CN112574159B

CN112574159B - Coumarin derivatives and analogs, and preparation method and application thereof

Info

Publication number: CN112574159B
Application number: CN202010967888.1A
Authority: CN
Inventors: 陈俐娟
Original assignee: Chengdu Zeiling Biomedical Technology Co ltd
Current assignee: Chengdu Zeiling Biomedical Technology Co ltd
Priority date: 2019-09-29
Filing date: 2020-09-15
Publication date: 2023-03-21
Anticipated expiration: 2040-09-15
Also published as: CN112574159A

Abstract

The invention relates to coumarin derivatives and analogs, and a preparation method and application thereof, and belongs to the field of chemical medicine. The invention provides a compound shown as a formula I or a pharmaceutically acceptable salt thereof. The invention also provides a preparation method and application of the compound. Biological experiments show that the compound has stronger in-vitro anti-fibrosis effect, can obviously reduce the deposition of intercellular collagen fibers on the NRK-49F cells induced by TGF-beta, and has inhibition on the migration of HUVEC cells. Meanwhile, the compounds with the structure have certain curative effect on mouse models of hepatic fibrosis induced by carbon tetrachloride and pulmonary fibrosis induced by bleomycin, have low toxicity, and are used for clinical treatment of hepatic fibrosis and pulmonary fibrosisTissue fibrosis diseases including fibrosis and renal fibrosis provide a new choice.

Description

Coumarin derivatives and analogs, and preparation method and application thereof

Technical Field

The invention relates to coumarin derivatives and analogs, and a preparation method and application thereof, and belongs to the field of chemical medicines.

Background

Fibrosis refers to the continuous repair of tissues or organs after the tissues or organs are damaged, and excessive fibrous connective tissues are formed due to excessive repair. It is manifested as a reactive, benign or pathological condition. The reactive process leading to injury, called scarring, if the fibrosis that occurs is derived from a single cell line, is called fibroma. Ideally, fibrosis occurs and deposits in connective tissue, gradually affecting the structure and function of normal tissues and organs. Fibrosis is a pathological condition that can be described by the use of over-deposited collagen. We usually define it as an excess of Extracellular matrix (ECM) deposition, fibrosis ultimately leads to scarring and thickening of the tissue, which is essentially an excessive injury repair response that ultimately affects normal organ function.

Renal fibrosis can be caused by different kidney diseases, eventually progressing to end stage renal disease. Although the pathogenesis of renal fibrosis has not been fully elucidated, it is usually manifested by hyperproliferation of renal resident cells (glomerular epithelial cells, mesangial cells and endothelial cells), repair of renal defects, abnormal activation of renal interstitial fibroblasts and excessive deposition of extracellular matrix, eventually leading to glomerulosclerosis and tubulointerstitial fibrosis.

Liver fibrosis occurs in different chronic lesions, including various viral hepatitis, alcohol abuse, drug causes, metabolic diseases, iron or copper overload, autoimmune attack on epithelial cells or congenital abnormalities of liver cells or bile ducts. Typically, the injury occurs months to years before scarring occurs. Liver fibrosis is reversible, while cirrhosis is the end-stage result of fibrosis, usually irreversible.

Pulmonary fibrosis is a fibrotic pulmonary disease characterized by interstitial pneumonia, which is a long-term, progressive disease caused by unknown reasons, and is manifested by symptoms such as dyspnea and shortness of breath. Of these, idiopathic Pulmonary Fibrosis (IPF) is the most common and severe form of pulmonary fibrosis, and its main features are dry cough, dyspnea, decreased lung function, etc. The incidence is about 6.8 to 8.8 per 10 million as shown by the us 2012 data, and the overall prevalence is 14 to 27.9 per 10 million. Through clinical research on patients, the IPF patients have the following characteristics: the incidence is high in men; the incidence of IPF increases with age, particularly after 50 years of age; IPF is more common in smokers, and is not improved even after smoking cessation; under CT the lungs of IPF patients will assume a honeycomb-like morphology. Although IPF is not generally prevalent, it is the most common and serious type of idiopathic interstitial pneumonia, and patients have a reduced quality of life, a low survival time and survival rate, and are difficult to diagnose accurately, which is an obstacle to the treatment of such diseases.

In recent years, there has been a shift in the study of the pathogenesis of IPF, from the inflammatory drive to epithelial diseases. It was previously classified as a chronic inflammatory disease, associated with the interaction of monocytes, fibroblasts and cytokines. IPF is a form of abnormal wound healing that involves fibrosis due to hyperproliferation of the interpulmonary spaces and alveolar spaces and myofibroblasts. That is, inflammation is not the primary cause of IPF, but it plays an important role in the pathogenesis and progression of the disease. Specific pathogenesis includes: first, the lungs are repeatedly attacked by micro-injury, such as smoking and viral infection on Alveolar Epithelial Cells (AECs). These injuries result in epithelial cell death and promote the wound healing process. Under normal conditions, apoptosis of epithelial cells initiates the wound healing process, increasing vascular permeability to proteins (fibrinogen and fibrin), and the formation of a wound clot. Damaged cells are removed by the action of inflammation, followed by the growth of fibroblasts to form a new extracellular matrix (ECM). After re-epithelialization, cells such as differentiated myofibroblasts, which participate in the wound healing process, undergo apoptosis. However, in IPF, the abnormal wound healing response leads to fibroblast proliferation and acute ECM deposition. This ultimately affects the balance between the fiber mediator and the anti-fibrotic mediator. Levels of active transforming growth factor (TGF-. Beta.) are elevated in patients with IPF. When this abnormal process continues, repeated lung remodeling eventually leads to the formation of cellulite cysts and destruction of the lung structure, ultimately resulting in the development and loss of function of pulmonary fibrosis.

Cell-mediated pathways associated with fibrosis include: (1) fibrosis-associated extracellular factors, mainly growth factors and cytokines. These factors act on adjacent or distant cells through specific signaling pathways. They bind to specific receptors on the cell membrane, facilitating intracellular conduction of extracellular signals, ultimately leading to a pre-fibrotic cellular response. (2) Intracellular influences, mainly involving multiple tyrosine kinases, can regulate many different intracellular signaling pathways through phosphorylation and dephosphorylation.

The only drugs currently approved by the FDA for IPF treatment are pirfenidone marketed in 2008 and nintedanib marketed in 2014. Nintedanib is a triple kinase inhibitor comprising: vascular Endothelial Growth Factor (VEGF); platelet growth factor (PDGF); fibroblast Growth Factor (FGFR). PDGFs can induce fibroblast chemotaxis and are the strongest stimulators of fibroblast proliferation. Therefore, they play an important role in the expansion of myofibroblasts and play a role in collagen synthesis. PDGF inhibitors like imatinib are reported to alter the fibrotic response, thereby reducing pulmonary fibrosis in animal studies. FGF channels are important signaling pathways that control angiogenesis, morphogenesis, and airway remodeling. VEGFR-2 antagonists may attenuate histopathological fibrosis and collagen deposition by modulating angiogenesis and inflammation. Thus, signaling of PDGF, FGF and VEGF has become a potential therapeutic target for IPF. Nintedanib inhibited significant anti-fibrotic effects by inhibiting the proliferation of primary human lung fibroblasts produced by IPF patients. However, nintedanib has poor selectivity and has certain inhibitory activity on other kinases, FLT3 and the like.

TGF-. Beta.1-4 is a family of multifunctional cytokines that bind to TGF-. Beta.receptors, consisting of TGF-. Beta.R 1 and R2. After the TGF-beta R2 is combined with TGF-beta, the TGF-beta R1 kinase can be phosphorylated, the cascade amplification of signals is induced, and S-mad protein is recruited and activated. They are transcribed with nuclear downstream regulatory protein effectors, leading to differentiation, chemotaxis, proliferation and activation of target cells. TGF-. Beta.1, as an inactive peptide, requires hydrolysis by proteases, including MMPs, to be activated. The key roles of TGF-beta 1 are: regulating the inflammatory process, the production of ECM, apoptosis and differentiation of T cells. During fibrosis, TGF- β signaling promotes silent fibroblasts to differentiate into myofibroblasts and secrete ECM, and the dependent S-mad3 pathway is particularly important. Therefore, inhibiting the binding of TGF- β 1 to its receptor and the associated S-mad3 pathway signaling becomes another goal of anti-fibrosis. The anti-fibrosis property of pirfenidone is attributed to its ability to reduce the expression of cytokines such as TGF-beta, which ultimately leads to the inhibition of fibrosis.

Disclosure of Invention

The invention aims to provide coumarin derivatives and analogues or pharmaceutically acceptable salts thereof, wherein the structures of the coumarin derivatives and analogues are shown as a formula I:

x is selected from O or NH;

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ independently selected from H, nitro, C ₁ ～C ₆ Alkoxy or

Y is selectedFrom O, S or NR ₁₂ ；R ₇ 、R ₈ 、R ₉ 、R ₁₀ 、R ₁₁ Independently selected from H, halogen or

R ₁₂ Selected from H, C ₁ ～C ₆ An alkyl group; r ₁₃ Selected from NR ₁₄ R ₁₅ Substituted or unsubstituted 5-6 membered heterocycle, heteroatom is N or O, the number of heteroatom is 1-2; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, substituted or unsubstituted 5-6 membered heterocycle, heteroatom is N or O, the number of heteroatom is 1-2.

As a preferred embodiment of the present invention, the above compound, R ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, substituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, substituted 6-membered heterocycle; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 5-membered heterocycle, substituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, substituted 6-membered heterocycle;

preferably, R ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, C ₁ ～C ₄ Alkyl-substituted 6-membered heterocycle, tert-butoxycarbonyl-substituted 6-membered heterocycle, -COR ₁₆ Substituted 6-membered heterocyclic ring, -COOR ₁₇ Substituted 6-membered heterocycles, -NR ₁₈ R ₁₉ A substituted 6-membered heterocyclic ring; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 6-membered heterocycle, tert-butoxycarbonyl substituted 6-membered heterocycle, -COOR ₁₇ A substituted 6-membered heterocyclic ring; r ₁₆ Is selected from C ₁ ～C ₃ Alkyl radical, C ₃ ～C ₆ A cycloalkyl group; r ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from C ₁ ～C ₄ An alkyl group;

further preferably, R ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, C ₁ ～C ₄ Alkyl-substituted 6-membered heterocyclic ring, tert6-membered heterocycle substituted by butoxycarbonyl, -COR ₁₆ Substituted 6-membered heterocyclic ring, -COOR ₁₇ Substituted 6-membered heterocycles, -NR ₁₈ R ₁₉ A substituted 6-membered heterocyclic ring; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 6-membered heterocycle, tert-butoxycarbonyl substituted 6-membered heterocycle, -COOR ₁₇ A substituted 6-membered heterocyclic ring; r ₁₆ Is selected from C ₁ ～C ₃ Alkyl radical, C ₃ ～C ₆ A cycloalkyl group; r ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from C ₁ ～C ₄ An alkyl group; the 5-membered heterocycle has 1 heteroatom, and the heteroatom is N;

more preferably, R ₁₃ Is selected from

Most preferably, R ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or halogen; r ₉ Is selected from

In a preferred embodiment of the present invention, when X is selected from O, the compound has a structural formula shown in formula ii below:

wherein Y is selected from O or NR ₁₂ ；R ₁ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₆ An alkoxy group; r ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or halogen; r ₁₂ Selected from H or methyl.

4. A compound according to claim 3, characterized in that: r ₁ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₃ An alkoxy group; preferably, R ₁ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Independently selected from H, nitro or methoxy; more preferably, R ₁ 、R ₃ 、R ₆ Are all selected from H, R ₄ 、R ₅ Independently selected from H, nitro or methoxy;

and/or, R ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or F; preferably, R ₇ 、R ₈ 、R ₁₁ Are all selected from H, R ₁₀ Selected from H or F;

and/or, R ₁₃ Is selected from

The compound has the following structural formula:

in a preferred embodiment of the present invention, in the above compound, when X is NH, Y is O, S or NR ₁₂ ，R ₂ 、R ₄ 、R ₅ Independently selected from H or

And R is ₂ 、R ₄ 、R ₅ One of which is

R ₁ 、R ₃ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₆ An alkoxy group; r ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or halogen; r is ₁₂ Selected from H or methyl.

Preferably, the above compound, R ₂ Is selected from

When Y is selected from O, S or NR ₁₂ ；R ₄ Or R ₅ Is selected from

When, Y is selected from O;

and/or, R ₁ 、R ₃ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₃ An alkoxy group; preferably, R ₁ 、R ₃ 、R ₆ Are all selected from H;

and/or, R ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or F; preferably, R ₇ Selected from H or F, R ₈ 、R ₁₀ 、R ₁₁ Are all selected from H;

and/or, R ₁₃ Is selected from

The compound has the following structural formula:

the invention also provides a preparation method of the compound, which mainly adopts the following synthetic routes:

route (i):

reaction reagents and reaction conditions: (a) Tetrabutylammonium bromide (TABA), P ₂ O ₅ Toluene (TOL), 90. + -. 5 ℃; (b) Et (Et) ₃ N, etOH,70 +/-5 ℃, 2-5 h; (c) Chloroacetyl chloride, et ₃ N, DMF, 0. + -. 5 ℃; (d) DMF, KI, room temperature.

Route (ii):

reaction reagents and reaction conditions: (a) TABA, P ₂ O ₅ ,TOL,90±5℃；(b)K ₂ CO ₃ Acetone (ACE), 65 ± 5 ℃; (c) Fe, HCl, meOH, H ₂ O, room temperature; (d) Chloroacetyl chloride, et ₃ N, DMF, 0. + -. 5 ℃; (e) DMF, KI, room temperature.

Route (iii):

reaction reagents and reaction conditions: (a) TABA, P ₂ O ₅ ,TOL,90±5℃；(b)hydrobromic acid,1,4-dioxane,90±5℃；(c)aniline,Xantphos,Pd ₂ (dba) ₃ t-BuOK, dioxane, 130. + -. 5 ℃; (d) trifluoroacetic acid, rt, stirring overnight; (e) Chloroacetyl chloride, et ₃ N,DMF,0-25℃；(f)RNH,Et ₃ N, DMF, rt, stirred overnight.

Route (iv):

reaction reagents and reaction conditions: (a) POCl ₃ ,100±5℃；(b)hydrochloric acid,1,4-dioxane,90±5℃；(c)4-aminothiopenenol,K ₂ CO ₃ DMF, 130. + -. 5 ℃; (d) Chloroacetyl chloride, et ₃ N,DMF,0±5℃,4h,0-25℃；(e)RNH ₂ ,Et ₃ N, DMF, rt, stir overnight.

Route (v):

reaction reagents and reaction conditions: (a) K ₂ CO ₃ ,DMF,100±5℃；(b)Fe,HCl,MeOH/H ₂ O =9/1,85 ± 5 ℃; (c) Chloroacetyl chloride, et ₃ N,DMF,0±5℃；(d)RNH ₂ ,Et ₃ N, DMF, stirred at room temperature.

Route (six):

reaction reagents and reaction conditions: (a) K ₂ CO ₃ DMF, room temperature; (b) Fe, HCl, meOH/H ₂ O =9/1,85 ± 5 ℃; (c) Chloroacetyl chloride, et ₃ N,DMF,0±5℃；(d)RNH ₂ ,Et ₃ N, DMF, stirred at room temperature.

