Novel PDE4 inhibitor and application thereof

Abstract

The invention discloses a novel PDE4 inhibitor and application thereof. The compound can be used for treating PDE4-mediated diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to difluoromethoxyphenyl derivatives with anti-inflammatory and anti-psoriatic properties. Background Art

[0002] Phosphodiesterases (PDEs) are hydrolases. Their function is to hydrolyze two second messenger biologically active cyclic nucleotides in cells - cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) - into biologically inactive linear nucleotides. Phosphodiesterases (PDEs) are divided into 11 subfamilies of PDEs, which play a key role in regulating cell function by metabolizing the 3′-cyclic phosphate bonds of cAMP and cGMP. PDE4 is a subtype of PDE.

[0003] PDE4 is a cAMP-specific enzyme that converts the second messenger cAMP into 5′-AMP. On the other hand, cAMP has a profound effect on multiple functions of inflammatory cell pathways. Increased intracellular cAMP levels inhibit T cell activation, can regulate macrophages and neutrophils, and cause bronchodilation. Increasing intracellular cAMP levels can also inhibit fibrosis, the release of inflammatory cytokines and chemokines, the biological activity of proteases, the generation of reactive oxygen systems, and the production of arachidonic acid metabolites. Inhibition of PDE4 increases cAMP levels, thereby relaxing airway smooth muscle and maintaining immune balance.

[0004] The PDE4 target has been developed for the treatment of a variety of inflammatory diseases, including respiratory diseases (chronic obstructive pulmonary disease, asthma), various skin diseases (such as psoriasis, atopic dermatitis, etc.), and immune system diseases (systemic lupus erythematosus, rheumatoid arthritis, etc.). Common PDE4 inhibitors include aprenomide for moderate to severe psoriasis and rheumatoid arthritis, crisaborole for systemic / topical use for mild to moderate psoriasis, and roflumilast for adjuvant treatment of chronic obstructive pulmonary disease (COPD). Currently, PDE4 inhibitors have become the first-line drugs for the treatment of psoriasis and some other chronic inflammatory diseases, and their safe and efficient efficacy has been widely recognized.

[0005] Inflammation is a common and frequently occurring disease that threatens human health. There are many causes of inflammation, including bacteria, viruses, rickettsial phages, mycoplasmas, fungi, etc. Inflammation caused by biological pathogens is also called infection. The human body has a complex structure, and different parts of the body will have different degrees of inflammation. For example, gastroenteritis, hepatitis, appendicitis, pancreatitis, pharyngitis, prostatitis, vaginitis, periarthritis of the shoulder, otitis media, etc. are all relatively representative.

[0006] Psoriasis is an immune-mediated disease. Psoriasis is divided into four types according to clinical characteristics: vulgaris, pustular, erythrodermic and articular. Among them, vulgaris is the most common. The onset of psoriasis is characterized by redness of the skin, large-scale distribution of scales, and distribution in the shape of water droplets on various parts of the body. It is also accompanied by severe itching and burning symptoms. The important cells in the pathogenesis of psoriasis are DC, Th17, Th1 and keratinocytes. DC is activated by various stimuli to overproduce and secrete TNF-α, IL-23, and IL-12. IL-23 induces T cells to differentiate into Th17. Activated Th17 cells overproduce IL-17 and IL-22. TNF-α and IL-17 activate keratinocytes, promote epidermal hyperplasia, promote inflammatory cells (such as neutrophils), and induce the production of antimicrobial peptides (AMPs). The IL-12 produced by DC also induces Th1 to produce the cytokine IFN-γ, which aggravates psoriasis.

[0007] Traditional therapeutic drugs have problems such as poor efficacy and toxic side effects. The demand for innovative drugs in clinical practice is constantly expanding, and more new small molecule compounds are urgently needed. Summary of the invention

[0008] One or more embodiments of the present application provide a compound of Formula I or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof.

[0009]

[0010] in

[0011] R 1 and R 2 Each independently is H, And R 1 and R 2 Not at the same time H;

[0012] X and Y are each independently N or NH, O or S, and at least one of X and Y is N;

[0013] Z is NH or O;

[0014] R 3 is C6-14 aryl, 5-14 membered heteroaryl, C6-10 cycloalkyl, or 5-14 membered heterocyclyl; optionally, the C6-14 aryl, 5-14 membered heteroaryl, C6-10 cycloalkyl, or 5-14 membered heterocyclyl is substituted by one or more substituents selected from halogen, halogenated C1-C6 alkyl, carbonyl, C1-C6 alkyl, and C1-C6 alkoxy; the 5-14 membered heteroaryl or 5-14 membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; preferably, the halogen is F, Cl, Br or I;

[0015] n is 0, 1, 2 or 3;

[0016] Each dashed line independently represents the presence or absence of a bond.

[0017] In one or more embodiments, R 1 for R 2 For H.

[0018] In one or more embodiments, R 1 for R 2 For H.

[0019] In one or more embodiments, R 1 for R 2 for

[0020] In one or more embodiments, R 1 for R 2 for

[0021] In one or more embodiments, X is N and Y is NH.

[0022] In one or more embodiments, X is N and Y is O.

[0023] In one or more embodiments, X is N and Y is S.

[0024] In one or more embodiments, R 3 is a C6-10 aryl, a 5-10 membered heteroaryl, a C6-10 cycloalkyl, or a 5-10 membered heterocyclyl; optionally, the C6-10 aryl, the 5-10 membered heteroaryl, the C6-10 cycloalkyl, or the 5-10 membered heterocyclyl is substituted by one or more substituents selected from F, a carbonyl, a C1-C4 alkyl, and a C1-C4 alkoxy group; the 5-10 membered heteroaryl or the 5-10 membered heterocyclyl contains 1, 2, or 3 heteroatoms selected from N, O and S.

[0025] In one or more embodiments, wherein R 3 for

[0026] In one or more embodiments, wherein Formula I is R 4 for

[0027]

[0028] n is 0 or 1.

[0029] In one or more embodiments, wherein Formula I is Where R 5 Each independently n is 0 or 1.

[0030] In one or more embodiments, wherein Formula I is Where R 5 for

[0031] In one or more embodiments, wherein Formula I is Where R 5 for

[0032] In one or more embodiments, wherein Formula I is R 6 for n is 0 or 1.

[0033] In one or more embodiments, wherein Formula I is R 7 for n is 0 or 1.

[0034] In one or more embodiments, wherein Formula I is R 8 for n is 0 or 1.

[0035] In one or more embodiments, wherein Formula I is R 9 for n is 0 or 1.

[0036] One or more embodiments of the present application provide a compound or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated substance thereof:

[0037]

[0038]

[0039] One or more embodiments of the present application provide an intermediate compound for preparing the compound of the present application, which has the following structure:

[0040]

[0041] One or more embodiments of the present application provide a method for preparing a compound of formula II,

[0042]

[0043] in

[0044] R 1 , R 2 , X, Y, R 3 , n as described above;

[0045] The preparation method comprises: (1)

[0047]

[0048] in

[0049] a) reacting 3,3-dibromo-1,1,1-trifluoro-2-one with sodium acetate at 80°C-120°C for 1-2 hours, adding R 1 , R 2 , difluoromethoxy-substituted benzaldehyde solution, react at 20°C-50°C for 1-8 hours;

[0050] b) hydrolyzing the product obtained in step a) at 50° C.-90° C. for 1-8 hours;

[0051] c) reacting the product obtained in step b) with R 3 -(CH 2 )m-NH 2 The reaction was carried out at 20°C-50°C for 5-7 hours; (2)

[0053]

[0054] in

[0055] d) will be R 1 , R 2 , difluoromethoxy-substituted benzoic acid with L-serine methyl ester hydrochloride and SOCl 2 React at 0°C for 10-14 hours;

[0056] e) reacting the product obtained in step d) with DAST at -78°C with stirring for 3-5 hours, adding an inorganic base, and reacting at room temperature for 24 hours;

[0057] f) reacting the product obtained in step e) with CBrCl 3 React with DBU at 0°C for 15-24 hours;

[0058] g) hydrolyzing the product obtained in step f) at 20° C.-50° C. for 1-8 hours;

[0059] h) reacting the product obtained in step g) with R 3 -(CH 2 ) n -NH 2 React at 20℃-50℃ for 3-8 hours;

[0060] or (3)

[0062]

[0063] Will be R 1 , R 2 , difluoromethoxy-substituted benzaldehyde and D-cysteine ​​methyl ester hydrochloride, K 2 CO 3 The reaction was stirred at 20°C-50°C for 20-30 hours, then stirred at -15°C, and then DBU and CBrCl were added at -20°C-0°C. 3 , react at 20℃-50℃ for 12-24 hours;

[0064] i) stirring the product obtained in step i) at 20° C.-50° C. for 8-12 hours to carry out a hydrolysis reaction;

[0065] k) reacting the product obtained in step j) with R 3 -(CH 2 ) n -NH 2 React at 20°C-50°C for 4-8 hours.

[0066] One or more embodiments of the present application provide a pharmaceutical composition comprising a compound of the present application and a pharmaceutically acceptable auxiliary material or excipient.

[0067] One or more embodiments of the present application provide use of the compound of the present application or the pharmaceutical composition of the present application in the preparation of a medicament for preventing and / or treating inflammatory diseases, respiratory diseases, skin diseases, or immune system diseases.

[0068] One or more embodiments of the present application provide a compound of the present application for use as a medicament.

[0069] One or more embodiments of the present application provide the pharmaceutical composition of the present application, which is used as a medicament.

[0070] One or more embodiments of the present application provide a compound or composition of the present application for preventing and / or treating inflammatory diseases, respiratory diseases, skin diseases, or immune system diseases.

[0071] One or more embodiments of the present application provide a compound or composition of the present application for preventing and / or treating a PDE4-mediated disease.

[0072] One or more embodiments of the present application provide a compound or composition of the present application for inhibiting PDE4.

[0073] One or more embodiments of the present application provide a method for preventing and / or treating inflammatory diseases, respiratory diseases, skin diseases, or immune system diseases, which comprises administering a compound or composition of the present application to a subject in need thereof.

[0074] One or more embodiments of the present application provide a method for preventing and / or treating a PDE4-mediated disease, which comprises administering a compound or composition of the present application to a subject in need thereof.

[0075] One or more embodiments of the present application provide a method for inhibiting PDE4, which comprises administering a compound or composition of the present application to a subject in need thereof.

[0076] In one or more embodiments, the inflammatory disease is an inflammatory skin disease.

[0077] In one or more embodiments, the respiratory disease is chronic obstructive pulmonary disease or asthma.

[0078] In one or more embodiments, the skin disease is psoriasis or atopic dermatitis.

[0079] In one or more embodiments, the immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

[0080] One or more embodiments of the present application provide use of the compound of the present application or the pharmaceutical composition of the present application in the preparation of a drug or a PDE4 inhibitor for preventing and / or treating a PDE4-mediated disease.

[0081] In one or more embodiments, the compounds of the present application have anti-inflammatory and psoriasis treatment uses. Biological experiments have shown that the compounds of the present application can significantly reduce the number of inflammatory cells in an inflammatory model, or reduce the levels of IL-1β, IL-6, TNF-α, and IL-17A in cells.

[0082] In one or more embodiments, the results of the psoriasis model mouse experiment show that the compounds of the present application can significantly improve the thickness, scaling, and severity of erythema of psoriasis in mice, indicating that the compounds of the present application can reduce the release of proinflammatory factors and the production of proinflammatory mediators to inhibit the occurrence and development of inflammatory reactions, and can also protect the skin of mice and play an anti-psoriatic effect.

[0083] The following is an explanation of the terms used in the technical solution of the present application. As used in the specification and the appended claims, unless otherwise specifically stated, the terms of the present application have the following meanings:

[0084] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0085] The term "amino" refers to -NH 2 .

[0086] The term "hydroxy" refers to -OH.

[0087] "Alkyl" refers to a straight or branched saturated aliphatic hydrocarbon group of 1 to 20 carbon atoms, preferably an alkyl group of 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8) carbon atoms, more preferably an alkyl group of 1 to 6 carbon atoms, and further preferably an alkyl group of 1 to 4 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, neobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and various branched isomers thereof; when the alkyl group is substituted, it may be optionally further substituted by one or more substituents.

[0088] "Alkoxy" refers to a group formed by replacing at least one carbon atom in an alkyl group with an oxygen atom. Non-limiting examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexyloxy, cyclopropyloxy and cyclobutyloxy. The definition of alkyl is the same as that of "alkyl" described above.

[0089] "Aryl" refers to a substituted or unsubstituted aromatic ring, which can be, for example, a monocyclic ring of 6 to 8 carbon atoms (e.g., 6, 7, 8 carbon atoms), a bicyclic ring of 6 to 12 carbon atoms (e.g., 6, 7, 8, 9, 10, 11, 12 carbon atoms), or a tricyclic ring system of 10 to 14 carbon atoms (e.g., 10, 11, 12, 13, 14 carbon atoms), which can be a bridged ring or a spirocyclic ring, and non-limiting examples include phenyl and naphthyl. The aryl group can be optionally further substituted by one or more substituents.

[0090] "Heteroaryl" refers to a substituted or unsubstituted aromatic ring, which can be, for example, a 5- to 8-membered (e.g., 5, 6, 7, 8-membered) monocyclic ring, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10- to 14-membered (e.g., 10, 11, 12, 13, 14-membered) tricyclic ring system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O or S, preferably a 5- to 8-membered heteroaryl. The 1 to 4 (e.g., 1, 2, 3, 4) N and S optionally substituted in the heteroaryl ring can be oxidized to various oxidation states. Heteroaryl can be connected to a heteroatom or a carbon atom, and can be a bridged ring or a spiro ring, and non-limiting examples include cyclopyridyl, furanyl, thienyl, pyranyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinyl benzimidazolyl, benzopyridinyl, pyrrolopyridinyl. Heteroaryl is optionally further substituted by one or more substituents.

[0091] "Heterocyclyl" or "heterocycle" refers to a saturated or unsaturated non-aromatic heterocycle, which can be, for example, a 5- to 10-membered (e.g., 5, 6, 7, 8, 9, 10-membered) monocyclic ring, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10- to 14-membered (e.g., 10, 11, 12, 13, 14-membered) tricyclic ring system, and contains 1 to 4 (e.g., 1, 2, 3, 4) heteroatoms selected from N, O, or S, preferably a 5- to 8-membered heterocyclyl. The 1 to 4 (e.g., 1, 2, 3, 4) N and S optionally substituted in the ring of the "heterocyclyl" or "heterocycle" can be oxidized to various oxidation states; the "heterocyclyl" or "heterocycle" can be attached to a heteroatom or a carbon atom; the "heterocyclyl" or "heterocycle" can be a bridged ring or a spiro ring. Non-limiting examples of “heterocyclyl” or “heterocycle” include oxirane, oxirane, aziridine, oxetanyl, azetidinyl, thietanyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxhexacyclyl, azepanyl, oxepinyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, pyridinyl, piperidinyl, homopiperidinyl, furanyl, pyridinyl, piperidinyl, homopiperidinyl, furanyl, pyridinyl, piperidinyl, piperidinyl, homopiperidinyl, furanyl, pyridinyl, piperidinyl, piperidinyl, homopiperidinyl, pyridinyl, piperidinyl, piperidinyl, py ... pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, pyridinyl, py pyranyl, thiophene, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, 1,3-dithianyl, dihydrofuranyl, dithiolanyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzimidazolyl, oxazolyl, benzopyridyl, pyrrolopyridyl, benzodihydrofuranyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-dioxolane, pyrazolinyl, dithianyl, dithiolanyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 1,2,3,4-tetrahydroisoquinolinyl, 3-azabicyclo[3 .1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 3H-indolylquinolizinyl, N-pyridylurea, 1,1-dioxothiomorpholinyl, azabicyclo[3.2.1]octanyl, azabicyclo[5.2.0]nonanyl, oxatricyclo[5.3.1.1]dodecyl, azaadamantyl and oxaspiro[3.3]heptanyl. The "heterocyclyl" or "heterocycle" may be optionally further substituted by one or more substituents.

[0092] "Cycloalkyl" refers to a saturated cyclic hydrocarbon group, which can be, for example, a monocyclic, bicyclic or polycyclic ring of 6 to 10 carbon atoms (e.g., 6, 7, 8, 9, 10 carbon atoms), preferably 6 to 8 carbon atoms. Non-limiting examples of "cycloalkyl" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. When the cycloalkyl is substituted, it can be optionally further substituted by one or more substituents.

[0093] "Pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" refers to a salt of the compound of the present invention that retains the biological effectiveness and properties of the free acid or free base, and the free acid is obtained by reacting with a non-toxic inorganic base or organic base, and the free base is obtained by reacting with a non-toxic inorganic acid or organic acid.

[0094] "Pharmaceutical composition" refers to a mixture of one or more compounds described herein, their pharmaceutically acceptable salts or prodrugs and other chemical components, wherein "other chemical components" refers to pharmaceutically acceptable carriers, excipients and / or one or more other therapeutic agents.

[0095] "Carrier" refers to a material that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.

[0096] "Excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, and disintegrants.

[0097] "Prodrug" refers to a compound of the present invention that can be converted into a biologically active compound through in vivo metabolism. The prodrug of the present invention is prepared by modifying the amino or carboxyl group in the compound of the present invention, and the modification can be removed by conventional operations or in vivo to obtain the parent compound. When the prodrug of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free amino or carboxyl group.

[0098] "Co-crystal" refers to a crystal formed by the active pharmaceutical ingredient (API) and the co-crystal former (CCF) under the action of hydrogen bonds or other non-covalent bonds, in which the pure state of API and CCF are solid at room temperature and there is a fixed stoichiometric ratio between the components. Co-crystal is a multi-component crystal, including binary eutectics formed between two neutral solids and multi-component eutectics formed between neutral solids and salts or solvates.

[0099] "Stereoisomers" refer to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and conformational isomers.

[0100] "Optional" or "optionally" or "selective" or "selectively" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, "heterocyclyl optionally substituted with alkyl" means that the alkyl group may but need not be present, and the description includes instances where the heterocyclyl group is substituted with alkyl group and instances where the heterocyclyl group is not substituted with alkyl group. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1 and Figure 2 In Example 8, the phenotypic manifestations of the back skin of each group (n=8, male) were treated with Compound A5 and Compound D2 for 7 days.

