Alkynamide compounds and their applications

Abstract

The present invention provides an alkyne amide compound having a structure represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and uses thereof. Specifically provided is the use of the alkyne amide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the selective modification of protein amino acid residues, preferably, the amino acid is aspartic acid, glutamic acid, and / or cysteine. Also provided is the use of the alkyne amide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a kinase inhibitor, wherein the kinase is EGFR, ALDH1, GAPDH, MEK2, ECH1, GAPDH, and / or ALDH1. Also provided is the use of the alkyne amide compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a drug for preventing and / or treating tumors.

Description

Technical Field

[0001] The present invention relates to the technical fields of chemical biology molecular probes and chemical medicine, and in particular to a class of alkyne amide compounds and applications thereof. Background Art

[0002] Covalent modification of proteins using chemical tools is an effective method for identifying, quantifying, and regulating these biomolecules. Small molecules are powerful tools for studying protein function. They can modulate protein function through covalent bond formation, potentially providing therapeutic benefits for several diseases, such as enhanced and sustained drug inhibition. However, most human proteins lack small molecule ligands, making them unsuitable for drug design. Selective modification of specific protein sites is one effective approach to address this issue. Currently, the most extensive selective modification efforts are based on small molecules modifying cysteine ​​and lysine residues. However, limited research has focused on the selective modification of aspartic acid (ASP) and glutamic acid (GLu), which comprise up to 12.2% of protein residues. Furthermore, limited research has been conducted on the design and synthesis of covalent inhibitors using acidic amino acid covalent warheads and their application in anti-tumor cell proliferation. Therefore, the design and development of small molecule covalent warheads targeting acidic amino acids is crucial for the application of chemical proteomics and the development of covalent inhibitors. Summary of the Invention

[0003] To address the above problems, the present invention provides a class of alkyne amide compounds that can selectively modify amino acid residues and effectively inhibit tumor cell proliferation, wherein the modified amino acids are aspartic acid, glutamic acid and / or cysteine.

[0004] The specific technical solutions include the following.

[0005] Use of an alkyne amide compound having a structure represented by formula (I) or a pharmaceutically acceptable salt or a stereoisomer thereof in selectively modifying amino acid residues of proteins,

[0006]

[0007] Wherein, R is selected from: sulfonyl, acyl or phosphoryl;

[0008] R1 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or R1, R and the nitrogen atom to which it is attached and R3 together form a substituted or unsubstituted heterocyclic ring;

[0009] R2 is selected from: H, substituted or unsubstituted aryl;

[0010] R3 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted aryl;

[0011] R4 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or R3, R and R4 together form a substituted or unsubstituted heterocycle; and, when R is sulfonyl and / or acyl, R4 is not.

[0012] In some embodiments, the amino acid is aspartic acid, glutamic acid and / or cysteine.

[0013] In some embodiments, R1 is selected from: R5 substituted or unsubstituted C1-C 12 Alkyl, R5 substituted or unsubstituted C6-C 18 Aryl, or R1, R and the nitrogen atom to which it is connected, and R3 together form a 5-10 membered heterocyclic ring which is substituted or unsubstituted with R5;

[0014] R5 is selected from: H, ethynyl, C6-C 10 Aryl, R6 substituted C6-C 10 aryl;

[0015] R6 is selected from: C3-C6 alkynyloxy, ethynyl.

[0016] In some embodiments, R1 is selected from: R5 substituted or unsubstituted C1-C6 alkyl, R5 substituted or unsubstituted C6-C 10 The aryl group, or R1, R and the nitrogen atom to which it is connected and R3 together form a 5-9 membered heterocyclic ring which is substituted or unsubstituted with R5.

[0017] In some embodiments, R5 is selected from: H, ethynyl, phenyl, R6 substituted phenyl; R6 is selected from: propargyloxy, ethynyl.

[0018] In some embodiments, R1 is selected from: methyl, ethyl, propyl, methyl substituted by propargyloxyphenyl, ethyl substituted by propargyloxyphenyl, 3-ethynylpropyl, 2-ethynylethyl, propargyl, or R1, R and the nitrogen atom to which it is attached and R3 together form the following group:

[0019]

[0020] In some embodiments, R2 is selected from: H, R7 substituted or unsubstituted C6-C 10 Aryl; R7 is selected from: H, ethynyl, phenyl, R6 substituted phenyl; R6 is selected from: propargyloxy, ethynyl.

[0021] In some embodiments, R2 is selected from the group consisting of: H, phenyl, and 4-ethynylphenyl.

[0022] In some embodiments, R3 is selected from: R8 substituted or unsubstituted C1-C 12Alkyl, R8 substituted or unsubstituted C6-C 18 aryl;

[0023] R4 is selected from: R8 substituted or unsubstituted C1-C 12 Alkyl, R8 substituted or unsubstituted C6-C 18 Aryl, or R3, R and R4 together form a 5-10 membered heterocyclic ring which is substituted or unsubstituted with R8;

[0024] R8 is selected from: H, ethynyl, C1-C 12 Alkyl, -OR9 substituted C1-C2 alkyl, -NR9R 10 Substituted C1-C2 alkyl, -OR9, C6-C 10 Aryl, -C(=O)OR 10 、-C(=O)NR9R 10 ;

[0025] R9 is selected from: R 11 Substituted or unsubstituted C1-C2 alkyl, R 11 Substituted or unsubstituted aryl, R 11 a substituted or unsubstituted heteroaryl group;

[0026] R 10 Selected from: C1-C6 alkyl;

[0027] R 11 Selected from: H, ethynyl, halogen, phenyl, C1-C6 alkyl, R 12 Substituted or unsubstituted phenoxy, -N(R 10 )2;

[0028] R 12 Selected from: H, ethynyl, C1-C6 alkyl, halogen.

[0029] In some embodiments, R3 is selected from: R8 substituted or unsubstituted C1-C6 alkyl, R8 substituted or unsubstituted C6-C 10 aryl;

[0030] R4 is selected from: R8 substituted or unsubstituted C1-C6 alkyl, R8 substituted or unsubstituted C6-C 10 aryl, or R3, R and R4 together form a 5-7 membered heterocyclic ring which is substituted or unsubstituted by R8.

[0031] In some embodiments, R8 is selected from: H, ethynyl, C1-C6 alkyl, -CH2-OR9, -CH2-NR9R 10 、-OR9、C6-C 10 Aryl, -C(=O)OR 10、-C(=O)NR9R 10 ;

[0032] R9 is selected from the group consisting of: benzyl, R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group;

[0033] R 10 Selected from: C1-C3 alkyl;

[0034] R 11 Selected from: H, ethynyl, halogen, C1-C6 alkyl, ethynyl-substituted phenoxy, phenoxy, -N(R 10 )2.

[0035] In some embodiments, R8 is selected from: H, ethynyl, C1-C3 alkyl, -CH2-OR9, -CH2-NR9CH3, phenoxy, phenyl, -C(=O)O CH3, -C(=O)NR9CH3;

[0036] R9 is selected from the group consisting of: benzyl, R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group;

[0037] R 11 Selected from: H, ethynyl, halogen, C1-C3 alkyl, ethynyl-substituted phenoxy, phenoxy, dimethylamino.

[0038] In some embodiments, the acetylene amide compound has a structure shown in the following formula (II):

[0039]

[0040] wherein R1 is selected from: C1-C3 alkyl; R2 is H;

[0041] R3 is selected from: R8 substituted or unsubstituted C6-C 10 aryl;

[0042] R8 is selected from the group consisting of: H, ethynyl, -CH2-OR9, -CH2-NR9CH3, phenoxy, phenyl, -C(=O)OCH3, -C(=O)NR9CH3;

[0043] R9 is selected from the group consisting of: benzyl, R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group;

[0044] R 11 Selected from: H, ethynyl, halogen, ethynyl-substituted phenoxy, phenoxy, dimethylamino.

[0045] In some embodiments, the alkyne amide compound is selected from the following compounds:

[0046]

[0047] The use of the alkyne amide compound or a pharmaceutically acceptable salt or a stereoisomer thereof in the preparation of a kinase inhibitor, wherein the kinase is EGFR, ALDH1, GAPDH, MEK2, ECH1, GAPDH and / or ALDH1.

[0048] Use of the alkyne amide compound or its pharmaceutically acceptable salt or its stereoisomer in the preparation of drugs for preventing and / or treating tumors.

[0049] In some embodiments, the tumor is liver cancer (HepG2), colon cancer (HT-29, HCT15, LOVO), breast cancer (MDA-MB-231), colorectal adenocarcinoma (NCI-H716), acute promyelocytic leukemia (HL60), lymphoma (U937) and / or lung cancer (H3255).

[0050] The present invention also provides a pharmaceutical composition for preventing and treating tumors, which is prepared from active ingredients and pharmaceutically acceptable carriers or excipients. The active ingredients include the alkyne amide compound or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0051] The present invention provides an alkyne amide compound that can selectively modify amino acid residues. This type of alkyne amide compound can selectively modify aspartic acid, glutamic acid and cysteine ​​residues, can be used as a molecular probe compound to selectively modify protein amino acid residues, can regulate the function of proteins by forming covalent bonds, and can be combined with affinity pharmacophores to form covalent inhibitors, providing benefits for the treatment of some diseases, especially effectively inhibiting the proliferation of tumor cells, and can be used to prepare anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is the result of covalent modification of the amino acid residues of pure protein by the compound.

