Toxin molecules suitable for antibody-drug conjugates

Abstract

The present application provides a toxin molecule suitable for an antibody-drug conjugate, in particular, the present application provides a compound as shown in the following formula (I), or a pharmaceutically acceptable salt or hydrate thereof. The compound of the present application can be used for preparing a medicament for treating a disease related to tumor cell proliferation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, and in particular, the present application provides a toxin molecule with tumor cell proliferation inhibitory activity. BACKGROUND

[0002] Antibody drug conjugate (ADC) connects monoclonal antibody or antibody fragment with cytotoxic substance with biological activity through stable chemical linker compound, which makes full use of the specificity of antibody binding to normal cell and tumor cell surface antigen and the high efficiency of cytotoxic substance, and avoids the defects of low efficacy of the former and excessive toxicity of the latter. This means that, compared with traditional chemotherapy drugs, antibody drug conjugate can more accurately bind to tumor cells and reduce the impact on normal cells.

[0003] At present, there are many ADC drugs used in clinical or clinical research, such as Kadcyla, which is an ADC drug formed by targeting Her2 trastuzumab and DM1. At the same time, there are patents of antibodies and ADC drugs targeting B7H3.

[0004] There are several categories of small molecules with cytotoxicity for antibody drug conjugates; one of them is camptothecin derivatives, which have anti-tumor effect by inhibiting topoisomerase I. Camptothecin derivative irinotecan has been reported to be used in antibody conjugate drugs (ADC), but the field still needs to further develop ADC drugs with better efficacy.

[0005] DNA topoisomerase (Topoisomerase, Topo) is a kind of essential enzyme widely existing in organisms, which is involved in all key nuclear processes such as DNA replication, transcription, recombination and repair. According to the different forms of instantaneous DNA strand breakage caused by topoisomerase, topoisomerase can be divided into two categories: topoisomerase I and topoisomerase II. Topoisomerase I and topoisomerase II jointly catalyze the unwinding of supercoiled DNA in the process of DNA replication, but topoisomerase II involves double-strand breakage, while topoisomerase I only causes single-strand breakage. Camptothecin and its analogues can reversibly bind to DNA topoisomerase I-DNA complex to form camptothecin and its analogue-DNA topoisomerase I-DNA ternary complex, which stops the progressive unwinding, and eventually causes the replication fork to collide with the ternary complex, resulting in irreparable DNA breakage and cell death. SUMMARY

[0006] The purpose of the present application is to provide a toxin molecule suitable for antibody conjugate drugs.

[0007] In a first aspect, the present application provides a compound of formula (I) or a pharmaceutically acceptable salt or hydrate thereof:

[0008]

[0009]

[0010] X is selected from the group consisting of H, OH, NH2, NHR 0 ;

[0011] Y is selected from the group consisting of (CR 1 R 2 ) n ;

[0012] Z is selected from the group consisting of a bond, C(O), C(S), C(NH), S(O)2, S(O);

[0013] R 0 is selected from the group consisting of C1-C8alkyl, C1-C8haloalkyl, C1-C8deuteroalkyl, C1-C8alkoxy, C1-C8hydroxyalkyl, C3-C8cycloalkyl or 3-12 membered heterocyclyl;

[0014] R 1 and R 2 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, C1-C8alkyl, C1-C8haloalkyl, C1-C8deuteroalkyl, C1-C8alkoxy, hydroxyl, amino, cyano, nitro, C1-C8hydroxyalkyl, C3-C8cycloalkyl or 3-12 membered heterocyclyl;

[0015] Alternatively, R 1 and R 2 together with the carbon atom to which they are attached form a C3-C8cycloalkyl or 3-12 membered heterocyclyl;

[0016] R 3 and R 4 are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, C1-C8alkyl, C1-C8haloalkyl, C1-C8deuteroalkyl;

[0017] Alternatively, R 3 and R 4 together with the carbon atom to which they are attached form a structure selected from the group consisting of a saturated or unsaturated 5-12 membered ring, a saturated or unsaturated 5-12 membered heterocyclic ring;

[0018] n is selected from 0, 1, 2 or 3 (preferably 0, 1 or 2);

[0019] and when n is 2, 3, each CR 1 R 2 may be the same or different.

[0020] Unless otherwise specified, the functional groups of this invention may be substituted by substituents selected from the group consisting of: halogen, nitrile, nitro, hydroxyl, amino, C1-C6 alkyl-amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halo-C1-C6 alkoxy, allyl, benzyl, C6-C 12 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.

[0021] In another preferred embodiment, the R 3 Selected from the following group: C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 deuteralkyl.

[0022] In another preferred embodiment, the R 4 Selected from the following groups: hydrogen atom, deuterium atom, halogen.

[0023] In another preferred embodiment, the compound has the structure shown in the following formula:

[0024]

[0025] In another preferred embodiment, Z is a chemical bond, C(O).

[0026] In another preferred embodiment, X is H, OH, or NH2.

[0027] In another preferred embodiment, Y is selected from the group consisting of: (CR) 1 R 2 ) n ;

[0028] Where R 1 and R 2 Each is independently selected from the following group: hydrogen atom, deuterium atom, halogen, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 deuteralkyl, C1-C8 hydroxyalkyl, C3-C6 cycloalkyl or 3-6 membered heterocyclic group; or, R 1 and R 2 Together with the carbon atom attached thereto, it forms a C3-C6 cycloalkyl or a 3-6 membered heterocyclic group; and the C1-C8 alkyl group may optionally be substituted by substituents selected from the group consisting of: C6-C 10 Aryl, 5-10 membered heteroaryl, C3-C6 cycloalkyl, 3-6 membered heterocyclic. n is selected from 0, 1, 2 or 3;

[0029] And when n is 2 or 3, each CR1 R 2 may be the same or different;

[0030] In another preferred embodiment, the compound has the structure of the following formula:

[0031]

[0032] In another preferred embodiment, the compound has the structure of the following formula:

[0033]

[0034]

[0035]

[0036] In a second aspect of the present application, there is provided a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, according to any one of the second aspect of the present application, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0037] In a third aspect of the present application, there is provided the use of a compound of formula I according to the first aspect of the present application, for the manufacture of a pharmaceutical composition for treating a disease associated with tumor cell proliferation.

[0038] In another preferred embodiment, the disease is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, lung cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, pharyngeal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, lymphoma.

[0039] In a fourth aspect of the present application, there is provided the use of a compound of formula I according to the first aspect of the present application, characterized in that it is used as a toxin in an antibody-drug conjugate for the manufacture of an antibody-drug conjugate.

[0040] It should be understood that, within the scope of the present application, each of the technical features described above and in the following (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. DETAILED DESCRIPTION

[0041] The present inventors have made a long-term and in-depth study and unexpectedly found a compound as shown in formula I. The compound has an unexpected activity in inhibiting tumor cell proliferation and can be used for treating diseases associated with tumor cell proliferation. Based on the above findings, the present inventors have completed the present application.

[0042] Definitions

[0043] As used herein, the term "alkyl" includes straight chain or branched chain alkyl groups. For example, C1-C8alkyl represents straight chain or branched chain alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, and the like.