[0102] Figure 3 and Figure 4 It shows the daily monitoring weight graph of Compound A5 and Compound D2 in Active Example 8, wherein erythema and thickness scores were monitored daily according to PASI, with scores ranging from 0 to 4, and cumulative scores were monitored daily according to PASI, with total scores ranging from 0 to 12.

[0103] Figure 5 and Figure 6 It shows the pathological changes of the skin tissue observed under a microscope by Compound A5 and Compound D2 in Activity Example 8.

[0104] Figure 7 and Figure 8 The graph shows the effects of Compound A5 and Compound D2 in Activity Example 8 on the spleen in a mouse psoriasis model.

[0105] Fig. 9 and Fig.10 The figure shows the immunohistochemical staining of Ki-67 antibody of compound A5 and compound D2 in Activity Example 8 on skin tissue in mouse psoriasis model.

[0106] Fig.11 and Fig.12 This indicates that compound A5 and compound D2 in Example 8 inhibit the expression of inflammatory factors in the skin tissue of the mouse psoriasis model. Specific embodiments

[0107] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only for the purpose of explanation and are not intended to limit the scope and essence of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0108] Example 1 Synthesis of Series A Compounds

[0109] (1) Synthesis of Compound 2:

[0110]

[0111] Weigh 3g of compound 3,4-dihydroxybenzaldehyde (21.72mmol, 1.0equiv.) and 6.91g of sodium carbonate (65.16mmol, 3.0equiv.), dissolve in 30mL N,N-dimethylformamide, and finally add 3.44g of ethyl difluorochloroacetate (21.72mmol, 1.0equiv.), react at 80°C for 8 hours, detect the complete reaction of the raw materials by TLC, and add water to quench the reaction. Use ethyl acetate to extract 3 times, and wash the organic phase with saturated sodium chloride solution. The organic phase is concentrated and 1.4g of white solid product (compound 2) is obtained by rapid column chromatography, with a yield of 34%.

[0112] The obtained compound 2 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (500 MHz, CDCl 3 )δ9.90 (s, 1H), 7.53 (d, J=2.0 Hz, 1H), 7.44 (dd, J=8.5, 2.0 Hz, 1H), 7.26 (d, J=8.5 Hz, 1H), 6.66 (t, J=73.5 Hz, 1H). ESI-HRMS m / z: calculated value is C 8 H 6 O 3 F 2 + [M+Na] + , 211.0177; the measured value is 211.0166.

[0113] (2) Synthesis of compound 3:

[0114]

[0115] Weigh 2.1g of compound 2 (11.16mmol, 1.0equiv.), add 3.09g of potassium carbonate (22.32mmol, 2.0equiv.), add 20mL of N,N-dimethylformamide, and finally add 2.26g of bromomethylcyclopropane (16.74mmol, 1.5equiv.), react at 80°C for 8 hours, detect the complete reaction of the raw materials by TLC, and quench the reaction by adding water. Use ethyl acetate to extract 3 times, and wash the organic phase with saturated sodium chloride solution. Concentrate the organic phase and obtain 2.3g of yellow oily product (compound 3) by rapid column chromatography, with a yield of 85%.

[0116] The obtained compound 3 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (500 MHz, DMSO-d 6)δ9.94 (s, 1H), 7.59-7.54 (m, 2H), 7.39 (d, J=8.0 Hz, 1H), 7.28 (t, J=73.5 Hz, 1H), 3.98 (d, J=7.0 Hz, 2H), 1.30-1.21 (m, 1H), 0.61-0.55 (m, 2H), 0.39-0.33 (m, 2H). ESI-MS m / z: calculated value is C 12 H 13 O 3 F 2 + [M+H] + , 243.1; the measured value is 243.1.

[0117] (3) Synthesis of compound 4:

[0118]

[0119] Weigh 4.46 g of 3,3-dibromo-1,1,1-trifluoro-2-one (16.52 mmol, 2.0 equiv.) and 2.7 g of sodium acetate (33.04 mmol, 4.0 equiv.) into a reaction bottle, add an appropriate amount of water to dissolve, stir at 100°C for 1 hour, cool to room temperature, add 2 g of compound 3 (8.26 mmol, 1.0 equiv.) in methanol and 4 mL of ammonia water to the above solution, stir at room temperature, and remove methanol by rotary evaporation when TLC detects that the raw material is no longer reduced. Extract with ethyl acetate 3 times, wash the organic phase once with saturated sodium chloride solution, and dry the organic phase with anhydrous sodium sulfate. Concentrate the organic phase and obtain 1.37 g of yellow solid compound 4 by rapid column chromatography, with a yield of 48%.

[0120] The obtained compound 4 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (400 MHz, CDCl 3 )δ12.56 (br, 1H), 7.50 (d, J = 2.0 Hz, 1H), 7.41 (s, 1H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.62 (t, J = 75.2 Hz, 1H), 3.72 (d, J = 7.2 Hz, 2H), 1.28-1.15 (m, 1H), 0.65-0.56 (m, 2H), 0.32-0.24 (m, 2H). ESI-MS m / z: calculated value is C 15 H 14 O 2 N 2 F 5 +[M+H] +, 349.1; the measured value is 349.1.

[0121] (4) Synthesis of Compound 5:

[0122]

[0123] 415 mg of compound 4 (1.19 mmol, 1.0 equiv.) was weighed and dissolved in a 1:1 mixed solvent of ethanol and water, 953 mg of sodium hydroxide (23.80 mmol, 20 equiv.) was added, and the mixture was stirred at 80°C for reaction, monitored by TLC, and after the reaction was complete, ethanol was removed by rotary evaporation, an appropriate amount of water was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The solid was filtered and dried to obtain 322 mg of yellow solid compound 5, with a yield of 83%.

[0124] The obtained compound 5 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (400 MHz, DMSO-d 6 )δ13.28 (br, 1H), 7.96-7.56 (m, 3H), 7.27 (d, J=8.4 Hz, 1H), 7.14 (t, J=74.4 Hz, 1H), 3.98 (d, J=6.8 Hz, 2H), 1.35-1.22 (m, 1H), 0.64-0.55 (m, 2H), 0.42-0.34 (m, 2H), OH (1H, not observed). ESI-HRMS m / z: calculated value is C 15 H 14 O 4 N 2 F 2 Na + [M+Na] + , 347.0814; the measured value is 347.0800.

[0125] (5) Synthesis of Compounds A1-A17:

[0126]

[0127] 50 mg of compound 5 (0.15 mmol, 1.0 equiv.) was weighed and dissolved in NN-dimethylformamide, 59 mg of HATU (0.15 mmol, 1.0 equiv.) and 60 mg of DIPEA (0.45 mmol, 3.0 equiv.) were added, stirred at room temperature for 20 min, the corresponding amine reagent (0.15 mmol, 1.0 equiv.) was added, and the reaction was carried out at room temperature for 6 h. Monitored by TLC, water was added to quench the reaction of the raw material after the reaction was complete, extracted with ethyl acetate, washed once with a saturated sodium chloride solution, the ethyl acetate layer was dried with anhydrous sodium sulfate, the solution was concentrated, and purified by column chromatography to obtain compounds A1-A17.

[0128] The compounds A1-A17 are arranged in the following order:

[0129]

[0130] Example 2 Synthesis of Compound A1:

[0131] The synthesis steps were as in Example 1, and the amine reagent in the last step was 2-aminothiazole to obtain compound A1 with a yield of 50%. 1 H NMR (500 MHz, DMSO-d 6 )δ13.27 (br, 1H), 11.54 (s, 1H), 8.19 (s, 1H), 7.82 (s, 1H), 7.65 (s, 1H), 7.53 (d, J = 3.5Hz, 1H), 7.30 (s, 1H), 7.25 (d, J =3.5Hz, 1H), 7.15 (t, J = 74.5Hz, 1H), 3.99 (d, J = 7.0Hz, 2H), 1.38-1.24 (m, 1H), 0.66-0.56 (m, 2H), 0.43-0.34 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ160.1, 157.9, 150.1, 146.0, 140.3, 137.7, 134.5, 127.9, 123.3, 121.3, 117.9, 116.7 (t, J = 256.5 Hz), 113.6, 111.4, 73.1, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 18 H 17 O 3 N 4 F 2 S + [M+H] + , 407.0984; the measured value is 407.0964.

[0132] Example 3 Synthesis of Compound A2:

[0133] The synthesis steps were as in Example 1, and 4-aminopyridine was selected as the amine reagent in the last step of the reaction to obtain compound A2 with a yield of 58%. 1 H NMR (400 MHz, DMSO-d 6 )δ13.26 (br, 1H), 10.17 (br, 1H), 8.46 (d, J = 5.6Hz, 2H), 8.08 (s, 1H), 7.88 (d, J = 4.8Hz, 2H), 7.81 (s, 1H), 7.69 (d, J = 8.4Hz, 1H) , 7.31 (d, J = 8.4Hz, 1H), 7.16 (t, J = 74.4Hz, 1H), 3.99 (d, J = 6.8Hz, 2H), 1.36-1.26 (m, 1H), 0.66-0.56 (m, 2H), 0.42-0.34 (m, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ161.6, 150.2 (2×C), 150.1, 145.7, 145.4, 140.2, 136.2, 128.0, 123.0, 121.3, 118.1, 116.7 (t, J=256.4 Hz), 113.9 (2×C), 111.4, 73.2, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 20 H 19 O 3 N 4 F 2 + [M+H] + , 401.1420; the measured value is 401.1402.

[0134] Example 4 Synthesis of Compound A3:

[0135] Synthesis steps refer to Example 1, the last step of the reaction of the amine reagent selected 2,6-difluorobenzylamine, to obtain compound A3, yield: 7%. 1 H NMR (600 MHz, MeOD-d 4)δ7.71 (s, 1H), 7.65 (d, J = 1.8Hz, 1H), 7.37 (d, J = 7.2Hz, 1H), 7.27-7.20 (m, 1H), 7.17 (d, J = 8.4Hz, 1H), 6.82 (s, 2H), 6.77 (t, J=75.0Hz, 1H), 4.58 (s, 2H), 3.64 (s, 2H), 1.21-1.12 (m, 1H), 0.56 (d, J=7.2Hz, 2H), 0.23 (s, 2H), NH (2H, not observed). 13 C NMR (151 MHz, MeOD-d 4 )δ164.7, 162.9 (dd, J = 247.5, 7.5 Hz, 2×C), 152.2, 147.7, 142.4, 137.5, 131.1 (t, J = 10.5 Hz), 129.3, 123.3, 122.4, 118.7, 117.9 (t, J = 256.5 Hz), 114.5 (t, J = 18.0 Hz), 112.6, 112.2 (dd, J = 21.0, 6.0 Hz, 2×C), 74.6, 31.5, 10.8, 3.5 (2×C). ESI-HRMS m / z: calculated value is C 22 H 20 O 3 N 3 F 4 + [M+H] + , 450.1435; the measured value is 450.1408.

[0136] Example 5 Synthesis of Compound A4:

[0137] The synthesis steps were as in Example 1. The amine reagent in the last step was 2-methylaminopyrimidine (CAS No. 75985-45-4) to obtain compound A4 with a yield of 14%. 1 H NMR (400 MHz, DMSO-d 6 ) δ13.02 (br, 1H), 8.77 (d, J = 4.8Hz, 2H), 8.46 (br, 1H), 7.82 (s, 1H), 7.76 (s, 1H), 7.63 (d, J = 8.0Hz, 1H), 7.40 (t, J = 4.8Hz, 1H), 7.28 (d, J = 8.4Hz, 1H), 7.14 (t, J=74.4Hz, 1H), 4.68 (d, J=6.0Hz, 2H), 3.97 (d, J=6.8Hz, 2H), 1.33-1.25 (m, 1H), 0.62-0.58 (m, 2H), 0.40-0.36 (m, 2H).13 C NMR (101 MHz, DMSO-d 6 )δ166.9, 162.2, 157.4 (2×C), 150.1, 147.1, 144.9, 140.0, 136.9, 121.4, 120.9, 119.9, 117.7, 116.7 (t, J=256.4 Hz), 111.1, 73.2, 44.5, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 20 H 20 O 3 N 5 F 2 + [M+H] + , 416.1529; the measured value is 416.1530.

[0138] Example 6 Synthesis of Compound A5:

[0139] The synthesis steps were as in Example 1. The amine reagent in the last step was 2,5-difluorobenzylamine to obtain compound A5 with a yield of 56%. 1 H NMR (600 MHz, MeOD-d 4 )δ7.74 (s, 1H), 7.72 (d, J=2.4Hz, 1H), 7.42 (dd, J=7.8, 1.8Hz, 1H), 7.18 (d, J=8.4Hz, 1H), 6.99-6.91 (m, 3H), 6.77 (t, J= 75.6Hz, 1H), 4.47 (s, 2H), 3.69 (d, J=5.4Hz, 2H), 1.20-1.15 (m, 1H), 0.57-0.53 (m, 2H), 0.22 (d, J=4.8Hz, 2H), NH (2H, not observed). 13 C NMR (151 MHz, MeOD-d 4 )δ165.1, 160.0 (dd, J = 240.0, 3.0 Hz), 157.9 (dd, J = 240.0, 3.0 Hz), 152.2, 147.9, 142.5, 137.3, 129.3, 128.5 (dd, J = 18.0, 7.5 Hz), 123.4, 122.5, 118.7, 117.9 (t, J = 256.5 Hz), 117.4 (dd, J = 24.0, 4.5 Hz), 116.9 (dd, J = 33.0, 9.0 Hz), 116.3 (dd, J = 33.0, 9.0 Hz), 112.6, 74.7, 37.2, 10.8, 3.5 (2×C). ESI-HRMS m / z: calculated for C 22H 20 O 3 N 3 F 4 + [M+H] + , 450.1435; the measured value is 450.1413.

[0140] Example 7 Synthesis of Compound A6:

[0141] The synthesis steps were as in Example 1. The amine reagent in the last step was 2,4-difluorobenzylamine to obtain compound A6 with a yield of 53%. 1 H NMR (500 MHz, MeOD-d 4 )δ7.73 (s, 1H), 7.69 (d, J=1.5Hz, 1H), 7.47-7.42 (m, 1H), 7.34 (q, J=7.5, 7.0Hz, 1H), 7.21 (d, J=8.0Hz, 1H), 6.87-6.80 (m, 2H), 6.80 (t, J=75.5Hz, 1H), 4.54 (s, 2H), 3.84 (d, J=6.0Hz, 2H), 1.27-1.24 (m, 1H), 0.65-0.55 (m, 2H), 0.35-0.28 (m, 2H), NH (2H, not observed). 13 C NMR (151 MHz, MeOD-d 4 )δ165.2, 163.7 (dd, J = 246, 12.0 Hz), 162.2 (dd, J = 248, 12.0 Hz), 152.2, 147.8, 142.5, 137.6, 132.0 (dd, J = 9.0, 6.0 Hz), 129.4, 123.4, 122.9 (dd, J = 15.0, 4.5 Hz), 122.3, 118.9, 117.9 (t, J = 256.5 Hz), 112.7, 112.1 (dd, J = 25.5, 4.5 Hz), 104.5 (t, J = 27.0 Hz), 74.9, 37.1, 10.9, 3.5 (2×C). ESI-HRMS m / z: calculated for C 22 H 20 O 3 N 3 F 4 + [M+H] + , 450.1435; the measured value is 450.1415.

[0142] Example 8 Synthesis of Compound A7:

[0143] The synthesis steps were as in Example 1. The amine reagent in the last step was 3,4-difluorobenzylamine to obtain compound A7 with a yield of 23%. 1 H NMR (400 MHz, DMSO-d 6 )δ13.02 (br, 1H), 8.66 (br, 1H), 7.81 (s, 1H), 7.75 (s, 1H), 7.61 (d, J = 8.0Hz, 1H), 7.39-7.30 (m, 2H), 7.27 (d, J = 8.4Hz, 1H), 7.17-7.14 (m, 1H), 7.12 (t, J=74.4Hz, 1H), 4.42 (d, J=6.4Hz, 2H), 3.94 (d, J=7.2Hz, 2H), 1.33-1.24 (m, 1H), 0.64-0.54 (m, 2H), 0.38-0.32 (m, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ162.3, 150.1, 149.2 (dd, J = 244, 13 Hz), 148.3 (dd, J = 243, 16 Hz), 145.0, 140.0, 137.8, 136.8, 128.2, 123.9 (dd, J = 6.0, 3.0 Hz), 121.3, 120.9, 117.7, 117.1 (d, J = 17 Hz), 116.6 (t, J = 256.5 Hz), 116.3 (d, J = 17 Hz), 111.1, 73.1, 41.0, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated for C 22 H 20 O 3 N 3 F 4 + [M+H] + , 450.1435; the measured value is 450.1405.

[0144] Example 9 Synthesis of Compound A8:

[0145] The synthesis steps were as in Example 1. The amine reagent in the last step was aniline to obtain compound A8 with a yield of 23%. 1 H NMR (500 MHz, MeOD-d 4)δ7.83 (s, 1H), 7.76-7.65 (m, 3H), 7.52 (d, J=8.5Hz, 1H), 7.35 (t, J=8.0Hz, 2H), 7.24 (d, J=8.0Hz, 1H), 7.13 (t, J=7.5Hz , 1H), 6.83 (t, J=75.0Hz, 1H), 4.00 (d, J=7.0Hz, 2H), 1.38-1.29 (m, 1H), 0.68-0.64 (m, 2H), 0.42-0.39 (m, 2H), NH (2H, not observed). 13 C NMR (151 MHz, MeOD-d 4 )δ163.2, 152.3, 148.0, 142.6, 139.5, 130.8, 129.9 (2×C), 129.4, 125.3, 123.4, 122.9, 121.5 (2×C), 119.3, 118.0 (t, J=256.5 Hz), 112.9, 75.0, 11.0, 3.6 (2×C). ESI-HRMS m / z: calculated value is C 21 H 19 O 3 N 3 F 2 Na + [M+Na] + , 422.1287; the measured value is 422.1283.