[0053] Figure 2 It is the result of the covalent modification of the amino acid residues of tumor cell proteins by the compound.

[0054] Figure 3 This is the result of the compound's selective modification of the acidic amino acid residues of the pure protein.

[0055] Figure 4 This is the result of the compound's selective modification of the acidic amino acid residues of cells.

[0056] Figure 5 The results show the inhibitory activity of compound YN-4 on different kinases.

[0057] Figure 6 The results of proteomics experiments confirm the targets of compound YN-1 and compound YN-4.

[0058] Figure 7 Results of pull-down and western blotting experiments.

[0059] Figure 8 The results show that compound YN-4 inhibits the clone formation and proliferation of H3255 tumor cells.

[0060] Figure 9 The compound YN-4 inhibits the phosphorylation of EGFR kinase and its downstream pathway proteins in H3255 cells. DETAILED DESCRIPTION

[0061] In the compounds of the present invention, when any variable (such as R 10 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.

[0062] As used herein, the term "alkyl" is intended to include branched and straight-chain saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms in a straight or branched arrangement. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl. The term "heterocycle" is a saturated or partially unsaturated monocyclic or polycyclic cyclic substituent, wherein one or more ring atoms are selected from N, O, P(O) or S(O)m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon, and the attachment of the heterocyclic substituent can be achieved through carbon atoms or through heteroatoms.

[0063] As will be understood by those skilled in the art, "halo" or "halo" as used herein refers to chlorine, fluorine, bromine and iodine.

[0064] The present invention encompasses the free forms of the compounds of Formula I-II, as well as their pharmaceutically acceptable salts and stereoisomers. Included pharmaceutically acceptable salts include not only the exemplary salts of the specific compounds described herein, but also representative pharmaceutically acceptable salts of the free forms of all compounds of Formula I-II. The free forms of specific salts of the compounds described can be isolated using techniques known in the art. For example, the free forms can be regenerated by treating the salts with a suitable dilute aqueous base solution, such as dilute aqueous NaOH, dilute aqueous potassium carbonate, dilute aqueous ammonia, and dilute aqueous sodium bicarbonate. The free forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but for the purposes of the present invention, such acid and base salts are otherwise pharmaceutically equivalent to their respective free forms.

[0065] 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.

[0066] Therefore, pharmaceutically acceptable salts of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting a basic compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, and also include 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, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, and the like.

[0067] 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, guaiac, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

[0068] Berg et al., "Pharmaceutical Salts," J. Pharm. Sci. '1977: 66: 1-19, describes in more detail the preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts.

[0069] Since deprotonated acidic moieties such as carboxyl groups in the compounds may be anionic under physiological conditions, and this charge may then be balanced by internal cationic protonated or alkylated basic moieties such as tetravalent nitrogen atoms, it should be noted that the compounds of the present invention are potential inner salts or zwitterions.

[0070] In one embodiment, the present invention provides a method for treating tumor diseases in humans or other mammals using a compound having a structure represented by Formula I-II and a pharmaceutically acceptable salt thereof.

[0071] In one embodiment, the compounds of the present application and pharmaceutically acceptable salts thereof can be used to treat or control liver cancer, colon cancer, breast cancer, colorectal adenocarcinoma, acute promyelocytic leukemia, lymphoma and / or lung cancer.

[0072] Pharmaceutical composition

[0073] The present invention also provides a pharmaceutical composition comprising an active ingredient within a safe and effective amount, and a pharmaceutically acceptable carrier or excipient.

[0074] The "active ingredient" described in the present invention refers to the compound of formula I-II described in the present invention or a pharmaceutically acceptable salt or stereoisomer thereof.

[0075] The "active ingredient" and pharmaceutical composition of the present invention can be used as a protein kinase inhibitor and can be used to prepare drugs for preventing and / or treating tumors and.

[0076] A "safe and effective amount" refers to an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, a pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably 10-200 mg per dose. Preferably, "one dose" is one tablet.

[0077] "Pharmaceutically acceptable carrier or excipient" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must be sufficiently pure and have sufficiently low toxicity.

[0078] "Compatibility" herein means that the components of the composition can be mixed with the active ingredient of the present invention and with each other without significantly reducing the efficacy of the active ingredient.

[0079] Examples of pharmaceutically acceptable carriers or excipients include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers, Wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0080] In another preferred embodiment, the compound of formula I-II of the present invention can form a complex with a macromolecular compound or polymer through a non-bonding interaction. In another preferred embodiment, the compound of formula I-II of the present invention, as a small molecule, can also be linked to a macromolecular compound or polymer through a chemical bond. The macromolecular compound can be a biological macromolecule such as a polysaccharide, protein, nucleic acid, polypeptide, etc.

[0081] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0082] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0083] In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with:

[0084] (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid;

[0085] (b) binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic;

[0086] (c) humectants, for example, glycerin;

[0087] (d) disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate;

[0088] (e) buffering solvents, such as paraffin;

[0089] (f) absorption accelerators, for example, quaternary ammonium compounds;

[0090] (g) wetting agents, such as cetyl alcohol and glyceryl monostearate;

[0091] (h) adsorbents, for example, kaolin; and

[0092] (i) Lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffering agent.

[0093] The solid dosage forms can also be prepared using coatings and shells, such as enteric coatings and other materials known in the art. They can contain opacifying agents, and the release of the active ingredient in such compositions can be delayed in a certain portion of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes.

[0094] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, 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. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances.

[0095] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0096] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0097] The compounds of the present invention can be administered alone or in combination with other therapeutic drugs (such as hypoglycemic drugs).

[0098] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0099] Combination therapy

[0100] The compounds of Formula I-II can be used in combination with other drugs known to treat or improve similar conditions. When administered in combination, the original drug's mode of administration and dosage remain unchanged, while the compound of Formula I-II is taken simultaneously or subsequently. When the compound of Formula I-II is taken simultaneously with one or more other drugs, it is preferred to use a pharmaceutical composition containing one or more known drugs and the compound of Formula I-II. Drug combinations also include taking the compound of Formula I-II and one or more other known drugs during overlapping time periods. When the compound of Formula I-II is used in combination with one or more other drugs, the dosage of the compound of Formula I-II or the known drug may be lower than when the compound of Formula I-II or the known drug is taken alone.

[0101] Drugs or active ingredients that can be used in combination with the compounds of formula I-II include but are not limited to:

[0102] Estrogen receptor modulators, androgen receptor modulators, retinal-like receptor modulators, cytotoxins / cytostatics, antiproliferative agents, protein transferase inhibitors, HMG-CoA reductase inhibitors, HIV protein kinase inhibitors, reverse transcriptase inhibitors, angiogenesis inhibitors, cell proliferation and survival signal inhibitors, drugs that interfere with cell cycle checkpoints and apoptosis inducers, cytotoxic drugs, tyrosine protein inhibitors, EGFR inhibitors, VEGFR inhibitors, serine / threonine protein inhibitors, Bcr-Abl inhibitors, c-Kit inhibitors, Met inhibitors, Raf inhibitors, MEK inhibitors, MMP inhibitors, topoisomerase inhibitors, histidine deacetylase inhibitors, proteasome inhibitors, CDK inhibitors, Bcl-2 family protein inhibitors, MDM2 family protein inhibitors, IAP family protein inhibitors, STAT family protein inhibitors, PI3K inhibitors, AKT inhibitors, integrin blockers, interferon-α, interleukin-12, COX-2 inhibitors, p53, p53 activators, VEGF antibodies, EGF antibodies, JAK inhibitors, etc.

