N3O 15 m / z[M+H] + 1038.4885, found [M+H] + 1038.4886.

[0179] Example 11 Synthesis of Compound 10d

[0180]

[0181] After compound 1 (1 eq) and compound 2 (2 eq) were stirred in DCM, Et3N (2.2 eq) was slowly added at 0°C and stirred at room temperature for 5 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0182] After compound 3 (1 eq) and compound 4 (1.2 eq) were stirred in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 5. The crude compound was then purified by flash column chromatography to obtain the desired product 5.

[0183] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0184] 1 H NMR (400MHz, DMSO) δ8.23(dt,J=2.3,0.9Hz,0H),7.41(dd,J=8.4,2.4Hz,0H),5.37(dd,J=3.8,1.1Hz,1H),5.01(d,J=0.9Hz,1H),4. 23(d,J=11.5Hz,1H),4.16(s,1H),3.98(d,J=11.3Hz,1H),2.11–1.92(m,8H),1.79–1.67(m,3H),1.08(s,1H),0.90(t,J=8.0Hz,1H).

[0185] 13 C NMR (101MHz, DMSO) δ177.20,172.62,157.50,153.56,150.32,150.05,149.28,146.70,142.66,131.42,131.15,130.61,126.72,124. 66,118.92,115.33,98.49,72.73,69.73,69.72,65.33,50.47,41.52,41.33,39.59,37.67,36.58,30.53,29.29,28.44,16.44,7.71.

[0186] ESI-HRMS calculation for C 48 H 54 N2O 11 m / z[M+H] + 1038.4885, found [M+H] + 835.3762.

[0187] Example 12 Synthesis of Compound 10e

[0188]

[0189] After compound 1 (1 eq) and compound 2 (2 eq) were stirred in DCM, Et3N (2.2 eq) was slowly added at 0°C and stirred at room temperature for 5 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 3. The crude compound was then purified by flash column chromatography to obtain the desired product 3.

[0190] After compound 3 (1 eq) and compound 4 (1.2 eq) were stirred in DCM, DMAP (1.5 eq) was slowly added at 0°C and stirred at room temperature for 6 h. After the reaction was completed under TLC monitoring, the reaction mixture was evaporated to obtain crude compound 5. The crude compound was then purified by flash column chromatography to obtain the desired product 5.

[0191] Compound 5 (1 eq) and compound 6 (1.1 eq) were stirred in DMF, then DIPEA (1.5 eq) was slowly added at 0°C and stirred at 50°C for 8 h. After TLC monitoring, the reaction was quenched by adding a saturated NaHCO3 solution and extracted three times with ethyl acetate. The organic phase was washed with brine, dried over anhydrous MgSO4, and evaporated to obtain the crude compound. The crude compound was then purified by flash chromatography to obtain the desired product.

[0192] 1 H NMR (400MHz, DMSO) δ7.31–7.17(m,1H),5.37(dd,J=3.8,1.1Hz,1H),4.99(d, J=0.7Hz,1H),4.23(d,J=11.5Hz,1H),4.16(s,1H),3.98(d,J=11.3Hz,1H),3. 09(qd,J=7.8,0.8Hz,1H),2.06(qd,J=5.7,4.6Hz,2H),2.00(d,J=5.0Hz,5H), 1.79–1.67(m,3H),1.29(t,J=7.8Hz,1H),1.08(s,1H),0.90(t,J=8.0Hz,1H).

[0193] 13 C NMR (101MHz, DMSO) δ177.20,172.62,157.50,153.21,151.43,150.05,147.06,146.49,144.71,134.01,131.38,128.45,126.68,123.86,118 .92,116.35,97.52,72.73,69.73,69.72,65.33,50.46,41.52,41.33, 39.59,37.67,36.58,30.53,29.29,28.44,24.77,16.44,14.39,7.71.

[0194] ESI-HRMS calculation for C 50 H 58 N2O 11 m / z[M+H] + 1038.4885, found [M+H] + 863.4070.

[0195] Biological Activity Example 1 Cytotoxicity Experiment

[0196] The antiproliferative abilities of compounds 4a-f and 10a-c against three cancer cell lines (human pancreatic cancer cell line MIA Paca2, human colon cancer cell line HCT 116, and human breast cancer cell line MCF-7) were evaluated.

[0197] All cell lines used in this study were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and grown in Dulbecco's Modified Eagle's Medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. All cells were cultured at 37°C in a humidified atmosphere with 5% CO2. The 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonatophenyl) 2H-tetrazolium monosodium salt (CCK-8) assay was used to detect cytotoxic compounds. Cells were seeded in 96-well plates at a concentration of 5 × 10 3 Cells / well were incubated with the test compound at 37°C in a 5% CO2 incubator for 72 h. The cells were then incubated with CCK-8 solution (10%) for 45 min, and the absorbance at 450 nm was measured on a microplate reader (BioRad Laboratories, Shanghai). 50 The values ​​were calculated by GraphPad Prism6 and are the mean of three independent experiments.

[0198] Experimental results: as shown in Table 1.