[0146] Example 10 Synthesis of Compound A9:

[0147] The synthesis steps were as shown in Example 1. The amine reagent for the last step was 2-(aminomethyl)naphthalene (CAS No.: 2018-90-8) to obtain compound A9 with a yield of 81%. 1 H NMR (600 MHz, DMSO-d 6 )δ8.71 (s, 1H), 7.90-7.83 (m, 3H), 7.82 (s, 1H), 7.78 (s, 2H), 7.62 (d, J=7.8Hz, 1H), 7.53-7.43 (m, 3H), 7.25 (s, 1H), 7.13 (t, J=74.4Hz, 1H), 4.63 (d, J=6.0Hz, 2H), 3.95 (d, J=6.6Hz, 2H), 1.32-1.24 (m, 1H), 0.62-0.55 (m, 2H), 0.40-0.29 (m, 2H), NH (not observed). 13 C NMR (151 MHz, DMSO-d 6)δ161.8, 150.1, 145.3, 140.0, 137.6, 132.9, 132.1, 129.7, 128.4, 127.8, 127.5 (2×C), 127.3, 126.2, 126.1, 125.6, 125.4, 121.3, 117.8, 116.7 (t, J=258.0 Hz), 111.2, 73.1, 42.0, 10.0, 3.0 (2×C). ESI-HRMS m / z: calculated value is C 26 H 23 O 3 N 3 F 2 Na + [M+Na] + , 486.1600; the measured value is 486.1582.

[0148] Example 11 Synthesis of Compound A10:

[0149] The synthesis steps were as in Example 1. The amine reagent in the last step was 2-methoxy-5-(aminomethyl)pyridine (CAS No.: 262295-96-5) to obtain compound A10 with a yield of 64%. 1 H NMR (600 MHz, DMSO-d 6 )δ13.06(br, 1H), 8.57(s, 1H), 8.10(s, 1H), 7.79(s, 1H), 7.73(s, 1H), 7.65( dd, J=8.4, 2.4Hz, 1H), 7.59 (d, J=8.4Hz, 1H), 7.24 (d, J=8.4Hz, 1H), 7.12 (t, J=74.4Hz, 1H), 6.75 (d, J=8.4Hz, 1H), 4.37 (d, J=6.0Hz, 2H), 3.92 (d, J=6.6H z, 2H), 3.81 (s, 3H), 1.32-1.24 (m, 1H), 0.62-0.56 (m, 2H), 0.38-0.32 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ162.7, 162.1, 150.1, 145.9, 145.0, 140.1, 139.0, 136.9, 128.5, 128.3, 121.4, 121.0, 117.8, 116.7 (t, J = 256.5 Hz), 111.2, 110.2, 73.2, 53.1, 39.0, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 22 H 22 O 4 N4 F 2 Na + [M+Na] + , 467.1501; the measured value is 467.1485.

[0150] Example 12 Synthesis of Compound A11:

[0151] The synthesis steps were as in Example 1. The amine reagent in the last step was 2-aminomethylpyrazine (Pyrazin-2-ylmethanamine, CAS No.: 20010-99-5) to obtain Compound A11 with a yield of 62%. 1 H NMR (500 MHz, DMSO-d 6 )δ13.04 (br, 1H), 8.66 (s, 1H), 8.61 (s, 1H), 8.58 (s, 1H), 8.53 (d, J = 2.5Hz, 1H), 7.83 (s, 1H), 7.75 (s, 1H), 7.61 (d, J = 7.0Hz, 1H), 7.28 (s, 1H), 7.13 (t, J=74.5Hz, 1H), 4.62 (d, J=6.0Hz, 2H), 3.96 (d, J=7.0Hz, 2H), 1.35-1.23 (m, 1H), 0.63-0.52 (m, 2H), 0.42-0.32 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ162.5, 154.5, 150.1, 145.0, 143.9, 143.4, 143.1, 140.0, 136.7, 128.3, 121.4, 121.1, 117.7, 116.7 (t, J = 258.0 Hz), 111.2, 73.2, 42.0, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 20 H 19 O 3 N 5 F 2 Na + [M+Na] + , 438.1348; the measured value is 438.1304.

[0152] Example 13 Synthesis of Compound A12:

[0153] The synthesis steps were as in Example 1, and benzylamine was selected as the amine reagent in the last step to obtain compound A12 with a yield of 57%. 1 H NMR (400 MHz, DMSO-d 6)δ13.01 (br, 1H), 8.52 (t, J=6.4Hz, 1H), 7.82 (s, 1H), 7.73 (d, J=1.6Hz, 1H), 7.59 (dd, J=8.4, 1.6Hz, 1H), 7.34-7.30 (m, 4H), 7.28-7.25 (m, 1H), 7 .25-7.20(m, 1H), 7.14(t, J=74.4Hz, 1H), 4.46(d, J=6.4Hz, 2H), 3.96(d , J=6.8Hz, 2H), 1.32-1.28(m, 1H), 0.65-0.57(m, 2H), 0.39-0.34(m, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ162.2, 150.1, 144.9, 140.1, 140.0, 137.0, 128.4, 128.3 (2×C), 127.4 (2×C), 126.7, 121.3, 120.9, 117.7, 116.7 (t, J=258.0 Hz), 111.1, 73.1, 41.8, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 22 H 21 O 3 N 3 F 2 Na + [M+Na] + , 436.1443; the measured value is 436.1435.

[0154] Example 14 Synthesis of Compound A13:

[0155] The synthesis steps were as in Example 1. The amine reagent in the last step was 2-aminomethylpyridine (CAS No.: 3731-51-9) to obtain compound A13 with a yield of 46%. 1 H NMR (400 MHz, DMSO-d 6 )δ13.02 (br, 1H), 8.59 (br, 1H), 8.53-8.50 (m, 1H), 7.84 (s, 1H), 7.76-7.70 (m, 2H), 7.60 (d, J=8.0Hz, 1H), 7.33-7.25 (m, 3H), 7.14 (t, J=74.4Hz, 1H), 4.56 (d, J=6.0Hz, 2H), 3.97 (d, J=6.8Hz, 2H), 1.34-1.29 (m, 1H), 0.63-0.58 (m, 2H), 0.40-0.35 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6)δ162.3, 158.7, 150.1, 148.8, 144.9, 140.0, 136.9, 136.7, 128.3, 122.0, 121.4, 121.0, 120.9, 117.7, 116.7 (t, J = 258.0 Hz), 111.2, 73.1, 43.8, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 21 H 20 O 3 N 4 F 2 Na + [M+Na] + , 437.1396; the measured value is 437.1382.

[0156] Example 15 Synthesis of Compound A14:

[0157] The synthesis steps were as in Example 1. The amine reagent in the last step was 4-aminomethyltetrahydropyran (CAS No.: 130290-79-8) to obtain compound A14 with a yield of 55%. 1 H NMR (600 MHz, DMSO-d 6 )δ12.98(br, 1H), 7.98(s, 1H), 7.77(s, 1H), 7.74(s, 1H), 7.59(d, J=7.8Hz, 1H), 7 .27 (d, J=7.8Hz, 1H), 7.06 (t, J=74.4Hz, 1H), 3.93 (d, J=5.4Hz, 2H), 3.82 (d, J=10. 2Hz, 2H), 3.23 (t, J=11.4Hz, 2H), 3.18-3.08 (m, 2H), 1.75 (s, 1H), 1.53 (d, J=12.6 Hz, 2H), 1.30-1.25 (m, 1H), 1.21-1.10 (m, 2H), 0.59 (q, J=5.4Hz, 2H), 0.35 (s, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ162.2, 150.1, 144.8, 140.0, 137.2, 128.3, 121.3, 120.6, 117.7, 116.7 (t, J = 258.0 Hz), 111.2, 73.1, 66.8 (2×C), 43.9, 35.1, 30.5 (2×C), 10.0, 3.0 (2×C). ESI-HRMS m / z: calculated value is C 21 H 25 O 4 N 3 F 2 Na+ [M+Na] + , 444.1705; the measured value is 444.1693.

[0158] Example 16 Synthesis of Compound A15:

[0159] The synthesis steps were as in Example 1. The amine reagent in the last step was 1-methyl-4-aminopyrazole (CAS No.: 69843-13-6) to obtain compound A15 with a yield of 40%. 1 H NMR (500 MHz, DMSO-d 6 ) δ13.08 (br, 1H), 9.99 (s, 1H), 8.01 (s, 1H), 7.90 (s, 1H), 7.79 (s, 1H), 7.66 (d, J = 13.5Hz, 2H), 7.30 (t, J = 4.0Hz, 1H), 7.15 (t, J=74.5Hz, 1H), 3.99 (d, J=7.0Hz, 2H), 3.82 (s, 3H), 1.36-1.27 (m, 1H), 0.66-0.56 (m, 2H), 0.44-0.31 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ159.3, 150.1, 145.0, 140.0, 136.8, 130.3, 129.7, 128.2, 121.6, 121.4, 121.3, 117.9, 116.7 (t, J = 258.0 Hz), 111.3, 73.2, 38.7, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 19 H 19 O 3 N 5 F 2 Na + [M+Na] + , 426.1348; the measured value is 426.1337.

[0160] Example 17 Synthesis of Compound A16:

[0161] The synthesis steps were as in Example 1. The amine reagent in the last step was N-methyl-3-aminopyrazole (CAS No.: 1904-31-0) to obtain compound A16 with a yield of 62%. 1 H NMR (500 MHz, DMSO-d 6)δ13.17 (br, 1H), 9.68 (s, 1H), 7.97 (s, 1H), 7.80 (s, 1H), 7.65-7.61 (m, 1H), 7.60 (d, J=2.0Hz, 1H), 7.28 (d, J=5.5Hz, 1H), 7.13 (t,, J=74.5Hz, 1H), 6.56 (s, 1H), 4.00 (d, J=7.0Hz, 2H), 3.77 (s, 3H), 1.37-1.25 (m, 1H), 0.63-0.51 (m, 2H), 0.46-0.33 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ159.5, 150.1, 146.4, 145.2, 140.1, 136.1, 131.2, 128.1, 121.7, 121.3, 117.8, 116.7 (t, J = 256.5 Hz), 111.4, 96.5, 73.2, 38.3, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 19 H 19 O 3 N 5 F 2 Na + [M+Na] + , 426.1348; the measured value is 426.1348.

[0162] Example 18 Synthesis of Compound A17:

[0163] The synthesis steps were as in Example 1. The amine reagent in the last step was 5-aminoindole to obtain compound A17 with a yield of 32%. 1 H NMR (400 MHz, DMSO-d 6 )δ13.13 (br, 1H), 11.04 (br, 1H), 9.60 (s, 1H), 8.02 (s, 1H), 7.96 (s, 1H), 7.84 (d, J = 1.6Hz, 1H), 7.68 (dd, J = 8.4, 1.6Hz, 1H), 7.45-7.41 (m, 1 H), 7.39-7.27 (m, 3H), 7.16 (t, J=74.4Hz, 1H), 6.40 (s, 1H), 3.99 (d, J=6.8Hz, 2H), 1.36-1.29 (m, 1H), 0.66-0.57 (m, 2H), 0.44-0.35 (m, 2H). 13 C NMR (101 MHz, DMSO-d 6)δ160.3, 150.2, 145.0, 140.1, 137.3, 132.9, 130.7, 128.3, 127.5, 126.0, 121.4, 121.3, 117.8, 116.8 (t, J = 256.3 Hz), 115.6, 111.4, 111.3, 111.1, 101.1, 73.2, 10.1, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 23 H 20 O 3 N 4 F 2 Na + [M+Na] + , 461.1396; the measured value is 461.1394.

[0164] Example 19 Synthesis of Series B Compounds

[0165] (1) Synthesis of compound 7:

[0166]

[0167] 1.0 g of compound 3 (4.13 mmol, 1.0 equiv.) was dissolved in a mixture of methanol and water, 1.32 g of sodium hydroxide (33.04 mmol, 8.0 equiv.) and 1.32 g of 30% hydrogen peroxide solution (20.65 mmol, 5.0 equiv.) were added, and the mixture was stirred at 50° C. and monitored by TLC. Then, methanol was removed by rotary evaporation, pH was adjusted to 6 with dilute hydrochloric acid, and the solid was washed with water 3 times to obtain intermediate 7 (867.5 mg) with a yield of 89%.

[0168] The obtained compound 7 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (400 MHz, CDCl 3 )δ7.74 (dd, J=8.4, 2.0 Hz, 1H), 7.67 (d, J=2.0 Hz, 1H), 7.25 (d, J=8.4 Hz, 1H), 6.74 (t, J=74.8 Hz, 1H), 3.95 (d, J=6.8 Hz, 2H), 1.37-1.30 (m, 1H), 0.72-0.63 (m, 2H), 0.42-0.35 (m, 2H), OH (1H, not observed). ESI-MS m / z: calculated value is C 12 H 13 O 4 F 2 + [M+Na] +, 259.0; the measured value is 259.0.

[0169] (2) Synthesis of Compound 8:

[0170]

[0171] Compound 7 (0.57 mmol, 1.0 equiv.) was dissolved in thionyl chloride and stirred at room temperature for 0.5 h. The solvent was removed by rotary evaporation to obtain the acid chloride intermediate. Triethylamine (1.14 mmol, 2.0 equiv.) was added to a dichloromethane solution of L-serine methyl ester hydrochloride (0.57 mmol, 1.0 equiv.) and the mixture was stirred for 0.5 h. The dichloromethane solution containing the acid chloride intermediate was added dropwise at 0 ° C. The reaction mixture was stirred for 12 h. The reaction solution was concentrated and purified by column chromatography to obtain compound 8. The yield was 45%.

[0172] The obtained compound 8 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (400 MHz, CDCl 3 )δ7.47 (d, J = 2.0 Hz, 1H), 7.32 (dd, J = 8.4, 2.0 Hz, 1H), 7.20-7.13 (m, 2H), 6.69 (t, J = 75.2 Hz, 1H), 4.86-4.80 (m, 1H), 4.08 (dd, J = 11.6, 3.6 Hz, 1H), 4.02 (dd, J = 11.6, 3.6 Hz, 1H), 3.91 (d, J = 6.8 Hz, 2H), 3.81 (s, 3H), 2.51 (br, 1H), 1.33-1.27 (m, 1H), 0.68-0.60 (m, 2H), 0.38-0.31 (m, 2H). ESI-HRMS m / z: calculated value is C 16 H 20 O 6 NF 2 + [M+H] + , 360.1253; the measured value is 360.1261.

[0173] (3) Synthesis of compound 9:

[0174]

[0175] Diethylaminosulfur trifluoride (20.51 mmol, 3.5 equiv.) was added dropwise to a dichloromethane solution of intermediate 8 (5.86 mmol, 1.0 equiv.). After stirring the reaction at -78°C for 4 hours, potassium carbonate was added. The reaction mixture was then allowed to warm to room temperature and stirred for 24 hours. After adding water, it was extracted with dichloromethane, the organic layer was washed with brine, and dried over sodium sulfate. Purification by column chromatography gave intermediate 9. Yield 52%.

[0176] The obtained compound 9 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (400 MHz, CDCl 3 )δ7.58 (d, J=1.6 Hz, 1H), 7.54 (dd, J=8.4, 1.6 Hz, 1H), 7.18 (d, J=8.4 Hz, 1H), 6.69 (t, J=75.2 Hz, 1H), 4.94 (dd, J=10.4, 8.0 Hz, 1H), 4.68 (t, J=8.0 Hz, 1H), 4.61 (dd, J=10.4, 8.0 Hz, 1H), 3.94 (d, J=6.8 Hz, 2H), 3.84 (s, 3H), 1.32-1.29 (m, 1H), 0.68-0.60 (m, 2H), 0.37-0.32 (m, 2H). ESI-MS m / z: calculated value is C 16 H 18 O 5 NF 2 + [M+H] + , 342.0; the measured value is 342.0.

[0177] (4) Synthesis of compound 10:

[0178]

[0179] Bromotrichloromethane (5.90 mmol, 5.0 equiv.) was added to a DMF solution of compound 9 (1.18 mmol, 1.0 equiv.) and DBU (2.95 mmol, 2.5 equiv.), and the reaction mixture was stirred at 0°C for 20 hours. The resulting precipitate was collected by filtration to give compound 10. Yield 65%.

[0180] The obtained compound 10 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (600 MHz, DMSO-d 6)δ8.97(s, 1H), 7.64-7.58(m, 2H), 7.36(s, 1H), 7.22(t, J=73.8Hz, 1H), 4.00(d, J= 7.2Hz, 2H), 3.85(s, 3H), 1.32-1.22(m, 1H), 0.63-0.55(m, 2H), 0.42-0.36(m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ161.0, 160.6, 150.2, 145.9, 142.1, 133.5, 123.9, 121.2, 119.1, 116.5 (t, J = 256.5 Hz), 111.7, 73.3, 51.9, 9.9, 3.0 (2×C). ESI-HRMS m / z: calculated value is C 16 H 15 O 5 NF 2 Na + [M+Na] + , 362.0811; the measured value is 362.0822.

[0181] (5) Synthesis of compound 11:

[0182]

[0183] 3.0 g of compound 10 (8.84 mmol, 1.0 equiv.) was weighed and dissolved in 15 mL of a 1:1 mixed solvent of ethanol and water, 2.12 g of sodium hydroxide (53.04 mmol, 6.0 equiv.) was added, and the mixture was stirred at room temperature for reaction, and monitored by TLC. After the reaction was complete, the ethanol was removed by rotary evaporation, an appropriate amount of water was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The solid was filtered and dried to obtain 2.8 g of yellow solid compound 11, with a yield of 97%.

[0184] The obtained compound 11 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (400 MHz, CDCl 3 )δ8.39 (s, 1H), 7.71 (d, J=2.0Hz, 1H), 7.66 (dd, J=8.4, 2.0Hz, 1H), 7.26 (d, J=8.4Hz, 1H), 6.72 (t , J=74.8Hz, 1H), 3.96 (d, J=6.8Hz, 2H), 1.38-1.28 (m, 1H), 0.71-0.64 (m, 2H), 0.40-0.35 (m, 2H). 13 C NMR (101 MHz, CDCl 3)δ165.7, 162.1, 151.0, 145.3, 142.9, 133.8, 124.5, 122.8, 120.0, 115.9 (t, J = 259.3 Hz), 112.6, 74.4, 10.1, 3.4 (2×C). ESI-MS m / z: calculated value is C 15 H 13 O 5 NF 2 + [M+H] + , 326.1; the measured value is 326.1.