[0044] As used herein, the term "alkenyl" includes straight chain or branched chain alkenyl groups. For example, C2-C6alkenyl refers to straight chain or branched chain alkenyl groups having 2 to 6 carbon atoms, such as ethenyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0045] As used herein, the term "alkynyl" includes straight chain or branched chain alkynyl groups. For example, C2-C6alkynyl refers to straight chain or branched chain alkynyl groups having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

[0046] As used herein, the term "C3-C 10 Cycloalkyl" refers to cyclic alkyl groups having 3 to 10 carbon atoms. It can be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It can also be in the form of a bicyclic ring, such as a bridged or spirocyclic ring.

[0047] As used herein, the term "C1-C8alkylamino" refers to an amine group substituted with a C1-C8alkyl group, which can be mono- or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, t-butylamino, dimethylamino, diethylamino, dipropylamino, diisopropylamino, dibutylamino, diisobutylamino, di-t-butylamino, and the like.

[0048] As used herein, the term "C1-C8alkoxy" refers to straight chain or branched chain alkoxy groups having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy, and the like.

[0049] As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S, and O" refers to a saturated or partially saturated cyclic group having 3 to 10 atoms and wherein 1 to 3 atoms are heteroatoms selected from the group consisting of N, S, and O. It can be monocyclic, or it can be in the form of a bicyclic ring, such as a bridged or spirocyclic ring. Specific examples can be oxetanyl, azetidinyl, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.

[0050] As used herein, the term "C6-C 10 Aryl" refers to aryl groups having 6 to 10 carbon atoms, such as phenyl or naphthyl, and the like.

[0051] As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a cyclic aromatic group having 5-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It can be monocyclic or in the form of a fused ring. Specific examples can be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.

[0052] Unless otherwise specifically noted, the groups described herein are "substituted or unsubstituted". The groups of the present application can be substituted with a substituent selected from the group consisting of halogen, nitrile, nitro, hydroxy, amino, C1-C6alkyl-amine, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, haloC1-C6alkyl, haloC2-C6alkenyl, haloC2-C6alkynyl, haloC1-C6alkoxy, allyl, benzyl, C6-C10aryl, C1-C6alkoxy-C1-C6alkyl, C1-C6alkoxy-carbonyl, phenoxycarbonyl, C2-C6alkynyl-carbonyl, C2-C6alkenyl-carbonyl, C3-C6cycloalkyl-carbonyl, C1-C6alkyl-sulfonyl and the like. 12 Unless otherwise specifically noted, the groups described herein are "substituted or unsubstituted". The groups of the present application can be substituted with a substituent selected from the group consisting of halogen, nitrile, nitro, hydroxy, amino, C1-C6alkyl-amine, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6alkoxy, haloC1-C6alkyl, haloC2-C6alkenyl, haloC2-C6alkynyl, haloC1-C6alkoxy, allyl, benzyl, C6-C10aryl, C1-C6alkoxy-C1-C6alkyl, C1-C6alkoxy-carbonyl, phenoxycarbonyl, C2-C6alkynyl-carbonyl, C2-C6alkenyl-carbonyl, C3-C6cycloalkyl-carbonyl, C1-C6alkyl-sulfonyl and the like.

[0053] As used herein, "halogen" or "halo" means F, Cl, Br, and I. More preferably, the halogen or halo is selected from F, Cl and Br. "Halo" means substituted with an atom selected from F, Cl, Br, and I.

[0054] Unless otherwise specifically noted, the structural formulae described herein are intended to include all isomeric forms (e.g., enantiomeric, diastereomeric, and geometric (or conformational) isomers): e.g., the R, S configurations for asymmetric centers, the (Z), (E) isomers for double bonds, and the like. Thus, individual stereochemical isomers or mixtures thereof (e.g., enantiomeric, diastereomeric, or geometric (or conformational) isomers) of the compounds of the present application are within the scope of the present application.

[0055] As used herein, the term "tautomer" indicates that structural isomers of different energy can interconvert over a low energy barrier. For example, prototropic tautomers (i.e., proton shift) include interconversion by proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion by reorganization of some of the bonding electrons.

[0056] As used herein, the term "hydrate" means a complex formed by coordination of a compound of the present application with water.

[0057] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by a combination of the specific embodiments set forth below with other chemical synthetic methods well known in the art, and equivalents thereof as appreciated by those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present application.

[0058] The solvents used in the present application can be obtained commercially, and the compounds can be prepared by either synthetic or Software nomenclature, commercially available compounds use the supplier catalog name.

[0059] Preparation of compounds

[0060] The compounds of the present application can be prepared by the following general methods:

[0061] Preparation Method 1:

[0062]

[0063] The compound of formula I is obtained by reacting the compound of formula Ia with phosphorus pentasulfide or Lawesson's reagent.

[0064] Preparation Method 2:

[0065]

[0066] The hydroxyl group in the compound of formula IIa is protected with a protecting group (PG 1 ) and reacted with phosphorus pentasulfide or Lawesson's reagent to obtain the compound of formula IIc. Then IIc and IId are subjected to dehydrative condensation and deprotection of the protecting groups (PG 1 and PG 2 ) to obtain the compound of formula IIf. Finally, the compound of formula I is obtained by transformation from IIf.

[0067] Pharmaceutical compositions and methods of administration

[0068] Since the compounds of the present application have excellent inhibitory activity against tumor cell proliferation, the compounds of the present application and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present application as the main active ingredient can be used for the prevention and / or treatment (stabilization, alleviation or cure) of diseases associated with tumor cell proliferation.

[0069] The pharmaceutical compositions of the present application comprise a safe and effective amount of a compound of the present application in combination with a pharmaceutically acceptable carrier. By "safe and effective amount" is meant an amount of the compound sufficient to significantly induce a desired effect, while not causing serious side effects. Typically, the pharmaceutical composition comprises from 1 to 2000 mg of a compound of the present application per dose, more preferably from 1 to 200 mg of a compound of the present application per dose. Preferably, the "dose" is a capsule or tablet.

[0070] "Pharmaceutically acceptable carrier" means one or more compatible solid or liquid filler or gel materials which are suitable for human use and which are nontoxic to the recipient at the dosages employed. By "compatible" is meant that the carrier is not chemically reactive with the compound of the present application and does not adversely affect the therapeutic efficacy of the medicament. Examples of pharmaceutically acceptable carriers suitable for use in the present application include, but are not limited to, one or more of the following: binders such as carboxymethylcellulose, ethylcellulose, cellulose acetate, gelatin, talc, solid lubricants such as stearic acid, magnesium stearate, calcium stearate, polyethylene glycol, sodium lauryl sulfate, or mixtures thereof; disintegrants such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; humectants such as glycerin; lubricants such as stearic acid, magnesium stearate, calcium stearate, and sodium stearyl fumarate; glidants such as colloidal silicon dioxide; colorants; flavorants; stabilizers; antioxidants; preservatives; and pyrogen-free water.

[0071] The mode of administration of the compounds or pharmaceutical compositions of the present application is not critical. Representative modes of administration include, but are not limited to, oral, parenteral (intravenous, intramuscular, or subcutaneous), or topical.

[0072] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, such as hydroxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; e) solution retarders, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents, such as kaolin and bentonite clay; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage form can also comprise buffering agents.

[0073] ​Solid dosage forms such as tablets, sugar coated tablets, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other well-known materials. They can contain opacifying agents, and can be of a composition that they release the active compound or compounds in a certain part of the digestive tract. Examples of embedding compositions that can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0074] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, and the like, together with

[0075] Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0076] Suspensions, in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, and agar-agar, as well as mixtures thereof and the like.