[0199] Table 1. Antiproliferative activity of compounds against three types of tumor cells a

[0200]

[0201]

[0202] a Cells were treated with various concentrations of compounds for 48 h, and cell viability was determined using the CCK-8 assay as described in Materials and Methods. b IC 50 Values ​​are expressed as mean ± SD (standard deviation) of at least three independent experiments.

[0203] Combine Figure 1 From Table 1, it can be found that among these compounds, 4b showed the most effective anti-cell proliferation effect on human pancreatic cancer cells, IC 50 The IC value of SN38 on MIA Paca2 cells was 6 nM, which was comparable to that of the positive control SN38. 50 =19.67nM, its anticancer activity was significantly improved, and for other cancer cells, IC 50 The values ​​were 41.12nM, 89.66nM, and 113.66nM, respectively, which were equivalent to or better than the positive control group SN38. In addition, the other compounds also showed significant anti-cell proliferation effects on different cell lines, which were stronger than the positive control group SN38.

[0204] Biological Activity Example 2 Cell Apoptosis Experiment

[0205] In order to investigate whether the mechanism of the cytotoxic activity of the compounds was the induction of cell apoptosis, the apoptosis-inducing ability of the compounds was further evaluated.

[0206] MIA Paca2 cells were incubated with the compounds and then stained with annexin V fluorescein isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI). The cell cycle arrest effect of compound 4b on MIA Paca2 cells was assessed by flow cytometry according to the manufacturer’s protocol (Beyotime, Jiangsu, China). 5 Cells were seeded / well in a 6-well plate and treated with DMSO or the desired concentration of drug at 37°C, 5% CO2 for 24 or 48 hours. The cells were then washed three times with ice-cold PBS and centrifuged at 1200 rpm for 5 minutes. The cells were then resuspended in 200 μL (1x) binding buffer at a density of ~5×10 5Cells were stained with 5 μL Annexin V-FITC and 15 μL DAPI for 20 minutes at room temperature in the dark. Finally, apoptotic cells were quantified by flow cytometry (CytoFLEX, Beckman Coulter).

[0207] Experimental results: Figure 2 As shown, MIA Paca2 cells were treated with 4b for 24h and 48h. As the concentration of 4b increased from 10nM to 100nM, the percentage of cells in the apoptotic part increased. The percentages of the apoptotic population in the 24h treatment group were 41.06 and 41.66, respectively, and the percentages of the apoptotic population in the 48h treatment group were 61.08 and 59.8, respectively. The number of apoptotic cells was much higher than that in the SN38 control group.

[0208] Biological Activity Example 3 Topo1 enzyme inhibition activity experiment

[0209] In order to evaluate the inhibitory activity of the compounds on Topo1 enzyme, human pancreatic cancer cells MIAPaca2 that highly express Topo I were used to measure protein levels by western blot.

[0210] Cells were treated with the desired concentration of drug or DMSO for 24 hours. After treatment, cells were collected and lysed in a western blot and IP cell lysis buffer kit according to the manufacturer's instructions. Protein concentration was determined using a BCA protein assay kit. Total cell protein extracts were separated using sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked with rapid blocking solution for 20 minutes and air-cleared with the appropriate antibodies. Subsequently, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Finally, protein bands were visualized using enhanced chemiluminescence (ECL) detection reagents on the ChemiDoc system.

[0211] Experimental results: Figure 3 As shown in Figure 2, compound 4b exhibited strong anti-proliferative activity in MIA Paca2, with IC 50 The value was 5.595nM (Table 1). It also reduced the protein level of Topo1 in a concentration-dependent manner. Compared with the SN38 control group at the same concentration, 4b showed a more significant inhibitory effect on Topo1 expression, which is consistent with the anti-cell proliferation effect. Figure 4 As shown, at the same concentration, 10a-c containing two adamantanes showed a more obvious inhibitory effect on the expression of Topo1 than 4b. The results showed that multivalent adamantane, as a hydrophobic tag, is beneficial to promote the degradation of related proteins.

[0212] Biological Activity Example 4: Mouse Tumor Inhibition Experiment

[0213] Since compound 4b showed the strongest biological activity in vitro, HCT 116 and MIA Paca2 xenograft models were established, and in vivo efficacy studies were performed to evaluate its antitumor activity.