The method for synthesizing the compound 29-1 or the compound 29-2 is the same as that of the compound 29, except that the starting compound 10 is replaced with the compound 10-1 or the compound 10-2.

In all the above synthetic schemes, R ₁₃ Selected from NR ₁₄ R ₁₅ Substituted or unsubstituted 5-6 membered heterocycle, heteroatom is N or O, the number of heteroatom is 1-2; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, substituted or unsubstituted 5-6 membered heterocycle, heteroatom is N or O, the number of heteroatom is 1-2.

Preferably, R ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, substituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, substituted 6-membered heterocycleA ring; r is ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 5-membered heterocycle, substituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, substituted 6-membered heterocycle.

Further preferably, R ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, C ₁ ～C ₄ Alkyl-substituted 6-membered heterocycle, tert-butoxycarbonyl-substituted 6-membered heterocycle, -COR ₁₆ Substituted 6-membered heterocyclic ring, -COOR ₁₇ Substituted 6-membered heterocycles, -NR ₁₈ R ₁₉ A substituted 6-membered heterocyclic ring; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 6-membered heterocycle, tert-butoxycarbonyl substituted 6-membered heterocycle, -COOR ₁₇ A substituted 6-membered heterocyclic ring; r ₁₆ Is selected from C ₁ ～C ₃ Alkyl radical, C ₃ ～C ₆ A cycloalkyl group; r ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from C ₁ ～C ₄ An alkyl group.

More preferably, R ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, C ₁ ～C ₄ Alkyl-substituted 6-membered heterocycle, tert-butoxycarbonyl-substituted 6-membered heterocycle, -COR ₁₆ Substituted 6-membered heterocyclic ring, -COOR ₁₇ Substituted 6-membered heterocycles, -NR ₁₈ R ₁₉ A substituted 6-membered heterocyclic ring; r is ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 6-membered heterocycle, tert-butoxycarbonyl substituted 6-membered heterocycle, -COOR ₁₇ A substituted 6-membered heterocycle; r ₁₆ Is selected from C ₁ ～C ₃ Alkyl radical, C ₃ ～C ₆ A cycloalkyl group; r ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from C ₁ ～C ₄ An alkyl group; the 5-membered heterocyclic ring has 1 heteroatom number and N heteroatoms.

Most preferably, R ₁₃ Is selected from

The invention also provides a pharmaceutical composition which is prepared by taking the compound, the pharmaceutically acceptable salt or the pharmaceutically acceptable hydrate as an active ingredient and adding pharmaceutically acceptable auxiliary ingredients.

The invention also provides the application of the compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing a medicament for treating the fibrotic disease.

Further, the medicine is used for inhibiting the expression of COL1A1, alpha-SMA and p-Smad3 protein.

Further, the above use, wherein the drug is a drug inhibiting TGF-. Beta./Smad 3 pathway.

Further, in the above use, the fibrotic disease is pulmonary fibrosis, hepatic fibrosis or renal fibrosis.

Definition of terms:

the compounds and derivatives provided by the present invention may be named according to the IUPAC (international union of pure and applied chemistry) or CAS (chemical abstracts service, columbus, OH) naming system.

The term "alkyl" is a radical of a straight or branched chain saturated hydrocarbon group. C ₁ ～C ₆ Examples of alkyl groups include, but are not limited to, methyl (C) ₁ ) Ethyl (C) ₂ ) N-propyl (C) ₃ ) Isopropyl (C) ₃ ) N-butyl (C) ₄ ) Tert-butyl (C) ₄ ) Sec-butyl (C) ₄ ) Isobutyl (C) ₄ ) N-pentyl group (C) ₅ ) 3-pentyl radical (C) ₅ ) Pentyl group (C) ₅ ) Neopentyl (C) ₅ ) 3-methyl-2-butyl (C) ₅ ) Tert-amyl (C) ₅ ) And n-hexyl (C) ₆ )。

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group, with or without heteroatoms, which may be a single ring structure or two or more rings, wherein the heteroatoms are selected from phosphorus, sulfur, oxygen and/or nitrogen.

The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

The term "pharmaceutically acceptable salts" refers to acid and/or base salts of the compounds of the present invention with inorganic and/or organic acids and bases, and also includes zwitterionic salts (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound may be obtained by appropriately (e.g., equivalent) mixing the above compound with a certain amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered by evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization. The salt in the invention can be hydrochloride, sulfate, citrate, benzene sulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or solubilizers, for example, starch, lactose, sucrose, glucose, mannitol, and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) Disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such a composition may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, especially cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

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 nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The pharmaceutically acceptable auxiliary components of the invention refer to substances contained in the dosage form in addition to the active ingredients, such as cyclodextrin, arginine or meglumine. The cyclodextrin is selected from alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and (C) _1-4 Alkyl) -alpha-cyclodextrin, (C) _1-4 Alkyl) -beta-cyclodextrin, (C) _1-4 Alkyl) -gamma-cyclodextrin, (hydroxy-C) _1-4 Alkyl) -alpha-cyclodextrin, (hydroxy-C) _1-4 Alkyl) -beta-cyclodextrin, (hydroxy-C) _1-4 Alkyl) -gamma-cyclodextrin, (carboxy-C) _1-4 Alkyl) -alpha-cyclodextrin, (carboxy-C) _1-4 Alkyl) -beta-cyclodextrin, (carboxy-C) _1-4 Alkyl) -gamma-cyclodextrin, saccharide ethers of alpha-cyclodextrin, saccharide ethers of beta-cyclodextrin, saccharide ethers of gamma-cyclodextrin, sulfobutyl ethers of alpha-cyclodextrin, sulfobutyl ethers of beta-cyclodextrin and sulfobutyl ethers of gamma-cyclodextrin. The auxiliary components also comprise a pharmaceutically acceptable carrier, adjuvant or vehicle. Can be used in pharmaceutically acceptable pharmaceutical composition, such as ion exchanger, aluminum oxide, aluminum stearate, and lecithin; buffer substances include phosphate, glycine, arginine, sorbic acid, and the like.

The invention provides a coumarin derivative with a novel structure. Biological experiments show that the compound has a strong in-vitro anti-fibrosis effect, can obviously reduce the deposition of intercellular collagen fibers on the NRK-49F cells induced by TGF-beta, and has an inhibitory effect on the migration of HUVEC cells. Meanwhile, the compounds with the structure have certain curative effect on mouse models of hepatic fibrosis induced by carbon tetrachloride and pulmonary fibrosis induced by bleomycin, have low toxicity, and provide a new choice for clinically treating fibrotic diseases including hepatic fibrosis and pulmonary fibrosis.

Drawings

FIG. 1 shows scratch test of

compounds

21a, 25k, 29f, 29i, and 29 j;

FIG. 2 is a graph of the inhibitory effect of Compound 9d on COL1A1, α -SMA, and p-Smad3 protein expression;

FIG. 3 is a graph of the inhibitory effect of Compound 29f on COL1A1, α -SMA, and p-Smad3 protein expression;

figure 4 is the in vivo effect of compound 9d in pulmonary fibrosis mice;

figure 5 is the in vivo effect of compound 29f in pulmonary fibrosis mice;

figure 6 is a graph of the in vivo effect of compound 9d in mice model of acute liver injury.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The abbreviations for the starting materials or reagents used in the examples are now set forth in the following tables.

Reagent	Abbreviations	Reagent	Abbreviations
				Tetrabutylammonium bromide	TBAB	Methylene dichloride	DCM
Ethyl acetate	EtOAc	Tris (dibenzylidene-BASE acetone) dipalladium (0)	Pd ₂ (dba) ₃
				Methanol	MeOH	4, 5-bis-diphenylphosphino-9, 9-dimethylxanthene	Xantphos
Ethanol	EtOH	Acetone (II)	ACE
				Isopropanol (I-propanol)	IPA	Toluene	TOL
N, N-dimethylformamide	DMF	Reduced iron powder	Fe
				Phosphorus pentoxide	P ₂ O ₅	Water (W)	H ₂ O
Hydrochloric acid solution	HCl	Potassium iodide	KI
				Aqueous hydrobromic acid solution	HBr	Potassium tert-butoxide	tBu-OK
Potassium carbonate	K ₂ CO ₃	Palladium on carbon (10%)	Pd/C
				Nitrogen gas	N ₂	Nitrogen gas	N ₂
Petroleum ether	PE	Hydrogen gas	H ₂
				Methane acyl chloride	MsCl	Phosphorus oxychloride	POCl ₃
Triethylamine	Et ₃ N

Example 1

4-Hydroxycoumarin (4.86g, 3 mol) was added to a reaction flask, 400ml of toluene was added as a solvent, tetrabutylammonium bromide (TBAB, 14.49g,4.5 mol) was added thereto with stirring at room temperature, and then the temperature was slowly raised to 90 ℃ from room temperature. At 90 ℃ adding P ₂ O ₅ (17.04g, 12mol) was added in three portions over an hour, and stirring was continued for about 6-8 hours at a temperature of 90 ℃ and the reaction was checked by spotting. And (3) cooling the reaction liquid to room temperature, carrying out rotary evaporation to obtain a black viscous liquid, adding 1.2L of ice water and 400ml of ethyl acetate for extraction, combining three organic phases, and carrying out rotary drying. Adding n-heptane at room temperature, heating to 90 deg.C for dissolving completely, vacuum filtering to obtain yellowish liquid, cooling to room temperature to obtain yellow crystal, vacuum filtering, and vacuum drying to obtain yellow product with yield of 58%.

¹ H NMR(400MHz,DMSO-d ₆ )δ:6.9(s,1H),7.29(m,1H),7.4(m,2H),7.80(m,1H).MS(ESI),m/z:224.94[M+H] ⁺ .

The following compound 2-1 was synthesized with reference to example 1, except that the starting material 4-hydroxycoumarin in example 1 was replaced with 4-hydroxy-7-methoxycoumarin.

¹ H NMR(400MHz,DMSO)δ7.72–7.67(m,1H),7.02(dd,J＝7.5,2.2Hz,2H),6.87(s,1H),3.88(s,3H).MS(ESI),m/z:254.95[M+H] ⁺ .

The following compound 2-2 was synthesized with reference to example 1, except that the starting material 4-hydroxycoumarin in example 1 was replaced with 4-hydroxy-6-nitrocoumarin.

¹ H NMR(400MHz,DMSO-d ₆ )δ:8.63(d,J＝2.9Hz,1H),8.35(d,J＝2.9Hz,1H),8.33(d,J＝2.9Hz,1H),7.43(d,J＝9.0Hz,1H).MS(ESI),m/z:269.93[M+H] ⁺ .

Example 2

4-bromocoumarin (1g, 3.93mmol) and p-diphenylamine (0.43g, 3.93mmol) were placed in a 50mL dry clean round bottom flask, an appropriate amount of ethanol was added as a reaction solvent, and Et was then added ₃ N (1.1mL, 7.88mmol) is dripped into a reaction bottle, the reaction bottle is lifted into a heating reactor, after stirring for 3h at 65 ℃, the reaction is lifted to room temperature, stirring is continued for two hours, a large amount of solid is separated out, filtration is carried out, ethanol is recrystallized to obtain the target product, the yield is 57%, and white powdery solid is obtained.

¹ H NMR(400MHz,DMSO-d6)δ:1H NMR(400MHz,DMSO)δ8.96(s,1H),8.11(d,J＝9.0Hz,1H),6.96(m,3H),6.90(d,J＝2.5Hz,1H),6.64(m,2H),5.21(s,2H),4.87(s,1H),3.86(s,3H).MS(ESI),m/z:253.09[M+H] ⁺ .

The following synthesis of compound 3-1 refers to example 2 except that the starting compound 2 in example 2 is replaced with compound 2-1.

¹ H NMR(400MHz,DMSO-d ₆ )δ:1H NMR(400MHz,DMSO)δ8.96(s,1H),8.11(d,J＝9.0Hz,1H),6.96(m,3H),6.90(d,J＝2.5Hz,1H),6.64(m,2H),5.21(s,2H),4.87(s,1H),3.86(s,3H).MS(ESI),m/z:283.10[M+H] ⁺ .

The following synthesis of compound 3-2 refers to example 2 except that the starting compound 2 in example 2 is replaced with compound 2-2.

¹ H NMR(400MHz,DMSO-d ₆ )δ:9.52(s,1H),9.28(d,J＝2.1Hz,1H),8.45(dd,J＝9.1,2.2Hz,1H),7.57(d,J＝9.1Hz,1H),7.00(d,J＝8.4Hz,2H),6.67(d,J＝8.4Hz,2H),5.27(s,2H),5.12(s,1H).MS(ESI),m/z:298.08[M+H] ⁺ .

Synthesis of the following compounds 3-3 reference is made to example 2 except that p-diphenylamine as a raw material in example 2 is replaced with 2-fluoro-1, 4-phenylenediamine.

¹ H NMR(400MHz,DMSO-d ₆ )δ:9.11(s,1H),8.19(d,J＝7.8Hz,1H),7.64(t,J＝7.6Hz,1H),7.37(dd,J＝10.9,8.3Hz,2H),7.04(d,J＝12.0Hz,1H),6.87(m,2H),5.28(s,2H),5.07(s,1H).MS(ESI),m/z:271.08[M+H] ⁺ .

Example 3

Placing 4-bromocoumarin (5g, 0.022mol), p-hydroxyphenol (4.65g, 0.033mol) and potassium carbonate (7.7g, 0.055mol) in a round-bottom flask of a proper size, adding 300ml of acetone as a solvent, heating to 65 ℃ and stirring, monitoring the reaction by TLC, removing the acetone by rotary evaporation, extracting by an EA/H2O system, combining EA layers, washing the EA layers by 10 percent NaOH for three times, drying anhydrous Na2SO4, and carrying out rotary evaporation to obtain a solid, and recrystallizing the solid by ethanol to finally obtain the target product with the yield of 72% as a yellow powdery solid. 1H NMR (400MHz, DMSO-d 6) delta: 8.39 (m, 2H), 7.90 (d, J =8.8Hz, 1H), 7.65 (m, 2H), 7.11 (d, J =2.4Hz, 1H), 7.05 (dd, J =8.8,2.4Hz, 1H), 5.35 (s, 1H), 3.90 (s, 3H). MS (ESI), m/z: 284.05M + H] ⁺ .

The following compounds were synthesized with reference to example 3, except that the starting compound 2 in example 3 was replaced with compound 2-1.

¹ H NMR(400MHz,DMSO-d ₆ )δ:8.39(m,2H),7.90(d,J＝8.8Hz,1H),7.65(m,2H),7.11(d,J＝2.4Hz,1H),7.05(dd,J＝8.8,2.4Hz,1H),5.35(s,1H),3.90(s,3H).MS(ESI),m/z:314.06[M+H] ⁺ .

Example 4

Intermediate 6 (3 g, 0.01mol) was dispersed well in 200ml of 10% aqueous methanol solution, then reduced iron powder was added at room temperature, concentrated hydrochloric acid (5 ml) was added dropwise when the temperature was raised to 85 ℃, reflux was maintained at 85 ℃, after completion of the reaction monitoring by spotting, yellow filtrate was obtained by suction filtration while hot, and the solvent was removed by rotary evaporation. Then adding 50ml of water, pulping for 10 minutes, filtering to obtain a solid, and drying in vacuum to obtain a yellow solid.