[0103] In one embodiment, the drugs or active ingredients that can be used in combination with the compound of formula I-II include but are not limited to: aldesleukin, alendronic acid, interferon, atrenoin, allopurinol, allopurinol sodium, palonosetron hydrochloride, hexamethylmelamine, aminoglutethimide, amifostine, amrubicin, anacrine, anastrozole, dolasetron, aranesp, arglabin, arsenic trioxide, arnosine, 5-azacytidine, azathioprine, BCG or tice BCG, betadine, betamethasone acetate, betamethasone sodium phosphate preparation, bexarotene, bleomycin sulfate, bromourea, bortezomib, busulfan, calcitonin, alezotocin injection, capecitabine, carboplatin, cascade, cefospermumab injection, ne, simoleukin, daunorubicin, chlorambucil, cisplatin, cladribine, cladribine, clodronate, cyclophosphamide, cytarabine, dacarbazine, actinomycin D, liposomal daunorubicin, dexamethasone, dexamethasone phosphate, estradiol valerate, denileukin-2, depomet, deslorelin, delazoxane, diethylstilbestrol, diflucan, docetaxel, doxorubicin, dronabinol, chin-166-chitosan complex, eligard, rasburicase, epirubicin hydrochloride, aprepitant, epirubicin, epoetin alfa, erythropoietin, epoetin, levamisole tablets, estradiol preparations, 17-beta-estradiol, estramustine sodium phosphate, ethinyl estradiol, amifostine, hydroxyphosphatase, fenbifurcated, etoposide, fadrozoles, tadalafil Moxifen preparations, filgrastim, finasteride, firaprestin, floxuridine, fluconazole, fludarabine, 5-fluorodeoxyuridine monophosphate, 5-fluorouracil, fluoxymesterone, flutamide, formestane, 1-β-D-arabinofuranosylcytosine-5'-stearoyl phosphate, fotemustine, fulvestrant, immunoglobulin, gemcitabine, gemtuzumab tuzumab, imatinib mesylate, carmustine rice paper capsules, goserelin, glaniciron hydrochloride, histrelin, homicide, hydrocortisone, erythro-hydroxynonyl adenine, hydroxyurea, tentanamib, idarubicin, ifosfamide, interferon α, interferon-α2, interferon α-2A, interferon α-2B, interferon α-nl, interferon α-n3, interferon β, interferon gamma-la, interleukin-2, intron A, Iressa, irinotecan, Kateri, lentinan sulfate, letrozole, leucovorin, leuprorelin acetate, levamisole, levofolinate calcium salt, levothyroxine sodium, levothyroxine sodium preparations, lomustine, lonidamine, dronabinol, nitrogen mustard, methylcobalamin, medroxyprogesterone acetate, megestrol acetate, melphalan, esterified estrogen, 6-mercaptopurine, mesna, methotrexate, methyl aminolevulinate, miltefosine, minocycline, mitomycin C, mitotane, mitoxantrone, trilostane, liposomal doxorubicin citrate, nedaplatin, pegfilgrastim, oprelleukin, neupogen, nilutamide, tamoxifen,NSC-631570, recombinant human interleukin-1-beta, octreotide, ondansetron hydrochloride, prednisone oral solution, oxaliplatin, paclitaxel, prednisone sodium phosphate, pegaspargase, PEGASYS, pentostatin, streptozotocin, pilocarpine hydrochloride, pirarubicin, plicamycin, porfimer sodium, prednimustine, stiprenisolone, prednisone, premarin, procarbazine, recombinant human erythropoietin, raltitrexed, rebifurcation, rhenium-186 etidronate, rituximab, dapoxetine-A, romostatide, pilocarpine hydrochloride tablets, octreotide, sarmustine, semustine, sizolan, sobuzosine, methylprednisolone sodium, paphos acid, stem cell therapy, streptozocin, strontium-89 chloride, levothyroxine sodium, tamoxifen Fen, tamsulosin, tasofenamin, tastolactone, taxotere, tesifuzine, temozolomide, teniposide, testosterone propionate, methyltestosterone, thioguanine, thiotepa, thyroid-stimulating hormone, tiludronic acid, topotecan, toremifene, tositumomab, trastuzumab, treosulfan, tretinoin, methotrexate tablets, trimethylmelamine, trimetrexate, triptorelin acetate, triptorelin pamoate, eufotaxime, uridine, valrubicin, veslanone, vinblastine, vincristine, vinsamide, vinorelbine, virulizine, dextromethorphan, statins, zofran, paclitaxel protein-stabilized formulation, acolbifene, interferon r-lb, affinitak, aminopterin, arzoxifene, as oprisnil, atamestane, atrasentan, BAY43-9006, Avastin, CCI-779, CDC-501, Celebrex, cetuximab, crisentan, cyproterone acetate, decitabine, DN-101, doxorubicin-MTC, dSLIM, dutasteride, edotecarin, eflornithine, exitecan, fenretinide, histamine dihydrochloride, histrelin hydrogel implant, holmium-166DOTMP, ibandronic acid, interferon gamma, intron-PEG, ixabepilone, keyhole limpet hemocyanin, L-651582, lanreotide, lasofoxifene, libra, lonafamib, miproxifene, minodecan, MS-2 09. Liposomal MTP-PE, MX-6, nafarelin, nemorubicin, nevastatin, noratriptide, oblimersen, onco-TCS, osidem, paclitaxel polyglutamate, sodium pyrimidine, PN-401, QS-21, quasitan, R-1549, raloxifene, ranpirnase, 13-cis retinoic acid, satraplatin, ciocalcitol, T-138067, tarceva, paclitaxel docosahexaenoic acid, thymosin α1, gazofurin, tipifarnib, tirapazamine, TLK-286, toremifene, trans-MID-107R, valspodar, vapreotide, vatalanib, verteporfin, vinflunine, Z-100, and zoledronic acid, or a combination thereof.

[0104] The present invention will be further described below in conjunction with specific examples. 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, were 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.

[0105] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0106] The reagents used in the following examples are all commercially available.

[0107] The compounds synthesized in the following examples and their abbreviations are as follows:

[0108]

[0109] Example 1

[0110]

[0111] In the first step, a dry round-bottom flask was charged with 4-bromo-N-methylbenzenesulfonamide (248.9 mg, 1.0 mmol), PdCl2(PPh3)2 (10% mmol), and CuI (20 mol%). Ultra-dry THF (5 ml) and Et3N (157.7 mg, 1.55 mmol) were then added under nitrogen. After stirring at room temperature for 30 minutes, TMS acetylene was slowly added, and the mixture was stirred at room temperature for 24 hours. The reaction was monitored by thin-layer chromatography until completion. The mixture was diluted with 5 ml of ethyl acetate and filtered through celite. The filtrate was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to yield compound 2 (221.9 mg, 83% yield). 1 H NMR (400MHz, CDCl3) δ7.76 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 8.6 Hz, 2H), 4.36 (dd, J = 10.6, 5.3 Hz, 1H), 2.63 (d, J = 5.4 Hz, 3H), 0.23 (s, 9H). 13 C NMR (101MHz, CDCl3) δ138.4,132.7,128.1,127.3,103.3,98.8,29.5.

[0112] In the second step, compound 2 was dissolved in methanol, and anhydrous KCO (103.7 mg, 1.5 eq) was added under N2 protection. The reaction mixture was stirred at room temperature for 5 h. After completion of the reaction, the mixture was filtered through celite, and the filtrate was evaporated under reduced pressure. Purification by silica gel column chromatography afforded compound 3 (94.7 mg, 97% yield). 1 H NMR (400MHz, CDCl3) δ7.85-7.79(m,2H), 7.64-7.60(m,2H), 4.58(d,J=4.9Hz,1H), 3.26(s,1H), 2.67(d,J=5.4Hz,3H). 13 C NMR (101MHz, CDCl3) δ139.0,132.9,129.0,127.4,82.2,80.9,29.5.

[0113] In the third step, compound 3 (47 mg, 0.24 mmol) and 60% NaH (300 mg, 1.5 mmol) were added to a Schlenk tube equipped with a stirrer. 2 mL of DMSO was added to the tube via syringe. The reaction mixture was immediately stirred for 15 minutes, heated to 70°C, and 1,1-dichloroethylene (58.2 mg, 0.6 mmol) was slowly added. The mixture was stirred at 70°C for an additional 24 hours before being cooled to room temperature. The mixture was then quenched with cold water and extracted three times with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate and evaporated under vacuum. Purification by silica gel column chromatography afforded the desired product, YN-1, as a pale yellow solid (40.0 mg, 76% yield). 1 H NMR (400MHz, CDCl3) δ7.87 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 3.31 (s, 1H), 3.09 (s, 3H), 2.70 (s, 1H). 13 C NMR(101MHz, CDCl3)δ136.1,133.0,128.3,127.9,81.9,81.7,77.5,58.0,39.2.HRMS(ESI)calcd.for C 11 H9NO2S 220.0427[M+H] + ,found 220.0436.

[0114] The method for synthesizing YN-5 is similar to the above method. YN-5 (light yellow solid, yield 91%). 1H NMR (400MHz, CDCl3) δ7.80 (d, J = 8.3Hz, 2H), 7.37 (d, J = 8.0Hz, 2H), 3.06 (s, 3H), 2.68 (s, 1H), 2.46 (s, 3H). 13 C NMR (101MHz, CDCl3) δ145.1,133.4,130.0,128.077.8,57.7,39.1,21.9.

[0115] Example 2

[0116]

[0117] In the first step, a solution of PPh3 (2.1 g, 4.0 mmol) and CBr4 (1.3 g, 8.0 mmol) in DCM (10 mL) was added to a solution of 4-alkynylbenzaldehyde (260.3 mg, 2.0 mmol) in DCM (10 mL) at 0°C. The mixture was stirred at room temperature until the reaction was complete as determined by TLC. The reaction mixture was filtered, concentrated, and purified by column chromatography to afford compound 5 (520.5 mg, 91% yield).