[0077] The compositions for parenteral injection can contain 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 vehicles include water, ethanol, polyols and suitable mixtures thereof.

[0078] The compounds of the present application can be administered alone, or in combination with other pharmaceutically acceptable therapeutic agents.

[0079] In combination therapy, the pharmaceutical composition further comprises one or more (2, 3, 4, or more) other pharmaceutically acceptable therapeutic agents. One or more (2, 3, 4, or more) of the other pharmaceutically acceptable therapeutic agents can be used simultaneously, separately or sequentially with the compounds of the present application to prevent and / or treat cytokine and / or interferon-mediated diseases.

[0080] The pharmaceutical compositions are used in a safe and effective amount of the compound of the present application for a mammal (e.g., human) in need of treatment, wherein the dosage is administered in a pharmaceutically effective amount, and for a 60 kg body weight human, the daily dosage is usually 1-2000 mg, preferably 1-500 mg. Of course, the specific dose will also take into account a route of administration, the patient's health status, and the like, which are all within the skill of a trained physician.

[0081] The present application is further illustrated by the following examples. It is to be understood that these examples are merely illustrative of the present application and do not in any way limit the scope of the application. The experimental procedures in the following examples, unless otherwise indicated, were carried out under conventional conditions or as suggested by the manufacturer. Unless otherwise indicated, percentages and parts are by weight.

[0082] Examples

[0083] Example 1

[0084]

[0085]

[0086] First step

[0087] Into a three-necked flask (100 mL) were sequentially placed 1a (1.95 g, 7.40 mmol), imidazole (2.52 g, 37.00 mmol), after nitrogen replacement, anhydrous N,N-dimethylformamide (30 mL) was added into the flask, the reaction system was cooled to 0 °C, triethylsilyl chloride (4.45 g, 29.60 mmol) and 4-dimethylaminopyridine (0.90 g, 7.40 mmol) were sequentially added into the reaction system, the reaction was continued at 0 °C for 2 hours, the reaction system was diluted with ethyl acetate (200 mL) and washed with saturated brine (25 mL x 4), the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 0-100%) to obtain 1b-1 (0.90 g) and 1b-2 (1.50 g), total yield: 73%.

[0088] 1b-1:

[0089] MS-ESI calculated value [M+H] + 378, measured value 378.

[0090] 1b-2:

[0091] MS-ESI calculated value [M+H] + 492, measured value 378 (lose one molecule of TES).

[0092] Second step

[0093] Into a three-necked flask (100 mL) was placed 1b-2 (1.10 g, 2.20 mmol) and Lawesson's reagent (1.80 g, 4.40 mmol) under nitrogen. After the flask was charged with anhydrous toluene (30 mL), the mixture was heated to 90 °C and stirred for 5-6 h. The reaction mixture was diluted with ethyl acetate (200 mL), washed with saturated brine (25 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a residue, which was purified by column chromatography on silica gel (ethyl acetate: petroleum ether = 0-100%) to give 1c (620 mg) in 51% yield.

[0094] MS-ESI calculated [M+H] + 394, found 394.

[0095] Third step

[0096] Into a single-necked flask (100 mL) was placed 1c (620 mg, 1.10 mmol), 1d (380 mg, 1.54 mmol), and pyridine-p-toluenesulfonic acid salt (166 mg, 0.66 mmol) under nitrogen. After the flask was charged with anhydrous toluene (30 mL), the mixture was heated to 120 °C and stirred for 24 h. To the reaction mixture was added 1d (60 mg) and pyridine-p-toluenesulfonic acid salt (90 mg), and the mixture was heated to 120 °C and stirred for another 20 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography on silica gel (methanol:dichloromethane = 0-100%) to give 1e (260 mg) in 48% yield.

[0097] MS-ESI calculated [M+H] + 494, found 494.

[0098] Fourth step

[0099] Into a single-necked flask (100 mL) was placed 1e (80 mg, 0.16 mmol) under nitrogen. To the flask was added 6N aqueous hydrochloric acid (27 mL), and the mixture was heated to 110 °C and stirred for 4 h. The reaction mixture was filtered to remove insoluble materials, and the filtrate was concentrated under reduced pressure to give crude 1f (50 mg).

[0100] MS-ESI calculated [M+H] + 452, found 452.

[0101] Fifth step

[0102] Into a single-necked flask (25 mL) was added 1f (20 mg, 0.04 mmol) and 1.2 mg / mL glycolic acid in N,N-dimethylformamide solution (2.00 mL, 0.032 mmol) sequentially, the reaction system was cooled to 0 °C, N,N-diisopropyl ethylamine (11 mg, 0.08 mmol) was added to the reaction system sequentially, 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (16 mg, 0.04 mmol) was added, after 0.5 h, 1.2 mg / mL glycolic acid in N,N-dimethylformamide solution (0.20 mL, 0.003 mmol) and 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1 mg) were added, the reaction was continued for 1 h, the reaction system was diluted with ethyl acetate (80 mL), the organic phase was washed with 0.5N dilute hydrochloric acid (5 mL x 1), saturated sodium bicarbonate solution (10 mL x 2) and saturated brine (10 mL x 5) sequentially, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain a residue, the residue was purified by preparative thin layer chromatography (methanol: dichloromethane) to obtain compounds 1-1 (2 mg) and 1-2 (5 mg), total yield: 34%.

[0103] 1-1:

[0104] MS-ESI calculated value [M+H] + 510, measured value 510.

[0105] 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 10.8 Hz, 1H), 7.80 (s, 1H), 5.92 (d, J = 16.8 Hz, 1H), 5.69 - 5.60 (m, 1H), 5.53 (d, J = 20.4 Hz, 1H), 5.52 (d, J = 16.4 Hz, 1H), 5.35 (d, J = 20.0 Hz, 1H), 4.15 - 3.98 (m, 2H), 3.30 - 3.22 (m, 1H), 3.19 - 3.09 (m, 1H), 2.40 (s, 3H), 2.26 - 2.14 (m, 2H), 1.96 - 1.84 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

[0106] 1-2:

[0107] MS-ESI calculated value [M+H] + 510, measured value 510.

[0108] 1H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 9.2 Hz, 1H), 7.81 (d, J = 11.2 Hz, 1H), 7.80 (s, 1H), 6.69 (s, 1H), 5.92 (d, J = 16.8 Hz, 1H), 5.69 - 5.62 (m, 1H), 5.62 - 5.56 (m, 1H), 5.55 - 5.47 (m, 2H), 5.33 (d, J = 20.0 Hz, 1H), 4.16 - 3.98 (m, 2H), 3.30 - 3.20 (m, 1H), 3.16 - 3.07 (m, 1H), 2.39 (s, 3H), 2.26 - 2.15 (m, 2H), 1.95 - 1.85 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

[0109] Example 2

[0110]

[0111]

[0112] First step

[0113] The crude product 1f (15.00 g) was purified by preparative HPLC to give 2-1 (2.80 g) and 2-2 (4.00 g), preparative yield: 45%.

[0114] 2-1:

[0115] MS-ESI calculated [M+H] 452, found 452. + 452, found 452.

