Patent Description:
The human body's immune system can generally be divided into "natural immunity" system and "adaptive immunity" system. The natural immune system plays an important role in resisting infections, inhibiting tumor growth and the pathogenesis of autoimmune diseases. It mainly recognizes pathogenic microorganisms and cancer cell components through pattern recognition receptors, initiates downstream signal pathways, and finally induces cytokine expression, kill pathogenic microorganisms and cancer cell components, and adapt to the immune system to promote the production of antibodies and specific T lymphocytes.

STING (interferon gene stimulating factor, TMEM173, MITA, etc.) is a key node molecule for intracellular response to DNA invasion. Under the stimulation of cytoplasmic DNA, it recognizes the signal of cytoplasmic DNA receptor and plays a key role in the process of inducing interferon production. After the host cell's DNA recognition receptor recognizes exogenous or endogenous "non-self" DNA, it transmits the signal to the node molecule STING, and then STING rapidly dimerizes and transfers from the endoplasmic reticulum to the perinuclear body. The activation of STING leads to the up-regulation of IRF3 and NKκB pathways, which leads to the induction of interferon-β and other cytokines.

Compounds that induce human interferon can be used to treat various diseases (including allergic diseases and other inflammatory diseases, allergic rhinitis and asthma, infectious diseases, neurodegenerative diseases, precancerous syndrome and cancer), and can also be used as immunization composition or vaccine adjuvant. Therefore, the development of new compounds that are capable of activating STING is an effective method for the treatment of type <NUM> IFN pathway diseases (including inflammatory diseases, allergic and autoimmune diseases, infectious diseases, cancer, and precancerous syndromes).

<CIT> discloses heterocyclic amides that are useful as modulators of STING.

In order to solve the above-mentioned problems, the present disclosure provides an immunomodulator.

The present invention provides a compound, or the stereoisomer, or the pharmaceutically acceptable salt thereof, wherein the compound is represented by formula II as defined by claim <NUM>
<CHM>.

The present invention further provides, a compound, or the stereoisomer, or the pharmaceutically acceptable salt thereof, wherein the compound is:
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>.

The present invention further provides a compound, or the stereoisomer, or the pharmaceutically acceptable salt thereof, wherein the compound is represented by formula III as defined in claim <NUM>.

The present invention further provides the compound, or the stereoisomer, or the pharmaceutically acceptable salt thereof, wherein the compound is:
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>.

The present invention also provides the above-mentioned compound, or a stereoisomer, or a pharmaceutically acceptable salt thereof for use in treating diseases related to STING activity that are one or more of diseases related to inflammatory diseases, autoimmune diseases, infectious diseases, cancer, and precancerous syndrome.

The present invention also provides the above-mentioned compound, or a stereoisomer, or a pharmaceutically acceptable salt thereof for use as an immune adjuvant.

The present invention also provides a medicament, which is a preparation prepared from the above-mentioned compound, or a stereoisomer, or a pharmaceutically acceptable salt thereof as the active ingredient, plus pharmaceutically acceptable auxiliary materials.

The diseases related to STING activity defined in the present invention are diseases in which STING plays an important role in the pathogenesis of the disease.

Diseases related to STING activity include inflammatory diseases, allergic diseases and autoimmune diseases, infectious diseases, cancer, and precancerous syndrome.

"Cancer" or "malignant tumor" refers to any of a variety of diseases characterized by uncontrolled abnormal cell proliferation, and the ability of affected cells to spread to other locations locally or through the bloodstream and lymphatic system of the body (i.e., metastasis) and any of many characteristic structural and/or molecular characteristics. "Cancer cells" refer to cells that undergo multiple stages of tumor progression in the early, middle, or late stages. Cancers include sarcoma, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer, and prostate cancer. In some embodiments, the compound of formula I is used to treat a cancer selected from colon cancer, brain cancer, breast cancer, fibrosarcoma, and squamous cell carcinoma. In some embodiments, the cancer is selected from melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer. In some embodiments, the cancer being treated is a metastatic cancer.

Inflammatory diseases include a variety of conditions characterized by histopathological inflammation. Examples of inflammatory diseases include acne vulgaris, asthma, enterocoelia diseases, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammation, reperfusion injury, rheumatoid arthritis, sarcoidosis, vasculitis, airway inflammation and interstitial cystitis caused by house dust mites. There is a significant overlap between inflammatory diseases and autoimmune diseases. Some embodiments of the present invention relate to the treatment of asthma, an inflammatory disease. The immune system is usually involved in inflammatory diseases, which are manifested in allergic reactions and some myopathy. Many immune system diseases lead to abnormal inflammation.

The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) naming system.

Regarding the definition of terms used in the present invention: Unless otherwise specified, the initial definitions of radical groups or terms provided herein are applicable to the radical groups or terms throughout the specification; for terms not specifically defined herein, it should be, based on the disclosure and context, given the meaning that those skilled in the art can give them.

"Substitution" refers to the replacement of a hydrogen atom in a molecule by a different atom or molecule.

The minimum and maximum content of carbon atoms in a hydrocarbon group is indicated by a prefix. For example, the prefix (Ca-Cb) alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, (C<NUM>-C<NUM>) alkyl refers to an alkyl group containing <NUM> to <NUM> carbon atoms.

In the present invention, "alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. For example, C<NUM>-C<NUM> alkyl refers to an alkyl group having <NUM> to <NUM> carbon atoms. Alkyl groups can be linear or branched. Representative branched alkyl groups have one, two, or three branches. The alkyl group may be optionally substituted with one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl. The alkyl group may also be a part of another group, such as a C<NUM>-C<NUM> alkoxy group.

In the present invention, the Ca-Cb alkoxy group refers to a group obtained by connecting an alkyl group containing "a" to "b" carbon atoms to the corresponding oxygen atom.

In the present invention, "C<NUM>-C<NUM> alkylene" refers to a divalent saturated aliphatic hydrocarbon group having <NUM> to <NUM> carbon atoms. Alkylene groups include branched and straight chain hydrocarbyl groups. For example, "(C<NUM>-C<NUM>)alkylene" is meant to include ethylene, propylene, <NUM>-methylpropylene, dimethylethylene, pentylene and the like.

Similarly, "C<NUM>-C<NUM> alkenylene" refers to a divalent aliphatic hydrocarbon group having <NUM> to <NUM> carbon atoms and containing one or more carbon-carbon double bonds. Alkenylene groups include branched and straight chain groups. The carbon-carbon double bond in the alkenylene group includes a cis double bond and a trans double bond.

Similarly, "C<NUM>-C<NUM> alkynylene group" refers to a divalent aliphatic hydrocarbon group having <NUM> to <NUM> carbon atoms and containing one or more carbon-carbon triple bonds. Alkynylene groups include branched and straight chain groups.

In the present invention, the -C(O)- in -C(O)NRaRb, -NRaC(O)Rb, -C(O)Ra, -C(O)ORa and -C(O)Rf means a carbonyl group <IMG> composed of carbon and oxygen in which the atoms are connected by a double bond.