[0118] In the second step, under N₂ protection, a 10 mL test tube was charged with p-toluenesulfonamide (185.2 mg, 1.0 mmol), compound 5 (314.6 mg, 1.1 mmol), CsCO₃ (1.96 g, 6.0 mmol), N,N'-dimethylethylenediamine (88.2 mg, 1.0 mmol), and CuI (34.2 mg, 0.18 mmol). Dry, degassed DMF (5 mL) was then added. The mixture was heated to 70°C and stirred for 24 h. The mixture was then filtered and washed with ethyl acetate. After evaporation of the solvent, YN-2 was purified by column chromatography to afford a white solid (275.3 mg, 89% yield). 1 HNMR (400MHz, CDCl3) δ7.85(d,J=8.3Hz,2H),7.41(dd,J=11.3,8.3Hz,4H),7.35–7.25(m,2H),3.18(s,3H),3.17(s,1H),2.48(s,3H). 13 C NMR(101MHz, CDCl3)δ145.2,133.4,132.2,131.2,130.1128.0,123.5,121.5,86.1,83.5,78.9,69.0,39.5,21.9.HRMS(ESI)calcd.forC 18 H 15 NO2S 310.0756[M+H] + ,found 310.0722.

[0119] The method for synthesizing YN-6 was similar to that described above. YN-6 (white solid, 89% yield). 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J = 8.3 Hz, 2H), 7.43–7.34 (m, 4H), 7.31–7.27 (m, 3H), 3.15 (s, 3H), 2.46 (s, 3H). 13 C NMR (101MHz, CDCl3) δ145.0,133.3,131.58,130.0,128.5,128.0,122.8,84.1,69.2,39.5,21.9.

[0120] Example 3

[0121]

[0122] In the first step, a mixture of N,4-dimethylbenzenesulfonamide (555.7 mg, 3.0 mmol), NBS (356.0 mg, 2.0 mmol), and AIBN (492.6 mg, 3.0 mmol) was heated to reflux in acetonitrile (10 mL) for 7 h. The mixture was then filtered and concentrated to afford compound 7. The crude product was used in the next step without further purification.

[0123] In the second step, compound 7 (264.1 mg, 1.0 mmol), K2CO3 (276.4 mg, 2 mmol), and p-hydroxybenzylene (177.2 mg, 1.5 mmol) were added to 5.0 mL of DMF solvent. The mixture was stirred at 80°C for 4 hours. After the reaction was complete, 10.0 mL of water was added. The mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After evaporation of the solvent, the product was purified by column chromatography to obtain compound 8 (283.3 mg, 94% yield).

[0124] In the third step, under N₂ protection, 1,10-phenanthroline monohydrate (36.0 mg, 0.2 mmol), compound 8 (301.4 mg, 1.0 mmol), K₂CO₃ (276.4 mg, 2.0 mmol), CuSO₄·5H₂O (21.2 mg, 0.1 mmol), and 2-bromoethynyltriisopropylsilane (522.2 mg, 2.0 mmol) were added. The mixture was stirred at 85°C for 12 h, filtered, and washed with diethyl ether. After evaporation of the solvent, the mixture was purified by column chromatography to obtain the crude product. The product was then dissolved in THF (10 mL), and TBAF (2.0 eq) was added. The mixture was stirred at room temperature for 2 h, quenched with water (10.0 mL), and extracted with diethyl ether (3 × 10.0 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated in vacuo to afford YN-3 as a white solid (211.5 mg, 65% yield). 1 H NMR (400MHz, CDCl3) δ7.97(d,J=8.5Hz,2H),7.67(d,J=8.6Hz,2H),7.48(d,J=8.9Hz,2H),7.28( s,2H),6.94(d,J=8.9Hz,2H),5.19(s,2H),3.12(s,3H),3.04(s,1H),2.73(s,1H),1.58(s,2H). 13 C NMR (400MHz, CDCl3) δ158.5,143.0,135.7,133.8,128.2,127.7,115.1,114.8,8 3.3,77.4,77.3,77.3,77.0,76.8,76.3,68.9,57.7,38.9.HRMS(ESI)calcd.for C 18 H 15 NO3S 326.0845[M+H] + ,found 326.0843.

[0125] The method for synthesizing YN-7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 / 16 / 17 / 18 is similar to the above method.

[0126] YN-7 (white solid, yield 47%), 1 H NMR(400MHz,DMSO-d6)δ8.04–7.97(m,2H),7.99–7.92(m,2H),7.81–7.75(m,2H),7 .58–7.49(m,2H),7.51–7.43(m,1H),3.82(d,J=1.5Hz,1H),3.08(d,J=1.6Hz,3H).

[0127] YN-8 (white solid, yield 34%), 1 H NMR(300MHz,DMSO-d6)δ7.87–7.80(m,2H),7.51–7.45(m,2H),7.21–7.13(m, 2H),6.73–6.66(m,2H),4.71(s,2H),3.76(s,1H),3.05(s,3H),3.02(s,3H).

[0128] YN-9 (yellow solid, yield 55%), 1 H NMR (400MHz, DMSO-d6) δ7.90–7.84(m,2H),7.53–7.46(m,2H),7.32–7.27(m,1H),7.23–7.16(m,4H),3.80(s,1H),3.02(s,3H).

[0129] YN-10 (white solid, yield 73%), 1 H NMR (400MHz, DMSO-d6) δ8.61(d,J=2.0Hz,1H),8.26(dd,J=14.0,8.4Hz,2H),8.12(d,J=8.1Hz,1H),7.88(dd,J= 8.7, 2.0Hz, 1H), 7.79 (ddd, J=8.3, 6.8, 1.4Hz, 1H), 7.73 (ddd, J=8.2, 6.9, 1.4Hz, 1H), 3.79 (s, 1H), 3.10 (s, 3H).

[0130] YN-11 (white solid, yield 82%), 1 H NMR(400MHz, DMSO-d6)δ8.64(dd,J=8.5,1.2Hz,1H),8.39(d,J=8.1Hz,1H),8.26(dd ,J=7.4,1.3Hz,1H),8.20–8.13(m,1H),7.81–7.69(m,3H),3.84(s,1H),3.09(s,3H).

[0131] YN-12 (white solid, yield 69%), 1 H NMR(400MHz,DMSO-d6)δ8.04–7.97(m,2H),7.99–7.92(m,2H),7.81–7.75(m,2H),7 .58–7.49(m,2H),7.51–7.43(m,1H),3.82(d,J=1.5Hz,1H),3.08(d,J=1.6Hz,3H).

[0132] YN-13 (white solid, yield 46%), 1 H NMR(400MHz,DMSO-d6)δ9.21(s,1H),8.00(d,J=8.9Hz,1H),7.95–7.91(m,2H),7.85(d,J=2.6H z,1H),7.83–7.79(m,2H),7.26(dd,J=8.9,2.6Hz,1H),5.36(s,2H),3.79(s,1H),3.05(s,3H).

[0133] YN-14 (yellow oil, yield 22%), 1 H NMR(400MHz,Chloroform-d)δ8.15(s,1H),7.97–7.91(m,2H),7.72–7.67(m,2H),7.33(dt,J=8.8,0.7Hz,1H),7.21(t,J=2.8Hz,1 H),7.16(d,J=2.4Hz,1H),6.95(dd,J=8.8,2.5Hz,1H),6.49(ddd,J=3.1,2.0,0.9Hz,1H),5.21(s,2H),3.09(s,3H),2.70(s,1H).

[0134] YN-15 (white solid, yield 62%), 1 H NMR(400MHz,DMSO-d6)δ7.91–7.87(m,2H),7.77–7.71(m,2H),6.94–6.86(m, 2H),6.75–6.68(m,2H),5.16(s,2H),3.80(s,1H),3.04(s,3H),2.79(s,6H).

[0135] YN-16 (white solid, yield 57%), 1 H NMR (400MHz, Chloroform-d) δ8.30–8.18(m,2H),8.02–7.96(m,2H),3.98(d,J=1.2Hz,3H),3.11(d,J=1.2Hz,3H),2.71(d,J=1.2Hz,1H).

[0136] YN-17 (white solid, yield 56%), 1H NMR (400MHz, DMSO-d6) δ7.71(d,J=7.9Hz,2H),7.55(d,J=8.0Hz,2H),7.23(dd,J=22.2,8.3Hz,5H),3.78(s,1H),3.40(s,3H),2.96(s,3H).

[0137] YN-18 (white solid, yield 76%), 1 H NMR (400MHz, DMSO-d6) δ7.85 (dd, J=8.3, 2.5Hz, 2H), 7.68 (dd, J=8.4, 2.5Hz, 2H), 7.42–7.30 (m, 4H), 7.25 (s, 1H), 3.76(d,J=2.5Hz,1H), 3.62(d,J=2.6Hz,2H), 3.54(d,J=2.6Hz,2H), 3.02(d,J=2.5Hz,3H), 2.10(d,J=2.6Hz,3H).

[0138] Example 4

[0139]

[0140] In the first step, a mixture of 2-amino-5-hydroxybenzoic acid (1.53 g, 10.0 mmol) and formamide (2.25 g, 50.0 mmol) was heated to 160°C and stirred for 1 hour. The mixture was cooled to room temperature and filtered. The solid was then rinsed with methanol and dried to afford compound 9-2 (90% yield).

[0141] In the second step, a mixture of compound 9-2 (810.7 mg, 5.0 mmol) and acetyl chloride (471 mg, 6.0 mmol) was dissolved in DMF (20 mL), and then TEA (6.0 mmol) and DMAP (0.5 mmol) were added at 0°C. The mixture was heated to room temperature and stirred for 1 h to obtain compound 9-3 (yield 97%).