[0116] 1 H NMR (400 MHz, DMSO-d6) δ 7.82 - 7.73 (m, 2H), 6.70 (s, 1H), 5.92 (d, J = 16.8 Hz, 1H), 5.86 (d, J = 20.4 Hz, 1H), 5.59 (d, J = 20.4 Hz, 1H), 5.52 (d, J = 16.4 Hz, 1H), 4.45 - 4.37 (m, 1H), 3.30 - 3.19 (m, 1H), 3.10 - 2.98 (m, 1H), 2.40 (s, 3H), 2.25 - 2.12 (m, 1H), 2.08 - 1.96 (m, 1H), 1.95 - 1.82 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

[0117] 2-2:

[0118] MS-ESI calculated [M+H] 452, found 452. + 452, found 452.

[0119] 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 4.4 Hz, 1H), 7.92 (d, J = 10.4 Hz, 1H), 7.84 (s, 1H), 6.02 (d, J = 20.0 Hz, 1H), 5.95 (d, J = 16.4 Hz, 1H), 5.70 (d, J = 20.0 Hz, 1H), 5.53 (d, J = 16.8 Hz, 1H), 5.23 - 5.15 (m, 1H), 3.36 - 3.25 (m, 1H), 3.20 - 3.07 (m, 1H), 2.60 - 2.51 (m, 1H), 2.42 (s, 3H), 2.25 - 2.12 (m, 1H), 1.98 - 1.82 (m, 1H), 0.86 (t, J = 7.2 Hz, 3H).

[0120] Example 3

[0121]

[0122] First step

[0123] Into a single necked flask (25 mL) was added 2-1 (20 mg, 0.04 mmol), dichloromethane (2 mL), triethylamine (25 μL, 0.18 mmol) sequentially, then acetic anhydride (19 μL, 0.20 mmol) was added dropwise, the reaction was stirred at room temperature for 1 hour. The reaction was filtered directly, the filter cake was rinsed with dichloromethane solution (5 mL), the filter cake was collected and dried to give compound 3 (10 mg), yield: 46%.

[0124] MS-ESI calculated [M+H] 494, found 494. + 494, found 494.

[0125] 1 H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 8.8 Hz, 1H), 7.84 (d, J = 10.8 Hz, 1H), 7.79 (s, 1H), 6.71 (s, 1H), 5.91 (d, J = 16.8 Hz, 1H), 5.62 - 5.57 (m, 1H), 5.56 - 5.48 (m, 2H), 5.41 (d, J = 20.0 Hz, 1H), 3.26 - 3.12 (m, 2H), 2.41 (s, 3H), 2.22 - 2.10 (m, 2H), 2.00 (s, 3H), 1.92 - 1.86 (m, 2H), 0.85 (t, J = 7.2 Hz, 3H).

[0126] Example 4

[0127]

[0128] First step

[0129] Into a single neck flask (100 mL), 2-2 (4.00 g, 8.86 mmol), anhydrous dichloromethane (30 mL) and triethylamine (3.58 g, 35.44 mmol) were added in sequence, acetic anhydride (3.70 g, 36.28 mmol) was added dropwise into the above reaction solution, and the reaction was stirred at room temperature for 1.5 hours. The reaction solution was directly filtered, the filter cake was washed with dichloromethane (20 mL), and the filter cake was collected and dried to obtain compound 4 (4.26 g), yield: 96%.

[0130] MS-ESI calculated value [M+H] + 494, found 494.

[0131] 1 H NMR (400 MHz, DMSO-d6) δ 8.58 (d, J = 8.8 Hz, 1H), 7.83 - 7.77 (m, 2H), 6.70 (s, 1H), 5.91 (d, J = 16.8 Hz, 1H), 5.61 - 5.55 (m, 1H), 5.55 - 5.51 (m, 1H), 5.49 (d, J = 12.0 Hz, 1H), 5.32 (d, J = 19.6 Hz, 1H), 3.28 - 3.07 (m, 2H), 2.37 (s, 3H), 2.26 - 2.04 (m, 2H), 2.01 (s, 3H), 1.96 - 1.84 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[0132] Example 5

[0133]

[0134]

[0135] First step

[0136] Into a single neck flask (25 mL), 2-1 (20 mg, 0.04 mmol), 2-hydroxy-2-phenylacetic acid (13 mg, 0.09 mmol) and N,N-dimethylformamide (2 mL) were added in sequence, N,N-diisopropylethylamine (21 μL, 0.12 mmol) was added into the reaction system at room temperature, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23 mg, 0.06 mmol) was added into the reaction system, and the reaction was stirred for 1.5 hours. The reaction system was quenched with water (50 mL), dichloromethane and methanol mixed solvents (V 二氯甲烷 :V 甲醇= 10:1, 15 mL x 4) extraction, the organic phase was combined, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure to obtain a residue, the residue was purified by preparative thin layer chromatography (methanol: dichloromethane) to obtain compound 5 (5 mg) with a yield of 19%.

[0137] MS-ESI calculated value [M+H] + 586, the measured value is 586.

[0138] 1 H NMR (400 MHz, DMSO-d6) δ 8.76 - 8.70 (m, 1H), 7.88 - 7.82 (m, 1H), 7.803 (s, 0.5H), 7.800 (s, 0.5H), 7.56 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 6.8 Hz, 1H), 7.35 - 7.31 (m, 2H), 7.29 - 7.26 (m, 1H), 6.72 (s, 1H), 6.33 - 6.18 (m, 1H), 5.93 (d, J = 16.8 Hz, 0.5H), 5.92 (d, J = 16.4 Hz, 0.5H), 5.56 - 5.47 (m, 4H), 5.17 - 5.02 (m, 1H), 3.17 - 3.13 (m, 2H), 2.41 (s, 3H), 2.17 - 2.10 (m, 2H), 1.94 - 1.87 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0139] Example 6

[0140]

[0141] First step

[0142] A single-mouth flask (25 mL) was sequentially added with 2-2 (60 mg, 0.13 mmol), 2-hydroxy-2-phenylacetic acid (40 mg, 0.27 mmol) and anhydrous N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (52 mg, 0.40 mmol) was sequentially added into the reaction system at room temperature, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (76 mg, 0.20 mmol) was sequentially added into the reaction system, the reaction was stirred for 1.5 hours. The reaction system was quenched by adding water (10 mL), extracted with ethyl acetate (200 mL x 1), the organic phase was washed with saturated brine (25 mL x 4), dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure to obtain a residue, the residue was purified by preparative thin layer chromatography (methanol: dichloromethane) to obtain compound 6 (65 mg) with a yield of 83%.

[0143] MS-ESI calculated value [M+H] +586, found 586.

[0144] 1 H NMR (400 MHz, DMSO-d6) δ 8.80 - 8.70 (m, 1H), 7.87 - 7.81 (m, 1H), 7.81 (s, 0.5H), 7.80 (s, 0.5H), 7.57 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 7.2 Hz, 1H), 7.37 - 7.30 (m, 2H), 7.30 - 7.23 (m, 1H), 6.71 (s, 1H), 6.35 - 6.17 (m, 1H), 5.95 (d, J = 16.4 Hz, 0.5H), 5.93 (d, J = 16.4 Hz, 0.5H), 5.65 - 5.35 (m, 4H), 5.15 - 5.02 (m, 1H), 3.22 - 3.06 (m, 2H), 2.39 (s, 3H), 2.19 - 2.05 (m, 2H), 1.96 - 1.85 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0145] Example 7

[0146]

[0147] First step

[0148] Into a single neck flask (25 mL), 2-1 (20 mg, 0.04 mmol), 2-hydroxy-3- phenylpropionic acid (15 mg, 0.09 mmol) and N,N-dimethylformamide (2 mL) were added successively, into the reaction system, N,N-diisopropylethylamine (21 μL, 0.12 mmol), 2-(7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (23 mg, 0.06 mmol) were added successively at room temperature, the reaction was stirred for 1.5 hours. Water (50 mL) was added to the reaction system, the mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 15 mL x 4) was extracted, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to obtain compound 7 (15 mg) with a yield of 56%.