[0185] (6) Synthesis of Compounds B1-B12:

[0186]

[0187] 100 mg of compound 11 (0.31 mmol, 1.0 equiv.) was dissolved in DMF, and then 117 mg of HATU (0.31 mmol, 1.0 equiv.), amine reagent (0.31 mmol, 1.0 equiv.) and 80 mg of DIPEA (0.62 mmol, 2 equiv.) were added. The reaction was stirred at room temperature and monitored by TLC. When there was no starting material, water was added to quench the reaction, and the mixture was extracted once with ethyl acetate. The organic phase was washed three times with water and once with saturated sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and silica gel was added to mix the sample. The product B1-B10 was purified by column chromatography. Different amine reagents (0.31 mmol, 1.0 equiv.) and thionyl chloride were added to the DCM solution of intermediate 11 (0.31 mmol, 1.0 equiv.). The reaction was stirred at 40°C for 8 hours. After adding water, the mixture was extracted with ethyl acetate, the organic layer was washed with brine, and Na 2 SO 4 The residue was dried and purified by column chromatography to obtain Compound B11 or Compound B12.

[0188] The compounds B1-B12 are arranged in the following order:

[0189]

[0190] Example 20 Synthesis of Compound B1:

[0191] The synthesis steps were as in Example 19. The amine reagent used in the last step was 2,6-difluorobenzylamine to obtain compound B1 with a yield of 86%. 1 H NMR (400 MHz, CDCl 3)δ8.23 (s, 1H), 7.60-7.57 (m, 2H), 7.32-7.22 (m, 3H), 6.95-6.90 (m, 2H), 6.70 (t, J=75.2Hz, 1H), 4. 74 (d, J=6.0Hz, 2H), 3.95 (d, J=7.2Hz, 2H), 1.36-1.29 (m, 1H), 0.70-0.64 (m, 2H), 0.41-0.36 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ161.7 (dd, J=248,8 Hz, 2×C), 160.7, 160.2, 150.9, 142.6, 141.4, 137.2, 129.9 (t, J=10.3 Hz), 124.9, 122.9, 119.8, 116.0 (t, J=259.2 Hz), 113.6 (t, J=19 Hz), 112.3, 111.6 (dd, J=18,6 Hz, 2×C), 74.2, 31.0, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated value is C 22 H 18 O 4 N 2 F 4 Na + [M+Na] + , 473.1095; the measured value is 473.1097.

[0192] Example 21 Synthesis of Compound B2:

[0193] Synthesis steps: Refer to Example 19, the amine reagent in the last step of the reaction is 2-methylaminopyrimidine (CAS No.: 75985-45-4), and compound B2 is obtained with a yield of 86%; 1 H NMR (400 MHz, benzene-d 6 )δ8.36 (t, J=4.8Hz, 1H), 8.23-8.15 (m, 3H), 7.56-7.47 (m, 2H), 7.06 (d, J=8.4Hz, 1H), 6.48 (t, J=75.2Hz, 1H), 6.33- 6.29 (m, 1H), 5.08 (d, J = 5.2Hz, 2H), 3.47 (d, J = 6.8Hz, 2H), 1.46-1.35 (m, 1H), 0.45-0.38 (m, 2H), 0.20-0.12 (m, 2H). 13 C NMR (101 MHz, methanol-d 4)δ167.5, 163.2, 162.4, 158.7 (2×C), 152.2, 144.2, 143.0, 138.4, 126.0, 123.3, 121.2, 120.6, 117.8 (t, J=255.9 Hz), 113.4, 75.1, 45.8, 11.0, 3.6 (2×C). ESI-HRMS m / z: calculated value is C 20 H 18 O 4 N 4 F 2 Na + [M+Na] + , 439.1188; the measured value is 439.1185.

[0194] Example 22 Synthesis of Compound B3:

[0195] The synthesis steps were as in Example 19. The amine reagent in the last step was 6-aminobenzothiazole to obtain compound B3 with a yield of 75%. 1 H NMR (400 MHz, CDCl 3 )δ8.97(br, 1H), 8.95(s, 1H), 8.74(d, J=2.4Hz, 1H), 8.36(s, 1H), 8.11(d , J=8.8Hz, 1H), 7.68 (dd, J=8.4, 2.0Hz, 1H), 7.65 (d, J=2.0Hz, 1H), 7.59 (d d, J=8.8, 2.0Hz, 1H), 7.30 (d, J=8.0Hz, 1H), 6.73 (t, J=75.2Hz, 1H), 4.01( d, J=7.2Hz, 2H), 1.40-1.34(m, 1H), 0.74-0.67(m, 2H), 0.45-0.40(m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ160.9, 158.6, 153.7, 151.0, 150.3, 142.8, 141.9, 137.5, 135.2, 135.0, 124.7, 123.9, 123.0, 120.0, 119.2, 115.9 (t, J = 259.4 Hz), 112.7, 112.4, 74.3, 10.2, 3.5 (2×C). ESI-HRMS m / z: calculated value is C 22 H 18 O 4 N 3 SF 2 + [M+Na] +, 458.0981; the measured value is 458.0970.

[0196] Example 23 Synthesis of Compound B4:

[0197] The synthesis steps were as in Example 19. The amine reagent in the last step was 2,4-difluorobenzylamine to obtain compound B4 with a yield of 38%. 1 H NMR (400 MHz, CDCl 3 )δ8.24 (s, 1H), 7.61-7.56 (m, 2H), 7.45-7.37 (m, 2H), 7.24 (d, J=8.0Hz, 1H), 6.88-6.79 (m, 2H), 6.70 (t, J=74. 8Hz, 1H), 4.6 (d, J=6.0Hz, 2H), 4.0 (d, J=7.2Hz, 2H), 1.37-1.30 (m, 1H), 0.70-0.65 (m, 2H), 0.41-0.36 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ162.6 (dd, J=247, 11.9 Hz), 160.8, 160.7, 161.1 (dd, J=247, 11.9 Hz), 150.9, 142.6, 141.3, 137.2, 131.4 (dd, J=9.6, 5.8 Hz), 124.8, 122.9, 121.1 (dd, J=15.0, 3.7 Hz), 119.8, 115.9 (t, J=259.3 Hz), 112.3, 111.6 (dd, J=21.0, 3.7 Hz), 104.1 (t, J=25.4 Hz), 74.2, 36.5, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated for C 22 H 18 O 4 N 2 F 4 Na + [M+Na] + , 473.1095; the measured value is 473.1094.

[0198] Example 24 Synthesis of Compound B5:

[0199] The synthesis steps were as in Example 19. The amine reagent in the last step was 2-aminothiazole to obtain compound B5 with a yield of 40%. 1 H NMR (500 MHz, DMSO-d 6) δ12.37 (br, 1H), 9.03 (s, 1H), 7.73 (d, J = 2.0Hz, 1H), 7.66 (dd, J = 8.5, 2.0Hz, 1H), 7.56 (d, J = 3.5Hz, 1H), 7.38 (d, J = 8.0Hz, 1H) , 7.30 (d, J=3.5Hz, 1H), 7.23 (t, J=74.0Hz, 1H), 4.02 (d, J=6.5Hz, 2H), 1.39-1.23 (m, 1H), 0.66-0.51 (m, 2H), 0.45-0.33 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ160.3, 158.4, 157.9, 150.1, 143.9, 142.1, 137.6, 135.6, 124.0, 121.2, 119.2, 116.5 (t, J = 256.5 Hz), 114.1, 111.9, 73.3, 9.9, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 18 H 15 O 4 N 3 F 2 SNa + [M+Na] + , 430.0644; the measured value is 430.0656.

[0200] Example 25 Synthesis of Compound B6:

[0201] The synthesis steps were as in Example 19. The amine reagent used in the last step was 2,4,6-trifluorobenzylamine to obtain compound B6 with a yield of 80%. 1 H NMR (400 MHz, CDCl 3 )δ8.23 (s, 1H), 7.60-7.56 (m, 2H), 7.29 (t, J=6.0Hz, 1H), 7.23 (d, J=8.4Hz, 1H), 6.88-6.51 (m, 2H), 6.70 (t, J=73 .2Hz, 1H), 4.68 (d, J=6.0Hz, 2H), 3.94 (d, J=6.8Hz, 2H), 1.35-1.29 (m, 1H), 0.70-0.65 (m, 2H), 0.40-0.36 (m, 2H). 13 C NMR (101 MHz, CDCl 3)δ162.5 (dt, J=248, 16 Hz), 161.9 (ddd, J=242, 15, 11 Hz, 2×C), 160.8, 160.3, 150.9, 142.7, 141.4, 137.1, 124.9, 122.9, 119.9, 116.0 (t, J=259.3 Hz), 112.3, 110.1 (td, J=19.8, 4.7 Hz), 100.6 (ddd, J=28, 25, 3 Hz, 2×C), 74.3, 30.7, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated for C 22 H 17 O 4 N 2 F 5 Na + [M+Na] + , 491.1001; the measured value is 491.1018.

[0202] Example 26 Synthesis of Compound B7:

[0203] The synthesis steps were as in Example 19. The amine reagent in the last step was 2-aminobenzothiazole to obtain compound B7 with a yield of 47%. 1 H NMR (400 MHz, CDCl 3 )δ10.33 (br, 1H), 8.42 (s, 1H), 7.88-7.83 (m, 2H), 7.66 (d, J = 2.0Hz, 1H), 7.61 (dd, J = 8.4, 2.0Hz, 1H), 7.50-7.46 (m, 1H), 7.37-7.33 ( m, 1H), 7.29 (d, J=8.4Hz, 1H), 6.74 (t, J=75.2Hz, 1H), 4.02 (d, J=6.8Hz, 2H), 1.41-1.34 (m, 1H), 0.75-0.70 (m, 2H), 0.48-0.44 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ161.4, 158.3, 157.0, 151.1, 148.7, 142.9, 142.8, 135.7, 132.5, 126.6, 124.4, 124.3, 123.0, 121.7, 121.3, 119.7, 115.9 (t, J = 259.4 Hz), 112.3, 74.4, 10.2, 3.5 (2×C). ESI-HRMS m / z: calculated value is C 22 H 17 O 4 N 3 F 2SNa + [M+Na] + , 480.0800; the measured value is 480.0795.

[0204] Example 27 Synthesis of Compound B8:

[0205] The synthesis steps were as shown in Example 19. Aniline was used as the amine reagent in the last step to obtain compound B8 with a yield of 85%. 1 H NMR (400 MHz, CDCl 3 )δ8.77(br, 1H), 8.33(s, 1H), 7.74-7.72(m, 2H), 7.69-7.62(m, 2H), 7.39(t, J=7.6Hz, 2H), 7.28(d, J=8.4Hz, 1H), 7.17( t, J=7.6Hz, 1H), 6.73 (t, J=74.8Hz, 1H), 4.01 (d, J=7.2Hz, 2H), 1.39-1.32 (m, 1H), 0.74-0.67 (m, 2H), 0.45-0.38 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ160.8, 158.5, 151.0, 142.8, 141.8, 137.7, 137.5, 129.3 (2×C), 124.8 (2×C), 123.0, 120.0 (2×C), 119.9, 116.0 (t, J=258.2 Hz), 112.4, 74.3, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated value is C 21 H 18 O 4 N 2 F 2 Na + [M+Na] + , 423.1127; the measured value is 423.1135.

[0206] Example 28 Synthesis of Compound B9:

[0207] The synthesis steps were as in Example 19. The amine reagent in the last step was benzylamine, and compound B9 was obtained with a yield of 68%. 1 H NMR (400 MHz, CDCl 3)δ8.26 (s, 1H), 7.61-7.56 (m, 2H), 7.41-7.29 (m, 6H), 7.24 (d, J=8.4Hz, 1H), 6.70 (t, J=75.2Hz, 1H), 4 .66 (d, J=6.0Hz, 2H), 3.95 (d, J=7.2Hz, 2H), 1.36-1.30 (m, 1H), 0.70-0.64 (m, 2H), 0.41-0.36 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ160.7, 160.6, 150.9, 142.6, 141.3, 138.1, 137.4, 128.9 (2×C), 128.1 (2×C), 127.8, 124.9, 122.9, 119.8, 116.0 (t, J=258.0 Hz), 112.3, 74.2, 43.2, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated value is C 22 H 20 O 4 N 2 F 2 Na + [M+Na] + , 437.1283; the measured value is 437.1299.

[0208] Example 29 Synthesis of Compound B10:

[0209] The synthesis steps were as shown in Example 19. The amine reagent in the last step was 4-aminopyridine to obtain compound B10 with a yield of 100%. 1 H NMR (400 MHz, CDCl 3 )δ8.87 (br, 1H), 8.57 (d, J = 6.4Hz, 2H), 8.36 (s, 1H), 7.70-7.61 (m, 4H), 7.29 (d, J = 8.0Hz, 1H), 6.73 (t, J=74.8Hz, 1H), 4.01 (d, J=6.8Hz, 2H), 1.38-1.32 (m, 1H), 0.74-0.66 (m, 2H), 0.44-0.38 (m, 2H). 13 C NMR (101 MHz, CDCl 3)δ161.1, 159.0, 151.0 (2×C), 144.4, 142.9, 142.4 (2×C), 137.0, 124.5, 123.0, 120.1, 115.9 (t, J=259.6 Hz), 113.8 (2×C), 112.4, 74.4, 10.2, 3.5 (2×C). ESI-MS m / z: calculated value is C 20 H 17 O 4 N 3 F 2 + [M+Na] + , 424.1; the measured value is 424.1.

[0210] Example 30 Synthesis of Compound B11:

[0211] The synthesis steps were as in Example 19. The reagent for the last step was N-hydroxysuccinimide to obtain compound B11 with a yield of 85%. 1 H NMR (400 MHz, CDCl 3 )δ8.50 (s, 1H), 7.70 (d, J=2.0Hz, 1H), 7.66 (dd, J=8.0, 2.0Hz, 1H), 7.31-7.27 (m, 1H), 6.72 (t, J=74.8 Hz, 1H), 3.97 (d, J=6.8Hz, 2H), 2.92 (s, 4H), 1.35-1.31 (m, 1H), 0.70-0.65 (m, 2H), 0.41-0.36 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ168.9 (2×C), 162.6, 156.5, 151.0, 146.4, 143.1, 130.0, 124.1, 122.9, 120.1, 115.9 (t, J=258.2 Hz), 112.8, 74.4, 25.8 (2×C), 10.2, 3.4 (2×C). ESI-MS m / z: calculated value is C 19 H 17 O 7 N 2 F 2 + [M+H] + , 423.1; the measured value is 423.1.

[0212] Example 31 Synthesis of Compound B12:

[0213] The synthesis steps were as in Example 19. The reagent for the last step was 1H-benzo[d][1,2,3]triazole to obtain compound B12 with a yield of 53%. 1 H NMR (400 MHz, CDCl 3 )δ9.15 (s, 1H), 8.47 (d, J = 8.4Hz, 1H), 8.19 (d, J = 8.4Hz, 1H), 7.81 (d, J = 2.0Hz, 1H), 7.77-7.72 (m, 2H), 7.61-7.56 (m, 1H), 7. 30 (d, J=8.4Hz, 1H), 6.74 (t, J=74.8Hz, 1H), 4.01 (d, J=7.2Hz, 2H), 1.41-1.32 (m, 1H), 0.73-0.66 (m, 2H), 0.43-0.38 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ161.8, 158.1, 151.0, 147.9, 146.0, 143.0, 133.9, 132.0, 131.2, 126.9, 124.3, 122.9, 120.5, 120.2, 116.0 (t, J = 258.2 Hz), 114.9, 112.8, 74.5, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated value is C 21 H 16 O 4 N 4 F 2 + [M+H] + , 449.1032, the measured value is 449.1057.

[0214] Example 32 Synthesis of C series compounds

[0215] (1) Synthesis of compound 12:

[0216]

[0217] 1.0 g of compound 1 (7.24 mmol, 1.0 equiv.) was dissolved in 10 mL of DMF solvent, and then 0.72 g of sodium hydroxide (18.10 mmol, 2.5 equiv.) and 2.52 g of ethyl difluorochloroacetate (15.93 mmol, 2.2 equiv.) were added, and the mixture was heated to 80° C. for 12 h and monitored by thin layer chromatography. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate, and the organic phase was concentrated. 0.93 g of compound 12 was obtained by flash column chromatography with a yield of 54%.

[0218] The obtained compound 12 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (400 MHz, CDCl 3 )δ9.91(s,1H),7.75-7.73(m,2H),7.39(d,J=8.8Hz,1H),6.64(t,J=72.8Hz,1H),6.59(t,J=72.8Hz,1H).ESI-MS m / z: calculated value is C 9 H 7 O 3 F 4 + [M+H] + , 239.0; the measured value is 239.0.

[0219] (2) Synthesis of compound 13:

[0220]

[0221] 3,3-Dibromo-1,1,1-trifluoro-2-one (42.0 mmol, 2.0 equiv.) was added to an aqueous sodium acetate solution (84.0 mmol, 4.0 equiv.), stirred at 100°C for 1 hour, and after cooling to room temperature, a mixed solution of intermediate 12 (21.0 mmol, 1.0 equiv.) in methanol and aqueous ammonia was added. The reaction mixture was stirred at room temperature overnight. The methanol was then removed by rotary evaporation. It was extracted with ethyl acetate three times, washed with a saturated sodium chloride solution, and subjected to column chromatography to obtain intermediate 13 with a yield of 64%.

[0222] The obtained compound 13 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (500 MHz, CDCl 3 )δ12.06 (br, 1H), 7.58 (d, J=2.0 Hz, 1H), 7.53 (dd, J=8.5, 2.0 Hz, 1H), 7.49 (d, J=1.0 Hz, 1H), 7.12 (d, J=8.5 Hz, 1H), 6.48 (t, J=73.0 Hz, 1H), 6.39 (t, J=73.0 Hz, 1H). ESI-HRMS m / z: calculated value is C 12 H 8 O 2 N 2 F 7 + [M+H] + , 345.0469; the measured value is 345.0429.