[0142] In the third step, under argon, the crude product 9-3 was dissolved in 10 mL of thionyl chloride, and 0.05 mL of DMF was added dropwise. The mixture was stirred in toluene at 50°C for 2 hours. The solvent was co-evaporated with toluene at room temperature to remove excess thionyl chloride. CHCl and HO were then added, and the organic layer was dried over MgSO. The solvent was removed under vacuum, and the product was purified by column chromatography to obtain compound 9-4 (yield 65%). 1 H NMR (400MHz, DMSO) δ8.83(s,1H),7.99(d,J=9.0Hz,1H),7.89(d,J=2.3Hz,1H),7.80–7.70(m,1H),2.34(s,3H).

[0143] In the fourth step, compound 9-4 (1.11 g, 5.0 mmol) was dissolved in NH3 (17.8 mL, 124 mmol, 7 N in CH3OH) under argon. The mixture was stirred at room temperature for 1.5-2 hours. After completion of the reaction, the solvent was removed under vacuum. The solid was triturated with diethyl ether and filtered to obtain compound 9 in a 70% yield.

[0144] In the fifth step, compound 9 (180.6 mg, 1 mmol) was dissolved in DMF (5.0 mL), and KCO (276.3 mg, 2 mmol) and 4-(bromoethyl)-N-methylbenzenesulfonamide (396.2 mg, 1.5 mmol) were added. The mixture was stirred at 50°C for 2 h, and 10.0 mL of water was added. The mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After evaporation of the solvent, the product was purified by column chromatography to obtain compound 10.

[0145] In the sixth step, compound 10 (363.4 mg, 1.0 mmol), K2CO3 (276.4 mg, 2 mmol), and p-hydroxybenzylene (177.2 mg, 1.5 mmol) were added to a 5.0 mL solution of DMF. The mixture was stirred at 50°C for 12 h, and 10.0 mL of water was added. The mixture was extracted with ethyl acetate, and the organic layer was washed with brine and dried over anhydrous sodium sulfate. After evaporation of the solvent, the product was purified by column chromatography to obtain compound 11 (271.7 mg, 61% yield).

[0146] In the seventh step, (bromoethyl)triisopropylsilane (313.5 mg, 1.2 mmol) and 1,10-phenanthroline monohydrate (36.0 mg, 0.2 mmol) were added to a solution of compound 11 (445.5 mg, 1.0 mmol), K2CO3 (276.4 mg, 2.0 mmol), CuSO4·5H2O (21.2 mg, 0.1 mmol), and toluene (5 mL). The mixture was stirred at 120°C for 6 h, filtered, and washed with diethyl ether. After evaporation of the solvent, the mixture was purified by column chromatography to yield compound 12.

[0147] In the eighth step, compound 12 was dissolved in 10 ml of THF, 2.0 eq of TBAF was added, and the mixture was stirred at 0°C for 0.5 h. The mixture was quenched with water (10.0 mL) and extracted with ether (3 × 10.0 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to afford YN-4 as a pale yellow solid (164.3 mg, 35% yield). 1H NMR (400MHz, CDCl3) δ8.72(s,1H),8.01(dd,J=8.3,1.3Hz,3H),7.76(d,J=8.5Hz,2H),7.71-7.67(m,2H),7. 51–7.45(m,2H),7.44–7.41(m,1H),7.33–7.29(m,1H),5.37(s,2H),3.16(s,1H),3.13(s,3H),2.74(s,1H). 13 C NMR (101MHz, CDCl3) δ165.9,157.1,152.4,152.1,148.0,142.5,135.9,130.0,129.9,129.8,128.3,127 .9,126.7,125.6,123.9,122.8,117.0,102.3,82.5,78.4,77.3,69.4,57.8,39.0.HRMS(ESI)calcd.for C 26 H 19 N3O4S 470.1169[M+H] + ,found 470.1154.

[0148] Example 5

[0149]

[0150] In the first step, compound 13 (426 mg, 2.0 mmol), CuSO₄ (64 mg, 0.4 mmol), K₃PO₄ (828 mg, 4 mmol), and 1,10-phenanthroline monohydrate (180 mg, 1 mmol) were added to a dry round-bottom flask. Under nitrogen protection, toluene (10 ml) was added, followed by the slow addition of TIPS bromoacetylene and the heating to 110°C. The reaction was monitored by thin-layer chromatography until completion. After cooling to room temperature, the mixture was diluted with 5 ml of ethyl acetate and filtered through celite. The filtrate was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to afford compound 14 (422 mg, 53.7% yield). 1 H NMR (400MHz, DMSO-d6) δ7.63 (d, J = 2.1Hz, 4H), 3.25 (s, 3H), 0.90 (s, 21H). 13 C NMR (400MHz, DMSO-d6) δ170.20,133.25,131.54,130.44,125.14,100.58,69.65,37.51,18.76,11.11.

[0151] In the second step, 14 (242 mg, 0.62 mmol), PPh3 (3.2 mg, 0.0124 mmol), PdCl2(PPh3)2 (9 mg, 0.0124 mmol), CuI (5 mg, 0.0248 mmol), and Et3N (1.5 ml) were added to a dry round-bottom flask. Under N2 protection, ultra-dry ethyl acetate (10 ml) was added, followed by the slow addition of TIPS acetylene, and the mixture was heated to 60°C. The reaction was monitored by thin-layer chromatography until completion. After cooling to room temperature, the mixture was diluted with 5 ml of ethyl acetate and filtered through celite. The filtrate was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to afford compound 15 (175 mg, 57% yield). 1 H NMR (400MHz, CDCl3) δ7.75 (d, J = 8.4Hz, 2H), 7.47 (d, J = 8.4Hz, 2H), 3.36 (s, 3H), 1.15 (s, 21H), 0.97 (s, 21H). 13 C NMR (400MHz, CDCl3) δ170.7,133.0,131.4,128.5,126.4,106.3,99.8,93.2,70.3,37.8,18.7,18.5,11.3,11.2.

[0152] In the third step, compound 15 (145 mg, 0.3 mmol) was dissolved in THF (10 ml) and cooled on an ice bath. TBAF (1 M in THF) (164 μl, 0.6 mmol) was added and the reaction progress was monitored by thin-layer chromatography until completion. The product was then quenched with cold water and extracted three times with dichloromethane. The combined organic layer was washed three times with water, dried over anhydrous sodium sulfate, and evaporated under vacuum. Subsequent purification by silica gel column chromatography afforded the desired product, YN-19, as a white solid (40 mg, 72.9% yield). 1 H NMR (400MHz, CDCl3) δ7.75 (d, J = 8.4Hz, 2H), 7.52 (d, J = 8.4Hz, 2H), 3.34 (s, 3H), 3.19 (s, 1H), 2.79 (s, 1H). 13 CNMR(400MHz, CDCl3)δ170.7,133.4,131.8,128.9,125.5,83.0,79.7,77.4,60.5,37.6.HRMS(ESI)calcd.for C 12 H9NO 184.0684[M+H] + ,found 184.0757.

[0153] Example 6

[0154]

[0155] In the first step, 2-amino-1-(4-bromophenyl)ethanol (300.0 mg, 1.39 mmol) was added to a dry round-bottom flask. Then, under N2 protection, ultra-dry DCM (5 ml) and Et3N (310.0 mg, 3.05 mmol) were added. After stirring at room temperature for 10 minutes, methyl bis(trifluoroether)carbonate (110.0 mg, 555.0 mmol) was slowly added portionwise and stirred in an ice-water bath for 8 hours. The reaction was monitored by thin-layer chromatography until completion. The mixture was extracted with 20 ml × 3 of ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. After vacuum distillation, 310 mg of crude compound 17 was obtained, which was directly used in the next step.

[0156] In the second step, 200 mg of crude compound 17 was added to anhydrous CuSO₄ (26.4 mg, 0.2 eq), K₃PO₄ (350.8 mg, 2.0 eq), 1,10-phenanthroline monohydrate (74.4 mg, 0.5 eq), and 5 ml of toluene under N₂ protection. The mixture was stirred at room temperature for 10 minutes, and then (2-bromoethynyl)triisopropylsilane (260.0 mg, 1.2 eq) was added dropwise. The reaction mixture was refluxed and stirred for 4 hours. The reaction was monitored by thin-layer chromatography until completion. The mixture was extracted with 20 ml × 3 of ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure, and then purified by silica gel column chromatography using PE:EA = 7:1 as the eluent to obtain compound 18 (272.6 mg, 78% yield). 1 H NMR (400MHz, CDCl3) δ7.59(d,J=8.5Hz,2H),7.26(d,J=8.4Hz,2H),5.06(dd,J=8.7,7 .4Hz, 1H), 4.75 (t, J = 8.9Hz, 1H), 4.25 (dd, J = 9.0, 7.4Hz, 1H), 0.94 (d, J = 2.2Hz, 21H).