¹ H NMR(400MHz,DMSO-d ₆ )δ:8.01(d,J＝7.0Hz,1H),7.74(dd,J＝11.4,4.2Hz,1H),7.45(dd,J＝12.7,7.9Hz,2H),6.98(d,J＝8.7Hz,2H),6.70(m,2H),5.26(s,2H),5.18(s,1H).MS(ESI),m/z:254.07[M+H] ⁺ .

The following compounds were synthesized in accordance with example 4, and the compounds were substituted with the corresponding starting materials according to the structures of the compounds.

¹ H NMR(400MHz,DMSO-d ₆ )δ:7.90(d,J＝8.8Hz,1H),7.02(m,2H),7.01(m,2H),6.61(m,2H),5.24(s,2H),5.01(s,1H),3.95(m,3H).MS(ESI),m/z:284.09[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.60(s,1H),7.95(d,J＝7.7Hz,1H),7.60(t,J＝7.4Hz,1H),7.36(q,J＝8.8Hz,5H),7.25(t,J＝7.3Hz,1H),5.33(s,1H).MS(ESI),m/z:253.09[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.59(s,1H),7.97(d,J＝7.3Hz,1H),7.60(s,1H),7.44–7.10(m,3H),6.67(dd,J＝41.1,9.7Hz,2H),5.84(s,2H),5.33(s,1H).MS(ESI),m/z:271.08[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.87(s,1H),7.88(d,J＝9.5Hz,1H),7.45–7.35(m,2H),7.31(d,J＝6.8Hz,2H),7.25–7.12(m,2H),6.52(d,J＝9.4Hz,1H).MS(ESI),m/z:271.08[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.59(s,1H),7.86(d,J＝9.5Hz,1H),7.67(d,J＝8.5Hz,1H),7.33(dd,J＝15.3,8.0Hz,2H),7.14(d,J＝7.4Hz,1H),6.91–6.77(m,2H),6.38(d,J＝9.4Hz,1H).MS(ESI),m/z:271.08[M+H] ⁺ .

Example 5

4-hydroxy-2-quinolinone (8.05g, 5 mol) was charged into a reaction flask, 600ml of toluene was added as a solvent, tetrabutylammonium bromide (TBAB, 24.15g,7.5 mol) was added with stirring at room temperature, and then the temperature was slowly raised to 90 ℃ from room temperature. At 90 ℃ adding P ₂ O ₅ (28.40g, 20mol) was added in five portions over one hour, and stirring was continued at 90 ℃ for about 6 to 8 hours, and the reaction was checked to be complete by spotting. And (3) cooling the reaction liquid to room temperature, carrying out rotary evaporation to obtain a black viscous liquid, adding a proper amount of petroleum ether to fully dissolve the black viscous liquid, and carrying out suction filtration to obtain a light yellow filtrate and a brown solid. Adding proper amount of stoneAnd (3) sufficiently dissolving the filter residue by using the oil ether, combining the three filtrates, and carrying out spin drying and vacuum drying to obtain a yellow product with the yield of 40%.

1H NMR(400MHz,DMSO)δ7.82(dd,J＝8.1,1.0Hz,1H),7.61(ddd,J＝8.4,7.3,1.3Hz,1H),7.36(d,J＝8.2Hz,1H),7.33–7.28(m,1H),7.03(d,J＝1.5Hz,1H).MS(ESI),m/z:285.91[M+H] ⁺ .

Example 6

4-hydroxy-2-quinolinone (8.05g, 5 mol) is added into a reaction bottle, 200ml of phosphorus oxychloride is slowly dripped under stirring at room temperature, and a large amount of heat and gas are discharged during dripping. After the dropwise addition, the reaction solution is brown viscous, and then the temperature is slowly raised to 100 ℃ for reaction for about 6 hours. And (3) after the reaction is completely detected by a spot plate, cooling the reaction liquid to room temperature, slowly pouring the reaction liquid into a large amount of ice water to be fully cooled, adding ethyl acetate for extraction, combining three organic phases, and spin-drying. Adding a proper amount of 60-80 mesh silica gel to mix the sample, and purifying the mixture by passing through a column by using a rapid column chromatography machine to obtain a light yellow product, wherein the yield is 53%.

¹ H NMR(400MHz,DMSO)δ7.78(dd,J＝8.2,1.1Hz,1H),7.63(ddd,J＝8.3,7.2,1.3Hz,1H),7.33(d,J＝8.2Hz,1H),7.30–7.26(m,1H),7.08(d,J＝1.5Hz,1H).MS(ESI),m/z:197.98[M+H] ⁺ .

Example 7

2, 4-dibromoquinoline (2.5g, 8.77mmol) was dissolved in 60ml of 1, 4-dioxane solvent, the temperature was raised to 90 ℃ and then 10ml of an aqueous solution containing 40% HBr was added dropwise thereto, the reaction was maintained at 90 ℃ for about 6 hours, and after completion of the reaction was monitored by spotting, the solvent was removed by rotary evaporation. Then the appropriate amount of saturated NaHCO was added ₃ In the aqueous solution, a large amount of bubbles are generated, simultaneously, a white solid is precipitated, and a white solid product is obtained by suction filtration and drying, wherein the yield is 95%.

¹ H NMR(400MHz,DMSO)δ12.06(s,1H),7.82(dd,J＝8.1,1.0Hz,1H),7.61(ddd,J＝8.4,7.3,1.3Hz,1H),7.36(d,J＝8.2Hz,1H),7.33–7.28(m,1H),7.03(d,J＝1.5Hz,1H).MS(ESI),m/z:223.96[M+H] ⁺ .

Synthesis of the following compounds with reference to example 7, HBr was replaced by concentrated HCl.

¹ H NMR(400MHz,DMSO)δ12.05(s,1H),7.82(dd,J＝8.2,1.1Hz,1H),7.63(ddd,J＝8.3,7.2,1.3Hz,1H),7.40(d,J＝8.2Hz,1H),7.36–7.29(m,1H),7.11(d,J＝1.5Hz,1H).MS(ESI),m/z:180.01[M+H] ⁺ .

Example 8

4-bromo-2-quinolinone (2.2g, 9.8mmol), N-Boc-p-phenylenediamine (2.45g, 11.78mmol), potassium tert-butoxide (2.75g, 24.55mmol), pd ₂ (dba) ₃ (0.90g, 0.99mmol) and Xantphos (1.41g, 2.45mmol) were thoroughly dispersed in 100ml of 1, 4-dioxane, heated from room temperature to 100 ℃ under nitrogen, and the temperature was maintained for an additional 12 hours, and the reaction was monitored on a dot-and-dash basis for completion. And (3) carrying out suction filtration on the reaction solution to obtain brown filtrate, adding silica gel to mix with the sample, and purifying by using a rapid column chromatography to obtain a brown product with the yield of 60%.

¹ H NMR(400MHz,DMSO)δ10.95(s,1H),9.41(s,1H),8.52(s,1H),8.10(d,J＝7.9Hz,1H),7.57–7.45(m,3H),7.29–7.23(m,1H),7.23–7.13(m,3H),5.46(d,J＝0.9Hz,1H),1.49(s,9H).MS(ESI),m/z:423.18[M+H] ⁺ .MS(ESI),m/z:352.16[M+H] ⁺ .

Example 9

The compound (1.5g, 4.27mmol) was placed in a 50ml round bottom flaskIn a vial, 20ml of trifluoroacetic acid (TFA) was added dropwise at room temperature, followed by stirring at room temperature for about 4 hours, and the reaction was monitored on a spot-on-plate for completion. Then the trifluoroacetic acid is removed by rotary evaporation, and saturated NaHCO is added ₃ And (3) dispersing the brown solid in water until no air bubbles are generated in the water solution, performing suction filtration and drying to obtain the brown solid with the yield of 93%.

¹ H NMR(400MHz,DMSO)δ10.82(s,1H),8.32(s,1H),8.09(d,J＝7.8Hz,1H),7.46(dd,J＝11.3,4.1Hz,1H),7.28–7.21(m,1H),7.18–7.08(m,1H),6.94(d,J＝8.6Hz,2H),6.67–6.60(m,2H),5.25(s,1H),5.14(s,2H).MS(ESI),m/z:252.10[M+H] ⁺ .

Example 10

4-Aminobenzenethiol (7.0g, 0.06mol) and K ₂ CO ₃ (12.42g, 0.09mol) was placed in a round-bottom flask, followed by addition of DMF (200 ml). After heating the reaction mixture to 130 deg.C, 4-chloro-2-quinolinone (5.37g, 0.03mol) was added and stirred for an additional 6 hours. After the reaction was monitored by TLC, the mixture was cooled to room temperature and 400ml of water was added to form a suspension mixture. The solid was obtained by filtration and after purification on silica gel column, a brown solid was obtained in 58% yield.

¹ H NMR(400MHz,DMSO)δ11.53(s,1H),7.82(d,J＝7.6Hz,1H),7.59–7.52(m,1H),7.33(d,J＝8.1Hz,1H),7.23(t,J＝7.7Hz,3H),6.72(d,J＝8.5Hz,2H),5.74(s,2H),5.56(s,1H).MS(ESI),m/z:269.06[M+H] ⁺ .

Example 11

4-hydroxy-2-quinolinone (9.66g, 0.06mol) and K ₂ CO ₃ (12.42g, 0.09mol) was placed in a round bottom flask, then DMF (200 ml) was added and heated to 100 ℃. 1-fluoro-4-nitrobenzene (4.23g, 0.03mol) was dissolved in another 20ml of DMF and the solution was added dropwise to the preparation over a half hourIn the prepared mixture. The mixture was kept stirring at 100 ℃ for about 6 hours, and after monitoring the reaction by TLC, the mixture was cooled to room temperature and 400ml of water was added to form a suspension mixture. The solids were then collected by a filter. After drying by vacuum oven, a pale yellow solid was obtained in 72% yield).

¹ H NMR(400MHz,DMSO)δ11.75(s,1H),8.37(d,J＝7.4Hz,2H),7.87(d,J＝6.6Hz,1H),7.62(s,1H),7.55(d,J＝6.8Hz,2H),7.47–7.16(m,2H),5.72(s,1H).MS(ESI),m/z:283.06[M+H] ⁺ .

Example 12

4-hydroxy-2-quinolinone (9.66g, 0.06mol) and K ₂ CO ₃ (12.42g, 0.09mol) was placed in a round-bottom flask and dispersed in DMF (200 ml). 3, 4-difluoro-nitrobenzene (4.77g, 0.03mol) was dissolved in another 20ml of DMF and the solution was added dropwise to the previous mixture at room temperature with stirring. The mixture was kept stirring at room temperature for about 6 hours and after monitoring the reaction by TLC, 400ml of water was added to form a suspension mixture. The solids were then collected by a filter. After drying by vacuum oven, a pale yellow solid was obtained in 68% yield).

1H NMR(400MHz,DMSO)δ11.76(s,1H),8.45(dd,J＝10.4,2.7Hz,1H),8.22(ddd,J＝9.0,2.6,1.3Hz,1H),7.93(dd,J＝8.0,1.0Hz,1H),7.81–7.72(m,1H),7.68–7.58(m,1H),7.40(d,J＝8.2Hz,1H),7.32–7.23(m,1H),5.68(s,1H).MS(ESI),m/z:301.05[M+H] ⁺ .

Synthesis of the following Compounds reference example 12 is made, depending on the structure of the compound, to the corresponding starting materials

¹ H NMR(400MHz,DMSO)δ11.91(s,1H),8.34(dd,J＝10.8,2.7Hz,1H),8.09–8.02(m,1H),7.88(d,J＝9.6Hz,1H),7.56(d,J＝1.2Hz,1H),7.43(d,J＝2.3Hz,2H),7.13(t,J＝8.7Hz,1H),6.56(d,J＝9.6Hz,1H).MS(ESI),m/z:301.05[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.73(s,1H),8.40(dd,J＝10.7,2.7Hz,1H),8.13(ddd,J＝9.1,2.6,1.3Hz,1H),7.92(d,J＝9.6Hz,1H),7.76(d,J＝8.5Hz,1H),7.39(t,J＝8.6Hz,1H),7.04–6.97(m,2H),6.46(d,J＝9.5Hz,1H).MS(ESI),m/z:301.05[M+H] ⁺ .

Example 13

NaH (1.45g, 60.2mmol) and 7.5mL diethyl carbonate were placed in a round bottom flask of appropriate size, the flask was moved to a low temperature reactor and stirred at 0 ℃, then paeonol (2g, 12.04mmol) was dissolved in 10mL diethyl carbonate and placed in a 25mL constant pressure funnel, which was slowly dropped into the flask and stirred for twenty minutes while maintaining 0 ℃, the flask was moved to a heated reactor and stirred for 3 hours while monitoring the reaction by TLC, after the reaction was completed, heating was stopped, the flask was left at room temperature and carefully quenched with water, then excess diethyl carbonate was washed off with a large amount of ether (3X 25 mL), the ether layer was discarded, the aqueous layer was combined and acidified to pH 3 with 2N HCl (note: large amount of foam and white solid precipitated during acidification, which must be done with stirring), the cake was filtered and washed with water to obtain the desired product in a large amount, and vacuum dried to yield 69%, white solid was obtained as a powder.

¹ H NMR(400MHz,DMSO-d ₆ )δ:12.37(s,1H),7.72(d,J＝8.6Hz,1H),6.93(m,2H),5.47(s,1H),3.86(s,3H).MS(ESI),m/z:193.04[M+H] ⁺ .

Example 14

Sodium nitrate (0.52g and 6.17mmol) is placed in a round-bottom flask with a proper size, then 20mL of concentrated sulfuric acid is added into a reaction bottle, the reaction bottle is lifted into a low-temperature reactor and is stirred for ten minutes at 0 ℃, then a compound 4-hydroxycoumarin (1g and 6.17mmol) is added into the reaction bottle, the temperature is kept, the reaction is monitored by TLC (thin layer chromatography) after being continuously stirred for 1h, and after the reaction is finished, crushed ice is slowly added into the reaction bottle under low-temperature stirring until no excessive solid is separated out, the mixture is filtered, a filter cake is washed for a plurality of times by water and is dried in vacuum, and the target product is obtained, the yield is 42%, and white powdery solid is obtained.

¹ H NMR(400MHz,DMSO-d ₆ )δ:8.53(d,J＝2.6Hz,1H),8.45(dd,J＝9.1,2.7Hz,1H),7.63(dd,J＝17.8,8.2Hz,1H),5.71(s,1H).MS(ESI),m/z:208.02[M+H] ⁺ .

Example 15

Compound 3 (2.82g, 1.0 mol) was dissolved in 10mL of anhydrous DMF solution and placed therein, and then the reaction flask was taken up into a low-temperature reactor, stirred at 0 ℃ and dropped into Et ₃ N (1.81ml, 1.3 mol), continuously stirring for ten minutes while keeping the temperature after dripping, then dripping chloroacetyl chloride (960ul, 1.2mol), continuously stirring for 2 hours while keeping the temperature after dripping, extracting the reaction to room temperature, stirring for 2 hours at normal temperature, monitoring the reaction by TLC, adding water to quench the reaction after the reaction is finished, simultaneously precipitating a large amount of solid, filtering, washing the filter cake with water, and drying in vacuum to obtain the target product, wherein the yield is 85 percent, and the white powdery solid is obtained.