[0223] (3) Synthesis of compound 14:

[0224]

[0225] Intermediate 13 (0.13 mmol, 1.0 equiv.) was dissolved in a mixture of ethanol and water at a ratio of 1:1, sodium hydroxide (2.60 mmol, 20 equiv.) was added, stirred at 80°C, and monitored by TLC. After the reaction was completed, ethanol was removed by rotary evaporation. Intermediate 14 was obtained by column chromatography. Yield: 63%.

[0226] The obtained compound 14 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (500 MHz, DMSO-d 6 )δ13.23 (br, 1H), 8.13-7.66 (m, 4H), 7.46 (d, J=8.0 Hz, 1H), 7.27 (t, J=73.0 Hz, 2H). ESI-MS m / z: calculated value is C 12 H 9 O 4 N 2 F 4 + [M+H] + , 321.0; the measured value is 321.0.

[0227] (4) Synthesis of Compounds C1-C3:

[0228]

[0229] 50 mg of intermediate 14 (0.16 mmol, 1.0 equiv.) was dissolved in DMF, and HATU (0.16 mmol, 1.0 equiv.), amine reagent (0.16 mmol, 1.0 equiv.) and DIPEA (0.32 mmol, 2.0 equiv.) were added. The mixture was stirred at room temperature and monitored by TLC. The reactants were extracted with ethyl acetate and washed with Na 2 SO 4 After drying, the product was purified by column chromatography to obtain compounds C1-C3.

[0230]

[0231] Example 33 Synthesis of Compound C1:

[0232] The synthesis steps were as in Example 32. The amine reagent used in the last step was 2,5-difluorobenzylamine to obtain compound C1 with a yield of 25%. 1 H NMR (400 MHz, DMSO-d 6) δ13.24 (br, 1H), 8.64 (t, J = 6.4Hz, 1H), 7.98 (s, 1H), 7.94 (dd, J = 8.4, 1.6Hz, 1H), 7.86 (s, 1H), 7.49 (d, J = 8.4Hz, 1H), 7.28-7.22 (m, 1H), 7.28 (t, J=73.2Hz, 1H), 7.25 (t, J=73.2Hz, 1H), 7.17-7.07 (m, 2H), 4.48 (d, J=6.0Hz, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ162.4, 158.2 (dd, J = 239.6, 2.0 Hz), 156.0 (d, J = 235 Hz), 144.0, 142.1, 141.9, 136.9, 128.9 (dd, J = 17.6, 7.3 Hz), 128.3, 123.3, 121.6, 121.2, 118.3, 116.7 (dd, J = 24, 9 Hz), 116.6 (t, J = 258.6 Hz), 116.4 (t, J = 258.6 Hz), 115.5 (dd, J = 24.7, 4.9 Hz), 115.0 (dd, J = 23.8, 8.6 Hz), 38.3. ESI-HRMS m / z: calculated for C 19 H 13 O 3 N 3 F 6 Na + [M+Na] + , 468.0753; the measured value is 468.0755.

[0233] Example 34 Synthesis of Compound C2:

[0234] The synthesis steps were as in Example 32. The amine reagent in the last step was 2,4-difluorobenzylamine to obtain compound C2 with a yield of 29%. 1 H NMR (500 MHz, DMSO-d 6 )δ13.47 (br, 1H), 8.56 (s, 1H), 8.05 (s, 1H), 7.99 (d, J = 8.5Hz, 1H), 7.80 (s, 1H), 7.47 (d, J = 8.5Hz, 1H), 7.43-7.37 (m , 1H), 7.28 (td, J=73.0, 2.0Hz, 2H), 7.20 (td, J=9.5, 2.5Hz, 1H), 7.05 (td, J=8.5, 2.5Hz, 1H), 4.47 (d, J=6.0Hz, 2H). 13 C NMR (151 MHz, DMSO-d6 )δ162.2, 161.3 (dd, J = 243, 12.0 Hz), 159.8 (dd, J = 246, 12.0 Hz), 144.0, 141.9, 136.9, 130.6, 129.7, 128.2, 123.3, 123.0 (d, J = 13.5 Hz), 121.3, 121.2, 118.1, 116.5 (t, J = 258 Hz), 116.4 (t, J = 258 Hz), 111.3 (dd, J = 21.0, 3.0 Hz), 103.5 (t, J = 25.5 Hz), 35.2. ESI-HRMS m / z: calculated for C 19 H 13 O 3 N 3 F 6 Na + [M+Na] + , 468.0753; the measured value is 468.0755.

[0235] Example 35 Synthesis of Compound C3:

[0236] The synthesis steps were as in Example 32. The amine reagent was 2-methylaminopyrimidine (CAS No.: 75985-45-4) to obtain compound C3 with a yield of 30%. 1 H NMR (400 MHz, DMSO-d 6 ) δ13.23 (br, 1H), 8.78 (d, J = 4.8Hz, 2H), 8.48 (t, J = 5.6Hz, 1H), 7.99 (s, 1H), 7.95 (d, J = 8.8Hz, 1H), 7.85 (s, 1H), 7 .50 (d, J=8.8Hz, 1H), 7.41 (t, J=4.8Hz, 1H), 7.29 (t, J=73.2Hz, 1H), 7.28 (t, J=73.2Hz, 1H), 4.67 (d, J=5.6Hz, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ166.9, 162.1, 157.4 (2×C), 143.9, 142.1, 142.0, 137.1, 128.3, 123.4, 121.3, 121.3, 119.8, 118.1, 116.6 (t, J = 258.3 Hz), 116.4 (t, J = 258.3 Hz), 44.5. ESI-HRMS m / z: calculated value is C 17 H 13 O 3 N 5 F 4 Na +[M+Na] + , 434.0847; the measured value is 434.0843.

[0237] Example 36 Synthesis of D series compounds

[0238] (1) Synthesis of compound 15:

[0239]

[0240] D-cysteine ​​methyl ester hydrochloride (16.51 mmol, 1.0 equiv.) was added dropwise to intermediate 3 (16.51 mmol, 1.0 equiv.) in DMF. Potassium carbonate was added. Stirred at room temperature for 24 hours. Stirred at -15°C to turn orange-yellow, then bromochloromethane (33.02 mmol, 2.0 equiv.) and DBU (33.02 mmol, 2.0 equiv.) were added at -5°C, reacted for 1-2 hours, and quenched with water. The organic layer was washed with brine, dried over sodium sulfate, and concentrated. Purified by column chromatography to obtain intermediate 15. Yield 15%.

[0241] The obtained compound 15 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (400 MHz, CDCl 3 )δ8.17 (s, 1H), 7.66 (d, J=2.0Hz, 1H), 7.46 (dd, J=8.4, 2.0Hz, 1H), 7.22 (d, J=8.0Hz, 1H), 6.70 (t, J=75.2Hz, 1H), 4.00-3.98 (m, 5H), 1.38-1.27 (m, 1H), 0.70-0.64 (m, 2H), 0.41-0.36 (m, 2H). 13 C NMR (101 MHz, CDCl 3 )δ168.1, 162.0, 151.0, 147.8, 142.3, 131.3, 127.7, 122.9, 120.1, 116.0 (t, J = 258.8 Hz), 112.4, 74.4, 52.7, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated value is C 16 H 15 O 4 NF 2 SNa + [M+Na] + , 378.0582; the measured value is 378.0581.

[0242] (2) Synthesis of compound 16:

[0243]

[0244] Intermediate 15 (0.28 mmol, 1.0 equiv.) was added to a mixed solution of sodium hydroxide (1.40 mmol, 5.0 equiv.) in water and ethanol, and stirred at room temperature for 10 hours. HCl was added to adjust the pH to 6, and the resulting precipitate was collected by filtration to obtain compound 16. The yield was 96%.

[0245] The obtained compound 16 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (500 MHz, DMSO-d 6 )δ8.18 (s, 1H), 7.63 (s, 1H), 7.51 (d, J = 8.5Hz, 1H), 7.28 (d, J = 8.0Hz, 1H), 7.18 (t, J = 74.0Hz , 1H), 4.01 (d, J=7.0Hz, 2H), 1.32-1.24 (m, 1H), 0.62-0.53 (m, 2H), 0.45-0.33 (m, 2H), OH (not observed). 13 C NMR (101 MHz, CDCl 3 )δ167.6, 164.5, 150.7, 148.5, 142.1, 130.7, 127.7, 122.5, 119.7, 115.9 (t, J = 258.8 Hz), 112.0, 74.0, 10.0, 3.2 (2×C). ESI-MS m / z: calculated value is C 15 H 14 O 4 NF 2 S + [M+H] + , 342.1; the measured value is 342.1.

[0246] (3) Synthesis of compounds D1-D11:

[0247]

[0248] 50 mg of compound 16 (0.15 mmol, 1.0 equiv.) was weighed and dissolved in N, N-dimethylformamide, 59 mg of HATU (0.15 mmol, 1.0 equiv.) and 60 mg of DIPEA (0.45 mmol, 3.0 equiv.) were added, stirred at room temperature for 20 min, and a suitable amine reagent (0.15 mmol, 1.0 equiv.) was added, and the reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC, and water was added to quench the reaction after the reaction of the raw material was complete, extracted with ethyl acetate, washed once with a saturated sodium chloride solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate. The solution was concentrated and purified by column chromatography to obtain compounds D1-D11.

[0249]

[0250] Example 37 Synthesis of Compound D1:

[0251] The synthesis steps were as in Example 36. The amine reagent was 2,6-difluorobenzylamine to obtain compound D1 with a yield of 75%. 1 H NMR (600 MHz, DMSO-d 6 )δ8.85 (t, J=5.4Hz, 1H), 8.31 (s, 1H), 7.68 (d, J=2.4Hz, 1H), 7.62 (dd, J=8.4, 1.8Hz, 1H), 7.42-7.34 (m, 1H), 7.31 (s, 1H), 7.19 (t, J=7 4.4Hz, 1H), 7.08-7.04 (m, 2H), 4.59 (d, J=5.4Hz, 2H), 4.00 (d, J=7.2Hz, 2H), 1.32-1.21 (m, 1H), 0.63-0.54 (m, 2H), 0.43-0.26 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.3, 161.1 (dd, J = 246.0, 9.0 Hz, 2×C), 160.1, 150.3 (2×C), 141.7, 130.6, 129.8 (t, J = 10.5 Hz), 124.7, 121.3, 119.4, 116.6 (t, J = 258.0 Hz), 114.2 (t, J = 19.5 Hz), 112.2, 111.5 (dd, J = 21.0, 6.0 Hz, 2×C), 73.3, 31.0, 10.0, 3.0 (2×C). ESI-HRMS m / z: calculated value is C 22 H 18 O 3 N 2 F 4 SNa + [M+Na]+ , 489.0866; the measured value is 489.0849.

[0252] Example 38 Synthesis of Compound D2:

[0253] The synthesis steps were as in Example 36. The amine reagent was 2-methylaminopyrimidine (CAS No.: 75985-45-4) to obtain compound D2 with a yield of 88%. 1 H NMR (600 MHz, DMSO-d 6 )δ9.08 (t, J=6.0Hz, 1H), 8.78 (d, J=4.8Hz, 2H), 8.33 (s, 1H), 7.77 (s, 1H), 7.63 (d, J=8.4Hz, 1H), 7.41 (s, 1H), 7.33 (s, 1H), 7 .15 (t, J=74.4z, 1H), 4.73 (d, J=6.0Hz, 2H), 4.04 (d, J=6.6Hz, 2H), 1.33-1.25 (m, 1H), 0.66-0.57 (m, 2H), 0.43-0.36 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.7, 166.3, 160.6, 157.4 (2×C), 150.4, 150.3, 141.7, 130.7, 124.5, 121.4, 119.9, 119.3, 116.6 (t, J=258.5 Hz), 112.0, 73.3, 44.9, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 20 H 18 O 3 N 4 F 2 SNa + [M+Na] + , 455.0960; the measured value is 455.0950.

[0254] Example 39 Synthesis of Compound D3:

[0255] The synthesis steps were as in Example 36. The amine reagent was 2,4-difluorobenzylamine to obtain compound D3. The yield was 100%. 1 H NMR (600 MHz, DMSO-d 6)δ9.10 (t, J=6.0Hz, 1H), 8.23 ​​(s, 1H), 7.66 (d, J=2.4Hz, 1H), 7.52 (dd, J=8.4 , 1.8Hz, 1H), 7.35-7.29 (m, 1H), 7.23 (d, J=8.4Hz, 1H), 7.05 (t, J=73.2Hz, 1H ), 7.08-7.02 (m, 1H), 6.95 (dd, J=8.4, 2.4Hz, 1H), 4.46 (d, J=6.6Hz, 2H), 3.8 7(d, J=7.2Hz, 2H), 1.21-1.13(m, 1H), 0.57-0.47(m, 2H), 0.32-0.19(m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ167.3, 162.0 (dd, J = 243, 12.0 Hz), 161.6, 160.6 (dd, J = 246, 12.0 Hz), 150.8, 150.5, 142.2, 131.3 (dd, J = 9.0, 6.0 Hz), 131.1, 125.4, 122.6 (dd, J = 15.0, 4.5 Hz), 121.9, 120.1, 117.0 (t, J = 258.0 Hz), 112.4, 111.9 (dd, J = 24.0, 3.0 Hz), 104.2 (t, J = 25.5 Hz), 74.0, 36.4, 10.5, 3.6 (2×C). ESI-HRMS m / z: calculated for C 22 H 19 O 3 N 2 F 4 S + [M+H] + , 467.1047; the measured value is 467.1033.

[0256] Example 40 Synthesis of Compound D4:

[0257] The synthesis steps were as in Example 36, and 2-aminothiazole was selected as the amine reagent to obtain compound D4 with a yield of 81%; 1 HNMR (600 MHz, DMSO-d 6)δ8.57 (s, 1H), 7.84 (d, J = 2.4Hz, 1H), 7.63 (dd, J = 8.4, 1.8Hz, 1H), 7.56 (d, J = 3.6Hz, 1H), 7.30 (d, J = 3.6Hz, 1H), 7.29 (s, 1H) , 7.12 (t, J=74.4Hz, 1H), 4.00 (d, J=6.6Hz, 2H), 1.29-1.22 (m, 1H), 0.61-0.54 (m, 2H), 0.46-0.30 (m, 2H), NH (notobserved). 13 C NMR (151 MHz, DMSO-d 6 )δ167.5, 159.3, 158.2, 150.8, 148.5, 142.2, 138.3, 130.9, 127.8, 121.8, 120.2, 117.0 (t, J = 258.7 Hz), 115.0, 112.6, 73.9, 10.4, 3.6 (2×C). ESI-HRMS m / z: calculated value is C 18 H 16 O 3 N 3 F 2 S 2 + [M+H] + , 424.0596; the measured value is 424.0585.

[0258] Example 41 Synthesis of Compound D5:

[0259] Synthesis steps: Refer to Example 36, select 2,4,6-trifluorobenzylamine as the amine reagent to obtain compound D5, yield: 100%; 1 H NMR (600 MHz, DMSO-d 6 )δ8.89 (t, J=5.4Hz, 1H), 8.30 (s, 1H), 7.68 (s, 1H), 7.62 (dd, J=8.4, 1.8Hz, 1H), 7.31 (d, J=3.6Hz, 1H), 7.19-7.14 (m, 2H), 7. 18 (t, J=74.4Hz, 1H), 4.53 (d, J=6.0Hz, 2H), 4.01 (d, J=7.2Hz, 2H), 1.35-1.17 (m, 1H), 0.68-0.49 (m, 2H), 0.43-0.34 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6)δ166.3, 161.4 (dt, J = 246, 16.5 Hz), 161.3 (ddd, J = 248, 34.5, 30.0 Hz, 2×C), 160.2, 150.3, 150.2, 141.7, 130.7, 124.7, 121.4, 119.4, 116.6 (t, J = 258.0 Hz), 112.3, 111.1 (td, J = 19.5, 4.5 Hz), 100.5 (dd, J = 30.0, 25.5 Hz, 2×C), 73.4, 30.8, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 22 H 18 O 3 N 2 F 5 S + [M+H] + , 485.0953; the measured value is 485.0933.

[0260] Example 42 Synthesis of Compound D6:

[0261] The synthesis steps were as in Example 36, and 2-aminobenzothiazole was selected as the amine reagent to obtain compound D6 with a yield of 67%; 1 H NMR (600 MHz, DMSO-d 6 ) δ12.55 (s, 1H), 8.74 (s, 1H), 8.05 (d, J = 7.8Hz, 1H), 7.93 (d, J = 1.8Hz, 1H), 7.82 (d, J = 7.8Hz, 1H), 7.73 (dd, J = 8.4, 1.8Hz, 1H), 7.55-7 .46 (m, 1H), 7.38-7.31 (m, 2H), 7.22 (t, J=74.4Hz, 1H), 4.08 (d, J=6.6Hz, 2H), 1.35-1.27 (m, 1H), 0.66-0.57 (m, 2H), 0.48-0.32 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.8, 159.6, 157.9, 150.3, 148.5, 148.0, 141.9, 131.7, 130.5, 128.0, 126.3, 123.9, 121.8, 121.2, 120.5, 119.7, 116.6 (t, J = 258.0 Hz), 112.3, 73.4, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 22 H 18 O 3 N 3 F2 S 2 + [M+H] + , 474.0752; the measured value is 474.0753.

[0262] Example 43 Synthesis of Compound D7:

[0263] The synthesis steps were as in Example 36, and 4-aminopyridine was selected as the amine reagent to obtain compound D7 with a yield of 86%. 1 HNMR (500 MHz, DMSO-d 6 ) δ10.55 (s, 1H), 8.58 (s, 1H), 8.51 (d, J = 6.5Hz, 2H), 7.89 (d, J = 6.5Hz, 2H), 7.82 (d, J = 2.0Hz, 1H), 7.71 (dd, J = 8.0, 2.0Hz, 1H), 7.35 (d, J=8.5Hz, 1H), 7.22 (t, J=74.0Hz, 1H), 4.07 (d, J=7.0Hz, 2H), 1.35-1.25 (m, 1H), 0.65-0.56 (m, 2H), 0.46-0.32 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.6, 159.8, 150.3, 150.3 (2×C), 149.6, 145.2, 141.9, 130.4, 126.8, 121.3, 119.7, 116.6 (t, J=258.5 Hz), 114.3 (2×C), 112.4, 73.4, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 20 H 18 O 3 N 3 F 2 S + [M+H] + , 418.1031; the measured value is 418,1023.