[0149] MS-ESI calculated [M+H] + 600, found 600.

[0150] 1H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 8.8 Hz, 0.5H), 8.39 (d, J = 8.8 Hz, 0.5H), 7.85 - 7.82 (m, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.30 - 7.28 (m, 1H), 7.25 - 7.22 (m, 1H), 7.20 - 7.15 (m, 3H), 7.13 - 7.07 (m, 1H), 6.72 (d, J = 4.0 Hz, 1H), 5.94 - 5.89 (m, 1H), 5.56 - 5.51 (m, 2H), 5.43 - 5.38 (m, 2H), 4.36 - 4.31 (m, 0.5H), 4.24 - 4.19 (m, 0.5H), 3.18 - 3.05 (m, 4H), 2.40 (s, 3H), 2.17 - 2.12 (m, 2H), 1.93 - 1.87 (m, 2H), 0.88 - 0.85 (m, 3H).

[0151] Example 8

[0152]

[0153] First step

[0154] Into a single neck flask (25 mL) was added 2-2 (65 mg, 0.14 mmol), 2-hydroxy-3- phenylpropionic acid (48 mg, 0.29 mmol) and anhydrous dichloromethane (2 mL) successively, then N, N-diisopropylethylamine (55 mg, 0.43 mmol), 2-(7-azabenzotriazol- yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (84 mg, 0.22 mmol) were added successively into the reaction solution, and the reaction was stirred at room temperature for 1.5 hours. The reaction system was quenched with water (10 mL), extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 150 mL x 1) and the organic phase was washed with saturated brine (25 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to obtain compound 8 (55 mg) with a yield of 64%.

[0155] MS-ESI calculated value [M+H] + 600, the measured value is 600.

[0156] 1H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 9.2 Hz, 0.5H), 8.39 (d, J = 8.8 Hz, 0.5H), 7.85 - 7.76 (m, 2H), 7.34 - 7.07 (m, 6H), 6.71 (s, 0.5H), 6.70 (s, 0.5H), 5.98 - 5.87 (m, 1H), 5.75 - 5.50 (m, 3H), 5.42 - 5.38 (m, 1H), 4.40 - 4.30 (m, 0.5H), 4.26 - 4.15 (m, 0.5H), 3.21 - 3.04 (m, 4H), 2.38 (s, 3H), 2.20 - 2.00 (m, 2H), 1.96 - 1.82 (m, 2H), 0.91 - 0.81 (m, 3H).

[0157] Example 9

[0158]

[0159] First step

[0160] Into a single neck flask (25 mL) was added 2-1 (40 mg, 0.09 mmol), 2-methyl-2-hydroxypropanoic acid (14 mg, 0.13 mmol), anhydrous dichloromethane (5 mL), N,N-diisopropylethylamine (29 mg, 0.22 mmol) and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (51 mg, 0.13 mmol) sequentially. The reaction was stirred at room temperature for 1 hour. The reaction was quenched with water (10 mL) and extracted with dichloromethane (20 mL x 5). The organic phase was combined and dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative thin layer chromatography (methanol:dichloromethane) to give compound 9 (10 mg) with a yield of 21%.

[0161] MS-ESI calculated [M+H] + 538, found 538.

[0162] 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 9.2 Hz, 1H), 7.82 (d, J = 11.2 Hz, 1H), 7.79 (s, 1H), 6.71 (s, 1H), 5.90 (d, J = 16.4 Hz, 1H), 5.62 - 5.50 (m, 4H), 5.36 - 5.28 (m, 1H), 3.32 - 3.20 (m, 1H), 3.18 - 3.04 (m, 1H), 2.39 (s, 3H), 2.24 - 2.13 (m, 2H), 1.93 - 1.79 (m, 2H), 1.53 (s, 3H), 1.37 (s, 3H), 0.90 - 0.81 (m, 3H).

[0163] Example 10

[0164]

[0165] First step

[0166] Into a single neck flask (25 mL) was added 2-2 (50 mg, 0.11 mmol), 2-methyl-2-hydroxypropanoic acid (17 mg, 0.17 mmol), anhydrous dichloromethane (5 mL), N,N-diisopropylethylamine (36 mg, 0.28 mmol) and 2-(7-azabenzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (63 mg, 0.17 mmol) sequentially, the reaction was stirred at room temperature for 1 hour. The reaction was quenched with water (15 mL), extracted with dichloromethane (30 mL x 3), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure to give a residue, the residue was purified by preparative thin layer chromatography (methanol:dichloromethane) to give compound 10 (30 mg), yield: 51%.

[0167] MS-ESI calculated [M+H] + 538, found 538.

[0168] 1 H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 9.2 Hz, 1H), 7.83 - 7.74 (m, 2H), 6.71 (s, 1H), 5.91 (d, J = 16.4 Hz, 1H), 5.64 - 5.44 (m, 4H), 5.24 (d, J = 20.0 Hz, 1H), 3.29 - 3.18 (m, 1H), 3.17 - 3.08 (m, 1H), 2.37 (s, 3H), 2.22 - 2.09 (m, 2H), 1.96 - 1.85 (m, 2H), 1.55 (s, 3H), 1.37 (s, 3H), 0.87 (t, J = 7.2 Hz, 3H).

[0169] Example 11

[0170]

[0171] First Step

[0172] Into a single neck flask (25 mL) was added 2-1 (20 mg, 0.04 mmol), 3,3,3- trifluoro-2-methylpropanoic acid (13 mg, 0.09 mmol) and N,N-dimethylformamide (2 mL) successively, to the reaction system was added N,N-diisopropylethylamine (21 μL, 0.12 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23 mg, 0.06 mmol) successively at room temperature, the reaction was stirred for 1.5 hours. The reaction system was quenched with water (30 mL), extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 15 mL x 4), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to obtain compound 11-1 (2 mg) (small polarity) and 11-2 (5 mg) (large polarity), with a total yield of 27%.

[0173] 11-1:

[0174] MS-ESI calculated value [M+H] + 578, measured value 578.

[0175] 11-2:

[0176] MS-ESI calculated value [M+H] + 578, measured value 578.

[0177] Example 12

[0178]

[0179] First Step

[0180] Into a single neck flask (25 mL) was added 2-2 (50 mg, 0.11 mmol), 3,3,3-trifluoro-2- hydroxypropanoic acid (23.9 mg, 0.17 mmol) and anhydrous dichloromethane (4 mL) successively, to the reaction solution was added N,N-diisopropylethylamine (35.8 mg, 0.28 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (75 mg, 0.19 mmol) successively, the reaction was stirred for 4 hours at room temperature. The reaction system was quenched with water (15 mL), extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V甲醇 = 10:1, 10 mL x 6) extraction, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to obtain compound 12-1 (15 mg) (small polarity) and 12-2 (6 mg) (large polarity), total yield: 32%.