¹ H NMR(400MHz,DMSO-d ₆ )δ:9.06(s,1H),8.21(d,J＝7.5Hz,1H),7.63(t,J＝7.3Hz,1H),7.35(m,2H),6.99(d,J＝8.5Hz,2H),6.66(d,J＝8.5Hz,2H),5.23(s,2H),5.00(s,1H).MS(ESI),m/z:329.06[M+H] ⁺ .

The following compounds were synthesized in accordance with example 15, and the compounds were substituted with the corresponding starting materials according to the structures of the compounds.

¹ H NMR(400MHz,DMSO-d ₆ )δ:10.42(s,1H),9.19(s,1H),8.14(d,J＝9.0Hz,1H),7.70(d,J＝8.8Hz,2H),7.33(d,J＝8.8Hz,2H),6.99(dd,J＝8.9,2.5Hz,1H),6.94(d,J＝2.5Hz,1H),5.11(s,1H),4.27(s,2H),3.87(s,3H).MS(ESI),m/z:329.07[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ:10.17(s,1H),9.33(s,1H),8.21(d,J＝7.6Hz,1H),7.96(t,J＝8.7Hz,1H),7.67(t,J＝7.4Hz,1H),7.39(m,3H),7.24(d,J＝8.8Hz,1H),5.41(s,1H),4.37(s,2H).MS(ESI),m/z:347.05[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ:10.45(s,1H),9.74(s,1H),9.31(d,J＝2.5Hz,1H),8.48(dd,J＝9.1,2.6Hz,1H),7.73(d,J＝8.8Hz,2H),7.61(d,J＝9.1Hz,1H),7.37(d,J＝8.8Hz,2H),5.35(s,1H),4.28(s,2H).MS(ESI),m/z:374.05[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ:10.59(s,1H),8.04(d,J＝7.8Hz,1H),7.76(dd,J＝16.1,8.5Hz,3H),7.47(m,2H),7.35(m,2H),5.22(s,1H),4.31(s,2H).MS(ESI),m/z:330.05[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ:10.49(s,1H),7.94(d,J＝8.8Hz,1H),7.75(d,J＝8.8Hz,2H),7.33(d,J＝8.8Hz,2H),7.05(m,2H),5.05(s,1H),4.29(s,2H),3.89(s,3H).MS(ESI),m/z:360.06[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.02(s,1H),10.89(s,1H),8.71(s,1H),8.19(d,J＝8.1Hz,1H),7.72(d,J＝8.6Hz,2H),7.50(t,J＝7.6Hz,2H),7.33–7.27(m,3H),7.16(t,J＝7.5Hz,1H),5.58(s,1H),4.35(s,2H).MS(ESI),m/z:328.07[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.65(s,1H),10.76(s,1H),7.84(dd,J＝8.3,3.2Hz,3H),7.67–7.54(m,3H),7.36(d,J＝8.2Hz,1H),7.26(t,J＝7.5Hz,1H),5.58(s,1H),4.34(s,2H).MS(ESI),m/z:345.04[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.55(s,1H),10.47(s,1H),7.97(d,J＝8.1Hz,1H),7.74(d,J＝8.6Hz,2H),7.60(t,J＝7.7Hz,1H),7.35(d,J＝8.2Hz,1H),7.26(dd,J＝14.6,8.0Hz,3H),5.32(s,1H),4.29(s,2H).MS(ESI),m/z:329.06[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),10.69(s,1H),7.92(dd,J＝56.5,9.6Hz,2H),7.62(s,1H),7.47(s,2H),7.37(d,J＝7.3Hz,1H),7.27(s,1H),5.35(s,1H),4.31(s,2H).MS(ESI),m/z:347.05[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.80(s,1H),11.31(s,1H),7.83(dd,J＝17.0,5.7Hz,2H),7.46(d,J＝8.7Hz,1H),7.37(d,J＝8.9Hz,1H),7.25(dd,J＝8.9,2.5Hz,1H),7.18(dd,J＝15.0,5.7Hz,2H),6.48(d,J＝9.5Hz,1H),4.36(s,2H).MS(ESI),m/z:347.05[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.50(s,1H),11.01(s,1H),7.84(s,2H),7.64(d,J＝8.3Hz,1H),7.45(s,1H),7.33(d,J＝8.3Hz,1H),6.80(dd,J＝40.5,13.0Hz,2H),6.36(d,J＝8.9Hz,1H),4.34(s,2H).MS(ESI),m/z:347.05[M+H] ⁺ .

Example 16

Compound 4 (100mg, 0.3mmol) and KI (126mg, 0.75mmol) were dispersed in 5ml of anhydrous DMF, and pyrrolidine (21mg, 0.45mmol) was added dropwise with stirring at room temperature, followed by stirring at room temperature overnight. Detecting the reaction completely by a plate on the next day, adding 15ml of water while stirring, precipitating a large amount of white solid, performing suction filtration to obtain a solid, and drying at 50 ℃ to obtain a white powder solid.

¹ H NMR(400MHz,DMSO-d ₆ )δ:10.44(s,1H),9.28(s,1H),8.24(d,J＝7.7Hz,1H),7.68(m,3H),7.38(m,4H),5.24(s,1H),4.28(s,2H).MS(ESI),m/z:329.06[M+H] ⁺ .

The following compounds were synthesized in accordance with example 16, by substituting the corresponding starting materials according to the structures of the compounds.

¹ H NMR(400MHz,DMSO)δ9.79(s,1H),9.27(s,1H),8.24(d,J＝7.1Hz,1H),7.75(d,J＝8.8Hz,2H),7.65(m,1H),7.39(dd,J＝13.1,7.8Hz,2H),7.32(d,J＝8.7Hz,2H),5.21(s,1H),3.09(s,2H),2.49(s,4H),1.58(d,J＝4.8Hz,4H),1.41(s,2H).MS(ESI),m/z:378.19[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.87(s,1H),9.27(s,1H),8.24(d,J＝7.3Hz,1H),7.75(d,J＝8.8Hz,2H),7.66(m,1H),7.39(dd,J＝13.1,7.8Hz,2H),7.32(d,J＝8.8Hz,2H),5.21(s,1H),3.65(m,4H),3.15(s,2H),2.53(m,4H).MS(ESI),m/z:380.15[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.92(s,1H),9.41(d,J＝6.5Hz,1H),8.32(d,J＝8.0Hz,1H),7.77(dd,J＝14.6,8.7Hz,2H),7.66(t,J＝7.3Hz,1H),7.35(dd,J＝12.7,10.5,5.9Hz,4H),5.21(s,1H),3.15(s,2H),2.55(s,4H),2.42(s,4H),2.20(s,3H).MS(ESI),m/z:393.18[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.78(s,1H),9.27(s,1H),8.24(d,J＝7.2Hz,1H),7.76(d,J＝8.8Hz,2H),7.70–7.63(m,1H),7.39(dd,J＝13.1,7.8Hz,2H),7.32(d,J＝8.8Hz,2H),5.22(s,1H),3.10(s,2H),2.79(t,J＝8.8Hz,2H),2.08(td,J＝10.7,3.7Hz,1H),1.85–1.52(m,6H),0.85(d,J＝6.3Hz,4H).MS(ESI),m/z:392.19[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.84(s,1H),9.24(s,1H),8.18(d,J＝8.9Hz,1H),7.81(d,J＝8.7Hz,2H),7.29(d,J＝8.7Hz,2H),6.99(dd,J＝9.0,2.4Hz,1H),6.93(d,J＝2.4Hz,1H),5.08(s,1H),4.01(d,J＝5.9Hz,2H),3.87(s,3H),2.48(m,4H),1.24(m,4H).MS(ESI,m/z):394.10[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.77(s,1H),9.17(s,1H),8.14(d,J＝9.0Hz,1H),7.74(d,J＝8.8Hz,2H),7.29(d,J＝8.8Hz,2H),6.99(dd,J＝8.9,2.5Hz,1H),6.94(d,J＝2.5Hz,1H),5.08(s,1H),3.87(s,3H),3.08(s,2H),2.47(d,J＝5.2Hz,4H),1.57(dd,J＝10.9,5.6Hz,4H),1.41(d,J＝4.9Hz,2H).MS(ESI,m/z):408.01[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.85(s,1H),9.18(s,1H),8.14(d,J＝9.0Hz,1H),7.74(d,J＝8.8Hz,2H),7.30(d,J＝8.8Hz,2H),6.99(dd,J＝8.9,2.5Hz,1H),6.94(d,J＝2.5Hz,1H),5.08(s,1H),3.87(s,3H),3.65(m,4H),3.15(s,2H),2.53(d,J＝4.6Hz,4H).MS(ESI,m/z):410.10[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.80(s,1H),9.17(s,1H),8.14(d,J＝9.0Hz,1H),7.73(d,J＝8.8Hz,2H),7.29(d,J＝8.8Hz,2H),6.99(dd,J＝9.0,2.5Hz,1H),6.94(d,J＝2.5Hz,1H),5.08(s,1H),3.87(s,3H),3.13(s,2H),2.37(d,J＝24.4Hz,8H),2.19(s,3H).MS(ESI,m/z):423.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.77(s,1H),9.17(s,1H),8.14(d,J＝9.0Hz,1H),7.74(d,J＝8.8Hz,2H),7.29(d,J＝8.8Hz,2H),6.99(dd,J＝8.9,2.5Hz,1H),6.94(d,J＝2.5Hz,1H),5.08(s,1H),3.87(s,3H),3.09(s,2H),2.79(s,2H),2.07(d,J＝10.7,3.7Hz,2H),1.69(m,5H),0.85(d,J＝6.3Hz,3H).MS(ESI,m/z):422.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.81(s,1H),9.74(s,1H),9.32(d,J＝2.5Hz,1H),8.48(dd,J＝9.1,2.5Hz,1H),7.77(d,J＝8.8Hz,2H),7.62(t,J＝9.9Hz,1H),7.33(d,J＝8.8Hz,2H),5.34(d,J＝7.2Hz,1H),3.10(s,2H),2.79(t,J＝8.7Hz,2H),2.08(td,J＝10.7,3.6Hz,1H),1.67(m,6H),0.86(d,J＝6.3Hz,3H).MS(ESI),m/z:437.17[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.85(s,1H),8.36(s,1H),8.30(d,J＝9.8Hz,1H),7.52(d,J＝8.7Hz,2H),7.37(t,J＝8.2Hz,1H),7.08(d,J＝8.7Hz,2H),6.89(d,J＝8.2Hz,1H),6.77(d,J＝8.1Hz,1H),3.31(s,2H),2.05(m,4H),1.22(m,4H),0.85(d,J＝6.9Hz,2H).MS(ESI),m/z:423.16[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.83(s,1H),9.72(s,1H),9.31(d,J＝2.2Hz,1H),8.47(dd,J＝9.1,2.4Hz,1H),7.76(d,J＝8.7Hz,2H),7.58(t,J＝16.5Hz,1H),7.33(d,J＝8.7Hz,2H),5.33(s,1H),3.12(d,J＝15.0Hz,2H),2.54(s,4H),2.40(s,4H),2.19(s,3H).MS(ESI),m/z:438.17[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.89(s,1H),9.73(s,1H),9.32(d,J＝2.5Hz,1H),8.48(dd,J＝9.1,2.6Hz,1H),7.77(d,J＝8.8Hz,2H),7.61(d,J＝9.1Hz,1H),7.33(d,J＝8.8Hz,2H),5.33(s,1H),3.65(m,4H),3.16(s,2H),2.51(s,4H).MS(ESI),m/z:425.14[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.84(s,1H),9.72(s,1H),9.31(d,J＝2.5Hz,1H),8.48(dd,J＝9.1,2.5Hz,1H),7.78(d,J＝8.8Hz,2H),7.61(d,J＝9.1Hz,1H),7.32(d,J＝8.8Hz,2H),5.32(s,1H),3.27(d,J＝8.6Hz,2H),2.64(d,J＝26.1Hz,4H),1.76(t,J＝3.4Hz,4H).MS(ESI),m/z:409.14[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.68(s,1H),9.32(s,1H),8.21(d,J＝7.2Hz,1H),8.12(t,J＝8.8Hz,1H),7.67(t,J＝7.8Hz,1H),7.38(m,3H),7.23(d,J＝8.7Hz,1H),5.36(s,1H),3.13(s,2H),2.50(s,4H),1.58(m,4H),1.43(d,J＝5.0Hz,2H).MS(ESI),m/z:382.15[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.68(s,1H),9.32(s,1H),8.21(d,J＝7.2Hz,1H),8.12(t,J＝8.8Hz,1H),7.67(t,J＝7.8Hz,1H),7.38(m,3H),7.23(d,J＝8.7Hz,1H),5.36(s,1H),3.13(s,2H),2.50(s,4H),1.58(m,4H),1.43(d,J＝5.0Hz,2H).MS(ESI),m/z:396.16[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.66(s,1H),9.33(s,1H),8.21(d,J＝7.6Hz,1H),8.04(t,J＝8.7Hz,1H),7.67(t,J＝7.5Hz,1H),7.39(m,3H),7.23(d,J＝8.4Hz,1H),5.37(s,1H),3.65(s,4H),3.20(s,2H),2.56(s,4H).MS(ESI),m/z:398.14[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.64(s,1H),9.32(s,1H),8.21(d,J＝7.2Hz,1H),8.11(t,J＝8.8Hz,1H),7.67(t,J＝7.8Hz,1H),7.38(m,3H),7.23(d,J＝8.7Hz,1H),5.36(s,1H),3.17(d,J＝10.5Hz,2H),2.57(s,4H),2.39(s,4H),2.19(s,3H).MS(ESI),m/z:411.18[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.66(s,1H),9.32(s,1H),8.16(m,2H),7.67(t,J＝7.6Hz,1H),7.39(dt,J＝20.4,10.1Hz,3H),7.22(d,J＝8.4Hz,1H),5.36(s,1H),3.13(s,2H),2.80(d,J＝9.0Hz,2H),2.01(m,3H),1.61(d,J＝45.4Hz,4H),0.87(d,J＝6.1Hz,3H).MS(ESI),m/z:410.18[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ8.11(d,J＝8.3Hz,2H),7.58(m,2H),7.30(m,4H),5.12(s,1H),3.66(t,J＝6.3Hz,4H),3.31(s,2H),1.95(m,4H).MS(ESI),m/z:365.14[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.86(s,1H),8.05(dd,J＝7.9,1.3Hz,1H),7.77(m,3H),7.48(dd,J＝13.7,7.6Hz,2H),7.38(d,J＝8.8Hz,2H),5.20(s,1H),4.13(s,2H),3.45(s,2H),3.07(s,3H),1.77(s,5H). ¹³ CNMR(101MHz,DMSO):δ168.26,166.50,162.78,154.20,151.61,133.90,126.92,123.02,121.20,117.08,115.21,98.09,63.46,51.10,25.12,24.54.MS(ESI),m/z:379.16[M+H] ⁺

¹ H NMR(400MHz,DMSO)δ9.94(s,1H),8.04(dd,J＝7.9,1.3Hz,1H),7.82(d,J＝9.0Hz,2H),7.76(m,1H),7.47(dd,J＝14.0,7.5Hz,2H),7.32(d,J＝9.0Hz,2H),5.20(s,1H),3.65(m,4H),3.16(s,2H),2.53(d,J＝4.6Hz,4H).MS(ESI),m/z:381.14[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.89(s,1H),8.04(m,1H),7.77(m,3H),7.47(dd,J＝14.2,7.6Hz,2H),7.31(d,J＝8.9Hz,2H),5.21(s,1H),3.14(s,2H),2.60(d,J＝58.5Hz,4H),2.39(s,4H),2.18(s,3H).MS(ESI),m/z:394.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ9.94(s,1H),8.03(dd,J＝7.9,1.3Hz,1H),7.72(d,J＝9.0Hz,2H),7.65(m,1H),7.47(dd,J＝14.0,7.5Hz,2H),7.32(d,J＝9.0Hz,2H),5.20(s,1H),3.16(s,2H),2.80(d,J＝9.0Hz,2H),2.53(d,J＝4.6Hz,4H),2.01(m,3H),1.61(d,J＝45.4Hz,4H),0.87(d,J＝6.1Hz,3H).MS(ESI),m/z:393.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ:9.93(s,1H),7.94(d,J＝8.8Hz,1H),7.80(d,J＝8.9Hz,2H),7.29(d,J＝8.9Hz,2H),7.05(m,2H),5.04(s,1H),3.89(s,3H),3.27(d,J＝8.6Hz,2H),2.64(d,J＝26.1Hz,4H),1.76(t,J＝3.4Hz,4H).MS(ESI),m/z:395.15[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ:9.02(s,1H),7.88(d,J＝8.8Hz,1H),7.76(d,J＝8.9Hz,2H),7.49(d,J＝8.9Hz,2H),7.05(m,2H),5.02(s,1H),3.89(s,3H),3.45(s,2H),3.07(s,3H),1.77(s,5H).MS(ESI),m/z:409.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ:9.93(s,1H),7.94(d,J＝8.8Hz,1H),7.80(d,J＝8.9Hz,2H),7.29(d,J＝8.9Hz,2H),7.05(m,2H),5.04(s,1H),3.89(s,3H),3.62(m,8H),3.14(d,J＝15.3Hz,2H).MS(ESI),m/z:411.15[M+H] ⁺ .