[0264] Example 44 Synthesis of Compound D8:

[0265] The synthesis steps were as in Example 36, and 2,3-difluorobenzylamine was selected as the amine reagent to obtain compound D8 with a yield of 100%. 1 H NMR (500 MHz, DMSO-d 6)δ9.14 (t, J=6.5Hz, 1H), 8.35 (s, 1H), 7.73 (d, J=2.0Hz, 1H), 7.64 (dd, J=8.5, 2.5Hz, 1H), 7.36-7.25 (m, 2H), 7.22-7.15 (m, 2H) , 7.19 (t, J=74.5Hz, 1H), 4.60 (d, J=6.0Hz, 2H), 4.02 (d, J=7.0Hz, 2H), 1.33-1.24 (m, 1H), 0.63-0.56 (m, 2H), 0.40-0.36 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.3, 160.7, 150.3, 150.2, 149.6 (dd, J = 244.5, 13.5 Hz), 147.6 (dd, J = 244.5, 13.5 Hz), 141.7, 130.6, 128.8 (d, J = 12.0 Hz), 124.8, 124.6 (dd, J = 7.5, 4.5 Hz), 124.5 (t, J = 3.0 Hz), 121.3, 119.4, 116.6 (t, J = 258.0 Hz), 115.9 (d, J = 16.5 Hz), 112.1, 73.3, 35.8, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 22 H 18 O 3 N 2 F 4 SNa + [M+Na] + , 489.0866; the measured value is 489.0853.

[0266] Example 45 Synthesis of Compound D9:

[0267] The synthesis steps were as shown in Example 36. The amine reagent was 2-(aminomethyl)naphthalene (CAS No.: 2018-90-8) to obtain compound D9 with a yield of 100%. 1 H NMR (500 MHz, DMSO-d 6)δ9.21(t, J=6.5Hz, 1H), 8.36(s, 1H), 7.91-7.85(m, 3H), 7.82(s, 1H), 7.75(d, J=2.0Hz, 1H), 7.64 (dd, J=8.5, 2.0Hz, 1H), 7.53 (dd, J=8.5, 1.5Hz, 1H), 7.50-7. 46 (m, 2H), 7.32 (d, J = 8.5Hz, 1H), 7.19 (t, J = 74.5Hz, 1H), 4.70 (d, J = 6.5Hz, 2H), 4.02 (d, J=7.0Hz, 2H), 1.32-1.24 (m, 1H), 0.64-0.55 (m, 2H), 0.42-0.29 (m, 2H). 13 C NMR (151 MHz, DMSO-d 6 )δ166.3, 160.6, 150.5, 150.3, 141.7, 137.1, 132.9, 132.1, 130.7, 127.9, 127.5, 127.5, 126.2, 126.0, 125.6, 125.4, 124.6, 121.3, 119.4, 116.6 (t, J = 258.3 Hz), 112.1, 73.3, 42.5, 10.0, 3.0 (2×C). ESI-HRMS m / z: calculated for C 26 H 22 O 3 N 2 F 2 SNa + [M+Na] + , 503.1211; the measured value is 503.1191.

[0268] Example 46 Synthesis of Compound D10:

[0269] The synthetic steps were as in Example 36, and benzylamine was selected as the amine reagent to obtain compound D10 with a yield of 49%; 1 H NMR (500 MHz, DMSO-d 6 )δ9.10 (t, J=6.5Hz, 1H), 8.33 (s, 1H), 7.74 (d, J=2.0Hz, 1H), 7.63 (dd, J=8.0, 2.0Hz, 1H), 7.36-7.31 (m, 5H), 7.27-7.22 (m, 1H) , 7.19 (t, J=74.0Hz, 1H), 4.53 (d, J=6.5Hz, 2H), 4.02 (d, J=7.0Hz, 2H), 1.32-1.24 (m, 1H), 0.65-0.54 (m, 2H), 0.43-0.30 (m, 2H). 13C NMR (101 MHz, CDCl 3 )δ167.3, 161.1, 151.0, 150.8, 142.3, 138.3, 131.3, 128.9 (2×C), 128.0 (2×C), 127.7, 123.7, 123.1, 119.9, 116.0 (t, J=259.1 Hz), 112.2, 74.3, 43.5, 10.2, 3.4 (2×C). ESI-HRMS m / z: calculated value is C 22 H 21 O 3 N 2 F 2 S + [M+H] + , 431.1235; the measured value is 431.1229.

[0270] Example 47 Synthesis of Compound D11:

[0271] The synthetic steps were as in Example 36. Aniline was selected as the amine reagent to obtain compound D11 with a yield of 46%. 1 H NMR (600 MHz, DMSO-d 6 )δ10.22 (s, 1H), 8.48 (s, 1H), 7.87-7.82 (m, 2H), 7.71 (dd, J=8.4, 1.8Hz, 1H), 7.42-7.36 (m, 2H), 7.33 (d, J=2.4Hz, 1H), 7.3 6-7.06 (m, 2H), 7.15 (tt, J=7.2, 1.2Hz, 1H), 4.07 (d, J=6.6Hz, 2H), 1.35-1.26 (m, 1H), 0.67-0.58 (m, 2H), 0.49-0.36 (m, 2H). 13 CNMR (151 MHz, DMSO-d 6 )δ166.4, 159.0, 150.4, 150.3, 141.8, 138.3, 130.6, 128.7 (2×C), 125.6, 124.1, 121.3, 120.7 (2×C), 119.6, 116.6 (t, J=256.5 Hz), 112.4, 73.4, 10.0, 3.1 (2×C). ESI-HRMS m / z: calculated value is C 21 H 19 O 3 N 2 F 2 S + [M+H] + , 417.1079; the measured value is 417.1058.

[0272] Example 48 Synthesis of E series compounds

[0273] (1) Synthesis of Compound 18:

[0274]

[0275] Weigh 200 mg of raw material 17 (1.10 mmol, 1.0 equiv.), add 116.2 mg of sodium carbonate (1.10 mmol, 1.0 equiv.), add 2.5 mL of reagent N, N-dimethylformamide, and finally add 174 mg of ethyl difluorochloroacetate (1.10 mmol, 1.0 equiv.). After reacting at 80°C for 12 hours, TLC detected that the raw material reacted completely, and aqueous solution was added to quench the reaction. Use ethyl acetate to extract 3 times, and wash the organic phase with saturated sodium chloride solution. The organic phase is concentrated and subjected to rapid column chromatography to obtain 60 mg of the product with a yield of 24%.

[0276] The obtained compound 18 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (500 MHz, CDCl 3 )δ9.92(s,1H),7.16(s,2H),6.66(t,J=76.0Hz,1H),3.96(s,6H). ESI-MSm / z: calculated value is C 10 H 11 O 2 F 2 + [M+H] + , 233.1; the measured value is 233.1.

[0277] (2) Synthesis of Compound 19:

[0278]

[0279] Weigh 1.76 g of 3,3-dibromo-1,1,1-trifluoro-2-one (8.6 mmol, 2.0 equiv.) and 1.42 g of sodium acetate (17.2 mmol, 4.0 equiv.) into a reaction bottle, add an appropriate amount of water to dissolve, stir at 100°C for 1 hour, cool to room temperature, add a methanol solution containing 1 g of compound 18 (4.3 mmol, 1.0 equiv.) and 4 mL of ammonia water to the above solution, stir at room temperature, and remove methanol by rotary evaporation when TLC detects that the raw material is no longer reduced. Add water, extract with ethyl acetate 3 times, wash the organic phase with saturated sodium chloride solution once, and dry the organic phase with anhydrous sodium sulfate. Concentrate the organic phase and obtain 890 mg of a yellow solid product (compound 19) by rapid column chromatography, with a yield of 61%.

[0280] The obtained compound 19 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (500 MHz, DMSO-d 6 )δ13.26 (br, 1H), 7.98 (s, 1H), 7.36 (s, 2H), 6.89 (t, J=75.5 Hz, 1H), 3.89 (s, 6H). ESI-MS m / z: calculated value is C 13 H 12 O 2 N 2 F 5 + [M+H] + , 339.1; the measured value is 339.1.

[0281] (3) Synthesis of Compound 20:

[0282]

[0283] 1.6 g of compound 19 (4.73 mmol, 1.0 equiv.) was weighed and dissolved in a 1:1 mixed solvent of ethanol and water, 3.8 g of sodium hydroxide (94.6 mmol, 20 equiv.) was added, and the mixture was stirred at 80°C for reaction, monitored by TLC, and after the reaction was complete, the ethanol was removed by rotary evaporation, an appropriate amount of water was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The solid was filtered and dried to obtain 778 mg of a yellow solid product with a yield of 52%.

[0284] The obtained compound 20 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (500 MHz, DMSO-d 6 )δ7.88(s, 1H), 7.48(s, 2H), 6.90(t, J=75.0Hz, 1H), 3.89(s, 6H).OH(1H, not observed), NH(1H, not observed). ESI-MS m / z: calculated value is C 13 H 13 O 5 N 2 F 2 + [M+H] + , 315.0; the actual measured value is 315.0.

[0285] (4) Synthesis of Compounds E1-E3:

[0286]

[0287] 50 mg of compound 20 (0.09 mmol, 1.0 equiv.) was dissolved in DMF, and then 53 mg of HATU (0.09 mmol, 1.0 equiv.), 15 mg of amine reagent (0.1 mmol, 1.1 equiv.) and 36.5 mg of DIPEA (0.27 mmol, 3.0 equiv.) were added. The reaction was stirred at room temperature and monitored by TLC. Water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic phase was washed with water three times and with saturated sodium chloride solution once. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated, and silica gel was added to mix the sample. The product E1-E3 was purified by column chromatography.

[0288]

[0289] Example 49 Synthesis of Compound E1:

[0290] The synthesis steps were as in Example 48. The amine reagent was 2-methylaminopyrimidine (CAS No.: 75985-45-4) to obtain compound E1 with a yield of 19%. 1 H NMR (500 MHz, DMSO-d 6 ) δ 8.77 (d, J = 4.5Hz, 2H), 7.77 (s, 1H), 7.54 (s, 2H), 7.40 (t, J = 5.0Hz, 1H), 6.88 (t, J = 75.0Hz, 1H), 4.67 (d, J = 6.0Hz, 2H), 3.90 (s, 6H), NH (2H, not observed). 13 C NMR (101 MHz, DMSO-d 6 )δ167.1, 162.3, 157.4 (2×C), 152.8 (2×C), 145.0, 137.0, 128.7, 128.3, 121.1, 119.8, 117.4 (t, J=258.2 Hz), 102.4 (2×C), 56.3 (2×C), 44.5. ESI-HRMS m / z: calculated value is C 18 H 17 O 4 N 5 F 2 Na + [M+Na] + , 428.1141; the measured value is 428.1141.

[0291] Example 50 Synthesis of Compound E2:

[0292] The synthesis steps were as in Example 48, and 6-aminobenzothiazole was selected as the amine reagent to obtain compound E2 with a yield of 9%. 1H NMR (600 MHz, DMSO-d 6 )δ13.26(br, 1H), 10.10(s, 1H), 9.28(s, 1H), 8.72(s, 1H), 8.11-8.02(m, 2H), 7.99-7.88(m, 1H), 7.52(s, 2H), 6.91(t, J=75.0Hz, 1H), 3.92(s, 6H). 13 CNMR (151 MHz, DMSO-d 6 )δ160.9, 154.6, 152.8 (2×C), 149.2, 145.3, 136.7 (2×C), 134.1, 128.8, 128.1, 122.8, 122.3, 119.7, 117.4 (t, J=256.5 Hz), 112.4, 102.7 (2×C), 56.4 (2×C). ESI-HRMS m / z: calculated value is C 20 H 16 O 4 N 4 SF 2 Na + [M+Na] + , 469.0753; the measured value is 469.0737.

[0293] Example 51 Synthesis of Compound E3:

[0294] The synthesis steps were as in Example 48, and 2,6-difluorobenzylamine was selected as the amine reagent to obtain compound E3 with a yield of 11%; 1 H NMR (500 MHz, DMSO-d 6 ) δ13.04 (br, 1H), 8.26 (t, J = 246Hz, 1H), 7.84 (t, J = 211Hz, 1H), 7.38 (s, 3H), 7 .13-7.02 (m, 2H), 6.87 (t, J=75.5Hz, 1H), 4.54 (d, J=5.5Hz, 2H), 3.86 (s, 6H). 13 C NMR (151 MHz, DMSO-d 6 )δ161.7, 161.1 (dd, J = 246.0, 7.5 Hz, 2×C), 152.7 (2×C), 145.0, 136.8, 129.7, 128.7, 128.2, 121.1, 117.3 (t, J = 258.0 Hz), 117.3, 114.6, 111.5 (dd, J = 21, 6.0 Hz, 2×C), 102.4, 56.3 (2×C), 30.3. ESI-HRMS m / z: calculated value is C 20 H17 O 4 N 3 F4F + [M+Na] + , 462.1047; the measured value is 462.1046.

[0295] Example 52 Synthesis of F series compounds

[0296] (1) Synthesis of Compound 22:

[0297]

[0298] Weigh 200 mg of 3,4,5-trihydroxybenzaldehyde (1.29 mmol, 1.0 equiv.), add 195 mg of sodium carbonate (1.29 mmol, 1.0 equiv.), add 2.5 mL of solvent NN-dimethylformamide, and finally add 205.7 mg of ethyl difluorochloroacetate (1.29 mmol, 1.0 equiv.). After reacting at 70°C for 12 hours, TLC detected that the raw material reacted completely, and water was added to quench the reaction. Use ethyl acetate to extract 3 times, and wash the organic phase with saturated sodium chloride solution. The organic phase is concentrated and subjected to rapid column chromatography to obtain 58 mg of the product as a white solid with a yield of 22%.

[0299] The obtained compound 22 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (500 MHz, DMSO-d 6 )δ10.33 (br, 2H), 9.77 (s, 1H), 6.96 (t, J=75.5 Hz, 1H), 6.93 (s, 2H). ESI-MS m / z: calculated value is C 8 H 7 O 4 F 2 + [M+H] + , 205.0; the measured value is 205.0.

[0300] (2) Synthesis of compound 23:

[0301]

[0302] 180 mg of compound 22 (0.88 mmol, 1.0 equiv.) was weighed, 305 mg of potassium carbonate (2.2 mmol, 2.5 equiv.) was added, 10 mL of solvent N, N-dimethylformamide was added, and finally 261.9 mg of bromomethylcyclopropane (1.94 mmol, 2.2 equiv.) was added. After reacting at 80°C for 5 hours, TLC detected that the raw material reacted completely, and water was added to quench the reaction. Ethyl acetate was used for extraction 3 times, and the organic phase was washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to obtain 169 mg of the product (compound 23), with a yield of 62%.

[0303] The obtained compound 23 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (400 MHz, CDCl 3 )δ9.86 (s, 1H), 7.10 (s, 2H), 6.74 (t, J = 74.4 Hz, 1H), 3.95 (d, J = 6.8 Hz, 4H), 1.34-1.30 (m, 2H), 0.68-0.64 (m, 4H), 0.40-0.36 (m, 4H). ESI-MS m / z: calculated value is C 16 H 19 O 4 F 2 + [M+H] + , 313.1; the measured value is 313.1.

[0304] (3) Synthesis of compound 24:

[0305]

[0306] Weigh 1.3 g of 3,3-dibromo-1,1,1-trifluoro-2-one (4.8 mmol, 1.5 equiv.) and 1.1 g of sodium acetate (12.8 mmol, 4.0 equiv.) into a reaction bottle, add an appropriate amount of water to dissolve, stir at 100°C for 1 hour, cool to room temperature, add a methanol solution containing 1 g of compound 23 (3.2 mmol, 1.0 equiv.) and 4 mL of ammonia water to the above solution, stir at room temperature, detect by TLC, and remove methanol by rotary evaporation. Add water, extract with ethyl acetate 3 times, wash the organic phase with saturated sodium chloride solution once, and dry the organic phase with anhydrous sodium sulfate. Concentrate the organic phase and obtain 1.0 g of yellow solid product by rapid column chromatography, with a yield of 74%.

[0307] The obtained compound 24 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (400 MHz, DMSO-d 6)δ13.21 (br, 1H), 7.98 (s, 1H), 7.30 (s, 2H), 6.90 (t, J = 75.2 Hz, 1H), 3.95 (d, J = 6.8 Hz, 4H), 1.31-1.22 (m, 2H), 0.61-0.55 (m, 4H), 0.39-0.34 (m, 4H). ESI-MS m / z: calculated value is C 19 H 20 O 3 N 2 F 5 + [M+H] + , 419.3; the measured value is 419.3.

[0308] (4) Synthesis of Compound 25:

[0309]

[0310] 63 mg of compound 24 (0.27 mmol, 1.0 equiv.) was weighed and dissolved in a 1:1 mixed solvent of ethanol and water, 21.7 mg of sodium hydroxide (0.54 mmol, 2.0 equiv.) was added, and the reaction was stirred at 80°C and monitored by TLC. After the reaction was complete, the ethanol was removed by rotary evaporation, an appropriate amount of water was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The solid was filtered and dried to obtain 42 mg, with a yield of 39%.

[0311] The obtained compound 25 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (500 MHz, DMSO-d 6 )δ13.05 (br, 1H), 7.96 (br, 1H), 7.61 (br, 1H), 7.51 (s, 1H), 7.33 (s, 1H), 6.89 (t, J = 74.5 Hz, 1H), 3.95 (d, J = 5.5 Hz, 4H), 1.30-1.27 (m, 2H), 0.60-0.56 (m, 4H), 0.39-0.35 (m, 4H). ESI-MS m / z: calculated value is C 19 H 21 O 5 N 2 F 2 + [M+H] + , 395.1; the measured value is 395.1.

[0312] (5) Synthesis of Compound F1:

[0313]

[0314] 50 mg of compound 25 (0.13 mmol, 1.0 equiv.) was dissolved in DMF, and then 50 mg of HATU (0.13 mmol, 1.0 equiv.), amine reagent (0.14 mmol, 1.1 equiv.) and 50 mg of DIPEA (0.39 mmol, 3.0 equiv.) were added. The reaction was stirred at room temperature and monitored by TLC. When there was no starting material, water was added to quench the reaction, and the mixture was extracted once with ethyl acetate. The organic phase was washed three times with water and once with saturated sodium chloride solution. The ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated, and silica gel was added to mix the sample. The product was purified by column chromatography to obtain a yellow solid product.