In the present invention, "halogen" refers to a halogen group: fluorine, chlorine, bromine or iodine.

The immune adjuvant in the present invention is an immunomodulator, which refers to a substance that has the function of enhancing and regulating immunity in terms of anti-infection, anti-virus, anti-tumor, anti-allergic reaction, anti-asthma, etc. It is mainly used clinically for adjuvant treatment of inflammation, autoimmune diseases, infectious diseases, cancer, and precancerous syndromes. The term "pharmaceutically acceptable" refers to a certain carrier, vehicle, diluent, excipient, and/or the salt formed is usually chemically or physically compatible with other ingredients constituting a certain pharmaceutical dosage form, and physiologically compatible with the receptor.

The terms "salts" and "pharmaceutically acceptable salts" refer to the above-mentioned compounds or their stereoisomers, or acid salts and/or basic salts formed with inorganic and/or organic acids and bases, and also include zwitterionic salts (inner salt), also include quaternary ammonium salts, such as alkyl ammonium salts. These salts can be directly obtained in the final isolation and purification of the compound. They can also be obtained by appropriately mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base (for example, equivalent). These salts may form a precipitate in the solution and be collected by filtration, or recovered after evaporation of the solvent, or prepared by freeze-drying after reacting in an aqueous medium. The salt in the present invention can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compounds.

In certain embodiments, one or more compounds of the present invention may be used in combination with each other. Optionally, the compound of the present invention can be used in combination with any other active agent to prepare drugs or pharmaceutical compositions for regulating cell function or treating diseases. If a group of compounds are used, these compounds can be administered to the subject simultaneously, separately or sequentially.

The compound provided by the present invention can effectively combine with STING, has a good STING protein agonistic function, shows a good inhibitory effect on a variety of tumors, and can also activate the immune memory mechanism of mice and inhibit tumor re-stimulation. Therefore, the compounds of the present invention can be used as STING agonists and used to treat various related diseases. The compounds of the present invention have very good application prospects in the manufacture of a medicament for the treatment of diseases related to STING activity (especially medicaments for the treatment of inflammatory diseases, allergic diseases, autoimmune diseases, infectious diseases, cancer or precancerous syndromes) and in the manufacture of immune adjuvants, thus provide a new choice for clinical screening and/or manufacture of a medicament for treating diseases related to STING activity.

Apparently, according to the above-mentioned content of the present invention, according to common technical knowledge and conventional means in the field, various other modifications, replacements or changes can be made without departing from the above-mentioned basic technical idea of the present invention.

Hereinafter, the above-mentioned content of the present invention will be further described in detail through specific implementations in the form of examples.

The raw materials and equipments used in the specific embodiments of the present invention are all known products, which can be obtained by purchasing commercially available products.

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or (and) mass spectrometry (MS). The NMR shift (δ) is given in unit of <NUM>-<NUM> (ppm). NMR was measured with (Bruker Avance III <NUM> and Bruker Avance <NUM>) nuclear magnetic instrument, the solvent was deuterated dimethyl sulfoxide (DMSO-d<NUM>), deuterated chloroform (CDCl<NUM>), deuterated methanol (CD<NUM>OD), and the internal standard was tetramethylsilane (TMS).

The LC-MS was measured with Shimadzu LC-MS (Shimadzu LC-MS <NUM> (ESI)).

The Shimadzu high pressure liquid chromatograph (Shimadzu LC-20A) was used for the HPLC measurement.

Gilson GX-<NUM> reversed-phase preparative chromatograph was used for reversed-phase preparative chromatography.

The thin layer chromatography silica gel plate used was Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, and the specifications for thin layer chromatography separation and purification products were <NUM>-<NUM>.

Column chromatography generally used Yantai Huanghai silica gel of <NUM>-<NUM> mesh as the carrier.

The known starting materials of the present invention can be synthesized by using or according to methods known in the art, or can be purchased from Anaiji Chemical, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, J & K Scientific and other companies.

The hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about <NUM>.

The hydrogenation reaction is usually performed by vacuumizing, and filling with hydrogen, and the operations are repeated <NUM> times.

Unless there are special instructions in the examples, the reaction is carried out under a nitrogen atmosphere.

Unless there are special instructions in the examples, the solution refers to an aqueous solution.

Unless there are special instructions in the examples, the reaction temperature is room temperature.

Unless there are special instructions in the examples, M is mole per liter.

Room temperature is the most suitable reaction temperature, which is <NUM>-<NUM>.

PE refers to petroleum ether; EA refers to ethyl acetate; DCM refers to dichloromethane; MeOH refers to methanol; DMF refers to N,N-dimethylformamide; DMSO refers to dimethyl sulfoxide; DMAP refers to <NUM>-dimethylaminopyridine; DIPEA refers to diisopropylethylamine; Boc refers to tert-butyloxycarbonyl; TFA refers to trifluoroacetic acid; DBU refers to <NUM>,<NUM>-diazabicycloundec-<NUM>-ene; HATU refers to <NUM>-(<NUM>-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

The following is the preparation of intermediate compounds:.

<NUM>-Chloro-<NUM>-methoxy-<NUM>-nitrobenzyl ester (<NUM>, <NUM> mmol) was added to a single-neck flask containing ammonium hydroxide (<NUM>) and stirred at <NUM> for <NUM>. The reaction solution was concentrated to <NUM>, cooled and filtered, and the solid was washed with ice water. After drying, <NUM>-chloro-<NUM>-methoxy-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol) was obtained as a brown solid.

Intermediate compound: Synthesis of <NUM>-chloro-<NUM>-hydroxy-<NUM>-nitrobenzamide
<CHM>.

In an ice bath, <NUM>-chloro-<NUM>-methoxy-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol) was dispersed in dry DCM (<NUM>), to which boron tribromide (<NUM> , <NUM>) was then slowly added dropwise. After the addition, the ice bath was removed, and the reaction was performed overnight at room temperature under protection of nitrogen. After the reaction was completed, the reaction solution was poured into ice water, stirred vigorously for <NUM> minutes, filtered, and the filter cake was washed with water. After the filter cake was dried, <NUM>-chloro-<NUM>-hydroxy-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol, <NUM>% yield) was obtained as pale yellow solid.

Intermediate compound: Synthesis of <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate
<CHM>.

<NUM>-Ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carboxylic acid (<NUM>, <NUM> mmol) was dispersed in dry DCM (<NUM>), to which was added oxalyl chloride (<NUM>, <NUM> mmol) and a catalytic amount of DMF. After reacting for <NUM> hour at room temperature, the volatiles were removed by rotary evaporation under reduced pressure. DCM (<NUM>) was added to the crude product, and the solvent was removed by rotary evaporation to obtain <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl chloride (<NUM>, <NUM>% yield), which was used directly for the next reaction.