¹ H NMR(400MHz,DMSO-d6)δ:10.03(s,1H),7.98(d,J＝8.8Hz,1H),7.72(d,J＝8.9Hz,2H),7.39(d,J＝8.9Hz,2H),7.05(m,2H),5.02(s,1H),3.89(s,3H),2.60(d,J＝58.5Hz,4H),2.39(s,4H),2.18(s,3H).MS(ESI),m/z:424.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ:9.93(s,1H),7.94(d,J＝8.8Hz,1H),7.80(d,J＝8.9Hz,2H),7.29(d,J＝8.9Hz,2H),7.05(m,2H),5.04(s,1H),3.89(s,3H),3.62(m,9H),3.14(d,J＝15.3Hz,3H).MS(ESI),m/z:423.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.96(s,1H),9.74(s,1H),8.55(s,1H),8.10(d,J＝7.8Hz,1H),7.70(d,J＝8.8Hz,2H),7.49(t,J＝7.3Hz,1H),7.26(t,J＝8.8Hz,3H),7.17(t,J＝7.6Hz,1H),5.54(s,1H),3.25(s,2H),2.60(d,J＝5.3Hz,4H),1.80–1.72(m,4H).MS(ESI),m/z:363.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.96(s,1H),9.72(s,1H),8.55(s,1H),8.10(d,J＝7.9Hz,1H),7.68(d,J＝8.8Hz,2H),7.49(t,J＝7.2Hz,1H),7.26(dd,J＝8.1,6.2Hz,3H),7.17(t,J＝7.6Hz,1H),5.54(d,J＝1.3Hz,1H),3.12(s,2H),2.51(s,4H),2.36(d,J＝24.3Hz,4H),2.18(s,3H).MS(ESI),m/z:392.22[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.96(s,1H),9.87(s,1H),8.55(s,1H),8.10(d,J＝7.8Hz,1H),7.69(d,J＝8.8Hz,2H),7.49(t,J＝7.2Hz,1H),7.26(dd,J＝7.9,5.2Hz,3H),7.17(t,J＝7.6Hz,1H),5.54(s,1H),3.84(dt,J＝11.4,3.4Hz,2H),3.33(s,2H),3.28(dd,J＝11.5,2.1Hz,2H),2.68–2.58(m,1H),1.77(d,J＝12.5Hz,2H),1.31(ddd,J＝14.5,11.5,3.7Hz,2H).MS(ESI),m/z:393.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.97(s,1H),9.71(s,1H),8.56(s,1H),8.11(d,J＝7.8Hz,1H),7.69(d,J＝8.8Hz,2H),7.49(dd,J＝11.3,4.1Hz,1H),7.27(t,J＝7.6Hz,3H),7.20–7.13(m,1H),5.55(s,1H),3.07(s,2H),2.50(td,J＝3.9,2.1Hz,4H),1.62–1.53(m,4H),1.45–1.37(m,2H).MS(ESI),m/z:377.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.98(s,1H),9.82(s,1H),8.57(s,1H),8.11(d,J＝7.7Hz,1H),7.70(d,J＝8.8Hz,2H),7.55–7.44(m,1H),7.27(dd,J＝7.9,5.2Hz,3H),7.20–7.13(m,1H),5.55(s,1H),3.50(dd,J＝10.1,5.8Hz,4H),3.18(s,2H),2.58–2.52(m,2H),2.50–2.46(m,2H),2.00(s,3H).MS(ESI),m/z:420.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.00(s,1H),9.93(s,1H),8.65(s,1H),8.16(d,J＝8.1Hz,1H),7.71(d,J＝8.8Hz,2H),7.50(t,J＝7.7Hz,1H),7.28(dd,J＝11.4,8.6Hz,3H),7.17(t,J＝7.3Hz,1H),5.56(s,1H),3.39(s,4H),3.19(s,2H),2.51–2.47(m,4H),1.41(s,9H).MS(ESI),m/z:478.24[M+H] ⁺ .

¹ H NMR(400MHz,MeOD)δ8.13–8.08(m,1H),7.69(d,J＝8.8Hz,2H),7.58(t,J＝7.7Hz,1H),7.41–7.26(m,4H),5.86(s,1H),3.49(s,2H),2.88(q,J＝7.2Hz,4H),1.20(t,J＝7.2Hz,6H).MS(ESI),m/z:365.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.98(s,1H),9.71(s,1H),8.57(s,1H),8.12(d,J＝8.0Hz,1H),7.70(d,J＝8.7Hz,2H),7.50(t,J＝7.4Hz,1H),7.27(t,J＝8.1Hz,3H),7.18(t,J＝7.5Hz,1H),5.56(s,1H),3.10(s,2H),2.85(d,J＝11.4Hz,2H),2.14(t,J＝10.7Hz,2H),1.60(d,J＝11.4Hz,2H),1.33–1.21(m,3H),0.92(d,J＝6.0Hz,3H).MS(ESI),m/z:391.21[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.97(s,1H),9.74(s,1H),8.56(s,1H),8.11(d,J＝8.0Hz,1H),7.69(d,J＝8.5Hz,2H),7.50(t,J＝7.4Hz,1H),7.27(t,J＝7.4Hz,3H),7.18(t,J＝7.4Hz,1H),5.55(s,1H),3.13(s,2H),2.52(s,4H),2.45(s,4H),2.34(dd,J＝14.1,7.0Hz,2H),1.00(t,J＝7.1Hz,3H).MS(ESI),m/z:406.22[M+H] ⁺ .