[0315]

[0316] Example 53 Synthesis of Compound F1:

[0317] The synthesis steps were as in Example 52. The amine reagent was 2-methylaminopyrimidine (CAS No.: 75985-45-4) to obtain compound F1 with a yield of 8%. 1 H NMR (500 MHz, DMSO-d 6 ) δ13.53 (br, 1H), 8.76 (d, J=4.5Hz, 2H), 8.52 (s, 1H), 7.78 (s, 1H), 7.50 (s, 2H), 7.39 (t, J=4.5Hz, 1H), 6.89 (t, J=7 5.5Hz, 1H), 4.66 (d, J=6.0Hz, 2H), 3.97 (d, J=7.0Hz, 4H), 1.30-1.23 (m, 2H), 0.61-0.55 (m, 4H), 0.38-0.35 (m, 4H). 13 C NMR (101 MHz, DMSO-d 6 )δ167.1, 162.4, 157.4 (2×C), 152.2 (2×C), 145.2, 136.8, 129.3, 128.2, 120.9, 119.9 (2×C), 117.5 (t, J=256.4 Hz), 103.7, 73.3 (2×C), 44.5, 10.1 (2×C), 3.1 (4×C). ESI-HRMS m / z: calculated value is C 24 H 25 O 4 N 5 F 2 Na + [M+Na] + , 508.1767; the measured value is 508.1760.

[0318] Example 54 Synthesis of G series compounds

[0319] (1) Synthesis of compound 27:

[0320]

[0321] Weigh 200 mg of 3,3-dibromo-1,1,1-trifluoro-2-one (0.74 mmol, 1.12 equiv.) and 193 mg of sodium acetate (1.48 mmol, 2.24 equiv.) into a reaction bottle, add an appropriate amount of water to dissolve, stir at 100°C for 1 hour, cool to room temperature, add a methanol solution containing 88.8 mg of compound 26 (0.66 mmol, 1.0 equiv.) and 4 mL of ammonia water to the above solution, stir at room temperature, detect by TLC, and remove methanol by rotary evaporation. Add water, extract with ethyl acetate 3 times, wash the organic phase with saturated sodium chloride solution once, and dry the organic phase with anhydrous sodium sulfate. Concentrate the organic phase and obtain 83 mg of yellow solid product by rapid column chromatography, with a yield of 52%.

[0322] The obtained compound 27 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1 H NMR (500 MHz, DMSO-d 6 )δ13.04 (br, 1H), 7.85 (s, 1H), 7.77 (s, 1H), 7.68 (dd, J=7.5, 2.0 Hz, 1H), 7.23 (d, J=8.0 Hz, 1H), 2.28 (s, 3H), 2.25 (s, 3H). ESI-HRMS m / z: calculated value is C 12 H 12 N 2 F 3 + [M+H] + , 241.0947; the measured value is 241.0908.

[0323] (2) Synthesis of compound 28:

[0324]

[0325] 39 mg of compound 27 (0.16 mmol, 1.0 equiv.) was weighed and dissolved in a 1:1 mixed solvent of ethanol and water, 8.9 mg of sodium hydroxide (0.23 mmol, 1.4 equiv.) was added, and the reaction was stirred at 80°C and monitored by TLC. After the reaction was complete, the ethanol was removed by rotary evaporation, an appropriate amount of water was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The solid was filtered and dried to obtain 30 mg, with a yield of 86%.

[0326] The obtained compound 28 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification result was: 1H NMR (400 MHz, DMSO-d 6 )δ7.89(s,1H),7.84-7.75(m,2H),7.22(d,J=8.0Hz,1H),2.26(s,3H),2.24(s,3H),OH(1H,not observed).NH(1H,not observed). ESI-MS m / z: calculated value is C 12 H 12 O 2 N 2 + [M+H] + , 217.0; the measured value is 217.0.

[0327] (3) Synthesis of Compound G1:

[0328]

[0329] 100 mg of compound 28 (0.31 mmol, 1.0 equiv.) was dissolved in DMF, and then 117 mg of HATU (0.31 mmol, 1.0 equiv.), 44 mg of 4-aminopyridine (0.31 mmol, 1.0 equiv.) and 80 mg of DIPEA (0.62 mmol, 2.0 equiv.) were added. The reaction was stirred at room temperature and monitored by TLC. When there was no starting material, water was added to quench, and the mixture was extracted three times with ethyl acetate, washed once with saturated sodium chloride solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and silica gel was added to mix the sample. Purification by column chromatography gave 78 mg of yellow solid product G1 with a yield of 86%.

[0330] The obtained compound G1 was identified by nuclear magnetic resonance spectroscopy and mass spectrometry, and the identification results were: 1 H NMR (500 MHz, DMSO-d 6 )δ13.41(br, 1H), 10.23(br, 1H), 8.46(d, J=5.5 Hz, 2H), 8.00(s, 1H), 7.94(s, 1H), 7.91(d, J=5.5 Hz, 2H), 7.85(d, J=7.5 Hz, 1H), 7.24(d, J=7.5 Hz, 1H), 2.29(s, 3H), 2.26(s, 3H). ESI-HRMS m / z: calculated value is C 17 H 17 ON 4 + [M+H] + , 293.1397; the measured value is 293.1390.

[0331] Active Examples

[0332] Activity Example 1 PDE4D enzyme activity test

[0333] The following methods were used to carry out biological tests on the compounds of the present application:

[0334] 1. Preparation of reaction buffer and reaction stop solution (reagents see Table 1)

[0335] (1) Preparation of 1x reaction buffer

[0336] IMAP reaction buffer (provided by IMAP FP IPP Explorer Kit) containing 0.1% BSA (5×) was diluted into 1× reaction buffer containing 1 mM DTT.

[0337] (2) Preparation of reaction termination solution

[0338] The reaction termination solution was prepared by mixing IMAP Progressive Binding Buffer A (5×), IMAP Progressive Binding Buffer B (5×), and IMAP Progressive Binding Reagent (provided by IMAP FP IPP Explorer Kit) according to the instruction manual.

[0339] 2. Compound Preparation

[0340] (1) Compound dilution

[0341] Prepare a solution with a concentration of 100 times the final concentration of the compound to be tested. Use an automatic microporous pipette (PrecisionPRC384U) to dilute the compound gradient to the set number of concentration points as follows: For a 5-fold dilution, add 50 μL of the starting concentration of the compound DMSO solution to well A2 on the Echo 384-well plate, and add 40 μL of 100% DMSO to wells A3-A11; take 10 μL of the compound from well A2 and add well A3, mix well, and make 5-fold dilutions in turn, diluting 10 concentration points; add 40 μL of 100% DMSO to wells A1 and A12.

[0342] (2) Transfer compounds to 384 reaction plates

[0343] Use the Echo550 instrument to transfer 200 nL of the diluted compound from the Echo384-well plate to a 384-well reaction plate, and transfer 200 nL of 100% DMSO to both the negative control and the positive control.

[0344] 3. Enzymatic reaction

[0345] (1) Prepare 2x enzyme solution

[0346] PDE4D was added to 1x reaction buffer to form a 2x enzyme solution (final concentration of PDE4D: 0.00625 μg / ml).

[0347] (2) Prepare 2 times the substrate solution

[0348] For the enzyme PDE4D, FAM-labeled cAMP was added to 1x reaction buffer to form a 2x substrate solution (final concentration of FAM-cAMP: 0.1 μM).

[0349] (3) Add enzyme solution to the 384-well plate

[0350] Add 10 μL of 2x enzyme solution to each well of a 384-well reaction plate. For the no-enzyme control well, replace the enzyme solution with 10 μL of 1x reaction buffer. Centrifuge at 1000 rpm for 1 min and incubate at room temperature for 15 min.

[0351] (4) Add substrate solution to the 384-well plate to start the enzymatic reaction

[0352] Add 10 μL of 2x substrate solution to each well of a 384-well reaction plate, centrifuge at 1000 rpm for 1 min, and react at 25°C for 30 min.

[0353] (5) Termination of enzyme reaction

[0354] 60 μL of the reaction stop solution was added to each well of the 384-well reaction plate to terminate the reaction, and the plate was incubated at room temperature for 60 minutes on a shaker at 600 rpm in the dark.

[0355] 4. Read data and calculate data using EnVision

[0356] Readings were taken with EnVision.

[0357] 5. Calculation of Inhibition Rate and IC 50 Curve Fitting

[0358] Data were copied from EnVision, where the maximum value refers to the reading of the DMSO control and the minimum value refers to the reading of the no enzyme activity control. Inhibition rate (%) = (maximum value - sample value) / (maximum value - minimum value) × 100%.

[0359] The data were imported into MS Excel and IC fitted using XLFit excel add-in version 5.4.0.8 50 value;

[0360] Fitting formula: Y = Bottom + (Top-Bottom) / (1 + (IC50 / X)^HillSlope)

[0361] Table 1 Reagent information

[0362] name brand Part Number PDE4D BPS 60048 Trequinsin TOCRIS 2337 / 10 IMAP FP IPP Kit Molecular Device R8124 FAM-CAMP Molecular Device R7506

[0363] The inhibitory effect of the compounds provided in the examples on the PDE4D enzyme was determined according to the above method. The results are shown in Table 2. Table 2 shows the results of the determination of the inhibitory effect of the compounds of the present application on the PDE4D enzyme.

[0364] Table 2 Inhibition rate of compounds on PDE4D

[0365]

[0366]

[0367] IC of the compound of Activity Example 2 for PDE4D 50 Detection

[0368] The implementation steps refer to the active example 1 to obtain IC 50 value.

[0369] IC of the compound 50 The values ​​are shown in Table 3.

[0370] Table 3 Compound IC 50 value

[0371] Compound <![CDATA[PDE4D IC 50 (nM)]]> Compound <![CDATA[PDE4D IC 50 (nM)]]> A3 15.93±1.08 A14 <32 A4 0.5±0.19 B1 27.31±1.07 A5 2.41±1.07 B2 18.24±5.04 A6 4.74±1.87 D1 9.36±1.67 A7 14.95±2.59 D2 0.57±0.09 A10 <32 D5 <160 A11 <32 D7 <160 A12 <32 D9 25.9±13.85 A13 <32 D11 <160

[0372] Activity Example 3 Study on the Inhibition of RAW 264.7 Cell Inflammation by the Compounds of the Application

[0373] The test method adopts the conventional ELISA method to carry out the inhibition test of the expression level of cellular inflammatory factors on the compounds of the present application.

[0374] 1. Cell Culture

[0375] Mouse mononuclear macrophage Raw 264.7 cells were cultured in DMEM high-glucose medium supplemented with 10% (V / V) FBS, 100 μg / mL penicillin, and 100 μg / mL streptomycin. The culture conditions were 37°C, 5% CO 2 The cells were passaged when they reached 80% confluence.

[0376] 2. Cellular administration and induction of cellular inflammation

[0377] The experiment was conducted using cells in the logarithmic growth phase. 5 / well were inoculated in a 12-well plate and incubated at 37°C and 5% CO 2Culture in an environment until the cells grow to 70% and set aside. Set up groups: blank group, LPS stimulation group, positive control group (Roflumilast) and drug-treated group (compound of the present application). Carefully remove the culture medium, add fresh complete culture medium containing the compound to the positive control group and the drug-treated group, and add an equal volume of DMSO to the blank group and LPS stimulation group. After 1 hour, 1 μg / mL LPS was added to all groups except the blank group for 4 hours to induce cell inflammation. Take out the well plate and perform the ELISA experiment according to the instructions of the kit.

[0378] Collect the culture medium supernatant: collect the supernatant into a 1.5 mL EP tube, centrifuge at 1000 rpm for 10 minutes, and use the supernatant for ELISA detection of cytokines.

[0379] The secretion of mouse TNF-α was detected using an enzyme-linked immunosorbent assay (ELISA) kit. The specific steps are as follows:

[0380] (1) Reagent preparation

[0381] ①. Take out the refrigerator and place at room temperature to balance for 20 minutes. ②. Dilute the washing solution (20X) to 1X with double distilled water to prepare the required washing solution. ③. Add the standard diluent to 1 bottle of standard according to the volume marked on the standard label and incubate at room temperature for 15 minutes. ④. Take 5 clean 1.5mL centrifuge tubes, add 250μL of standard diluent to each tube in advance, and dilute the standard in multiples to obtain a total of six standard concentrations. Finally, add the diluted standards to the pre-coated plate wells in turn, and add the standard diluent directly as 0pg / mL concentration, for a total of seven standard concentrations.

[0382] (2) Operation steps

[0383] ①. Calculate the number of pre-coated strips required for one experiment, take out the required strips and place them in the 96-well frame.

[0384] ②. Add samples or standards of different concentrations into the corresponding wells at 100 μL / well, seal the wells with a sealing film (transparent), and incubate at room temperature for 120 minutes.

[0385] ③. Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0386] ④. Add 100 μL / well of biotinylated antibody, seal the reaction wells with a sealing film (transparent), and incubate at room temperature for 60 minutes.

[0387] ⑤. Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0388] ⑥. Add 100 μL / well of horseradish peroxidase-labeled Streptavidin, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes.

[0389] ⑦. Wash the plate 5 times and pat dry on thick absorbent paper for the last wash.

[0390] ⑧. Add 100 μl / well of TMB solution, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 20 minutes.

[0391] ⑨. Add 50 μL / well of stop solution, mix well and measure the A450 value immediately.

[0392] The test results are shown in Table 4. For the tested mouse mononuclear macrophage RAW 264.7 and combined with the enzyme activity results, compounds A5, D2, and A4 can effectively inhibit the expression level of TNF-α. The above experimental results show that compounds A5, D2, and A4 of the present invention have good anti-inflammatory activity and can be used for the research of anti-inflammatory drugs.

[0393] Table 4 Anti-inflammatory activity results of the compounds of the present invention on RAW 264.7 cells

[0394]

[0395]

[0396] Active Example 4

[0397] The compounds A4, A5, D1 and D2 with better anti-inflammatory and enzyme activities of the present invention were tested for cytotoxicity and inflammatory factor IC 50 Detection.

[0398] 1. Experimental Materials

[0399] (1) Experimental cells

[0400] Human immortalized epidermal cells HaCaT, mouse monocyte macrophage Raw 264.7

[0401] (2) Cell culture

[0402] The cells were cultured in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) at 37°C in 5% CO2. The medium was renewed every 2 days and subcultured when the medium reached 90%.

[0403] (3) Experimental instruments

[0404] Heating stirrer, paraffin slicer, microscope, camera, syringe, microplate reader, balance, oven, refrigerated centrifuge, etc.

[0405] Table 5

[0406]

[0407] The results are shown in Table 5. Compounds A4, A5, D1, and D2 significantly downregulated the expression of TNF-α in Raw264.7 and HaCaT cells, indicating that compounds A4, A5, D1, and D2 have certain anti-inflammatory effects and are not toxic to cells.

[0408] Active Example 5

[0409] In vitro liver microsomal metabolic stability experiments were conducted on the 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide (A5, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide) and 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide (D2, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide of the present invention.

[0410] The 0.5 μM compound was incubated with liver microsomes (1 mg / mL) at 37°C. 100 μL of the reaction solution was taken at each time point of 0, 5, 15, 30, 45 and 60 minutes. 200 μL of acetonitrile containing internal standard was added to 100 μL of the reaction solution to extract the compound to be tested. The obtained mixture was centrifuged and the supernatant was analyzed by LC-MS / MS. The results are shown in Table 6. The half-life of D2 in human liver microsomes was 296.1 minutes, and the half-life in mouse liver microsomes was 28.6 minutes, which has good metabolic stability.

[0411] Table 6

[0412]

[0413] Active Example 6

[0414] In vitro Caco-2 permeability assay was performed on 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide (A5, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide) and 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide (D2, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide of the present invention.

[0415] Caco-2 monolayer analysis was performed for compounds A5 and D2, respectively, using standard procedures reported previously. Under the same conditions, the transport of compounds from the apical side to the basolateral side (AB) and from the opposite direction (BA) was measured simultaneously. Propranolol and nadolol were used as hyperosmotic and hypoosmotic controls, respectively. Digoxin was used as a positive control for pgp-mediated drug efflux. After washing the monolayer three times with Hanks balanced salt solution (HBSS, Sigma-Aldrich), compound A5 or D2 was added to the appropriate wells (apical side pH 6.8, basolateral pH 7.4). Incubate at 37°C for 95 min. Samples were collected from the donor side at 5 min and 95 min, and samples were collected from the acceptor side at 35 min and 95 min after incubation. The concentration of the samples was determined by liquid chromatography-mass spectrometry (LC-MS) / mass spectrometry. The average value is the average of three independent experiments, and each experiment was repeated three times.

[0416] Table 7

[0417]

[0418]

[0419] The results showed that compound D2 had moderate cell permeability to cells.

[0420] Note: a: The Papp value in A to B (AB) or B to A (BA) was calculated by the following formula: Papp = (VA / (area × time)) × (drug receptor / initial drug donor), where VA = volume in the receptor pore (in this assay: 0.1 on the apical side, 0.3 mL on the basolateral side), area = surface area of ​​the membrane (in this assay: 0.143 cm 2), time = total transport time in seconds (in this test: 7200s), Papp value expressed as 10 -6 cm / s. b: The BA / AB ratio is calculated by dividing the Papp value from B to A by the Papp from A to B as shown below. Outflow ratio (BA / AB) = Baptometry (B to A) / Baptometry (A to B).

[0421] Active Example 7

[0422] In vivo pharmacokinetic experiments were carried out on 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide (A5, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide) and 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide (D2, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide of the present invention.