At <NUM>, <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl chloride (<NUM>, <NUM> mmol) was dissolved in dry acetone (<NUM>) and added dropwise to a solution of potassium thiocyanate (<NUM>, <NUM> mmol) in acetone (<NUM>) and stirred at room temperature for <NUM>. The reaction system was filtered to remove inorganic salts. The crude product after concentration of the filtrate was purified by silica gel column (eluent: ethyl acetate/petroleum ether (v/v)=<NUM>/<NUM>), and <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol, <NUM>%) was obtained as a clear brown-yellow liquid.

Intermediate compound: Synthesis of <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate:
<CHM>.

<NUM>-Ethyl-<NUM>-methylthiazole-<NUM>-carboxylic acid (<NUM>, <NUM> mmol) was dispersed in dry DCM (<NUM>), to which oxalyl chloride (<NUM>, <NUM> mmol) and a catalytic amount of DMF were added dropwise under an ice bath. After reacting for <NUM> hour at room temperature, the volatiles were removed by rotary evaporation under reduced pressure. DCM (<NUM>) was added to the crude product, and the solvent was removed by rotary evaporation to obtain <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carboxylic acid chloride (<NUM>, <NUM>% yield), which was directly used in the next reaction.

At <NUM>, <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carboxylic acid chloride (<NUM>, <NUM> mmol) was dissolved in dry acetone (<NUM>) and added dropwise to a solution of potassium thiocyanate (<NUM>, <NUM> mmol) in acetone (<NUM>), stirred at room temperature for <NUM> hours. The reaction system was filtered to remove inorganic salts, the filtrate was concentrated and the crude product was purified by silica gel column (eluent: ethyl acetate/petroleum ether, v/v=<NUM>/<NUM>), and <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol, yield <NUM>%) was obtained as a clear brown liquid.

Intermediate compound: Synthesis of <NUM>-chloro-<NUM>-(<NUM>-morpholino)-<NUM>-nitrobenzamide
<CHM>.

To a solution of <NUM>-morpholinopropan-<NUM>-ol (<NUM>, <NUM> mmol) in dichloromethane (<NUM>) were added TEA (<NUM>, <NUM> mmol) and p-toluenesulfonyl chloride (<NUM>, <NUM> mmol). The mixture was reacted at room temperature for <NUM>, and water and dichloromethane were added for extraction, and the organic phase was washed with water and saturated brine respectively, dried with anhydrous sodium sulfate, filtered and concentrated to obtain <NUM>-morpholinopropyl <NUM>-methylbenzenesulfonate, which was used directly in the next reaction.

To a solution of <NUM>-chloro-<NUM>-hydroxy-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol) in DMF (<NUM>), potassium carbonate (<NUM>, <NUM> mmol), potassium iodide (<NUM>, <NUM> mmol) and <NUM>-morpholinopropyl <NUM>-methylbenzenesulfonate (<NUM>, <NUM> mmol) were added. The reaction was performed at <NUM> for <NUM>. After the reaction, the inorganic salts were removed by filtration. The filtrate was poured into water and extracted with ethyl acetate. The organic phase was washed with water and saturated brine successively, and then dried and concentrated to obtain <NUM>-chloro-<NUM>-(<NUM>-morpholino)-<NUM>-nitrobenzamide (<NUM>, <NUM>% yield).

Intermediate compound: synthesis of tert-butyl <NUM>-(<NUM>-(<NUM>-chloro-<NUM>-(methoxycarbonyl)-<NUM>-nitrophenoxy)propyl)piperazine-<NUM>-carboxylate
<CHM>.

Methyl <NUM>-chloro-<NUM>-methoxy-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) was dispersed in anhydrous dichloromethane (<NUM>). Boron tribromide (<NUM>, <NUM> mmol) was added slowly dropwise under an ice bath. After the addition, the mixture was slowly warmed to room temperature and stirred overnight. After the reaction was completed, methanol was slowly added dropwise to quench the reaction under an ice bath, and then it was spin-dried to dryness. Methanol (<NUM>) and concentrated sulfuric acid (<NUM>) were added to it, and the reaction solution was heated to <NUM> and stirred overnight. After cooling, the solvent was removed by concentration under reduced pressure, and <NUM> of water was added. After ultrasonic dispersion, the mixture was filtered, the solid was washed with water again, and the solid was dried to obtain methyl <NUM>-chloro-<NUM>-hydroxy-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol).

Methyl <NUM>-chloro-<NUM>-hydroxy-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol), tert-butyl <NUM>-(<NUM>-hydroxypropyl)piperazine-<NUM>-carboxylate (<NUM>, <NUM> mmol) ) and triphenylphosphine (<NUM>, <NUM> mmol) were dissolved in anhydrous THF (<NUM>) under nitrogen protection. Under an ice bath, DIAD (<NUM>, <NUM> mmol) was added dropwise to the above reaction solution. After the addition, the reaction solution was raised to room temperature and stirred for <NUM> hours. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and concentrated. The obtained crude product was purified by silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain the target compound as a pale yellow solid (<NUM>, yield <NUM>%).

Methyl <NUM>-chloro-<NUM>-hydroxy-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and <NUM>-morpholinopropyl <NUM>-methylbenzenesulfonate (<NUM>, <NUM> mmol) and K<NUM>CO<NUM> (<NUM>, <NUM> mmol) were added. The reaction solution was stirred at <NUM>° C for <NUM>, the inorganic salt was filtered out, and DMF was distilled off under reduced pressure. The crude product was dissolved in ethyl acetate, washed with water and saturated brine respectively, dried over anhydrous magnesium sulfate, filtered and concentrated to the remaining ethyl acetate (<NUM>), filtered to obtain the target compound as a yellow solid (<NUM>, yield <NUM>%).

Intermediate compound: synthesis of methyl <NUM>-fluoro-<NUM>-(methylthio)-<NUM>-nitrobenzoate
<CHM>.

<NUM>-Fluoro-<NUM>-nitrobenzoic acid (<NUM>, 270mmol) was dissolved in concentrated sulfuric acid (<NUM>), and NBS (<NUM>, 270mmol) was added. The mixture was warmed up to <NUM> and stirred overnight. After cooling to room temperature, the mixture was slowly poured into ice water and stirred. A pale yellow solid precipitated out. The solid was filtered and dried to obtain the target compound (<NUM>).

In an ice bath, thionyl chloride (<NUM>, <NUM>. 5mmol) was added dropwise to a solution of <NUM>-bromo-<NUM>-fluoro-<NUM>-nitrobenzoic acid (<NUM>, 249mmol) in methanol (<NUM>). The temperature was raised to <NUM>. The mixture was stirred overnight, and concentrated to about <NUM> of remaining solvent. After cooling, a solid continued to precipitate out. The solid was filtered and dried to obtain methyl <NUM>-bromo-<NUM>-fluoro-<NUM>-nitrobenzoate (<NUM>).