¹ H NMR(400MHz,MeOD)δ8.08(d,J＝7.9Hz,1H),7.68(d,J＝8.8Hz,2H),7.57(t,J＝7.2Hz,1H),7.40–7.26(m,5H),5.86(s,1H),3.82–3.74(m,4H),3.20(s,2H),2.65–2.58(m,4H).MS(ESI),m/z:379.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.96(s,1H),9.78(s,1H),8.57(s,1H),8.12(d,J＝8.0Hz,1H),7.70(d,J＝8.7Hz,2H),7.50(t,J＝7.6Hz,1H),7.27(t,J＝7.6Hz,3H),7.18(t,J＝7.6Hz,1H),5.55(s,1H),3.16(s,2H),3.02–2.95(m,2H),2.87–2.77(m,1H),2.57(s,6H),2.22(t,J＝11.2Hz,2H),1.89(d,J＝11.7Hz,2H),1.73–1.60(m,2H).MS(ESI),m/z:420.23[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ10.98(s,1H),10.35(s,1H),8.58(s,1H),8.11(d,J＝8.1Hz,1H),7.66(d,J＝8.7Hz,2H),7.51(t,J＝7.6Hz,1H),7.29(t,J＝9.0Hz,3H),7.18(t,J＝7.6Hz,1H),5.58(s,1H),3.80(s,2H),2.94(q,J＝7.1Hz,2H),1.18(dd,J＝9.0,5.4Hz,3H).MS(ESI),m/z:337.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.70(s,1H),10.53(s,1H),7.96(dd,J＝19.8,5.3Hz,2H),7.62(d,J＝7.1Hz,2H),7.43(t,J＝8.7Hz,2H),7.27(s,1H),5.34(s,1H),3.44(s,2H),2.70(s,4H),1.78(s,4H).MS(ESI),m/z:382.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.67(s,1H),10.28(s,1H),7.96(dd,J＝19.0,10.6Hz,2H),7.60(dd,J＝16.8,8.6Hz,2H),7.42(dd,J＝16.5,8.4Hz,2H),7.27(t,J＝7.5Hz,1H),5.34(s,1H),3.19(s,2H),2.55(s,4H),2.44(s,4H),2.21(s,3H).MS(ESI),m/z:411.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.61(s,1H),10.26(s,1H),7.97(dd,J＝9.0,6.3Hz,2H),7.62(dd,J＝12.0,4.8Hz,2H),7.41(q,J＝8.9Hz,2H),7.27(t,J＝8.0Hz,1H),5.34(s,1H),3.13(s,2H),2.49–2.42(m,4H),1.61–1.52(m,4H),1.40(d,J＝4.8Hz,2H).MS(ESI),m/z:396.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.69(s,1H),10.46(s,1H),7.97(t,J＝10.6Hz,2H),7.62(s,2H),7.47–7.39(m,2H),7.27(t,J＝7.5Hz,1H),5.34(s,1H),3.50(d,J＝3.8Hz,4H),3.26(s,2H),2.56(s,2H),2.50–2.46(m,2H),2.00(s,3H).MS(ESI),m/z:439.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.70(s,1H),10.42(s,1H),8.02–7.92(m,2H),7.66–7.57(m,2H),7.43(t,J＝9.0Hz,2H),7.30–7.23(m,1H),5.34(s,1H),3.36(s,4H),3.23(s,2H),2.63(s,6H),1.06(d,J＝5.1Hz,3H).MS(ESI),m/z:425.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),10.08(s,1H),7.95(dd,J＝30.0,10.2Hz,2H),7.58(dd,J＝20.2,7.5Hz,2H),7.49–7.19(m,3H),5.34(s,1H),3.40(s,4H),3.20(s,2H),2.50(s,4H),1.41(s,9H).MS(ESI),m/z:497.22[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.67(s,1H),10.39(s,1H),8.02–7.92(m,2H),7.66–7.57(m,2H),7.43(dd,J＝17.8,8.7Hz,2H),7.27(dd,J＝11.2,4.1Hz,1H),5.34(s,1H),3.24(s,2H),3.00(s,3H),2.66(s,6H),2.24(t,J＝11.4Hz,2H),2.00(d,J＝10.9Hz,2H),1.77(dd,J＝11.9,3.2Hz,2H).MS(ESI),m/z:439.21[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.67(s,1H),10.31(s,1H),7.97(ddd,J＝15.4,10.6,1.6Hz,2H),7.66–7.55(m,2H),7.42(dd,J＝18.4,9.1Hz,2H),7.31–7.21(m,1H),5.34(s,1H),3.70–3.58(m,4H),3.20(s,2H),2.57–2.52(m,4H).MS(ESI),m/z:398.15[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.67(s,1H),10.32(s,1H),7.97(ddd,J＝15.5,10.6,1.6Hz,2H),7.67–7.58(m,2H),7.48–7.37(m,2H),7.27(dd,J＝11.3,4.0Hz,1H),5.35(s,1H),2.70(d,J＝5.8Hz,4H),1.06(t,J＝7.1Hz,6H).MS(ESI),m/z:384.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.66(s,1H),10.36(s,1H),8.04–7.89(m,2H),7.61(dd,J＝14.2,7.3Hz,2H),7.42(dd,J＝20.1,8.7Hz,2H),7.27(t,J＝7.6Hz,1H),5.34(s,1H),3.17(d,J＝4.2Hz,2H),2.92(s,2H),2.26(s,2H),1.61(d,J＝11.8Hz,2H),1.30(dd,J＝21.9,10.5Hz,3H),0.92(d,J＝6.0Hz,3H).MS(ESI),m/z:410.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),10.16(s,1H),7.99(dd,J＝8.0,1.0Hz,1H),7.93(dd,J＝13.1,2.3Hz,1H),7.65–7.59(m,1H),7.54(dd,J＝8.9,1.4Hz,1H),7.44(t,J＝8.9Hz,1H),7.37(d,J＝8.1Hz,1H),7.27(dd,J＝11.6,4.5Hz,1H),5.34(s,1H),3.84(dt,J＝11.4,3.4Hz,2H),3.33(s,2H),3.28(dd,J＝11.5,2.0Hz,3H),2.64(ddd,J＝14.3,10.2,4.0Hz,1H),1.77(dd,J＝12.5,1.7Hz,2H),1.35–1.25(m,2H).MS(ESI),m/z:412.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),7.99(d,J＝7.3Hz,1H),7.92(dd,J＝13.1,2.3Hz,1H),7.66–7.59(m,1H),7.54(dd,J＝8.9,1.4Hz,1H),7.44(t,J＝8.9Hz,1H),7.37(d,J＝8.2Hz,1H),7.27(t,J＝7.4Hz,1H),5.34(s,1H),3.33(s,3H),2.60(q,J＝7.1Hz,2H),1.06(t,J＝7.1Hz,3H).MS(ESI),m/z:356.14[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),9.99(s,1H),7.90–7.82(m,3H),7.59(ddd,J＝10.9,6.2,1.6Hz,3H),7.35(d,J＝7.7Hz,1H),7.29–7.24(m,1H),5.58(s,1H),3.12(s,2H),2.50–2.45(m,4H),1.58(dt,J＝11.0,5.7Hz,4H),1.42(d,J＝5.0Hz,2H).MS(ESI),m/z:394.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),9.98(s,1H),7.86(dd,J＝10.6,8.0Hz,3H),7.62–7.55(m,3H),7.35(d,J＝7.7Hz,1H),7.29–7.23(m,1H),5.57(d,J＝1.1Hz,1H),3.13(s,2H),2.85(d,J＝11.6Hz,2H),2.14(t,J＝10.4Hz,2H),1.60(d,J＝10.2Hz,2H),1.36–1.21(m,3H),0.92(d,J＝6.1Hz,3H).MS(ESI),m/z:408.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.62(s,1H),10.04(s,1H),7.87(dd,J＝15.0,8.2Hz,3H),7.58(t,J＝7.8Hz,3H),7.35(d,J＝8.0Hz,1H),7.27(d,J＝8.0Hz,1H),5.57(s,1H),3.30(s,2H),2.61(s,4H),1.77(dd,J＝6.5,3.2Hz,4H).MS(ESI),m/z:380.14[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),10.09(s,1H),7.86(dd,J＝14.6,8.3Hz,3H),7.64–7.55(m,3H),7.35(d,J＝8.2Hz,1H),7.25(dd,J＝11.3,4.1Hz,1H),5.57(s,1H),3.51(dd,J＝10.1,6.2Hz,4H),3.23(s,2H),2.59–2.53(m,2H),2.49(d,J＝9.3Hz,2H),2.00(s,3H).MS(ESI),m/z:437.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.67(s,1H),10.19(s,1H),7.86(dd,J＝18.9,8.2Hz,3H),7.58(t,J＝8.3Hz,3H),7.38(d,J＝8.2Hz,1H),7.25(t,J＝7.6Hz,1H),5.57(s,1H),3.17(d,J＝7.6Hz,2H),2.54(s,4H),2.38(s,4H),2.18(s,3H).MS(ESI),m/z:409.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.59(s,1H),10.03(s,1H),7.82(dd,J＝11.4,8.6Hz,3H),7.55(dd,J＝12.6,8.0Hz,3H),7.31(d,J＝8.2Hz,1H),7.22(t,J＝7.6Hz,1H),5.53(s,1H),3.73–3.53(m,4H),3.15(s,2H),2.47(s,4H).MS(ESI),m/z:396.14[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),9.95(s,1H),7.87(dd,J＝18.0,8.4Hz,3H),7.63–7.54(m,3H),7.35(d,J＝8.2Hz,1H),7.30–7.21(m,1H),5.58(s,1H),3.21(s,2H),2.63(q,J＝7.1Hz,4H),1.04(t,J＝7.1Hz,6H).MS(ESI),m/z:382.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.64(s,1H),10.06(s,1H),7.87(dd,J＝12.9,8.5Hz,3H),7.64–7.54(m,3H),7.36(d,J＝8.2Hz,1H),7.26(t,J＝7.5Hz,1H),5.57(s,1H),3.18(s,2H),2.96(d,J＝11.2Hz,2H),2.52(s,1H),2.37(s,6H),2.19(t,J＝11.2Hz,2H),1.82(d,J＝11.0Hz,2H),1.66–1.52(m,2H).MS(ESI),m/z:437.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),10.07(s,1H),7.86(dd,J＝10.9,8.7Hz,3H),7.59(dd,J＝12.5,8.0Hz,3H),7.35(d,J＝8.2Hz,1H),7.26(t,J＝7.6Hz,1H),5.57(s,1H),3.40(m,4H),3.21(s,2H),2.49(m,4H),1.41(s,9H).MS(ESI),m/z:495.21[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),10.02(s,1H),7.86(t,J＝8.5Hz,3H),7.65–7.52(m,3H),7.35(d,J＝8.1Hz,1H),7.26(t,J＝7.6Hz,1H),5.57(s,1H),3.17(s,2H),2.55(s,4H),2.44(s,4H),2.34(q,J＝7.1Hz,2H),1.00(t,J＝7.2Hz,3H).MS(ESI),m/z:423.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.62(s,1H),10.03(s,1H),7.86(t,J＝9.1Hz,3H),7.67–7.54(m,3H),7.35(d,J＝8.1Hz,1H),7.25(dd,J＝11.3,4.1Hz,1H),5.57(s,1H),3.33(s,2H),2.60(q,J＝7.1Hz,2H),1.06(t,J＝7.1Hz,3H).MS(ESI),m/z:354.13[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.63(s,1H),10.16(s,1H),7.85(dd,J＝13.4,4.8Hz,3H),7.68–7.51(m,3H),7.35(d,J＝7.8Hz,1H),7.30–7.21(m,1H),5.57(s,1H),3.84(dt,J＝11.4,3.4Hz,2H),3.38(s,2H),3.28(m,3H),2.64(ddd,J＝14.3,10.2,4.0Hz,1H),1.82–1.74(m,2H),1.36–1.23(m,2H).MS(ESI),m/z:410.15[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.83(s,1H),7.98(d,J＝7.9Hz,1H),7.80(d,J＝8.9Hz,2H),7.60(t,J＝7.7Hz,1H),7.36(d,J＝8.2Hz,1H),7.25(t,J＝7.3Hz,3H),5.32(s,1H),3.10(s,2H),2.48(s,4H),1.64–1.54(m,4H),1.42(d,J＝4.8Hz,2H).MS(ESI),m/z:378.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.55(s,1H),9.82(s,1H),7.97(d,J＝7.8Hz,1H),7.80(d,J＝8.8Hz,2H),7.60(t,J＝7.4Hz,1H),7.36(d,J＝8.2Hz,1H),7.25(t,J＝7.1Hz,3H),5.32(s,1H),3.11(s,2H),2.85(d,J＝11.4Hz,2H),2.13(t,J＝11.0Hz,2H),1.59(d,J＝11.1Hz,2H),1.36–1.21(m,3H),0.91(d,J＝5.9Hz,3H).MS(ESI),m/z:392.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.89(s,1H),7.97(d,J＝7.3Hz,1H),7.78(d,J＝8.9Hz,2H),7.64–7.56(m,1H),7.36(d,J＝8.2Hz,1H),7.26(dd,J＝7.5,4.9Hz,3H),5.31(s,1H),3.19(s,2H),2.63(s,8H),2.35(s,3H).MS(ESI),m/z:393.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.55(s,1H),9.96(s,1H),7.97(d,J＝7.8Hz,1H),7.78(d,J＝8.9Hz,2H),7.61(t,J＝7.7Hz,1H),7.36(d,J＝8.2Hz,1H),7.26(t,J＝7.9Hz,3H),5.30(s,1H),3.31(s,2H),3.02(m,6H),2.83(m,4H),1.20(t,J＝7.1Hz,3H).MS(ESI),m/z:421.20[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.87(s,1H),7.98(d,J＝7.9Hz,1H),7.81(d,J＝8.8Hz,2H),7.60(t,J＝7.6Hz,1H),7.36(d,J＝8.2Hz,1H),7.25(t,J＝8.8Hz,3H),5.31(s,1H),3.27(s,2H),2.61(s,4H),1.76(s,4H).MS(ESI),m/z:364.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.90(s,1H),7.97(d,J＝8.5Hz,1H),7.79(d,J＝8.9Hz,2H),7.62–7.56(m,1H),7.35(d,J＝8.2Hz,1H),7.25(dd,J＝8.1,4.1Hz,3H),5.30(s,1H),3.39(m,4H),3.18(s,2H),2.49(m,4H),1.40(s,9H).MS(ESI),m/z:479.23[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.84(s,1H),7.97(d,J＝8.0Hz,1H),7.79(d,J＝8.8Hz,2H),7.60(t,J＝7.6Hz,1H),7.35(d,J＝8.2Hz,1H),7.25(t,J＝7.2Hz,3H),5.31(s,1H),3.12(s,2H),2.92(d,J＝11.5Hz,2H),2.51(m,1H),2.26(s,6H),2.16(t,J＝11.2Hz,3H),1.76(d,J＝11.7Hz,2H),1.54(dd,J＝20.4,11.4Hz,2H).MS(ESI),m/z:421.22[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.98(s,1H),7.97(d,J＝7.5Hz,1H),7.78(d,J＝8.9Hz,2H),7.59(t,J＝7.2Hz,1H),7.35(d,J＝8.2Hz,1H),7.25(dd,J＝7.8,5.5Hz,3H),5.31(s,1H),3.84(d,J＝11.3Hz,2H),3.34(d,J＝6.9Hz,2H),3.30–3.24(m,2H),2.64(m,J＝10.3,5.1Hz,1H),1.77(d,J＝11.4Hz,2H),1.31(dd,J＝19.3,11.2Hz,2H).MS(ESI),m/z:407.21[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.53(s,1H),9.89(s,1H),7.97(d,J＝8.0Hz,1H),7.78(d,J＝8.9Hz,2H),7.59(t,J＝7.7Hz,1H),7.35(d,J＝8.2Hz,1H),7.25(dd,J＝7.6,5.1Hz,3H),5.30(s,1H),3.69–3.61(t,4H),3.15(s,2H),2.53(t,J＝4.4Hz,4H).MS(ESI),m/z:380.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.92(s,1H),7.97(d,J＝7.7Hz,1H),7.79(d,J＝8.9Hz,2H),7.60(t,J＝7.2Hz,1H),7.35(d,J＝8.2Hz,1H),7.24(dd,J＝7.9,4.3Hz,3H),5.31(s,1H),3.51(d,J＝4.1Hz,4H),3.20(s,2H),2.57–2.53(m,2H),2.48(d,J＝4.8Hz,2H),2.00(s,3H).MS(ESI),m/z:447.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.79(s,1H),7.97(d,J＝7.3Hz,1H),7.80(d,J＝8.9Hz,2H),7.62–7.57(m,1H),7.35(d,J＝8.2Hz,1H),7.29–7.19(m,3H),5.31(s,1H),3.18(s,2H),2.62(q,J＝7.1Hz,4H),1.04(t,J＝7.1Hz,6H).MS(ESI),m/z:366.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.56(s,1H),10.45(s,1H),7.97(d,J＝7.3Hz,1H),7.76(d,J＝8.9Hz,2H),7.68–7.55(m,1H),7.36(d,J＝8.2Hz,1H),7.32–7.21(m,3H),5.31(s,1H),3.77(s,2H),2.91(q,J＝7.2Hz,2H),1.18(t,J＝7.2Hz,3H).MS(ESI),m/z:338.15[M+H] ⁺ .

1H NMR(400MHz,DMSO)δ10.04(s,1H),7.98(d,J＝8.0Hz,1H),7.91(d,J＝13.0Hz,1H),7.65–7.51(m,2H),7.47–7.33(m,2H),7.26(t,J＝7.6Hz,1H),5.34(s,1H),3.13(s,2H),2.79(s,4H),2.46(s,4H).MS(ESI),m/z:397.17[M+H] ⁺ .

1H NMR(400MHz,DMSO)δ11.68(s,1H),10.32(s,1H),7.96(t,J＝12.0Hz,2H),7.59(d,J＝7.9Hz,2H),7.41(d,J＝6.6Hz,2H),7.26(t,J＝7.5Hz,1H),5.33(s,1H),3.15(s,2H),2.50(m,8H),1.00(s,9H).MS(ESI),m/z:453.23[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.70(s,1H),10.53(s,1H),8.02–7.94(m,2H),7.62(t,J＝8.0Hz,2H),7.44(t,J＝8.7Hz,2H),7.27(t,J＝7.4Hz,1H),5.34(s,1H),3.74(s,2H),3.53(s,2H),3.27(s,2H),2.59(s,4H),1.98(ms,1H),0.71(m,J＝8.5Hz,4H).MS(ESI),m/z:465.19[M+H] ⁺ .