[0423] The pharmacokinetic experiment of each compound was arranged in 6 male ICR mice of 5-6 weeks, each weighing 18-22g. Specifically, 6 ICR mice were randomly divided into 2 groups. Compound A5 or D2 was first dissolved in phosphate buffered saline containing DMSO and 5% Tween-80, and then orally (PO, 10mg / kg) and intravenously (IV, 5mg / kg) after fasting for 12h, and fed 4h after administration. Blood was collected at each time point, IV blood collection point (5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h), PO blood collection point (15min, 30min, 1h, 2h, 4h, 6h, 8h, 24h). Blood was collected through the submandibular vein or other suitable methods, and each sample was collected about 30μL / time point, K2-EDTA anticoagulation, and placed on ice after collection. Blood samples were collected and placed on ice, and plasma was separated by centrifugation within 1 hour (centrifugation conditions: 6800g, 6 minutes, 2-8°C). Plasma samples were stored in a -80°C refrigerator before analysis. Plasma concentrations of ICR mice were analyzed using LC-MS / MS (Agilent 1260 Infinity LC system and Agilent 6460 triple quadruple mass spectrometer system).

[0424] Table 8 Pharmacokinetic data of compound A5 in ICR mice

[0425] parameter iv (5 mg / kg) parameter Po (10 mg / kg) <![CDATA[AUC (0-t) (mg / mL)]]> 5010±225 <![CDATA[AUC (0-t) (mg / mL)]]> 3567±232 <![CDATA[AUC (0-∞) (mg / mL)]]> 5020±226 <![CDATA[AUC (0-∞) (mg / mL)]]> 3931±444 <![CDATA[C 0 (ng / mL)]]> 7315±347 <![CDATA[C max (ng / mL)]]> 810±131 <![CDATA[T 1 / 2 (h)]]> 0.94±0.10 <![CDATA[T 1 / 2 (h)]]> 2.21±0.75 <![CDATA[V ss (L / kg)]]> 1017±81 <![CDATA[T max (h)]]> 1.00±0 CL(mL / min / kg) 997±44 F(％) 35.61±2.32

[0426] Table 9 Pharmacokinetic data of compound D2 in ICR mice

[0427] parameter iv (5 mg / kg) parameter Po (10 mg / kg) <![CDATA[AUC (0-t) (mg / mL)]]> 619±43 <![CDATA[AUC (0-t) (mg / mL)]]> 41±14 <![CDATA[AUC (0-∞) (mg / mL)]]> 620±43 <![CDATA[AUC (0-∞) (mg / mL)]]> 51±19 <![CDATA[C 0 (ng / mL)]]> 5242±126 <![CDATA[C max (ng / mL)]]> 31±8 <![CDATA[T 1 / 2 (h)]]> 0.27±0.08 <![CDATA[T 1 / 2 (h)]]> 1.08±0.53 <![CDATA[V ss (L / kg)]]> 957±25 <![CDATA[T max (h)]]> 0.50±0.43 CL(mL / min / kg) 8087±538 F(％) 3.31±1.18

[0428] Active Example 8

[0429] The in vivo efficacy evaluation study on the anti-psoriasis effect of 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide (A5, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2,5-difluorobenzyl)-1H-imidazole-4-carboxamide) and 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide (D2, 2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(pyrimidin-2-ylmethyl)thiazole-4-carboxamide of the present invention was carried out on mice.

[0430] 1. Purpose of the experiment

[0431] The therapeutic effects of compounds A5 and D2 on imiquimod-induced psoriasis in mice were investigated.

[0432] 2. Experimental Materials

[0433] Experimental animals: Balb / c male mice, 56 mice for each compound experiment, age: 6-8 weeks; body weight: about 18-22 g.

[0434] Animal grouping, modeling, and sampling

[0435] Experimental instruments: heating stirrer, paraffin slicer, microscope, syringe, microplate reader, balance, oven, refrigerated centrifuge, etc.

[0436] Experimental reagents: HE staining related reagents, PBS solution, etc., imiquimod, ELISA kits for IL-17A, IL-1β and IL-6.

[0437] 3. Animal Experiments

[0438] 1. Establishment of mouse psoriasis model and drug administration

[0439] ① Shave the 2cm×3cm area on the back of the mouse three days before modeling. Observe the shaving effect, and shave the villi and new hair at the beginning of modeling.

[0440] ② The mice were randomly divided into 7 groups: blank group, model group (5% IMQ 62.5 mg / d, for 9 consecutive days), compound group (0.3%, 0.6%, 1.2%), positive control group (roflumilast, 0.3%), and excipient group (only applied cream excipient formula), 8 mice in each group, for a total of 56 mice.

[0441] Blank group: Starting from the first day of psoriasis modeling, no treatment was given to the mice.

[0442] Model group: Starting from the first day, 62.5 mg of 5% IMQ was applied to the exposed skin on the back of mice every day for 9 consecutive days to establish a psoriasis model.

[0443] Positive treatment control group: Starting from the first day of psoriasis modeling, 62.5 mg 5% IMQ was applied to the exposed skin of the back of mice every day. Starting from the third day, 62.5 mg 5% IMQ was applied to the exposed skin of the back of mice in the morning. After 4 hours, 62.5 mg of roflumilast cream (0.3%) was applied to each mouse for 7 consecutive days.

[0444] Compound groups (0.3%, 0.6%, 1.2%): Starting from the first day of psoriasis modeling, 62.5 mg of 5% IMQ was applied to the exposed skin on the back of mice every day. Starting from the third day, 62.5 mg of 5% IMQ was applied to the exposed skin on the back of mice in the morning. After 4 hours, each group of mice was treated with low, medium and high doses of compound cream respectively for 7 consecutive days.

[0445] Excipient group: Starting from the first day of psoriasis modeling, 62.5 mg of the excipient formula of the cream was applied to the exposed skin on the back of the mice every day for 7 consecutive days.

[0446] 2. Experimental operation and results

[0447] 2.1 Scoring of Psoriasis Area and Severity Index (PSAI) in Mice

[0448] The PASI score was performed by observing the degree of scaling, the size of erythema, and the degree of hypertrophy of the skin on the back of the mice with the naked eye. The PASI score rule is to select three angles, erythema, scaling, and hypertrophy, to evaluate the severity of psoriasis. After scoring, the skin of the mice was photographed.

[0449] Depend on Figure 1 It can be seen that the mice in the model group showed a typical psoriasis-like dermatitis phenotype, characterized by erythema, scales and thickness. Applying compound A5 can significantly alleviate the symptoms of psoriasis.

[0450] Depend on Figure 2 It can be seen that the mice in the model group showed a typical psoriasis-like dermatitis phenotype, characterized by erythema, scales and thickness. Applying compound D2 can significantly alleviate the symptoms of psoriasis.

[0451] from Figure 3 It can be seen that compound A5 (1.2%) has the same efficacy as roflumilast in terms of erythema score, thickness, and total score. No significant weight loss was observed, which indicates that A5 has a lower risk of toxicity in vivo.

[0452] Depend on Figure 4 It can be seen that compound D2 (0.6%, 1.2%) has the same efficacy as roflumilast in terms of erythema score, thickness, and total score, with no significant change in body weight and a lower risk of in vivo toxicity.

[0453] 2.2 Mouse Sacrifice

[0454] Euthanasia was performed by intraperitoneal overdose of 0.3% sodium pentobarbital solution.

[0455] 2.3 Paraffin embedding and sectioning of skin tissue

[0456] 1) Cut the entire layer of skin parallel to the spine and fix it in 10% neutral formaldehyde solution for more than 24 hours;

[0457] 2) Place the trimmed tissue block in an embedding box and wash it with running water for 24 hours to completely remove residual formaldehyde;

[0458] 3) Dehydrating the tissue with alcohol gradient, the specific steps are: 70% alcohol overnight, 80% alcohol for 1.5 hours, 95% alcohol I for 45 minutes, 95% alcohol II for 30 minutes, 100% alcohol I for 25 minutes, 100% alcohol II for 20 minutes;

[0459] 4) After dehydration, place the tissue in an alcohol / xylene (1:1, v / v) solution for 20 min;

[0460] 5) Xylene transparent I, II 20min, 10min;

[0461] 6) Place the tissue in preheated melted paraffin I, II and embedding paraffin in an oven at 60-65°C for 1 hour each;

[0462] 7) Pour a small amount of embedding paraffin into the preheated metal embedding frame, place the skin tissue block in it, perpendicular to the embedding frame, pour paraffin again, embed the tissue, and cool it down;

[0463] 8) Paraffin sectioning: Fix the tissue paraffin block on the Leica slicer. Slice thickness is 5 μM, and slice continuously. Use toothless forceps to place the slices in 40℃ water for spreading, pick up the slices with a slide, bake the slices at 60℃ for 2h, and store them in a slice box at room temperature for later use.

[0464] 2.4 HE staining

[0465] 1) Dewaxing and hydration: Place the skin tissue paraffin sections in xylene I and II for 15 min each, then place them in 100% ethanol I and II for 3 min each, 95% ethanol I and II for 3 min each, 80% ethanol for 3 min, and double distilled water for 1 min;

[0466] 2) Hematoxylin staining for 15 min, then wash off the excess stain on the slide;

[0467] 3) 1% hydrochloric acid ethanol (99mL 70% ethanol + 1mL concentrated hydrochloric acid) color separation for 3s, the cell nucleus and chromatin should be clear under the microscope;

[0468] 4) Rinse with running water for 15 minutes, and then with distilled water for 1 minute;

[0469] 5) Eosin for 2 minutes, rinse with running water for 1 minute;

[0470] 6) Dehydrate with 80% and 100% ethanol for 2 seconds and 7 minutes respectively;

[0471] 7) Xylene I and II for 5 min each;

[0472] 8) Sealing: Take the slide out of xylene II, add neutral gum to the tissue, gently cover with a coverslip, and let it dry naturally.

[0473] 9) Observation: Observe pathological changes under a microscope, take photos and analyze.

[0474] like Figure 5 and Figure 6 As shown, the skin thickness of the model group was thickened and a large number of inflammatory cells infiltrated, and the drug-treated groups (A5, D2) had a dose-dependent protective effect on the skin.

[0475] 2.5 Take spleens from eight mice in each group, and take photos of A5 and D2.

[0476] The spleen is the largest immune organ in the human body and contains a large number of immune cells. The body's inflammatory response is closely related to the immune system to a certain extent. In the experiment, the spleens of each treatment group were observed and weighed. The spleens of the model group were enlarged, and those of the drug-treated group were significantly reduced. It can be seen that the drug-treated groups (A5, D2) have an inhibitory effect on the inflammatory infiltration of the spleen. Figure 7 and Figure 8 .

[0477] Note: Mouse spleen weight index: spleen index = spleen weight (mg) / body weight (g) on ​​the last day.

[0478] 2.6 Immunohistochemical staining with Ki-67 antibody

[0479] 1) Place the skin tissue paraffin sections in an oven and bake at 60°C for 1 hour.

[0480] 2) Dewaxing: xylene 10 min three times → anhydrous ethanol I 5 min → anhydrous ethanol II 5 min → 95% ethanol 5 min → 85% ethanol 5 min- → 75% ethanol 5 min- → ddH 2 O 5min.

[0481] 3) Antigen repair: Prepare 400 ml of antigen repair solution (800 mL dd H 2 (20 mL) was boiled with PBS (3 g sodium citrate + 400 mg citric acid and then fixed to 1000 mL), the slices were placed in a beaker and boiled for 20 min. After cooling to room temperature, the slices were rinsed with PBS 3 times, each time for 3 min.

[0482] 4) Remove PBS, take out the slices from PBS, shake them twice, mark them with an immunohistochemistry pen and put them into PBS, shake them again after marking, add 3% hydrogen peroxide, incubate at room temperature for 5 minutes, and wash the slices with PBS three times, each time for 3 minutes.

[0483] 5) Remove PBS and add 10% goat serum blocking solution at room temperature for 1 hour.

[0484] 6) Remove goat serum, shake dry and directly add primary antibody, incubate in a humidified box at 4°C (about 14 hours).

[0485] 7) Remove the slices, rewarm for 30 minutes, and rinse with PBS three times, 3 minutes each time.

[0486] 8) Remove PBS, add secondary antibody for immunohistochemistry to each sample, incubate at room temperature for 15 min, and rinse with PBS three times, 3 min each time.

[0487] 9) Dry the slices and immediately place them on a microscope. DAB was added for staining. After color development, the slices were rinsed with PBS for 5 minutes.

[0488] 10) Hematoxylin re-staining for 40 seconds → washing in running water for 2 minutes → blueing for 7 seconds → washing in running water for 10-15 minutes → dd H 2 O soak for 5 minutes.

[0489] 11) 95% ethanol (1 min) → anhydrous ethanol I (1 min) → anhydrous ethanol II (1 min) → xylene (1 min) → xylene (1 min) → xylene (1 min) and seal the slides with neutral gum.

[0490] The results showed that (see Fig. 9 and Fig.10 ) The expression of Ki-67 antibody in the model group was significantly increased, while the expression of Ki-67 antibody in the drug-treated groups (A5 and D2) was significantly decreased, indicating that the compound has a significant inhibitory effect on skin thickening.

[0491] 4. Expression of inflammatory factors in the skin of psoriasis mice after different treatments.

[0492] ① Protein levels of IL-17A, TNF-α, IL-6, and IL-1β in skin lesions

[0493] 1) Take 100 mg of lesioned skin in an EP tube, add 1 mL of saline, and grind it using a tissue grinder.

[0494] 2) Centrifuge at 3500 rpm and 12000 rpm at 4°C, take the supernatant, determine the protein concentration in the tissue grinding fluid by BCA method, and determine the content of IL-1β, IL-6, IL-17A and TNF-α by ELISA kit according to the instructions of the kit.

[0495] Results Fig.11 , showing that compound A5 has a down-regulating effect on inflammatory factors IL-1β, IL-6, IL-17A and TNF-α, indicating that compound A5 is effective in treating psoriasis-related inflammation.

[0496] Fig.12 The results showed that compound D2 had a down-regulating effect on IL-1β, IL-6, and IL-17A inflammatory factors, indicating that compound D2 is effective in treating psoriasis-related inflammation.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof in R1 and R2 are each independently H, and R1 and R2 are not H at the same time; X and Y are each independently N or NH, O or S, and at least one of X and Y is N; Z is NH or O; R3 is C6-14 aryl, 5-14 membered heteroaryl, C6-10 cycloalkyl, or 5-14 membered heterocyclyl; optionally, the C6-14 aryl, 5-14 membered heteroaryl, C6-10 cycloalkyl, or 5-14 membered heterocyclyl is substituted by one or more substituents selected from halogen, halogenated C1-C6 alkyl, carbonyl, C1-C6 alkyl, and C1-C6 alkoxy; the 5-14 membered heteroaryl or 5-14 membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; preferably, the halogen is F, Cl, Br or I; n is 0, 1, 2 or 3; Each dashed line independently represents the presence or absence of a bond.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof, wherein R1 is R2 is H; Preferably, R1 is R2 is H; Preferably, R1 is R2 is Preferably, R1 is R2 is 3. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof, wherein X is N and Y is NH; Preferably, X is N and Y is O; Preferably, X is N and Y is S.

4. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof, wherein R3 is C6-10 aryl, 5-10 membered heteroaryl, C6-10 cycloalkyl, or 5-10 membered heterocyclyl; optionally, the C6-10 aryl, 5-10 membered heteroaryl, C6-10 cycloalkyl, or 5-10 membered heterocyclyl is substituted with one or more substituents selected from F, carbonyl, C1-C4 alkyl, and C1-C4 alkoxy; the 5-10 membered heteroaryl or 5-10 membered heterocyclyl contains 1-3 heteroatoms selected from N, O and S; Preferably, R3 is 5. The compound of claim 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof, wherein Formula I is R4 is n is 0 or 1; Preferably, Formula I is Where R5 is independently n is 0 or 1; Preferably, Formula I is R6 is n is 0 or 1; Preferably, Formula I is R7 is n is 0 or 1; Preferably, Formula I is R8 is n is 0 or 1; Preferably, Formula I is R9 is n is 0 or 1.

6. A compound or a pharmaceutically acceptable salt, solvate, hydrate, prodrug, stereoisomer, polymorph, cocrystal or deuterated form thereof:

7. An intermediate compound, which is the following compound:

8. A method for preparing a compound of formula II, in R1, R2, X, Y, R3, n as described in any one of claims 1-5; The preparation method comprises: (1) in a) reacting 3,3-dibromo-1,1,1-trifluoro-2-one with sodium acetate at 80°C-120°C for 1-2 hours, adding a benzaldehyde solution substituted with R1, R2, and difluoromethoxy, and reacting at 20°C-50°C for 1-8 hours; b) hydrolyzing the product obtained in step a) at 50° C.-90° C. for 1-8 hours; c) reacting the product obtained in step b) with R3-(CH2) n -NH2 reacts at 20℃-50℃ for 5-7 hours; (2) in d) reacting benzoic acid substituted with R1, R2, and difluoromethoxy with L-serine methyl ester hydrochloride and SOCl2 at 0°C for 10-14 hours; e) reacting the product obtained in step d) with DAST at -78°C with stirring for 3-5 hours, adding an inorganic base, and reacting at 20°C-50°C for 24 hours; f) reacting the product obtained in step e) with CBrCl3 and DBU at 0°C for 15-24 hours; g) hydrolyzing the product obtained in step f) at 20° C.-50° C. for 1-8 hours; h) reacting the product obtained in step g) with R3-(CH2) n -NH2 reacts at 20℃-50℃ for 3-8 hours; or (3) i) reacting benzaldehyde substituted with R1, R2, and difluoromethoxy with D-cysteine ​​methyl ester hydrochloride and K2CO3 at 20°C-50°C for 20-30 hours with stirring, then stirring at -15°C, then adding DBU and CBrCl3 at -20°C-0°C, and reacting at 20°C-50°C for 12-24 hours; j) stirring the product obtained in step i) at 20° C.-50° C. for 8-12 hours to carry out a hydrolysis reaction; k) reacting the product obtained in step j) with R3-(CH2) n -NH2 reacts at 20℃-50℃ for 4-8 hours.

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 6 and a pharmaceutically acceptable adjuvant or excipient.

10. Use of the compound according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating inflammatory diseases, respiratory diseases, skin diseases, or immune system diseases; more preferably, the inflammatory disease is an inflammatory skin disease; more preferably, the respiratory disease is chronic obstructive pulmonary disease or asthma; more preferably, the skin disease is psoriasis or atopic dermatitis; more preferably, the immune system disease is systemic lupus erythematosus or rheumatoid arthritis.

11. Use of the compound according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing and / or treating a PDE4-mediated disease or a PDE4 inhibitor.

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