[0214] Female Balb / C mice, 6-7 weeks old, were housed under standard conditions (21±1°C, 50±10% relative humidity, 12-h light-dark cycle) with free access to food and water. Seven days after subcutaneous inoculation of HCT116 cells, visible tumors appeared at the inoculation site. HCT116 tumor-bearing mice were then randomly divided into four groups (3 mice per group): sample group 4b (5 mg / kg injected into the tail vein), a positive control group SN38 (10 mg / kg and 20 mg / kg injected into the tail vein), and a control group injected with an equal volume of solvent. MIAPaca2 tumor-bearing mice were randomly divided into three groups: sample group 4b (10 mg / kg injected into the tail vein), a positive control group irinotecan (30 mg / kg injected into the tail vein), and a control group injected with an equal volume of solvent. Dosing was performed once every three days via the tail vein. Daily body weight was measured before dosing, and clinical signs of toxicity were observed and recorded daily. 24 h after the last administration, the mice were killed by intraperitoneal injection of sodium pentobarbital (1%). Then, the main organs (heart, liver, spleen, lung and kidney) were removed. Histopathological examination was performed by formalin fixation, paraffin embedding, and H&E staining to evaluate tissue morphology. Statistical analysis. The data are expressed as mean ± standard deviation (SD). Except for tumor growth, the statistical differences between the two groups were evaluated by t-test. The inhibitory effect of tumor growth was analyzed by one-way analysis of variance (ANOVA) and least significant difference (LSD) method. P < 0.05 was considered statistically significant. All analyses were performed using SPSS (V19.0, SPSS Inc., Chicago, IL).

[0215] The experimental results of HCT116 tumor-bearing mice were as follows Figure 4 As shown, compared with the negative control group, the tumor tissue of the positive control group SN38 and the treatment group was reduced. The tumor volume of the two SN38 treatment groups was close to the treatment effect under the condition of far exceeding the dosage of sample 4b group ( Figure 4 A), indicating that the therapeutic effect of sample group 4b is far superior to that of SN38; at the same time, the body weight of mice in all drug-treated groups did not decrease significantly ( Figure 4 B), indicating that 4b is more potent in vivo than the clinically used drug SN38.

[0216] The results of the MIA Paca2 experiment are as follows Figure 6As shown, compared with the negative control group, the tumor tissues of the positive control group Iriontecan and the treatment group were reduced. The tumor volume of the positive control treatment group was still worse than that of the sample group (under the condition of far exceeding the dosage of sample 4b group). Figure 6 A), indicating that the therapeutic effect of sample group 4b is far superior to that of Iriontecan; at the same time, the body weight of mice in all treatment groups did not decrease significantly ( Figure 6 B), indicating that 4b is more effective in vivo than the clinically used drug iriontecan.

[0217] In addition, the results of H&E staining showed that the sections of colorectal cancer xenografts in mice in the SN38 or 4b groups showed significant morphological changes, with fewer cancer cells. The results of H&E staining showed that the blank group showed typical pathological nuclear division, which is consistent with the characteristics of cancer tissue. The number of cells in the transplanted tumors of all drug-treated groups decreased, the cytoplasm was lightly stained, the cell nucleus was lightly stained, some showed nuclear condensation, nuclear dissolution, cell disintegration, and structural disappearance, indicating that the drug-treated groups had good anti-tumor effects. For normal tissues, there was no obvious pathological tissue damage in the heart, liver, spleen, lungs, and kidneys.

[0218] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound represented by the following formula (I), or a pharmaceutically acceptable salt thereof, characterized in that: The compound has the structure shown in the following formula (I): wherein Y is a Top I inhibitor module, which is a camptothecin analog; A is selected from carbonate bonds NHC(O)NH, NHC(O), NH, OC(O), S, O, CRaRb, wherein Ra and Rb are each independently selected from the group consisting of H, deuterium, halogen, hydroxy, amino, nitro, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy; L1 is a divalent linking group having -(L3) x -Shown structure, wherein x is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; Each of the L3 is independently selected from the group consisting of a chemical bond, CH2, CHD, CD2, C=O, O, S, NH, SO, SO2, P=O, NHCO, NHSO2, OCH2, and a 5-7 membered heterocyclic group; wherein the heterocyclic group comprises 1, 2 or 3 heteroatoms selected from N, S or O as the ring skeleton; In addition to conventional substituents, the L1 may be optionally substituted by one -L2-Z substituent; L2 is a chemical bond, a C2-C3 amide group, or a C2-C3 ester group; L1 and L2 may each independently be substituted by one or more substituents selected from the group consisting of deuterium, halogen, hydroxy, amino, nitro, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 alkyl ester, C1-C4 alkanoyl, C1-C4 alkylamino, -C1-C4 alkyl-hydroxy; Z is adamantyl group, preferably 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The Y has a structure shown in the following formula (II): wherein R1 is selected from H, C1-C6 alkyl, NRaRb; Ra and Rb are each independently selected from H, C1-C4 alkyl; m is selected from 0, 1 or 2; R2 is selected from H, C1~C6 alkyl.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein Said Y has a structure selected from the following group:

4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L1 has -(L3) x -shown structure, wherein x is selected from the following group: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and each of the L3 is independently selected from the following group: chemical bond, CH2, C=O, O, S, NH, SO, SO2, NHCO, OCH2, 5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L1 is selected from the following group: -(CH2)n-, wherein n is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L2 is a chemical bond, 7. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein A is a carbonate bond NH, NHC(O)NH, O, S, CH2; preferably, a carbonate bond 8. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein The compound is selected from the following group:

9. The use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, characterized in that: Used for preparing a pharmaceutical composition for treating tumor diseases; the tumor is selected from the following group: gastric cancer, rectal cancer, lung cancer, cervical cancer and pancreatic cancer.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: a compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

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