1H NMR(400MHz,DMSO)δ11.74(s,1H),9.97(s,1H),7.84(d,J＝8.7Hz,2H),7.51–7.03(m,5H),6.49(d,J＝9.0Hz,1H),3.11(s,2H),2.83(d,J＝8.2Hz,2H),2.13(s,2H),1.58(d,J＝10.7Hz,2H),1.46–1.16(m,3H),0.91(s,3H).MS(ESI),m/z:410.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.73(s,1H),9.98(s,1H),7.83(t,J＝10.4Hz,2H),7.42(d,J＝8.6Hz,1H),7.35–7.22(m,2H),7.17(dd,J＝17.8,8.7Hz,2H),6.49(d,J＝9.5Hz,1H),3.38(s,4H),3.17(s,2H),2.48(d,J＝4.9Hz,4H),1.40(s,9H).MS(ESI),m/z:497.23[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.73(s,1H),9.99(s,1H),7.88–7.79(m,2H),7.43(d,J＝8.9Hz,1H),7.32(d,J＝8.9Hz,1H),7.26(dd,J＝8.9,2.6Hz,1H),7.22–7.13(m,2H),6.50(d,J＝9.6Hz,1H),3.74(s,2H),3.54(s,2H),3.20(s,2H),2.56(s,2H),2.50(s,2H),1.97(m,J＝12.6,7.7,4.8Hz,1H),0.77–0.69(m,4H).MS(ESI),m/z:465.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.72(s,1H),9.93(s,1H),7.82(dd,J＝18.7,5.9Hz,2H),7.41(d,J＝9.0Hz,1H),7.31(d,J＝8.9Hz,1H),7.25(dd,J＝8.9,2.6Hz,1H),7.21–7.11(m,2H),6.49(d,J＝9.6Hz,1H),3.14(s,2H),2.54(m,4H),2.42(m,4H),2.25(s,3H).MS(ESI),m/z:411.10[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.72(s,1H),9.87(s,1H),7.88–7.78(m,2H),7.43(d,J＝8.9Hz,1H),7.36–7.23(m,2H),7.22–7.11(m,2H),6.49(d,J＝9.6Hz,1H),3.07(s,2H),2.45(s,4H),1.62–1.51(m,4H),1.40(d,J＝4.9Hz,2H).MS(ESI),m/z:396.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.72(s,1H),9.96(s,1H),7.82(dd,J＝16.8,5.9Hz,2H),7.42(d,J＝8.8Hz,1H),7.35–7.22(m,2H),7.22–7.11(m,2H),6.49(d,J＝9.6Hz,1H),3.70–3.59(m,4H),3.14(s,2H),2.50(d,J＝1.1Hz,4H).MS(ESI),m/z:398.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.73(s,1H),9.98(s,1H),7.82(dd,J＝16.0,5.8Hz,2H),7.43(d,J＝8.9Hz,1H),7.35–7.23(m,2H),7.23–7.13(m,2H),6.50(d,J＝9.6Hz,1H),3.50(d,J＝4.3Hz,4H),3.18(s,2H),2.53(d,J＝4.6Hz,2H),2.48–2.44(m,2H),2.00(s,3H).MS(ESI),m/z:439.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.72(s,1H),9.85(s,1H),7.89–7.79(m,2H),7.46(d,J＝8.9Hz,1H),7.31(d,J＝8.9Hz,1H),7.25(dd,J＝8.9,2.6Hz,1H),7.22–7.11(m,2H),6.49(d,J＝9.6Hz,1H),3.17(s,2H),2.60(q,J＝7.1Hz,4H),1.02(t,J＝7.1Hz,6H).MS(ESI),m/z:384.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.72(s,1H),9.95(s,1H),7.90–7.77(m,2H),7.44(d,J＝8.8Hz,1H),7.36–7.22(m,2H),7.22–7.11(m,2H),6.49(d,J＝9.6Hz,1H),3.27(s,2H),2.60(s,4H),1.76(s,4H).MS(ESI),m/z:382.15[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.73(s,1H),9.95(s,1H),7.82(t,J＝12.2Hz,2H),7.48–7.05(m,5H),6.50(d,J＝9.4Hz,1H),3.18(s,2H),2.97(d,J＝10.6Hz,3H),2.77–2.54(m,6H),2.23(t,J＝11.2Hz,2H),1.90(d,J＝9.1Hz,2H),1.66(d,J＝10.5Hz,2H).MS(ESI),m/z:439.21[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.74(s,1H),10.72(s,1H),7.90–7.69(m,2H),7.36–7.15(m,5H),6.51(d,J＝9.5Hz,1H),3.31(s,2H),3.03(dd,J＝14.4,7.1Hz,2H),1.25–1.14(t,3H).MS(ESI),m/z:356.14[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.73(s,1H),10.00(s,1H),7.82(t,J＝12.0Hz,2H),7.47–7.08(m,5H),6.50(d,J＝9.6Hz,1H),3.48(s,2H),3.32(s,2H),3.09(s,4H),2.70(s,2H),1.34(s,9H).MS(ESI),m/z:453.23[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.48(s,1H),9.94(s,1H),7.87(dd,J＝17.8,5.9Hz,2H),7.64(d,J＝8.7Hz,1H),7.51(d,J＝8.8Hz,1H),7.29(t,J＝9.0Hz,1H),6.85(dd,J＝8.6,2.4Hz,1H),6.76(d,J＝2.1Hz,1H),6.36(d,J＝9.5Hz,1H),3.11(s,2H),2.82(d,J＝11.4Hz,2H),2.12(t,J＝11.2Hz,2H),1.59(d,J＝11.2Hz,2H),1.42–1.19(m,3H),0.91(d,J＝6.0Hz,3H).MS(ESI),m/z:410.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.49(s,1H),10.04(s,1H),7.91–7.80(m,2H),7.64(d,J＝8.7Hz,1H),7.50(d,J＝8.8Hz,1H),7.30(t,J＝9.0Hz,1H),6.84(dd,J＝8.6,2.4Hz,1H),6.76(d,J＝2.1Hz,1H),6.36(d,J＝9.5Hz,1H),3.70–3.61(m,4H),2.51(s,4H).MS(ESI),m/z:498.15[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.49(s,1H),10.07(s,1H),7.92–7.78(m,2H),7.65(d,J＝8.6Hz,1H),7.46(d,J＝8.6Hz,1H),7.31(t,J＝9.0Hz,1H),6.91–6.70(m,2H),6.36(d,J＝9.5Hz,1H),3.31(s,2H),3.17(s,4H),2.77-2.45(d,J＝20.0Hz,7H).MS(ESI),m/z:411.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.48(s,1H),10.05(s,1H),7.91–7.81(m,2H),7.63(t,J＝9.9Hz,1H),7.49(d,J＝8.8Hz,1H),7.30(t,J＝9.0Hz,1H),6.83(dt,J＝16.9,8.5Hz,1H),6.76(d,J＝2.2Hz,1H),6.36(d,J＝9.5Hz,1H),3.58–3.45(m,4H),3.20(s,2H),2.53(dd,J＝8.6,3.8Hz,2H),2.49–2.44(m,2H),2.01(d,J＝10.8Hz,3H).MS(ESI),m/z:439.18[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.48(s,1H),10.03(s,1H),7.86(dd,J＝11.5,5.6Hz,2H),7.64(d,J＝8.6Hz,1H),7.49(d,J＝8.9Hz,1H),7.30(t,J＝9.0Hz,1H),6.84(dd,J＝8.6,2.1Hz,1H),6.76(s,1H),6.36(d,J＝9.5Hz,1H),3.37(d,J＝13.9Hz,4H),3.19(s,2H),2.48(s,3H),1.40(s,9H).MS(ESI),m/z:497.22[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.49(s,1H),9.98(d,J＝24.3Hz,1H),7.92–7.74(m,2H),7.62(t,J＝13.5Hz,1H),7.51(d,J＝8.6Hz,1H),7.28(t,J＝8.9Hz,1H),6.84(d,J＝6.8Hz,1H),6.76(s,1H),6.35(d,J＝9.4Hz,1H),3.27(s,2H),2.59(s,4H),1.76(s,4H).MS(ESI),m/z:482.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.48(s,1H),9.95(s,1H),7.86(dd,J＝19.2,5.9Hz,2H),7.64(d,J＝8.7Hz,1H),7.50(dd,J＝8.8,1.0Hz,1H),7.29(t,J＝9.0Hz,1H),6.84(dd,J＝8.6,2.4Hz,1H),6.76(d,J＝2.1Hz,1H),6.36(d,J＝9.5Hz,1H),3.09(s,2H),2.46(s,4H),1.64–1.50(m,4H),1.40(d,J＝4.2Hz,2H).MS(ESI),m/z:396.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.50(s,1H),10.05(s,1H),7.89–7.80(m,2H),7.65(d,J＝8.7Hz,1H),7.48(d,J＝8.8Hz,1H),7.31(t,J＝9.0Hz,1H),6.83(dd,J＝8.6,2.4Hz,1H),6.77(d,J＝2.1Hz,1H),6.36(d,J＝9.5Hz,1H),3.59–3.39(m,2H),3.32(s,2H),3.11(d,J＝39.2Hz,4H),2.70(s,2H),1.32(s,9H).MS(ESI),m/z:453.23[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.48(s,1H),10.06(s,1H),7.91–7.81(m,2H),7.64(d,J＝8.7Hz,1H),7.50(d,J＝8.8Hz,1H),7.30(t,J＝9.0Hz,1H),6.84(dd,J＝8.6,2.3Hz,1H),6.76(d,J＝2.1Hz,1H),6.36(d,J＝9.5Hz,1H),3.74(s,2H),3.54(s,2H),3.21(s,2H),2.57(s,2H),2.50–2.45(m,2H),2.03–1.91(m,1H),0.78–0.64(m,4H).MS(ESI),m/z:465.19[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.48(s,1H),10.02(s,1H),7.94–7.76(m,2H),7.65(d,J＝8.6Hz,1H),7.48(d,J＝8.6Hz,1H),7.30(t,J＝9.0Hz,1H),6.84(dd,J＝8.6,2.2Hz,1H),6.76(s,1H),6.36(d,J＝9.5Hz,1H),3.20(s,2H),2.99(d,J＝11.3Hz,3H),2.71(d,J＝12.9Hz,6H),2.24(m,J＝11.3Hz,2H),1.92(d,J＝11.0Hz,2H),1.68(m,J＝20.6,10.2Hz,2H).MS(ESI),m/z:439.21[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.53–11.42(m,1H),9.98–9.87(m,1H),7.95–7.76(m,2H),7.68–7.61(m,1H),7.58–7.50(m,1H),7.32–7.24(m,1H),6.89–6.83(m,1H),6.79–6.71(m,1H),6.39–6.31(m,1H),3.18(s,2H),2.67–2.56(m,4H),1.04(d,J＝7.1Hz,6H).MS(ESI),m/z:384.17[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.50(s,1H),7.83(dd,J＝17.3,12.1Hz,2H),7.65(d,J＝8.7Hz,1H),7.42(d,J＝8.7Hz,1H),7.33(t,J＝8.9Hz,1H),6.84(dd,J＝8.6,2.0Hz,1H),6.77(s,1H),6.36(d,J＝9.5Hz,1H),3.73(s,2H),2.87(q,J＝7.3Hz,2H),1.16(t,J＝7.3Hz,3H).MS(ESI),m/z:354.14[M+H] ⁺ .

¹ H NMR(400MHz,MeOD)δ8.11(d,J＝7.3Hz,1H),7.77(d,J＝8.9Hz,2H),7.67–7.60(m,1H),7.39(d,J＝8.2Hz,1H),7.34(t,J＝7.7Hz,1H),7.23(d,J＝9.0Hz,2H),5.57(s,1H),3.30(s,2H),3.16(s,4H),2.86(s,4H),1.32(s,9H).MS(ESI),m/z:379.16[M+H] ⁺ .

¹ H NMR(400MHz,DMSO)δ11.54(s,1H),9.98(s,1H),7.97(d,J＝7.5Hz,1H),7.78(d,J＝8.9Hz,2H),7.59(t,J＝7.2Hz,1H),7.35(d,J＝8.2Hz,1H),7.25(dd,J＝7.8,5.5Hz,3H),5.31(s,1H),3.84(d,J＝11.3Hz,2H),3.34(d,J＝6.9Hz,2H),3.30–3.24(m,2H),2.64(m,J＝10.3,5.1Hz,1H),1.77(d,J＝11.4Hz,2H),1.31(dd,J＝19.3,11.2Hz,2H).MS(ESI),m/z:394.17[M+H] ⁺ .

Pharmacodynamic test section

The following representative experiments, without limitation, were used to analyze the biological activity of the compounds of the present invention

1. In vitro Activity

Excess extracellular matrix (ECM) is composed mainly of collagen and is a pathological marker of fibrotic diseases, and therefore detection of collagen synthesis is an effective index for evaluating fibrotic diseases. According to our previous studies and other reports, rat fibroblasts induced by TGF-. Beta.s (NRK-49F) produce a number of extracellular collagens with characteristics similar to fibrosis in vivo. It has therefore been considered as an effective, convenient in vitro anti-fibrotic screening model.

1.1 anti-fibrotic Activity

NRK-49 cells were cultured in 1640 medium containing 10% FBS, 50U/ml penicillin and 50. Mu.g/ml streptomycin, and cultured in an incubator at 37 ℃ in 5% CO2 for passaging. NRK-49F cells were plated on 96-well plates (104 cells/well), cultured in 1640+5% FBS medium for 3d, the cell supernatant was removed, and 1640+1 ITS was added for 2d, the cell supernatant was removed, and 1640+1% ITS medium containing TGF-. Beta.5 ng/ml and 10. Mu.M of the compound to be screened was added for 2d. Cell supernatants were removed, fixed with 100. Mu.l/well 4% paraformaldehyde for 30min, washed twice with PBS, incubated with 100. Mu.l/well 0.1% PSR staining solution at room temperature for 4h, the staining solution was removed, washed 3 times with 100. Mu.l/well 0.1% acetic acid, air dried and photographed under a microscope. Adding 100 μ l/well 0.1M NaOH, shaking at room temperature for 30min to dissolve precipitate, measuring OD value of each well at 540nm wavelength of microplate reader, and screening out compounds with collagen deposition inhibiting effect. Total collagen accumulation inhibition = (administration a value-control a value)/(model a value-control a value) × 100%. All assays were repeated three times. Nintedanib as a positive drug.

1.2 cytotoxicity

NRK-49F cells were cultured at 0.5X 10 ⁵ The cells/ml were seeded in 96-well plates and cultured overnight in 100. Mu.l suspension, and the experimental groups were dosed with drug at a concentration of 10. Mu.M. After 72 hours, 20. Mu.l of 5% (m/v) MTT solution was added to each well and incubated for 4 hours in an incubator. Then 150. Mu.l DMSO was added to each well. Finally, the absorbance (a value) of each well was measured on a microplate reader. Survival = dose-zero a value) /(blank A value-zero A value) × 100%. All assays were repeated three times. Nintedanib as a positive drug.

1.3 collagen deposition inhibition and cytotoxicity test results

TABLE 1 collagen inhibition and cytotoxicity of NH-4 substituted coumarin parent Compounds

TABLE 2 collagen inhibition and cytotoxicity of O-4 substituted coumarin parent nucleus compounds

TABLE 3 collagen inhibition and cytotoxicity of NH-4 and S-4 substituted 2-quinolinone core Compounds

TABLE 4 collagen inhibition and cytotoxicity of O-4 substituted 2-quinolinone core Compounds

TABLE 5 collagen inhibition and cytotoxicity of O-6-and O-7-substituted 2-quinolinone core Compounds

1.4 scratch test and results thereof

The occurrence and the exacerbation of inflammation are often accompanied by phenomena such as vascular proliferation and the like caused by the migration of fibroblasts, immune cells and the like and the healing of tissues. We selected several compounds with good collagen deposition inhibition and lower toxicity on TGF- β induced NRK-49F cells, followed by co-incubation with HUVEC cells, and investigated their effect on human venous endothelial cell migration capacity after 0h, 12h, 24 h.

The experiment comprises the following specific steps: 1. firstly, a marker pen is used at the back of the six-hole plate, 3 transverse lines are homogenized by a ruler and transversely penetrate through the holes; 2. cells grown in log phase were plated with complete medium at 5X10 per well ⁶ Individual HUVEC cells; 3. in the next day, after the cells overgrow, the sterilized 200uL gun tip is perpendicular to the transverse line scratch on the back as much as possible, and the gun tip is perpendicular and cannot be inclined; 4. washing the cells with PBS for 3 times, washing off the scratched cells, and changing into a serum-free culture medium; 5. the blank group was not treated, the induction group induced cell migration with 10ng/ml TGF-. Beta.and the administration group was treated with 10ng/ml TGF-. Beta.and a gradient concentration of compound,the photographing represents 0h;6. put into 37 degree 5% CO ₂ The incubator is used for culturing, and the samples are photographed for 12 and 24 hours.

As shown in fig. 1,

compounds

21a, 25k, 29f were most potent inhibitors of HUVEC cell de1 migration, suggesting that these compounds may be inhibitory to excessive tissue healing and repair during fibrosis.

1.5 inhibition of COL1A1, alpha-SMA, and p-Smad3 protein expression by Compounds

Collagen type I α 1 (COL 1 A1), α -smooth muscle actin (α -SMA), is overexpressed in fibrotic diseases, which is generally considered to be a fibrotic marker. In order to further study the anti-fibrotic activity of compounds with higher collagen inhibition rates, their ability to inhibit COL1A1, α -SMA protein expression in vitro was studied. TGF- β binds to its receptor and recruits downstream Smad3 proteins, causing differentiation of resting fibroblasts into myofibroblasts secreting ECM. Therefore, the TGF-. Beta./Smad 3 pathway has been the target of many attempts at fibrosis.

Protein lysates were harvested using RIPA buffer with 1mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail, and protein concentrations were determined by bicinchoninic acid (BCA) kit. Each sample of the same protein (30-40. Mu.g) was separated on a 10% -12% SDS/PAGE gel for 20 minutes at 80V and then converted to 120V for 1 hour. Proteins were transferred to PVDF membranes for 1 hour at 80-100V, membranes were blocked in 5% (wt/vol) dry milk in PBS containing 1% Tween 20, and then incubated overnight at 4 ℃ with the indicated primary antibody. After incubation with HRP-conjugated secondary antibody, immunoreactive bands were detected with SuperLumia ECL Plus HRP substrate kit solution (K22030, abbkinene). The primary antibody used was: α -SMA (251411, ZENBIO), collagen I (14695-1-AP, protein technology), p-smad3 (AF 3363, affinity), GAPDH (AB 0037, abways), secondary antibodies used were: goat Anti-Rabbit IgG (H + L) HRP (AB 0101, abways), goat Anti-Mouse IgG (H + L) HRP (AB 0102, abways).

The inhibitory activity of

compounds

9d and 29f on COL1A1, α -SMA, and p-Smad3 protein expression can be seen in FIGS. 2 and 3.

2. Pharmacokinetic experiments

The oral bioavailability of Nintedanib is 12%, and the low bioavailability limits its further clinical use. The oral bioavailability of

compounds

9d and 29f was 39.88% and 41.55%, respectively, showing good druggability. The specific data are shown in tables 6 and 7.

TABLE 6 drug metabolism parameters of Compound 9d

TABLE 7 drug metabolism parameters of Compound 29f

The experimental scheme is as follows: the pharmacokinetics of the test compounds were examined by oral intravenous administration in SD rats.

Sample preparation: approximately 15mg of the test compound is weighed, and 2% Tween80, 2% ethanol and physiological saline are added to prepare 1mg/ml of 9d or 29f compound solution for administration.