[0157] In the third step, compound 18 (140.0 mg, 0.33 mmol), cuprous iodide (1.3 mg, 0.006 mmol), triphenylphosphine (1.8 mg, 0.006 mmol), and triphenylphosphine palladium dichloride (4.7 mg, 0.006 mmol) were added to 3 ml of anhydrous ethyl acetate under N protection. TEA (495.8 mg, 4.9 mmol) and triisopropylsilyl acetylene (90.7 mg, 0.3 mmol) were added sequentially with stirring. After stirring at room temperature for 10 minutes, the reaction system was refluxed at 50°C for 6 hours. The reaction progress was monitored by thin-layer chromatography until completion. The mixture was extracted with 20 ml × 3 of ethyl acetate. The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure to obtain 160 mg of crude compound 19, which was directly used for the next step.

[0158] In the fourth step, 50 mg of crude compound 19 was added to 3 ml of anhydrous tetrahydrofuran under N2 protection. The mixture was stirred in an ice-water bath for 10 minutes. Subsequently, a 4 M solution of tetrabutylammonium fluoride in tetrahydrofuran (57 μl) was added and stirring continued in an ice-water bath for 10 minutes. The reaction was monitored by thin-layer chromatography until completion. The mixture was extracted with 10 ml of ethyl acetate (3 times ethyl acetate). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was removed by distillation under reduced pressure and purified by silica gel column chromatography using a PE:EA ratio of 3:1 as the eluent to obtain compound YN-20 as a white solid (15.6 mg, 65% yield). 1 H NMR (400MHz, CDCl3) δ7.60 (d, J = 8.2Hz, 2H), 7.37–7.33 (m, 2H), 5.12–5.07 (m, 1H), 4.80–4.75 (m, 1H), 4.28–4.23 (m, 1H), 3.17 (s, 1H), 2.76 (s, 1H). HRMS(ESI)calcd.for C 13 H9NO2212.0633[M+H] + ,found 212.0706.

[0159] Example 7

[0160]

[0161] In the first step, compound 20 (904 mg, 4 mmol), PdCl2(PPh3)2 (56 mg, 0.08 mmol), CuI (30 mg, 0.16 mmol), and Et3N (860 μl, 6.2 mmol) were added to a dry round-bottom flask. Under nitrogen, ultra-dry THF (20 ml) was added and heated to 70°C. TMS acetylene (706 μl, 5 mmol) was then slowly added. The reaction was monitored by thin-layer chromatography until completion. After cooling to room temperature, the mixture was diluted with 5 ml of ethyl acetate and filtered through celite. The filtrate was evaporated under reduced pressure, and the crude product was purified by silica gel column chromatography to afford compound 21 (900 mg, 92.6% yield). 1 H NMR (400MHz, DMSO-d6) δ11.50(s,1H),7.88–7.79(m,3H),0.27(s,9H). 13 C NMR (400MHz, DMSO-d6) δ169.0,168.8,137.8,133.6,132.7,128.2,126.0,123.8,103.85,0.27,0.2.

[0162] In the second step, compound 21 (243 mg, 1 mmol), Na2CO3 (212 mg, 2 mmol), Cu(OAc)2 (40 mg, 0.2 mmol), and 4A molecular sieves (400 mg) were added to a dry round-bottom flask. A pyridine (161 μL, 2 mmol)-toluene (10 mL) solution was added to the reaction flask, which was then purged with 3 volumes of O2. The reaction flask was stirred in an oil bath at 70°C for 1 h. Ethynyltrimethylsilane (216 μL, 1.2 mmol) was dissolved in dry toluene (1 mL) and slowly added to the reaction system. The reaction mixture was stirred for 4 h. The reaction mixture was filtered through celite, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain compound 22 (100 mg, 29.5% yield). 1 H NMR (400MHz, DMSO-d6) δ7.94–7.92(m,3H),0.27(s,9H),0.25(s,9H). 13 C NMR (400MHz, DMSO-d6) δ164.9,164.8,138.6,132.1,130.9,129.2,127.0,124.8,103.4,100.6,86.6,82.4,0.3,0.1.

[0163] In the third step, compound 22 (50 mg, 0.147 mmol) was dissolved in 2 mL of ultra-dry THF in a single-necked round-bottom flask with a magnetic stir bar. The mixture was cooled to 4°C in an ice bath. Acetic acid (16 μl, 0.294 mmol) was then added to the reaction solution and stirred for 5 minutes. TBAF (1 M in THF) (48 μl, 0.354 mmol) was then added dropwise over 30 minutes. The reaction was stirred at 4°C until the starting material was completely consumed as determined by thin-layer chromatography. The reaction flask was then returned to room temperature, transferred to a separatory funnel, quenched with cold water, and extracted three times with dichloromethane. The product was dried over anhydrous sodium sulfate and evaporated under vacuum. Purification by silica gel column chromatography afforded the desired product, YN-21, as a pale yellow solid (17 mg, 59.3% yield). 1 H NMR (400MHz, DMSO-d6) δ8.03–7.94(m,3H),4.72(s,1H),4.59(s,1H). 13 C NMR(400MHz,DMSO-d6)δ165.2,165.1,138.9,132.1,131.1,128.9,127.1,124.8,86.4,82.2,70.9,67.7.HRMS(ESI)calcd.for C 12 H5NO2196.0320[M+H] + ,found 196.0393.

[0164] Example 8

[0165]

[0166] Step 1: A mixture of diethyl phosphite (414.3 mg, 3.0 mmol), NCS (267.1 mg, 2.0 mmol), and AIBN (492.6 mg, 3.0 mmol) was heated to reflux in CCl4 (10 mL) for 7 h. The mixture was then filtered and concentrated to afford compound 24. The crude product was used in the next step without further purification.

[0167] Step 2: Compound 24 (738.2 mg, 4 mmol) was added to a dry 10 ml two-necked flask, followed by ultra-dry CH2Cl2 (10 ml) and Et3N (809.5 mg, 8 mmol) under N2 protection. After the reaction system cooled to 0°C, 4-hydroxybenzylamine (541.8 mg, 4.4 mmol) was slowly added dropwise. After the addition was complete, the mixture was returned to room temperature and stirred for 24 h. The reaction process was monitored by thin-layer chromatography until the reaction was complete. The reaction was quenched with 10 ml of water, extracted with dichloromethane, and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and the solvent was evaporated and purified by silica gel column chromatography to obtain compound 25 (862.0 mg, 79.4% yield).

[0168] Step 3: Compound 25 (850.0 mg, 3.1 mmol) was dissolved in DMF (10 ml). Anhydrous KCO (865.2 mg, 6.2 mmol) and 3-bromopropyne (559.2 mg, 4.7 mmol) were added under N2 protection. The reaction was stirred at room temperature for 5 h. After completion as monitored by TLC, the reaction was quenched with water (10 ml) and extracted 3-4 times with dichloromethane. The organic layers were combined, washed with brine, and dried over anhydrous sodium sulfate. After evaporation of the solvent, the mixture was purified by silica gel column chromatography to obtain compound 26 (762.2 mg, 78.6% yield).

[0169] Step 4: Compound 26 (200 mg, 0.65 mmol), anhydrous copper sulfate (20.75 mg, 0.13 mmol), 1,10-phenanthroline monohydrate (58.6 mg, 0.32 mmol), and anhydrous potassium phosphate (275.9 mg, 1.3 mmol) were added sequentially to a Schlenk tube equipped with a stirrer. Toluene (3 ml) and (bromoethynyl)triisopropylsilane (219.6 mg, 0.84 mmol) were then added under N2 protection. The reaction mixture was reacted at 80°C for 16 h. The reaction was monitored for completion by TLC. The mixture was filtered through celite and washed with dichloromethane. The filtrate was collected, evaporated under reduced pressure, and purified by silica gel column chromatography to afford compound 27 (164.1 mg, 51.8% yield).

[0170] Step 5: Compound 27 (160 mg, 0.32 mmol) was dissolved in ultra-dry THF (5 ml). TBAF (1 M in THF, 2.0 eq) was added dropwise at 0°C under N2 protection. The mixture was stirred at 0°C for 15 min and then slowly warmed to room temperature. The reaction was continued for 2 h. After the reaction was complete, the solvent was directly evaporated and the product YN-24 was purified by column chromatography as a white solid (55.0 mg, yield 50.9%). 1H NMR (400MHz, DMSO) δ7.32–7.23(m,2H),6.95(qd,J=5.0,2.5Hz,2H),5.38(td,J=12.4 ,6.3Hz,1H),4.94(s,1H),3.92–3.81(m,6H),3.77(s,1H),1.17(t,J=7.1Hz,6H).13C NMR(101MHz,DMSO)δ156.2,128.9,115.0,79.8,78.5,73.4,70.9,67.3,61.6,61.6,56.0,55.8,44.2,16.5,16.5.HRMS(ESI)calcd.for C 16 H 20 NO4P 321.1130[M+H] + ,found 322.1203.

[0171] The synthesis method of compound YN-22 / 23 / 25 / 26 is similar to the above method.

[0172] YN-22, (yellow oil, yield 50.9%). 1 H NMR (400MHz, DMSO) δ7.31(d,J=8.6Hz,2H),7.00(d,J=8.6Hz,2H),4.97(s,2H),4.32(d,J=9 .3Hz,2H),4.25–4.06(m,4H),3.80(s,1H),3.31(d,J=3.5Hz,1H),1.03(s,3H),1.02(s,3H). 13 C NMR (101MHz, DMSO) δ157.1,130.1,115.1,78.1,78.1,73.6,72.7,71.0,67.3,57.1,57.0,56.0,32.3,32.3,20.9,20.7.