To a solution of methyl <NUM>-bromo-<NUM>-fluoro-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) in toluene () and tert-butanol (mL), methyl mercaptan acetate (<NUM>, <NUM> mmol), Pd<NUM>(dba)<NUM> (<NUM>, <NUM> mmol), xantphos (<NUM>, <NUM> mmol), K<NUM>PO<NUM> (<NUM>, <NUM> mmol) were added. The mixture was reacted overnight at <NUM> under nitrogen protection, and the reaction was completed. After cooling to room temperature, the reaction solution was concentrated, and the crude product obtained was purified by normal phase column (eluent: PE/EA=<NUM>/<NUM>, v/v) to separate and obtain the target compound (g, yield <NUM>%).

Intermediate compound: Synthesis of methyl <NUM>-fluoro-<NUM>-((<NUM>-morpholinopropyl)thio)-<NUM>-nitrobenzoate
<CHM>.

To a solution of <NUM>-morpholinopropyl <NUM>-methylbenzenesulfonate (<NUM>, <NUM> mmol) in DMF (<NUM>) was added potassium thioacetate (<NUM>, <NUM> mmol), potassium carbonate (<NUM>, <NUM> mmol), the mixture was stirred at room temperature overnight. The inorganic salt was removed by filtration and the solvent was spin-dried. The crude product was purified by silica gel column (eluent: dichloromethane/methanol=<NUM>/<NUM>) to obtain the target compound (<NUM>, yield <NUM>%).

To a solution of methyl <NUM>-bromo-<NUM>-fluoro-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) in toluene (<NUM>) and tert-butanol (<NUM>) were sequentially added <NUM>-morpholinopropyl mercaptan acetate (<NUM>, <NUM> mmol), Pd<NUM>(dba)<NUM> (<NUM>, <NUM> mmol), xantphos (<NUM>, <NUM> mmol), K<NUM>PO<NUM> (<NUM>, <NUM> mmol). The mixture was reacted overnight at <NUM> under nitrogen protection, and the reaction was completed. After cooling to room temperature, the reaction solution was concentrated, and the crude product obtained was purified by normal phase column (eluent: PE/EA=<NUM>/<NUM>, v/v) to separate and obtain the target compound (<NUM>, yield <NUM>%).

Intermediate compound: Synthesis of <NUM>-ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate:
<CHM>.

<NUM>-Ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carboxylic acid (<NUM>, <NUM> mmol) was dispersed in dry DCM (<NUM>), to which oxalyl chloride (<NUM>, <NUM> mmol) and a catalytic amount of DMF were add dropwise under an ice bath. After reacting for <NUM> hour at room temperature, the volatiles were removed by rotary evaporation under reduced pressure. DCM (<NUM>) was added to the crude product, and the solvent was removed by rotary evaporation to obtain <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carboxylic acid chloride (<NUM>, <NUM>% yield), which was directly used in the next reaction.

At <NUM>, <NUM>-ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl chloride (<NUM>, <NUM> mmol) was dissolved in dry acetone (<NUM>) and added dropwise to a solution of potassium thiocyanate (<NUM>, <NUM> mmol) in acetone (<NUM>), and the mixture was stirred at room temperature for <NUM> hours. The reaction system was filtered to remove inorganic salts, and the crude product after concentration of the filtrate was purified by silica gel column (eluent: ethyl acetate/petroleum ether, v/v=<NUM>/<NUM>), and <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol, yield <NUM>%) was obtained as a clear brown-yellow liquid. MS (ESI) m/z =<NUM>[M+H]+.

The following is the preparation of the compounds of the present invention:.

<NUM>-Chloro-<NUM>-methoxy-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol) and tert-butyl (<NUM>-aminobut-<NUM>-en-<NUM>-yl)carbamate (<NUM>, <NUM> mmol) were dissolved in dimethyl sulfoxide (<NUM>), and triethylamine (<NUM>, <NUM> mmol) was added dropwise to the reaction solution. The reaction mixture was heated to <NUM> and stirred at this temperature overnight. After cooling to room temperature, the mixture was diluted with water, a large amount of orange solid precipitated, filtered, the filter cake was washed with water (<NUM> × <NUM>). Drying was performed by vacuum rotary evaporator to obtain (trans)-tert-butyl (<NUM>-(<NUM>-carbamoyl-<NUM>-methoxy-<NUM>-nitrophenyl)amino)n-but-<NUM>-enyl)carbamate (compound 1b) (<NUM>, yield <NUM>%) as orange-yellow solid.

Compound 1b (<NUM>, <NUM> mmol) was dissolved in methanol (<NUM>), the solution was cooled to <NUM>, and then aqueous ammonia (<NUM>, <NUM> mmol) and a solution of sodium dithionite (<NUM>, <NUM> mmol) in water (<NUM>) were added successively. The reaction mixture was stirred at <NUM> for <NUM>, and the color of the reaction liquid changed from orange-red to white. The reaction solution was rotated to remove methanol, then diluted with water, and extracted with ethyl acetate (<NUM> × <NUM>). The separated organic phase was washed with saturated brine (<NUM> × <NUM>), dried over anhydrous sodium sulfate, and then spin-dried to obtain (trans)-tert-butyl (<NUM>-((<NUM>-amino-<NUM>-carbamoyl-<NUM>-methoxyphenyl)amino)n-but-<NUM>-enyl)carbamate (compound 1c) (<NUM>, yield <NUM> %), as white solid.

Compound 1c (<NUM>, <NUM> mmol) and compound <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formyl isothiocyanate (<NUM>, <NUM> mmol) were dissolved in N,N-dimethylformamide (<NUM>), and the mixture was stirred at room temperature for <NUM>, then HATU (<NUM>, <NUM> mmol) and <NUM>,<NUM>-diazabicyclo[<NUM>. <NUM>]undec-<NUM>-ene (<NUM> , <NUM> mmol) were added to the reaction solution and stirred at room temperature for <NUM>. The reaction solution was diluted with water, and the white solid produced was collected by filtration and washed with water (<NUM>×<NUM>), and then separated by reverse phase column (eluent: acetonitrile/water = <NUM>/<NUM>, v/v) to obtain (trans)-tert-butyl (<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-1hydro-pyrazole-<NUM>-carboxamide))-<NUM>-methoxy-<NUM>-benzimidazolyl) -n-but-<NUM>-enyl)carbamate (compound 1d) (<NUM>, yield <NUM>%), as white solid.

Compound 1d (<NUM>, <NUM> mmol) was dissolved in methanol (<NUM>), and hydrochloric acid (6N aqueous solution, <NUM>, <NUM> mmol) was added dropwise. After the reaction solution was stirred at room temperature for <NUM> minutes, the temperature was raised to <NUM> and stirring was continued for <NUM>. The reaction solution was spin-dried, and the obtained crude product was separated and purified by reversed-phase column (eluent: water/acetonitrile = <NUM>/<NUM>, v/v) to obtain compound (trans)-<NUM>-(<NUM>-amino-n-but-<NUM>-ene)-<NUM>-(<NUM>-ethyl-<NUM>-methyl-1hydro-pyrazole-<NUM>-carboxamide) -<NUM>-methoxy-1hydro-benzimidazole-<NUM>-carboxamide (compound 1e) (<NUM>, yield <NUM>%), as pale yellow solid.