[0181] 12-1:

[0182] MS-ESI calculated value [M+H] + 578, measured value 578.

[0183] 1 H NMR (400 MHz, DMSO-d6) δ 9.01 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 10.8 Hz, 1H), 7.81 (s, 1H), 7.36 (br s, 1H), 6.70 (br s, 1H), 5.92 (d, J = 16.4 Hz, 1H), 5.67 - 5.59 (m, 2H), 5.52 (d, J = 16.4 Hz, 1H), 5.41 (s, 2H), 4.73 - 4.61 (m, 1H), 3.18 (t, J = 6.4 Hz, 2H), 2.40 (s, 3H), 2.31 - 2.21 (m, 1H), 2.20 - 2.10 (m, 1H), 1.96 - 1.84 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H).

[0184] 12-2:

[0185] MS-ESI calculated value [M+H] + 578, measured value 578.

[0186] 1 H NMR (400 MHz, DMSO-d6) δ 8.97 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 10.8 Hz, 1H), 7.80 (s, 1H), 7.17 - 7.09 (m, 1H), 6.72 (br s, 1H), 5.91 (d, J = 16.4 Hz, 1H), 5.68 - 5.59 (m, 1H), 5.58 - 5.33 (m, 3H), 4.66 - 4.55 (m, 1H), 3.20 - 3.12 (m, 2H), 2.40 (s, 3H), 2.28 - 2.09 (m, 2H), 1.93 - 1.83 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

[0187] Example 13

[0188]

[0189] First step

[0190] Into a single neck flask (25 mL), was added 2-1 (20 mg, 0.04 mmol), N-tert-butoxycarbonylglycine (16 mg, 0.09 mmol) and N,N-dimethylformamide (2 mL) successively, into the reaction system was added N,N-diisopropylethylamine (21 μL, 0.12 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23 mg, 0.06 mmol) successively at room temperature, the reaction was stirred for 1.5 hours. The reaction system was quenched by adding water (40 mL), extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 15 mL x 4), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to obtain compound 13a (15 mg) with a yield of 56%.

[0191] MS-ESI calculated value [M+H] + 609, the measured value was 609.

[0192] Second step

[0193] Into a single neck flask (25 mL), was added 13a (15 mg, 0.02 mmol), ethyl acetate (2 mL) successively, into the reaction system was added hydrogen chloride ethyl acetate solution (4.0 M, 2 mL) dropwise, the reaction was stirred at room temperature for 7 hours, then hydrogen chloride dioxane solution (4.0 M, 1 mL) was added dropwise, the reaction was continued to be stirred at room temperature for 1 hour. The reaction solution was slowly added into ice saturated aqueous sodium bicarbonate solution (50 mL), until no bubbles were generated, extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 30 mL x 6), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC to obtain compound 13 (2 mg) with a yield of 15%.

[0194] MS-ESI calculated value [M+H] + 509, the measured value was 509.

[0195] Example 14

[0196]

[0197] First step

[0198] Into a single-necked flask (25 mL), 2-2 (100 mg, 0.22 mmol), N-tert-butoxycarbonylglycine (77 mg, 0.44 mmol) and anhydrous dichloromethane (4 mL) were added successively, and into the reaction system, N,N-diisopropylethylamine (85 mg, 0.66 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (100 mg, 0.26 mmol) were added successively at room temperature, and the reaction was stirred for 1 h. The reaction system was quenched with water (10 mL), extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 150 mL x 1), and the organic phase was washed with saturated brine (25 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product 14a (140 mg).

[0199] MS-ESI calculated value [M+H] + 609, measured value 609.

[0200] Second step

[0201] The crude product 14a (125 mg) was dissolved in ethyl acetate (4 mL), and hydrogen chloride ethyl acetate solution (4.0 M, 4 mL) was added dropwise to the above solution, and the reaction was stirred at room temperature for 17 h. The reaction solution was slowly added to ice saturated aqueous sodium bicarbonate solution to adjust the pH to 8-9, extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 200 mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was slurried with acetonitrile (2 mL) to obtain 14 (60 mg), two-step yield: 54%.

[0202] MS-ESI calculated value [M+H] + 509, measured value 509.

[0203] 1 H NMR (400 MHz, DMSO-d6) δ 8.60 (br s, 1H), 7.82 (d, J = 10.8 Hz, 1H), 7.79 (s, 1H), 6.71 (br s, 1H), 5.90 (d, J = 16.8 Hz, 1H), 5.66-5.58 (m, 1H), 5.56-5.45 (m, 2H), 5.41-5.37 (m, 1H), 3.30-3.10 (m, 4H), 2.38 (s, 3H), 2.24-2.14 (m, 2H), 1.96-1.82 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

[0204] Example 15

[0205]

[0206] First step

[0207] Into a single-necked flask (25 mL) were added 2-1 (30 mg, 0.07 mmol), 4-(tert-butoxycarbonylamino)butyric acid (28 mg, 0.14 mmol) and N,N-dimethylformamide (3 mL) successively, into the reaction system were added N,N-diisopropylethylamine (37 μL, 0.21 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (40 mg, 0.11 mmol) successively, and the reaction was stirred at room temperature for 1.5 hours. The reaction system was added water (50 mL), and the mixture was extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 15 mL x 4), and the combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to obtain compound 15a (17 mg) with a yield of 40%.

[0208] MS-ESI calculated value [M+H] + 637, measured value 637.

[0209] Second step

[0210] Into a single-necked flask (25 mL) were added 15a (17 mg, 0.03 mmol), dioxane (1 mL) successively, and into the reaction system was added hydrogen chloride dioxane solution (4 M, 4 mL) dropwise, and the reaction was stirred at room temperature for 1 hour. The reaction solution was slowly added to ice saturated aqueous sodium bicarbonate solution (60 mL) until no gas bubbles were generated, and the mixture was extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 30 mL x 6), and the combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC to obtain compound 15 (3 mg) with a yield of 21%.

[0211] MS-ESI calculated value [M+H] + 537, measured value 537.

[0212] 1H NMR (400 MHz, DMSO-d6) δ 8.67 (d, J = 8.8 Hz, 1H), 8.37 (s, 1H), 7.85 (d, J = 10.8 Hz, 1H), 7.80 (s, 1H), 6.73 (br s, 1H), 5.91 (d, J = 16.8 Hz, 1H), 5.64 - 5.59 (m, 1H), 5.53 - 5.45 (m, 3H), 3.25 - 3.10 (m, 2H), 2.79 - 2.75 (m, 2H), 2.41 (s, 3H), 2.35 - 2.32 (m, 2H), 2.24 - 2.11 (m, 2H), 1.92 - 1.80 (m, 4H), 0.85 (t, J = 7.2 Hz, 3H).

[0213] Example 16

[0214]

[0215]

[0216] First step

[0217] Into a single neck flask (25 mL) was added 2-2 (100 mg, 0.22 mmol) and 4-(tert- butyloxycarbonylamino)butyric acid (90 mg, 0.44 mmol) and anhydrous dichloromethane (5 mL) successively, into the reaction solution was added N,N- diisopropylethylamine (86 mg, 0.66 mmol), 2-(7-azabenzotriazol)-N,N,N',N'- tetramethyluronium hexafluorophosphate (121 mg, 0.32 mmol) successively, the reaction was stirred at room temperature for 4 hours. The reaction system was quenched with water (30 mL), extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 50 mL x 4) successively, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by silica gel column chromatography (methanol:dichloromethane = 0-100%) to obtain 16a (106 mg) with a yield of 75%.