[0173] YN-23, (colorless oil, yield 50.8%): 1 H NMR (400MHz, DMSO) δ4.19-4.07(m,4H),3.39(d,J=3.6Hz,1H),3.26(dd,J=15.0,8.0Hz, 2H), 2.84(t,J=2.6Hz,1H),2.28–2.20(m,2H),1.84–1.74(m,2H),1.02(d,J=5.8Hz,6H). 13C NMR(101MHz,DMSO)δ83.8,78.1,78.0,72.2,32.3,32.3,27.4,20.9,20.6,15.2.HRMS(ESI)calcd.for C 12 H 18 NO3P 255.1024[M+H] + ,found 256.1097.

[0174] YN-25 (white solid, yield 78.6%): 1 H NMR (400MHz, DMSO) δ4.11 (d, J = 12.0Hz, 4H), 3.32 (d, J = 3.6Hz, 1H), 2.93 (d, J = 8.3Hz, 3H), 1.06 (s, 3H), 0.98 (s, 3H). 13 C NMR(101MHz,DMSO)δ78.1,78.0,54.6,54.6,37.83,37.8,32.3,32.3,21.0,20.5.HRMS(ESI)calcd.forC8H 14 NO3P 203.0711[M+H] + ,found 204.0784.

[0175] YN-26 (white oil, yield 45.45%): HRMS (ESI) calcd. for C7H 14 NO3P 190.0711[M+H] + ,found 192.0784.

[0176] Example 9 Covalent modification of amino acid residues of purified protein by compounds.

[0177] 10.0 μM molecular probe (i.e., the compound synthesized in Example 1-8) was incubated with bovine serum albumin (10 μL, 1 mg / mL) at 37°C for 3 h, and click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and fluorescent dye TARMA-Azide (0.1 mmol) were added to perform click chemistry reaction, and the reaction was carried out at room temperature for 2 h. 2 μL protein loading buffer solution (5×) was added and separated by polyacrylamide gel electrophoresis. Finally, the results were tested by multifunctional laser scanning imager Typhoon FLA 9500. Figure 1 As shown, molecular probes YN-1, YN-2, YN-3, and YN-4 can covalently label bovine serum albumin.

[0178] Example 10 Covalent modification of protein amino acid residues in tumor cells by the compound.

[0179] Human HepG2 hepatocellular carcinoma cells were cultured at 37°C, 5% CO₂, until the logarithmic growth phase and evenly distributed into 6-well plates. After 24 hours of attachment, 50 μM of the molecular probe was added to each well of the 6-well plate. The cells were incubated at 37°C for 3 hours, the culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). The cells were then lysed with RIPA cell lysis buffer containing 1% protease and phosphatase inhibitors. Protein concentration was quantified using a BCA protein quantification kit to a concentration of 1 mg / mL. A certain amount of cell lysate was added to the click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO₄ (1 mmol)] and the fluorescent dye TARMA-Azide (0.1 mmol) for a click chemistry reaction at room temperature for 2 hours. Subsequently, the protein was precipitated by adding ice-cold acetone solution and the organic solvent was removed by centrifugation. The resulting protein solid was denatured by adding protein loading buffer and boiling at 95°C for 10 minutes. The protein was then separated by polyacrylamide gel electrophoresis. Finally, the multifunctional laser scanning imager Typhoon FLA 9500 was used for testing. Figure 2 As shown, molecular probes YN-1, YN-2, YN-3, and YN-4 can covalently label proteins at the living cell level.

[0180] Example 11: Selective modification of acidic amino acid residues of purified protein by the compound.

[0181] 100 μM molecular probe YN-1 was incubated with bovine serum albumin (1 mL, 1 mg / mL) at 37°C for 0.5 h. Click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and DADPS (100 μM) were added for a click chemistry reaction at room temperature for 2 h. Subsequently, the protein was precipitated by adding pre-cooled acetone solution, and the organic solvent was removed by centrifugation. The protein sample was dissolved in 1% SDS and sonicated thoroughly. The supernatant was then added to Neutravidin agarose resin for protein enrichment. After incubation at room temperature for 4 h on a rotator, the supernatant was removed by centrifugation and washed sequentially with 1% SDS, 0.1% SDS, and PBS. The above-mentioned avidin protein agar was dissolved in 500 μL 6M urea PBS solution, 25 μL NH4HCO3 (25mM) buffer containing 100mM DTT was added, and the mixture was incubated at 37°C for 30 min. Subsequently, 25 μL NH4HCO3 (25mM) buffer containing 400mM IAA was added, and the mixture was reacted at room temperature in the dark for 30 min. The supernatant was removed by centrifugation and washed 3 times with PBS. 150 μL PBS containing 2M urea, 150 μL NH4HCO3 (50mM) buffer containing 1mM CaCl2, and 1.5 μL trypsin were added and incubated at 37°C overnight. The supernatant was removed by centrifugation and washed 3 times with double distilled water. 200 μL 10% formic acid aqueous solution was added to react for 2 h, centrifuged and washed 3 times with 50% acetonitrile aqueous solution, the eluate was combined and spin-dried, and the peptides were purified by C18. The obtained peptides were spin-dried for biological mass spectrometry analysis. Figure 3 As shown, the molecular probe YN-1 can selectively modify the acidic amino acid residues aspartic acid residues and glutamic acid residues.

[0182] Example 12: Selective modification of acidic amino acid residues in cells by the compound.

[0183] HepG2 cells were incubated with 100 μM of the molecular probe YN-1 at 37°C for 8 h. Click chemistry reactions were then performed with the addition of click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and DADPS (100 μM) for 2 h at room temperature. Subsequently, the protein was precipitated by adding pre-cooled acetone solution, and the organic solvent was removed by centrifugation. The protein sample was dissolved in 1% SDS and sonicated for thorough dissolution. The supernatant was then enriched with Neutravidin agarose resin. After incubation on a rotator at room temperature for 4 h, the supernatant was removed by centrifugation and washed sequentially with 1% SDS, 0.1% SDS, and PBS. The above-mentioned avidin protein agar was dissolved in 500 μL 6M urea PBS solution, 25 μL NH4HCO3 (25mM) buffer containing 100mM DTT was added, and the mixture was incubated at 37°C for 30 min. Subsequently, 25 μL NH4HCO3 (25mM) buffer containing 400mM IAA was added, and the mixture was reacted at room temperature in the dark for 30 min. The supernatant was removed by centrifugation and washed 3 times with PBS. 150 μL PBS containing 2M urea, 150 μL NH4HCO3 (50mM) buffer containing 1mMCaCl2, and 3.0 μL trypsin were added and incubated at 37°C overnight. The supernatant was removed by centrifugation and washed 3 times with double distilled water. 200 μL 10% formic acid aqueous solution was added to react for 2 h, centrifuged and washed 3 times with 50% acetonitrile aqueous solution, the eluate was combined and spin-dried, and the peptides were purified by C18. The obtained peptides were spin-dried for biological mass spectrometry analysis. Figure 4 As shown, the molecular probe YN-1 can selectively modify the acidic amino acid residues aspartic acid residues and glutamic acid residues.

[0184] Example 13 Inhibitory activity of YN-4 against different kinases.

[0185] Using Z'-LYTE TM Kinase inhibition of all probes was evaluated by fluorescence resonance energy transfer (FRET). TM The biochemical assay uses a FreeT-based coupled enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores, one at each end, which form a FRET pair. Compounds are diluted 3-fold in DMSO, starting from 5.1 × 10 -9 Dilute to 1×10 -4 M. Data were detected using EnVision reader (Perkin Elmer). Curve fitting and data presentation were performed using Graph Pad Prism 4.0. Figure 5 As shown, YN-4 selectively inhibited the kinase activity of EGFRL858R, and its IC 50It is 5.57μM.

[0186] The growth inhibitory activity of the compound in Example 14 on different tumor cells.

[0187] The CCK8 method was used to detect cell viability. 4000 cells per well were seeded in a 96-well plate and cultured in an incubator for 24 hours to maintain adhesion. YN-1 to YN-26 (0 μM to 100 μM) were dissolved in DMSO and added to each well of the cells, maintaining a final DMSO concentration of 0.1%. After incubation for 72 hours, 30 μL of CCK-8 reagent was added to each well and incubated for 2 hours. The absorbance was then measured using a plate reader at wavelengths of 450 nm and 650 nm. The cell viability was determined as VR = (A-A0) / (As-A0) × 100%, where A is the absorbance of the experimental group, As is the absorbance of the control group (using DMSO as the control), and A0 is the absorbance of the blank group (no cells). The IC50 value was calculated using GraphpadPris. The results are shown in Tables 1 to 3 below:

[0188] Table 1 Antiproliferation activity of alkyne amide compounds on different tumor cells

[0189]

[0190] Table 2 Antiproliferation activity of alkyne amide compounds on different tumor cells

[0191]

[0192] Table 3 Proliferation inhibition activity of alkyne amide compounds on different tumor cells

[0193]

[0194]

[0195] Example 15 Proteomic experiments for target confirmation of YN-1 / 4.