Compound 1e (<NUM>, <NUM> mmol), <NUM>-chloro-<NUM>-(<NUM>-morpholino)-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were dissolved in n-butanol (<NUM>), the reaction mixture was heated to <NUM> and stirred at this temperature for <NUM>. After cooling to room temperature, the mixture was diluted with water, extracted with ethyl acetate (<NUM> × <NUM>), and the combined organic phase was washed with saturated brine (<NUM> × <NUM>), dried with anhydrous sodium sulfate. The solvent was spin-dried, the crude product obtained was separated by reversed-phase column chromatography (eluent: acetonitrile/water = <NUM>/<NUM>, v/v) to obtain (E)-<NUM>-(<NUM>-((<NUM>-carbamoyl-<NUM>-(<NUM>-morpholino)-<NUM>-nitrophenyl)amino)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carboxamide)-<NUM>-methoxy-<NUM>-benzo[d]imidazole-<NUM>-carboxamide (compound 1f) (<NUM>, yield <NUM>%).

Under an ice bath, ammonium hydroxide (<NUM>) was added dropwise to a solution of compound 1f (<NUM>, <NUM> mmol) in methanol (<NUM>). After the reaction solution was stirred at <NUM> for <NUM> minutes, an aqueous solution (<NUM>) of sodium dithionite (<NUM>, <NUM> mmol) was slowly added dropwise. The reaction mixture was slowly heated to room temperature, and stirring was continued for <NUM>. The mixture was dilute with water, filtered, and the filtrate was spin-dried. The obtained crude product was separated by reverse phase column chromatography (eluent: acetonitrile/water=<NUM>/<NUM>, v/v) to obtain (E)-<NUM>-(<NUM>-((<NUM>-amino)-<NUM>-carbamoyl-<NUM>-(<NUM>-morpholino)phenyl) amino)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-ethyl -<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-methoxy-<NUM>-benzo[d] imidazole-<NUM>-carboxamide <NUM> (<NUM>, yield <NUM>%), as white solid.

Compound <NUM> (<NUM>, <NUM> mmol) and <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) were mixed and dissolved in DMF (<NUM>), and stirred at room temperature for <NUM>, then HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added successively, and stirring was continued at room temperature for <NUM>. The reaction solution was purified by reverse phase HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%) as white solid.

Methyl <NUM>-chloro-<NUM>-methoxy-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) was dispersed in n-butanol (<NUM>), then tert-butyl (E)-(<NUM>-aminobutane-<NUM>-en-<NUM>-yl)carbamate (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. The reaction solution was heated to <NUM> and stirred for <NUM>. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, and an appropriate amount of dilute hydrochloric acid (<NUM>) was added in an ice bath to adjust the pH to neutral. The separated organic phase was washed with water and saturated brine respectively, dried over anhydrous magnesium sulfate and spin-dried to obtain 2b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 2b (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and the mixture was slowly heated to room temperature and reacted for <NUM>. Inorganic salts were filtered off add ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, which was purified on a silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain compound 2c (<NUM>, yield <NUM> %). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added to a solution of 2c (<NUM>, <NUM> mmol) in DMF (<NUM>). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 2d (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, TFA (<NUM>) was added to a solution of 2d (<NUM>, <NUM> mmol) in DCM (<NUM>), and then the mixture was heated to room temperature and reacted for <NUM>. The solvent was spin-dried under reduced pressure, ethyl acetate was added and then free TFA was removed by spin-drying under reduced pressure to obtain compound 2e (<NUM>, yield <NUM>%). MS(ESI)m/z =<NUM>[M+H].

2e (<NUM>, <NUM> mmol) was dispersed in n-butanol (<NUM>), and methyl <NUM>-chloro-<NUM>-(<NUM>-morpholinopropoxy)-<NUM>-nitrobenzoate (<NUM> , <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. The reaction solution was heated to <NUM> and stirred for <NUM>. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated brine, dried over anhydrous magnesium sulfate and spin-dried, and purified by silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain 2f (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 2f (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and slowly warmed to room temperature to react for <NUM>. Inorganic salts were filtered off, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, and then purified with a silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain compound <NUM> (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, to a solution of <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>) was added <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, a yellow solid precipitated out. The solution was filtered and the solid was dried to obtain <NUM> (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

<NUM> (<NUM>, <NUM> mmol) was dissolved in a mixed solution (<NUM>) of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM> <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotary evaporation, the mixture was cooled to room temperature, and adjusted with dilute hydrochloric acid (<NUM>) in an ice bath until the solid no longer precipitated. The solid was filtered and dried to obtain compound 2i (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

2i (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, to a solution of <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>) was added <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 3a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>. <NUM>[M+H].

3a (<NUM>, <NUM> mmol) was dissolved in a mixed solution of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and hydrated lithium hydroxide (<NUM> <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotatory evaporation, the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) in an ice bath until the solid no longer precipitates, filtered, and the solid was dried to obtain compound 3b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

3b (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Methyl <NUM>-chloro-<NUM>-(<NUM>-morpholinopropoxy)-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) was dispersed in n-butanol (<NUM>), and then tert-butyl (E)-(<NUM>-aminobut-<NUM>-en-<NUM>-yl)carbamate (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. The reaction solution was heated to <NUM> and stirred for <NUM>. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, and an appropriate amount of dilute hydrochloric acid (<NUM>) was added in an ice bath to adjust the pH to neutral. The separated organic phase was washed with water and saturated brine respectively, dried over anhydrous magnesium sulfate and spin-dried to obtain 4b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 4b (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and the mixture was slowly heated to react at room temperature for <NUM>. Inorganic salts were removed by filtration, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, which was purified on a silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain compound 4c (<NUM>, yield <NUM> %). MS(ESI)m/z = <NUM> [M+H].