[0218] MS-ESI calculated value [M+H] + 637, the measured value was 637.

[0219] Second step

[0220] Into a single neck flask (25 mL) was added 16a (106 mg, 0.17 mmol) and ethyl acetate (5 mL) successively, hydrogen chloride ethyl acetate solution (4.0 M, 5 mL) was added dropwise into the above solution, the reaction was stirred at room temperature for 17 hours. The reaction was concentrated under reduced pressure to give a residue. The residue was added into saturated sodium carbonate solution to adjust pH to basic, the aqueous phase was extracted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 50 mL x 4) and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give a crude product. The crude product was separated and purified by preparative HPLC to give compound 16 (4 mg), yield: 4%.

[0221] MS-ESI calculated [M+H] + 537, found 537.

[0222] 1 H NMR (400 MHz, DMSO-d6) δ 8.73 (d, J = 8.8 Hz, 1H), 8.39 (br s, 1H), 7.83 (d, J = 10.8 Hz, 1H), 7.80 (s, 1H), 5.91 (d, J = 16.4 Hz, 1H), 5.64 - 5.56 (m, 1H), 5.55 - 5.34 (m, 3H), 3.19 - 3.10 (m, 2H), 2.80 (t, J = 7.6 Hz, 2H), 2.39 (s, 3H), 2.35 (t, J = 7.2 Hz, 2H), 2.25 - 2.05 (m, 2H), 2.04 - 1.93 (m, 1H), 1.94 - 1.80 (m, 3H), 0.86 (t, J = 7.2 Hz, 3H).

[0223] Example 17

[0224]

[0225] First Step

[0226] Into a single neck flask (25 mL) was added 1f (100 mg, 0.22 mmol), 1-hydroxy-1- cyclopropanecarboxylic acid (45 mg, 0.44 mmol), anhydrous dichloromethane (3 mL), N, N- diisopropylethylamine (85 mg, 0.66 mmol) and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (108 mg, 0.29 mmol) successively. The reaction was stirred at room temperature for 3 hours. The reaction was quenched by water (15 mL) and extracted with dichloromethane (150 mL x 1). The organic phase was combined and washed with saturated brine (15 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was separated and purified by preparative HPLC to give compound 17 (12 mg) in a yield of 10%.

[0227] MS-ESI calculated [M+H] + 536, found 536.

[0228] 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.8 Hz, 1H), 7.84 - 7.79 (m, 1H), 7.79 (s, 1H), 6.70 (s, 1H), 6.27 (s, 1H), 5.91 (d, J = 16.8 Hz, 1H), 5.66 - 5.57 (m, 1H), 5.56 - 5.46 (m, 2H), 5.35 - 5.24 (m, 1H), 3.32 - 3.21 (m, 1H), 3.20 - 3.05 (m, 1H), 2.39 (s, 3H), 2.35 - 2.15 (m, 2H), 1.38 - 1.15 (m, 2H), 1.04 - 0.90 (m, 2H), 0.85 (t, J = 7.2 Hz, 3H).

[0229] Example 18

[0230]

[0231] First step

[0232] Into a single necked flask (25 mL) was added 2-2 (70 mg, 0.16 mmol), 1- hydroxy-1-cyclopropanecarboxylic acid (33 mg, 0.32 mmol) and dry N,N- dimethylformamide (2 mL) successively, into the mixture was added N,N- diisopropylethylamine (60 mg, 0.47 mmol) and 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (91 mg, 0.24 mmol) successively, the reaction was stirred at room temperature for 3 hours. The reaction was quenched by water (15 mL), extracted with ethyl acetate (150 mL x 1), the organic phase was washed with saturated brine (25 mL x 5), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure to give a residue, the residue was purified by preparative thin layer chromatography (methanol: dichloromethane) to give compound 18 (28 mg), yield: 34%.

[0233] MS-ESI calculated [M+H] + 536, found 536.

[0234] 1 H NMR (400 MHz, DMSO-d6) δ 8.65 (d, J = 9.2 Hz, 1H), 7.81 (d, J = 11.2 Hz, 1H), 7.79 (s, 1H), 6.70 (s, 1H), 6.29 (s, 1H), 5.92 (d, J = 16.8 Hz, 1H), 5.66 - 5.58 (m, 1H), 5.56 - 5.46 (m, 2H), 5.92 (d, J = 20.0 Hz, 1H), 3.32 - 3.20 (m, 1H), 3.20 - 3.05 (m, 1H), 2.39 (s, 3H), 1.95 - 1.84 (m, 2H), 1.38 - 1.16 (m, 2H), 1.05 - 0.90 (m, 2H), 0.86 (t, J = 7.2 Hz, 3H).

[0235] Example 19

[0236]

[0237] First step

[0238] To a single neck flask (25 mL) was added 2-1 (30 mg, 0.07 mmol), 3-cyclopropyl-2-hydroxypropanoic acid (22 mg, 0.17 mmol), dry dichloromethane (3 mL), N,N-diisopropylethylamine (23 mg, 0.18 mmol) and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (42 mg, 0.11 mmol) successively. The reaction was stirred at room temperature for 1 h. The reaction was concentrated under reduced pressure to give a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to give compound 19 (6 mg) in 16% yield.

[0239] MS-ESI calculated [M+H] + 564, found 564.

[0240] 1 H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 8.8 Hz, 1H), 7.81 (d, J = 10.8 Hz, 1H), 7.79 (s, 1H), 6.71 (s, 1H), 5.90 (d, J = 16.4 Hz, 1H), 5.63 - 5.55 (m, 1H), 5.62 - 5.46 (m, 3H), 5.35 (d, J = 20.0 Hz, 1H), 4.27 - 4.05 (m, 1H), 3.29 - 3.20 (m, 1H), 3.19 - 3.05 (m, 1H), 2.40 (s, 3H), 2.29 - 2.08 (m, 2H), 1.74 - 1.63 (m, 2H), 1.72 - 1.62 (m, 1H), 1.62 - 1.49 (m, 1H), 0.97 - 0.89 (m, 1H), 0.85 (t, J = 7.4 Hz, 3H), 0.50 - 0.28 (m, 2H), 0.20 - 0.02 (m, 2H).

[0241] Example 20

[0242]

[0243]

[0244] First step

[0245] Into a single neck flask (25 mL) was added 20a (500 mg, 3.87 mmol), water (4 mL) and glacial acetic acid (930 mg, 15.48 mmol) sequentially, the mixture was cooled to 0-5 °C, sodium nitrite aqueous solution (2 mL, 7.7 mmol / mL) was added dropwise slowly, after the addition was completed, the reaction was continued to stir at room temperature for 3 hours. The reaction system was extracted with ethyl acetate (20 mL x 5), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product 20b (200 mg).

[0246] Second step

[0247] Into a single neck flask (25 mL) was added 2-2 (30 mg, 0.07 mmol), 20b (90 mg, 0.69 mmol), anhydrous dichloromethane (5 mL), N,N-diisopropyl ethylamine (15.2 mg, 0.28 mmol) and 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34 mg, 0.09 mmol) sequentially, the reaction was stirred at room temperature for 1 hour. The reaction system was quenched with water (10 mL), extracted with dichloromethane (30 mL x 3), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to obtain compound 20 (5 mg), yield: 13%.