[0196] HepG2 cells were incubated with 50 μM of the molecular probes YN-1 and YN-4 at 37°C for 5 h. The cells were then lysed with lysis buffer and sonicated. Equal volumes and concentrations of the lysed light and heavy cells were mixed and a click chemistry reaction was performed with the addition of click chemistry reagents [TBTA (100 μmol), TCEP (1 mmol), CuSO4 (1 mmol)] and Biotin-N3 (100 μM). The reaction was allowed to proceed at room temperature for 2 h. Subsequently, the proteins were precipitated by adding pre-cooled acetone solution and the organic solvent was removed by centrifugation. The protein samples were dissolved in 1% SDS and sonicated thoroughly. The supernatant was then added to Neutravidin agarose resin for protein enrichment. After incubation on a rotator at room temperature for 4 h, the supernatant was removed by centrifugation and the cells were washed sequentially with 1% SDS, 0.1% SDS, and PBS. The above-mentioned avidin protein agar was dissolved in 500 μL of 6M urea in PBS solution, and 25 μL of NH4HCO3 (25 mM) buffer containing 100 mM DTT was added. The mixture was incubated at 37°C for 30 min, followed by addition of 25 μL of NH4HCO3 (25 mM) buffer containing 400 mM IAA. The mixture was reacted at room temperature in the dark for 30 min, the supernatant was removed by centrifugation, and the mixture was washed three times with PBS. 150 μL of PBS containing 2M urea, 150 μL of NH4HCO3 (50 mM) buffer containing 1 mM CaCl2, and 1 μL of trypsin (1 μg / μl) were added and incubated at 37°C overnight. The reaction was quenched by adding pure TFA and the mixture was washed with C 18 The peptides were purified and dried to be used for mass spectrometry analysis. Figure 6 As shown, YN-1 can effectively identify 999 target proteins, and YN-4 can identify 428 target proteins, of which 211 target proteins overlap.

[0197] Example 16 Protein pull-down and protein immunoblotting experiments.

[0198] 50μM molecular probes YN-1 and YN-4 were incubated with HepG2 / H3255 cells at 37°C for 5h, and then the cells were lysed with lysis buffer, and click chemistry reagents [TBTA (100μmol), TCEP (1mmol), CuSO4 (1mmol)] and Biotin-N3 (50μM) were added to perform click chemistry reaction at room temperature for 2h. Subsequently, pre-cooled acetone solution was added to precipitate the protein and the organic solvent was removed by centrifugation. The protein sample was dissolved with 1% SDS and fully dissolved by ultrasound. The supernatant was taken and added to Neutravidin agarose resin to enrich the protein. After incubation on a rotator at room temperature for 4h (or overnight at 4°C), the supernatant was removed by centrifugation and washed with 1% SDS, 0.1% SDS and PBS in sequence. The magnetic beads were then boiled at 95°C for 30min, followed by immunoblotting and incubation with the corresponding antibodies, and finally developed. Figure 7 The results shown, such as Figure 7 As shown, YN-4 can target and bind to EGFR, ALDH1 and GAPDH; YN-1 can target and bind to MEK2, ECH1, GAPDH and ALDH1.

[0199] Example 17 YN-4 inhibits the clone formation and proliferation of H3255 tumor cells.

[0200] H3255 cells were seeded into 6-well plates at a density of 1000 cells / well. After overnight incubation, the cells were treated with different concentrations of YN-4 (0, 0.11, 0.33, 1.1, 3.3, and 10 μM) and replicated in three wells. The culture medium was discarded, and the cells were fixed with 4% polyoxymethylene and stained with Giemsa stain for 30 minutes. The culture dishes were washed and gently air-dried. Image J was used to count clone clusters and calculate the cell formation rate, as shown in Figure 5. Figure 8 As shown, YN-1 can effectively inhibit the colony formation of H3255 tumor cells at a concentration of 1.1 μM, and when the concentration of YN-4 reaches 10 μM, it can almost completely inhibit the proliferation of H3255 tumor cells.

[0201] Example 18 Inhibitory effect of YN-4 on the phosphorylation of EGFR kinase and its downstream pathway proteins in H3255 cells.

[0202] After H3255 cells grew to 80-90%, the culture medium was removed and treated with different concentrations of YN-4, with DMSO not exceeding 1%. After incubation for 3 hours, the culture medium was removed and the cells were washed twice with PBS to remove excess probes. The cells were lysed with RIPA buffer and centrifuged for 10 minutes (14000rpm, 4℃) to obtain a soluble protein solution. Finally, the protein concentration was detected with BCA protein and then diluted with PBS. 1× SDS loading buffer was added and boiled at 95℃ for 10 minutes. Then, the corresponding antibodies were incubated for immunoblotting and finally developed. Figure 9 The results shown, such as Figure 9 As shown, YN-4 can effectively inhibit the phosphorylation level of EGFR, and also inhibit the phosphorylation level of its downstream protein kinases, including AKT and ERK kinases.

[0203] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0204] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of an alkyne amide compound having a structure represented by formula (II) or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating tumors, in, R1 is selected from: C1-C6 alkyl; R2 is selected from: H; R3 is selected from: R8 substituted or unsubstituted C6-C 10 aryl; R8 is selected from: H, ethynyl, -CH2-OR9, -CH2-NR9R 10 , phenoxy, phenyl, -C(=O)OR 10 、-C(=O)NR9R 10 ; R9 is selected from: R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group; R 10 Selected from: C1-C3 alkyl; R 11 Selected from: H, ethynyl, halogen, C1-C6 alkyl, ethynyl-substituted phenoxy, phenoxy, -N(R 10 )2; The tumor is colon cancer, breast cancer, colorectal adenocarcinoma, acute promyelocytic leukemia, lymphoma and / or lung cancer.

2. The use according to claim 1, characterized in that R1 is selected from the group consisting of: methyl, ethyl, and propyl.

3. The use according to claim 1, characterized in that R8 is selected from the group consisting of: H, ethynyl, -CH2-OR9, -CH2-NR9CH3, phenoxy, phenyl, -C(=O)OCH3, -C(=O)NR9CH3; R9 is selected from: R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group; R 11 Selected from: H, ethynyl, halogen, C1-C3 alkyl, ethynyl-substituted phenoxy, phenoxy, dimethylamino.

4. The use according to claim 1, characterized in that R1 is selected from: C1-C3 alkyl; R2 is H; R3 is selected from: R8 substituted or unsubstituted C6-C 10 aryl; R8 is selected from the group consisting of: H, ethynyl, -CH2-OR9, -CH2-NR9CH3, phenoxy, phenyl, -C(=O)OCH3, -C(=O)NR9CH3; R9 is selected from: R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group; R 11 Selected from: H, ethynyl, halogen, ethynyl-substituted phenoxy, phenoxy, dimethylamino.

5. The use according to claim 1, characterized in that The alkyne amide compound is selected from the following compounds:

6. An alkynamide compound having a structure represented by formula (II) or a pharmaceutically acceptable salt thereof, in, R1 is selected from: C1-C6 alkyl; R2 is selected from: H; R3 is selected from: R8 substituted C6-C 10 aryl; R8 is selected from: ethynyl, -CH2-OR9, -CH2-NR9R 10 , phenoxy, phenyl, -C(=O)OR 10 、-C(=O)NR9R 10 ; R9 is selected from: R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group; R 10 Selected from: C1-C3 alkyl; R 11 Selected from: H, ethynyl, halogen, C1-C6 alkyl, ethynyl-substituted phenoxy, phenoxy, -N(R 10 )2.

7. The alkyne amide compound or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: R1 is selected from the group consisting of: methyl, ethyl, and propyl.

8. The alkyne amide compound or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: R8 is selected from: ethynyl, -CH2-OR9, -CH2-NR9CH3, phenoxy, phenyl, -C(=O)OCH3, -C(=O)NR9CH3; R9 is selected from: R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group; R 11 Selected from: H, ethynyl, halogen, C1-C3 alkyl, ethynyl-substituted phenoxy, phenoxy, dimethylamino.

9. The alkyne amide compound or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: R1 is selected from: C1-C3 alkyl; R2 is H; R3 is selected from: R8 substituted C6-C 10 aryl; R8 is selected from: ethynyl, -CH2-OR9, -CH2-NR9CH3, phenoxy, phenyl, -C(=O)OCH3, -C(=O)NR9CH3; R9 is selected from: R 11 Substituted or unsubstituted C6-C 10 Aryl, R 11 a substituted or unsubstituted 5-10 membered heteroaryl group; R 11 Selected from: H, ethynyl, halogen, ethynyl-substituted phenoxy, phenoxy, dimethylamino.

10. An alkyne amide compound or a pharmaceutically acceptable salt thereof, characterized in that: The alkyne amide compound is selected from the following compounds:

11. A pharmaceutical composition for preventing and treating tumors, characterized in that: The invention is prepared from active ingredients and pharmaceutically acceptable carriers or excipients, wherein the active ingredients include the alkyne amide compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 6 to 10.

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