Under an ice bath, to a solution of 4c (<NUM>, <NUM> mmol) in DMF (<NUM>) was added <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 4d (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, TFA (<NUM>) was added to 4d (<NUM>, <NUM> mmol) in DCM (<NUM>), and then heated to room temperature to react for <NUM>. The solvent was spin-dried under reduced pressure, ethyl acetate was added and then free TFA was removed by spin-drying under reduced pressure to obtain compound 4e (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

DIPEA (<NUM>, <NUM> mmol) and methyl <NUM>-fluoro-<NUM>-methylthio-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) were added to a solution of methyl (E)-<NUM>-(<NUM>-aminobut-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamino)-<NUM>-(<NUM>-morpholinepropoxy)-<NUM>-benzo[d]]imidazole-<NUM>-carboxylate (<NUM>, <NUM> mmol) in DMF (<NUM>), and the mixture was reacted at room temperature for <NUM> hours. The reaction solution was poured into water, extracted with ethyl acetate, washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was spin-dried under reduced pressure to obtain compound 4f (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 4f (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and the mixture was slowly heated to react at room temperature for <NUM>. Inorganic salts were removed by filtration, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, which was then purified with a silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain compound <NUM> (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, to a solution of <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>) was added <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol). After <NUM> of reaction, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain <NUM> (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

<NUM> (<NUM>, <NUM> mmol) was dissolved in a mixed solution of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The mixture was spin-dried to remove the organic solvent, cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) in an ice bath until the solid no longer precipitated, filtered, and the solid was dried to obtain compound 4i (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

4i (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added and stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Compound <NUM> was prepared by the same method as compound <NUM>, except that the ring-closure materials used in step <NUM> and step <NUM> were exchanged, that is, <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate was used in step <NUM> for ring closure, and <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate was used in step <NUM> for ring closure. The other steps were the same as the experiment method, and compound <NUM> (<NUM>) was obtained.

The compound <NUM> was prepared by the same method as the synthesis of compound <NUM>. Methyl <NUM>-chloro-<NUM>-(<NUM>-morpholinopropoxy)-<NUM>-nitrobenzoate used in the synthesis of compound <NUM> was replaced with methyl <NUM>-fluoro-<NUM>-((<NUM>-morpholinopropyl)thio)-<NUM>-nitrobenzoate, and other methods were the same.

Methyl (E)-<NUM>-(<NUM>-aminobut-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino) -<NUM>-methoxy-<NUM>-benzo[d]]imidazole-<NUM>-carboxylate (<NUM>, <NUM> mmol) was dispersed in n-butanol (<NUM>), and tert-butyl <NUM>-(<NUM>-(<NUM>-chloro-<NUM>-(methoxycarbonyl)-<NUM>-nitrophenoxy)propyl) piperazine-<NUM>-carboxylate (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. The reaction solution was heated to <NUM> and stirred for <NUM>. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated brine respectively, dried over anhydrous magnesium sulfate and spin-dried, and purified by silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain 7a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM> [M+H]+.

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 7a (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and the mixture was slowly heated to react at room temperature for <NUM>. Inorganic salts were filtered off, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, compound 7b (<NUM>, purity <NUM>%, yield <NUM>%). MS(ESI)m/z = <NUM> [M+H]+.

Under an ice bath, to a solution of 7b (<NUM>, <NUM> mmol) in DMF (<NUM>) was added <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol). After <NUM> of reaction, HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added, and then heated to room temperature for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 7c (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM> [M+H]+.

7c (<NUM>, <NUM> mmol) was dissolved in a mixed solution (<NUM>) of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotary evaporation, and the crude product was dissolved in water, extracted by adding ethyl acetate. The resulting aqueous phase obtained by separation was adjusted with dilute hydrochloric acid (<NUM>) in an ice bath until the solid did not precipitate, the solid was filtered and dried to obtain compound 7d (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM> [M+H]+.

7d (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was added with water to precipitate a solid, which was filtered, washed with pure water, and dried to obtain compound 7e (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM> [M+H]+.

Under ice bath, TFA(<NUM>) was added to a solution of compound 7e (<NUM>, <NUM> mmol) in DCM (<NUM>), the mixture was slowly warmed to room temperature, reacted for <NUM> and spin-dried to remove the solvent. The free TFA was removed under reduced pressure with an oil pump to obtain compound 7f (<NUM>, <NUM>% purity). MS(ESI)m/z = <NUM> [M+H].

<NUM>% formaldehyde solution (<NUM> drops) was added to a solution of compound 7f (<NUM>, <NUM> mmol) in methanol (<NUM>), acetic acid was added after <NUM> to adjust the pH to about <NUM>, then sodium borohydride (<NUM>) was added. After <NUM> of reaction, the reaction was quenched by adding water. After the reaction solution was concentrated, compound <NUM> (<NUM>, yield <NUM>%) was obtained by purification through preparative HPLC.

<NUM>H NMR (<NUM>, DMSO-d<NUM> + D<NUM>O) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>) , <NUM> (dt, J = <NUM>, <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Acetaldehyde (<NUM>) was added to a solution of compound 7f (<NUM>, <NUM> mmol) in methanol, and acetic acid was added after <NUM> to adjust the pH to about <NUM>, then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. After <NUM> of reaction, water was added to quench the reaction. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM>H NMR (<NUM>, MeOD) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>) , <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

Acetone (<NUM>) was added to a solution of compound 7f (<NUM>, <NUM> mmol) in methanol, and the pH was adjusted to about <NUM> with acetic acid. The mixture was heated to <NUM> and reacted for <NUM> hours, and the reaction solution was cooled to <NUM>, then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. The mixture was reacted overnight at room temperature, and the reaction was quenched by adding water. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM>H NMR (<NUM>, DMSO-d<NUM> + D<NUM>O) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> ( d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

<NUM>-ethoxy-<NUM>-trimethylsiloxycyclopropane (<NUM>, <NUM> mmol) was added to a solution of compound 7f (<NUM>, <NUM> mmol) in methanol, and the pH was adjusted to about <NUM> with acetic acid, and then sodium borohydride acetate (<NUM>, <NUM> mmol) was added and heated to <NUM> and reacted overnight. LCMS showed that all was converted to intermediate imine. After cooling to room temperature, sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. The reaction was quenched by adding water. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM>H NMR (<NUM>, DMSO-d<NUM>+D<NUM>O) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>).

TEA (<NUM>, <NUM> mmol) and acetic anhydride (<NUM>, <NUM> mmol) were added to a solution of 7f (<NUM>, <NUM> mmol) in DMF (<NUM>), and the mixture was reacted for <NUM> at room temperature. The reaction solution was purified by preparative HPLC and compound <NUM> was obtained (<NUM>, yield <NUM>%).

<NUM>H NMR (<NUM>, DMSO-d<NUM> + D<NUM>O) δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> ( d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m , <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>) , <NUM> (t, J = <NUM>, <NUM>).

According to the synthesis method of Example <NUM>, compounds <NUM>-<NUM> of Examples <NUM>-<NUM> were also prepared.