Collecting samples: 5 SD rats (Chengdou Shuo laboratory animals Co., ltd., license number: SCXK (Chuan) 2015-030), male and female half, respectively, were administered intravenously at 5mg/kg and orally at 10mg/kg, 0min before administration and 5min, 15min, 30min, 1h, 2h, 4h, 6h, 8h, 10h and 24h after administration. Approximately 0.2ml of blood was collected, and the collected blood 3500r9d or 29f compound was centrifuged for 15min, and the supernatant plasma was collected and frozen at-20 ℃ for testing.

And (3) sample determination: preparing corresponding instruments and equipment; preparing a standard solution, accurately sucking a certain amount of stock solution (1 mg/ml) of a compound to be detected by using a pipette, diluting the stock solution into an EP (EP) tube by using methanol, and preparing the stock solution into a solution with the concentration respectively as follows: 19.53 39.06, 78.125, 156.25, 312.5, 625, 1250, 2500, 5000, 10000ng/ml standard solution of the compound to be tested; and (3) establishing a standard curve, namely taking 45 mu l of blank plasma in each EP tube, respectively adding 5 mu l of 9d or 29f compound standard solution with each concentration, uniformly mixing by vortex, adding 200 mu l of methanol precipitated protein containing 12.5ng/ml internal standard SAHA, carrying out vortex for 3min, centrifuging (13000rpm, 15min), and taking 2 mu l of supernate for sample injection. Obtaining the solution to be tested with the theoretical concentrations of the compounds to be tested of 1.95,3.91,7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000ng/ml respectively; preparing a plasma sample, thawing the collected plasma sample at room temperature, and mixing uniformly by vortex. Adding 50 μ l of plasma into each EP tube, adding 200 μ l of methanol containing 12.5ng/ml internal standard SAHA to precipitate protein, vortexing for 3min, centrifuging (13000rpm, 15min), and sampling 2 μ l for analysis; searching liquid phase conditions; finally, the data is collated and the relevant PK value is calculated.

3. In vivo activity on bleomycin-induced pulmonary fibrosis model in mice

Pulmonary fibrosis is a long-term progressive lung disease with excessive deposition of the extracellular matrix, which is composed of collagen, fibronectin and other inflammatory cells. To explore the anti-fibrotic efficacy of

compounds

9d, 29f in vivo, a bleomycin-induced pulmonary fibrosis model was used. Upon injection of bleomycin as a positive drug, nintedanib (Nintedanib) was orally administered at a dose of 50 mg/kg/day, respectively. The compounds were also administered orally at doses of 50 and 100 mg/kg/day, respectively.

Modeling:

preparing a reagent: a. preparing chloral hydrate: chloral hydrate is prepared into 3% solution, the weight of each mouse is 20g according to the proportion of 1ml/100g, and about 200 mul is injected; b. and (3) bleomycin preparation: a bottle of the Bleomycin is 15U, 5ml of normal saline is added firstly to prepare 3U/ml of liquid, and then the liquid is diluted to 1U/ml of working solution for use. Each mouse weighed about 20g at a rate of 3.5mg/kg, and was injected with about 75. Mu.l.

Modeling: after anaesthetizing, the mice were attached to a dissecting wax plate with an adhesive tape, hairs between the larynx and the chest were shaved off, the skin of the larynx was cut open, muscles around the trachea were bluntly separated with forceps, the trachea was picked out, 75 μ l of the prepared bleomycin solution was injected into the trachea, the mice were turned upside down to uniformly distribute the bleomycin in the lung, and the muscle layer and the trachea layer were sutured.

Hydroxyproline is one of the biomarkers for collagen and is therefore measured on

days

14 and 28 of dosing. The content of collagen in the right lung of the mouse is measured by using a conventional hydroxyproline determination kit (Nanjing institute of bioengineering, A030-2). Briefly, the right lung was dried and acid hydrolyzed, and the residue was filtered and adjusted to pH 6.5-8.0. Hydroxyproline analysis was performed using chloramine-T spectrophotometric absorbance.

3.1 in vivo Effect of Compound 9d in pulmonary fibrosis mice

The hydroxyproline content at day 14 and day 28 of the model group was significantly increased compared to that of the sham-operated group, which means that the bleomycin-induced pulmonary fibrosis model was successfully established. The Nintedanib group was distinguished from the sham group on day 28, and chronic dosing may reduce the degree of fibrosis. At a dose of 50 mg/kg/day, the in vivo effect of inhibiting fibrosis between compound 9d and the Nintedanib group was not significant. However, compound 9d at a dose of 100 mg/kg/day showed better therapeutic effect due to its lower hydroxyproline content (fig. 4A). In addition, the survival rate of 9d was about 90% for both doses (50 mg/kg/day and 100 mg/kg/day), which was superior to the Nintedanib and model groups, indicating that 9d had lower toxicity and better efficacy.

Lung tissue sections from the sham group showed clear lung architecture, ordered arrangement of bronchial ciliated epithelium, and little edema in the bronchial and perivascular interstitial spaces. In contrast, alveolar structures in the model group were destroyed, and degeneration and necrosis of bronchial epithelial cells were clearly observed (fig. 4C ↓). There is a lot of inflammatory cell infiltration in the alveolar interstitium, mainly lymphocytes and neutrophils (fig. 4D right ↓). Drug treatment (Nintedanib group, 9d group) delayed the reduction of alveolar number and destruction of alveolar structure. The inflammatory cell infiltration was also reduced in the drug-treated group near the pulmonary artery, especially in the 9d-100mg/kg group (fig. 4).

3.2 in vivo Effect of Compound 29f in pulmonary fibrosis mice

The hydroxyproline content at day 14 and day 28 of the model group was significantly increased compared to that of the sham-operated group, which means that the bleomycin-induced pulmonary fibrosis model was successfully established.

The sections of the sham group showed clear lung architecture, the ciliated bronchial epithelium was well-aligned, and there was little edema in the bronchial and perivascular interstitial spaces. In contrast, alveolar structures in the model group were destroyed, and degeneration and necrosis of bronchial epithelial cells were clearly observed. There is a lot of inflammatory cell infiltration in the alveolar interstitium, mainly lymphocytes and neutrophils. Drug treatment (Nintedanib group, 29f group) delayed the reduction of alveolar number and destruction of alveolar structure. Inflammatory cell infiltration was also reduced in the drug-treated group near the pulmonary artery, especially at 100mg/kg in the 29f group (fig. 5).

4. In vivo activity in carbon tetrachloride-induced liver fibrosis model

Since fibrosis has a common pathological mechanism, in order to explore the effect of compounds on hepatic fibrosis occurring in the early stage of a mouse model with acute hepatic injury in vivo, the mouse is induced by CCl4 to obtain the model with acute hepatic injury. The levels of Aspartate transaminase (AST) and Alanine transaminase (ALT) are criteria for evaluating the degree of liver damage, and higher levels of these are indicative of higher degrees of liver damage. We assessed the severity of liver damage by detecting changes in both levels; and further performing Hematoxylin-eosin staining (H & E) on the liver tissue section to directly observe the liver injury condition.

Establishing an acute liver injury model:

on the first day, each mouse was intraperitoneally injected with a CCl4-Olive Oil solution at a dose of 1ml/kg, on the second to third days, each mouse was intraperitoneally injected with a compound at a dose of 5mg/kg, on the fourth day, the mice were sacrificed, peripheral blood and liver of the mobile phone, and the next experiment was performed.

Formal experiments: according to CCl ₄ Olive Oil = 1.5 formulation CCl ₄ 4ml, at a dose of 1ml/kg, i.p. 25. Mu.l per mouse (25 g), 5mg of Nintedanib and compound, respectively, were weighed out according to 30% PEG400+0.5% of the% Tween80+5% glycerol pylene +64.5% ₂ O preparation scheme, grinding with mortar, and preparing into 1mg/mlSolution in CCl ₄ 24h after injection, mice were given the compound, and each mouse (25 g) was injected intraperitoneally with 125. Mu.l for two consecutive days.

And (3) killing: on the third day, after the abdominal cavity administration of the mouse is completed, the mouse is fasted for 16 hours, the mouse is killed on the next day, blood is taken from the eye socket and all livers are taken out, the subsequent experiment is reserved, in the administration period, after the peripheral blood of the mouse is placed in a refrigerator at 4 ℃ overnight, the mouse is centrifuged for 15min under the conditions of 4 ℃ and 3500rpm, the supernatant is taken out after centrifugation, the supernatant is frozen and stored in the refrigerator at-80 ℃ for peripheral blood ALT detection, after the liver of the mouse is taken out, PBS is washed once, water is absorbed on paper, half of the mouse is placed in 4% paraformaldehyde, and after the mouse is fixed for more than 48 hours, the subsequent dehydration work and wax block embedding are carried out for H & E staining and immunohistochemical experiments.

4.1 in vivo Effect of Compound 9d in pulmonary fibrosis mice

Compound 9d and the positive drug Nintedanib stably sustained decreased ALT levels compared to the model group. Meanwhile, as reflected in H & E pathological sections, both compound 9d and Nintedanib can significantly inhibit liver tissue necrosis, delay the progression of liver fibrosis, and protect the liver from damage (fig. 6).

5 in vitro and in vivo Activity summary of Compounds

The invention relates to preparation and activity of 4-substituted coumarin, 4-substituted-2-quinolinone, 6-substituted-2-quinolinone and 7-substituted-2-quinolinone derivatives. The compounds have certain inhibition on TGF-beta induced collagen deposition of NRK-49F cells and simultaneously have low cytotoxicity. It was also observed by western blot analysis that compounds with high collagen inhibition also inhibited TGF-beta induced expression of COL1A1, α -SMA and p-Smad3 proteins in vitro, indicating that compounds reduced extracellular matrix ECM deposition by inhibiting TGF- β/Smad3 signaling pathways. The compound is in CCl ₄ Can obviously reduce ALT level and delay the damage of liver tissue structure on an induced acute liver injury mouse model. More importantly, the compounds reduce bleomycin-induced pulmonary fibrosis in mice by reducing hydroxyproline content and inflammatory cell infiltration in lung tissue, which may also be associated with their inhibition of the TGF- β pathway and anti-inflammation. In view of its good pharmacokineticsCharacteristically, anti-fibrotic effects and in vivo toxicity, this class of compounds is potentially active candidate compounds for the treatment of tissue fibrotic diseases including pulmonary fibrosis, liver fibrosis, kidney fibrosis.

Claims

1. A compound of formula I:

x is selected from O, and has a structural formula shown as the following formula II:

wherein Y is selected from O or NR ₁₂ ；R ₁ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₆ An alkoxy group; r ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or halogen; r ₁₂ Selected from H or methyl; r ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, C ₁ ～C ₄ Alkyl-substituted 6-membered heterocycle, tert-butoxycarbonyl-substituted 6-membered heterocycle, -COR ₁₆ Substituted 6-membered heterocyclic ring, -COOR ₁₇ Substituted 6-membered heterocycles, -NR ₁₈ R ₁₉ A substituted 6-membered heterocyclic ring; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 6-membered heterocycle, tert-butoxycarbonyl substituted 6-membered heterocycle, -COOR ₁₇ A substituted 6-membered heterocyclic ring; r ₁₆ Is selected from C ₁ ～C ₃ Alkyl radical, C ₃ ～C ₆ A cycloalkyl group; r ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from C ₁ ～C ₄ An alkyl group; the 5-membered heterocycle has 1 heteroatom, and the number of the heteroatoms is N.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein:

R ₁₃ is selected from

3. The compound of claim 2, wherein: r ₁ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₃ An alkoxy group.

4. A compound according to claim 3, characterized in that: r ₁ 、R ₃ 、R ₄ 、R ₅ 、R ₆ Independently selected from H, nitro or methoxy.

5. The compound of claim 4, wherein: r ₁ 、R ₃ 、R ₆ Are all selected from H, R ₄ 、R ₅ Independently selected from H, nitro or methoxy; r ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or F.

6. The compound of claim 5, wherein: r ₇ 、R ₈ 、R ₁₁ Are all selected from H, R ₁₀ Selected from H or F.

7. The compound according to any one of claims 1-6, wherein: r ₁₃ Is selected from

8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, having the structural formula:

9. the compound of claim 1, formula i or a pharmaceutically acceptable salt thereof, wherein:

x is selected from NH, R ₂ 、R ₄ 、R ₅ Independently selected from H or

And R is ₂ 、R ₄ 、R ₅ One of which is

R ₁ 、R ₃ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₆ An alkoxy group; r ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or halogen; y is selected from O, S or NR ₁₂ ，R ₁₂ Selected from H or methyl; r ₁₃ Selected from NR ₁₄ R ₁₅ Unsubstituted 5-membered heterocycle, unsubstituted 6-membered heterocycle, C ₁ ～C ₄ Alkyl-substituted 6-membered heterocycle, tert-butoxycarbonyl-substituted 6-membered heterocycle, -COR ₁₆ Substituted 6-membered heterocyclic ring, -COOR ₁₇ Substituted 6-membered heterocycles, -NR ₁₈ R ₁₉ A substituted 6-membered heterocyclic ring; r ₁₄ 、R ₁₅ Independently selected from H and C ₁ ～C ₆ Alkyl, unsubstituted 6-membered heterocycle, tert-butoxycarbonyl substituted 6-membered heterocycle, -COOR ₁₇ A substituted 6-membered heterocyclic ring; r ₁₆ Is selected from C ₁ ～C ₃ Alkyl radical, C ₃ ～C ₆ A cycloalkyl group; r ₁₇ 、R ₁₈ 、R ₁₉ Independently selected from C ₁ ～C ₄ An alkyl group; the 5-membered heterocycle has 1 heteroatom, and the number of the heteroatoms is N.

10. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein: r ₂ Is selected from

When Y is selected from O, S or NR ₁₂ ；R ₄ Or R ₅ Is selected from

When Y is selected from O.

11. The compound according to claim 10, or a pharmaceutically acceptable salt thereof, wherein: r ₁ 、R ₃ 、R ₆ Independently selected from H, nitro, C ₁ ～C ₃ An alkoxy group.

12. The compound according to claim 11, or a pharmaceutically acceptable salt thereof, wherein: r ₁ 、R ₃ 、R ₆ Are all selected from H.

13. The compound according to claim 9, or a pharmaceutically acceptable salt thereof, wherein: r ₇ 、R ₈ 、R ₁₀ 、R ₁₁ Independently selected from H or F.

14. The compound according to claim 13, or a pharmaceutically acceptable salt thereof, wherein: r ₇ Selected from H or F, R ₈ 、R ₁₀ 、R ₁₁ Are all selected from H.

15. The compound according to any one of claims 9-14, or a pharmaceutically acceptable salt thereof, wherein: r ₁₃ Is selected from

16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, having the formula:

17. a pharmaceutical composition comprising the compound of any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof as an active ingredient, together with pharmaceutically acceptable auxiliary ingredients.

18. Use of a compound according to any one of claims 1 to 16 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 17 in the manufacture of a medicament for the treatment of a fibrotic disease.

19. Use according to claim 18, characterized in that: the medicament for treating the fibrotic disease satisfies at least one of the following conditions:

the drug is a drug for inhibiting the expression of COL1A1, alpha-SMA and p-Smad3 protein;

the drug is a drug that inhibits the TGF-beta/Smad 3 pathway;

the fibrotic disease includes pulmonary fibrosis, hepatic fibrosis or renal fibrosis.