[0248] MS-ESI calculated value [M+H] + 564, found 564.

[0249] 1 H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 8.8 Hz, 1H), 7.78-7.65 (m, 2H), 6.69 (s, 1H), 5.86 (d, J = 16.4 Hz, 1H), 5.66 (d, J = 5.6 Hz, 1H), 5.60-5.34 (m, 3H), 5.20 (d, J = 20.0 Hz, 1H), 4.10-3.91 (m, 1H), 3.25-3.13 (m, 1H), 3.13-2.99 (m, 1H), 2.31 (s, 3H), 2.12 (q, J = 6.8 Hz, 2H), 1.99-1.75 (m, 3H), 1.63-1.42 (m, 1H), 0.95-0.86 (m, 1H), 0.83 (t, J = 7.3 Hz, 3H), 0.50-0.26 (m, 2H), 0.19-0.07 (m, 2H).

[0250] Example 21

[0251]

[0252]

[0253] First step

[0254] Into a single neck flask (25 mL) was added 21a (800 mg, 3.72 mmol) and ethyl acetate (4 mL) sequentially, hydrogen chloride ethyl acetate solution (4.0 M, 7.4 mL) was added dropwise into the above solution, the reaction was stirred at room temperature for 17 hours. The reaction was filtered directly, the filter cake was rinsed with ethyl acetate (5 mL), the filter cake was collected and dried to give compound 21b (570 mg), yield: 99%.

[0255] 1 H NMR (400 MHz, DMSO-d6) δ 13.63 (br s, 1H), 8.52 (s, 3H), 3.22 (d, J = 8.8 Hz, 1H), 1.16 - 1.02 (m, 1H), 0.68 - 0.50 (m, 4H).

[0256] Second step

[0257] Into a single neck flask (25 mL) was added 21b (330 mg, 2.20 mmol) and dilute sulfuric acid (2.0 N, 4.4 mL), the mixture was cooled to 0-5 °C, sodium nitrite aqueous solution (5 mL, 4.4 mmol / mL) was added dropwise slowly, after the addition was completed, the reaction was continued to stir at room temperature overnight. Sodium chloride was added to the reaction system to saturation, extracted with ethyl acetate (100 mL x 3), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product 21c (150 mg).

[0258] Third step

[0259] Into a single neck flask (25 mL) was added 2-2 (100 mg, 0.22 mmol), crude product 21c (130 mg) and anhydrous dichloromethane (5 mL) sequentially, N,N-diisopropyl ethylamine (113 mg, 0.88 mmol), 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (125 mg, 0.33 mmol) were added into the reaction system sequentially, the reaction was stirred at room temperature for 2 hours. The reaction system was diluted with a mixed solvent of dichloromethane and methanol (V 二氯甲烷 :V 甲醇 = 10:1, 200 mL), the organic phase was washed with dilute hydrochloric acid (1.0 N, 20 mL x 1), saturated brine (20 mL x 2) sequentially, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a residue, which was purified by preparative thin layer chromatography (methanol:dichloromethane) to give compound 21 (5 mg), yield: 4%.

[0260] MS-ESI calculated [M+H] + 550, found 550.

[0261] 1 H NMR (400 MHz, DMSO-d6) δ 8.75 (d, J = 8.8 Hz, 1H), 7.85 (d, J = 10.4 Hz, 1H), 7.80 (S, 1H), 6.69 (s, 1H), 5.92 (d, J = 16.4 Hz, 1H), 5.64 - 5.56 (m, 1H), 5.56 - 5.46 (m, 2H), 5.43 - 5.34 (m, 1H), 4.66 - 4.60 (m, 1H), 3.48 - 3.30 (m, 1H), 3.22 - 3.12 (m, 1H), 2.41 (s, 3H), 2.26 - 2.10 (m, 2H), 1.94 - 1.82 (m, 2H), 1.11 - 1.05 (m, 1H), 0.90 - 0.72 (m, 7H).

[0262] Biological activity test

[0263] Cell proliferation inhibition experiment

[0264] Logarithmic growth phase KPL-4 tumor cells were taken, the cells were resuspended with fresh RPMI1640 culture solution, counted and the cell suspension was adjusted to 2 x 10 4 The cell suspension was inoculated into a 96-well cell culture plate at 100 μL / well, and incubated in a carbon dioxide incubator (37°C, 5% CO2) overnight. The next day, one of the 96-well plates inoculated with cells was taken out, and after equilibration to room temperature, 100 μL of CellTiter-Glo reagent (Promega, USA) previously equilibrated to room temperature and mixed well was added to each well of the test plate. After incubation in the dark for 30 minutes, the luminescence value was read in a microplate reader (recorded as G0 value). Another parallel plate was taken and different concentrations of the test compound or DMSO (final concentration 0.5%) were added to the corresponding wells of the test plate. After incubation in a carbon dioxide incubator for 72 h, the test plate was equilibrated to room temperature and the cell activity was detected using CellTiter-Glo reagent, recorded as G3 value.

[0265] The cell proliferation rate was calculated according to the following formula: cell proliferation rate (%) = (average value of test compound well G3 - average value of G0) / (average value of DMSO control well G3 - average value of G0) * 100. The inhibition curve was fitted using Graphpad Prism software and the GI 50 value was calculated (see table below).

[0266] Examples GI 50 (nM) Example 1 : 1-1 13.9 Example 1 : 1-2 10.3 Example 2: 2-1 <1.52 Example 2: 2-2 2.47 Example 3 2.59 Example 4 1.55 Example 5 1.79 Example 6 5.31 Example 7 5.76 Example 8 4.95 Example 9 12.1 Example 10 3.54 Example 11 : 11-1 17.3 Example 11 : 11-2 23.5 Example 12: 12-1 5.8 Example 12: 12-2 6.4 Example 13 7.54 Example 14 6.14 Example 17 1.83 Example 18 3.89 Example 19 2.9 Example 20 4.79 Example 21 34.7 DXd 72

[0267] The results show that the compounds of the present application exhibit higher inhibitory activity on proliferation in the above-mentioned tumor cell proliferation inhibition experiment, and the activity is better than that of DXd (Exatecan derivative, structure as follows).

[0268]

[0269] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding benefits disclosed herein.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: I The R mentioned 1 and R 2 Each is independently selected from the group consisting of: hydrogen atom, deuterium atom, halogen, C1-C8 alkyl, C1-C8 haloalkyl, C1-C8 deuteralkyl, C1-C8 hydroxyalkyl or C3-C6 cycloalkyl; or, R 1 and R 2 The carbon atoms attached thereto form C3-C6 cycloalkyl groups; and the C1-C8 alkyl groups may optionally be substituted with substituents selected from the group consisting of: C6-C 10 Aryl, C3-C6 cycloalkyl.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compound has the structure shown in the following formula: 。 3. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.

4. Use of the compound of formula I as described in any one of claims 1-2, characterized in that, Used to prepare pharmaceutical compositions for treating diseases associated with tumor cell proliferation.

5. Use of the compound of formula I as described in any one of claims 1-2, characterized in that, Toxins are used as ingredients in antibody-drug conjugates to prepare antibody-drug conjugates.

6. An antibody-drug conjugate, characterized in that, This includes compounds of formula I as described in any one of claims 1-2 as toxins.

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