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-methoxy-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol (<NUM>) was added <NUM>% formaldehyde solution (<NUM> drops). After <NUM>, acetic acid was added to adjust the pH to about <NUM>, then sodium borohydride (<NUM>) was added, and the reaction was quenched by adding water after <NUM>. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM>H NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>) , <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM> ), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>) , <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-methoxy-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, acetaldehyde (<NUM>) was added, after <NUM> acetic acid was added to adjust the pH to about <NUM>, and then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. After reacting for <NUM>, the reaction was quenched by adding water. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM>H NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM> ), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM> ), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (t , J = <NUM>, <NUM>).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-methoxy-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, acetone (<NUM>) was added, and the pH was adjusted with acetic acid to about <NUM>, the mixture was heated to <NUM> and react for <NUM> hours. The reaction solution was cooled to <NUM>, then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added, reacted overnight at room temperature, and the reaction was quenched by adding water. After concentrating the reaction solution, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM> NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>) , <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m , <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-methoxy-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, <NUM>-ethoxy-<NUM>-trimethylsilyloxycyclopropane (<NUM>, <NUM> mmol) was added, the pH was adjusted to about <NUM> with acetic acid, then sodium borohydride acetate (<NUM>, <NUM> mmol) was added, and the mixture was heated to <NUM> and reacted overnight. LCMS showed that all was converted to intermediate imine. After cooling to room temperature, sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. The reaction was quenched by adding water. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM> NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> ( s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

To a solution of compound (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, <NUM>% formaldehyde solution (<NUM> drops) was added, after <NUM>, acetic acid was added to adjust the pH to about <NUM>, then sodium borohydride (<NUM>, <NUM> mmol) was added, and the reaction was quenched by adding water after <NUM>. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, acetaldehyde (<NUM>) was added. After <NUM>, acetic acid was added to adjust the pH to about <NUM>, and then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. After reacting for <NUM>, the reaction was quenched by adding water. After concentrating the reaction solution, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM> NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>) , <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s , <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>) , <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

To a solution of ((E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, acetone (<NUM>) was added, and then the pH was adjusted to about <NUM> with acetic acid, the mixture was heated to <NUM> and reacted for <NUM> hours. The reaction solution was cooled to <NUM>, then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. The mixture was reacted overnight at room temperature, and water was added to quench the reaction. The reaction solution was concentrated, then it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, <NUM>%).

<NUM> NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>) , <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>,<NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM> ), <NUM> (q, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, <NUM>-ethoxy-<NUM>-trimethylsiloxycyclopropane (<NUM>, <NUM> mmol) was added, the pH was adjusted to about <NUM> with acetic acid, then sodium borohydride acetate (<NUM>, <NUM> mmol) was added, the mixture was heated to <NUM> and reacted overnight. LCMS showed that all was converted to intermediate imine. The mixture was cooled to room temperature and sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. The reaction was quenched by adding water. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, <NUM>% formaldehyde solution (<NUM> drops) was added. After <NUM>, acetic acid was added to adjust the pH to about <NUM>, then sodium borohydride (<NUM>, <NUM> mmol) was added, and the reaction was quenched with water after <NUM>. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, acetaldehyde (<NUM>) was added. After <NUM>, acetic acid was added to adjust the pH to about <NUM>, and then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added. After reacting for <NUM>, the reaction was quenched by adding water. After concentrating the reaction solution, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

<NUM> NMR (<NUM>, MeOD-d<NUM>) δ <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>( m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>(s, <NUM>), <NUM>-<NUM>(m, <NUM>), <NUM>- <NUM>(m, <NUM>), <NUM>-<NUM>(m, <NUM>).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, acetone (<NUM>) was added, and then the pH was adjusted with acetic acid to about <NUM>, the mixture was heated to <NUM> to react for <NUM> hours, the reaction solution was cooled to <NUM>, then sodium cyanoborohydride (<NUM>, <NUM> mmol) was added, the mixture was reacted overnight at room temperature, and water was added to quench the reaction. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, <NUM>%).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide (<NUM>, <NUM> mmol) in methanol, <NUM>-ethoxy-<NUM>-trimethylsiloxycyclopropane (<NUM>, <NUM> mmol) was added, the pH was adjusted to about <NUM> with acetic acid, then sodium acetate borohydride (<NUM>, <NUM> mmol) was added, the mixture was heated to <NUM> and reacted overnight, then water was added to quench the reaction. After the reaction solution was concentrated, it was purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-methoxy-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide in DMS, TEA and the corresponding acid chloride was added, the mixture was reacted at room temperature for <NUM>. The reaction solution was purified by preparative HPLC, and Compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM> were obtained. The compound structures and characterization are shown in Table <NUM> below:.

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-methoxy-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide in DMF, TEA and the corresponding acid chloride were added, the mixture was reacted at room temperature for <NUM>, and the reaction solution was purified by preparative HPLC. Compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> were obtained. The compound structures and characterization are shown in Table <NUM> below:.

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide in DMF, TEA and the corresponding acid chloride were added, and the reaction was performed for <NUM> at room temperature. The reaction solution was purified by preparative HPLC. Compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> were obtained. The compound structures and characterization are shown in Table <NUM> below:.

To a solution of (E)-N-(<NUM>-carbamoyl-<NUM>-(<NUM>-(<NUM>-carbamoyl-<NUM>-(<NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-formylamino)-<NUM>-(<NUM>-(piperazin-<NUM>-yl)propoxy)-<NUM>-benzo[d]imidazol-<NUM>-yl)but-<NUM>-en-<NUM>-yl)-<NUM>-(methylthio)-<NUM>-benzo[d]imidazol-<NUM>-yl)-<NUM>-ethyl-<NUM>-methylthiazole-<NUM>-formylamide in DMF, TEA and the corresponding acid chloride were added, and the reaction was performed for <NUM> at room temperature. The reaction solution was purified by preparative HPLC to obtain compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. The compound structures and characterization are shown in Table <NUM> below:.

4e (<NUM>, <NUM> mmol) was dispersed in n-butanol (<NUM>), and methyl <NUM>-chloro-<NUM>-(<NUM>-morpholinopropoxy)-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. The reaction solution was heated to <NUM> and stirred for <NUM>. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated brine respectively, dried over anhydrous magnesium sulfate and spin-dried, and purified by silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain 47a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 47a (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and the mixture was slowly heated to react at room temperature for <NUM>. Inorganic salts were removed by filtration, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain compound 47b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, <NUM>-ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added to a solution of 47b (<NUM>, <NUM> mmol) in DMF (<NUM>). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 47c (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

47c (<NUM>, <NUM> mmol) was dissolved in a mixed solution (<NUM>) of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotatory evaporation, the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) under an ice bath until no solid precipitated, the solid was filtered and dried to obtain compound 47d (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

47d (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, <NUM>-ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added to a solution of <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 48a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

48a (<NUM>, <NUM> mmol) was dissolved in a mixed solution (<NUM>) of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and hydrated lithium hydroxide (<NUM>, <NUM> mmoL) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotary evaporation, the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) under an ice bath until no solid precipitated. The solid was filtered and dried to obtain compound 48b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

48b (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, <NUM>-ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added to a solution of <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 49a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

49a (<NUM>, <NUM> mmol) was dissolved in a mixed solution of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and hydrated lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotary evaporation, the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) under an ice bath until no solid precipitated. The solid was filtered and dried to obtain compound 49b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

49b (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, to a solution of <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>), <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added. After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 50a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

50a (<NUM>, <NUM> mmol) was dissolved in a mixed solution of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and hydrated lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotatory evaporation, the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) under an ice bath until no solid precipitated. The solid was filtered and dried to obtain compound 50b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

50b (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, <NUM>-ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added to a solution of 4c (<NUM>, <NUM> mmol) in DMF (<NUM>). After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 51a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, TFA (<NUM>) was added to 51a (<NUM>, <NUM> mmol) in DCM (<NUM>), and then the mixture was heated to room temperature for <NUM>. The solvent was spin-dried under reduced pressure, ethyl acetate was added and then the free TFA was removed by spin-drying under reduced pressure to obtain compound 51b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

To a solution of 51b (<NUM>, <NUM> mmol) in DMF (<NUM>), DIPEA (<NUM>, <NUM> mmol) and methyl <NUM>-fluoro-<NUM>-methylthio-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) were added, the mixture was reacted at room temperature for <NUM>. The reaction solution was poured into water, extracted with ethyl acetate, washed with saturated brine and dried over anhydrous sodium sulfate, and the solvent was spin-dried under reduced pressure to obtain compound 51c (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 51c (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and the mixture was slowly heated to react at room temperature for <NUM>. Inorganic salt was removed by filtration, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, and then purified with a silica gel column (eluent: petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain compound 51d (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, to a solution of 51d (<NUM>, <NUM> mmol) in DMF (<NUM>), <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added. After <NUM> of reaction, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 51e (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

51e (<NUM>, <NUM> mmol) was dissolved in a mixed solution of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotatory evaporation, the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) in an ice bath until no solid was precipitated. The solid was filtered and dried to obtain compound 51f (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

51f (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added and stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, to a solution of 51d (<NUM>, <NUM> mmol) in DMF (<NUM>), <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added. After <NUM> of reaction, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 52a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

52a (<NUM>, <NUM> mmol) was dissolved in a mixed solution of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotatory evaporation, and the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) under an ice bath until no solid precipitated. The solid was filtered and dried to obtain compound 52b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

52b (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, <NUM>-ethyl-<NUM>-fluoro-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added to a solution of 51d (<NUM>, <NUM> mmol) in DMF (<NUM>). After <NUM> of reaction, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 53a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

53a (<NUM>, <NUM> mmol) was dissolved in a mixed solution of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotatory evaporation, the mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) under an ice bath until no solid precipitated. The solid was filtered and dried to obtain compound 53b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

53b (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%).

Under an ice bath, to a solution of 4c (<NUM>, <NUM> mmol) in DMF (<NUM>), <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added. After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 54a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, TFA (<NUM>) was added to 54a (<NUM>, <NUM> mmol) in DCM (<NUM>), and then heated to room temperature for <NUM>. The solvent was spin-dried under reduced pressure, ethyl acetate was added and then the free TFA was removed by spin-drying under reduced pressure to obtain compound 54b (<NUM>), which was directly used in the next step. MS(ESI)m/z = <NUM>[M+H].

To a solution of 54b (<NUM>, crude product, <NUM> mmol) in DMF (<NUM>), DIPEA (<NUM>, <NUM> mmol) and methyl <NUM>-fluoro-<NUM>-methylthio-<NUM>-nitrobenzoate (<NUM>, <NUM> mmol) were added, the mixture was reacted at room temperature for <NUM>. After the reaction was completed, the mixture was poured into water, filtered, washed with water and dried to obtain 54c as a yellow solid (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 54c (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and the mixture was slowly heated to react at room temperature for <NUM>. Inorganic salt was removed by filtration and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, which was purified by silica gel column (eluent: dichloromethane/methanol=<NUM>/<NUM>, v/v) to obtain compound 54d (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

Under an ice bath, to a solution of 54d (<NUM>, <NUM> mmol) in DMF (<NUM>), <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added. After <NUM> of reaction, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the temperature was raised to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a white solid precipitated out. The solid was filtered and dried to obtain 54e (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

54e (<NUM>, <NUM> mmol) was dissolved in a mixed solution (<NUM>) of methanol, tetrahydrofuran and water (volume ratio: <NUM>/<NUM>/<NUM>), and lithium hydroxide (<NUM>, <NUM> mmol) was added. The temperature was raised to <NUM> and the reaction was stirred overnight. The organic solvent was removed by rotatory evaporation. The mixture was cooled to room temperature, adjusted with dilute hydrochloric acid (<NUM>) under an ice bath until no solid precipitated. The solid was filtered and dried to obtain compound 54f (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H].

54f (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and HATU (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. After <NUM>, ammonium bicarbonate (<NUM>, <NUM> mmol) was added, and the mixture was stirred at room temperature for <NUM>. The reaction solution was concentrated and purified by preparative HPLC to obtain compound <NUM> (<NUM>, yield <NUM>%) as a white solid.

Under an ice bath, to a solution of compound <NUM> (<NUM>, <NUM> mmol) in tetrahydrofuran (<NUM>), triethylamine (<NUM>, <NUM> mmol) and methanesulfonyl chloride (<NUM>, <NUM> mmol) were added. The temperature was raised to room temperature and reacted for <NUM>. LCMS showed that the reaction was completed. Potassium carbonate (<NUM>, <NUM> mmol) and <NUM>-oxa-<NUM>-azabicyclo[<NUM>. <NUM>]octane (<NUM>, <NUM> mmol) were added to the reaction solution, and the temperature was raised to <NUM> for reaction for <NUM>, and the solvent was spin-dried. The crude product was purified by preparative HPLC to obtain compound <NUM> (<NUM>).

According to the preparation method of compound <NUM>, using compound <NUM> and the corresponding amine as raw materials, compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> were obtained.

At room temperature, <NUM>% palladium on carbon (<NUM>) was added to a solution of <NUM>-fluoro-<NUM>-methoxy-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol) in methanol (<NUM>), and hydrogenation was performed at room temperature for <NUM>. The palladium carbon was filtered off, and the filtrate was spin-dried to obtain compound 65a (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H]+.

Compound 65a (<NUM>, <NUM> mmol) was dissolved in DMF (<NUM>), and potassium carbonate (<NUM>, <NUM> mmol) and tert-butyl (E)-(<NUM>-bromobut-<NUM>-en-<NUM>-yl)carbamate (<NUM>, <NUM> mmol) were added sequentially. The mixture was heated to <NUM> and reacted for <NUM> hours. After cooling, inorganic salts were removed by filtration. The filtrate was spin-dried, dissolved with ethyl acetate, washed with water and saturated brine respectively. The organic phase was spin-dried and purified by silica gel column (petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain compound 65b (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H]+.

Compound 65b (<NUM>, <NUM> mmol) was dissolved in acetic anhydride (<NUM>), and when it was cooled to <NUM> under an ice bath, nitric acid (<NUM>, <NUM> mmol, <NUM>% purity) was added dropwise to the reaction solution. The reaction was maintained at <NUM> for <NUM>. The reaction solution was poured into water, extracted with ethyl acetate, washed with saturated brine. The organic phase was concentrated and purified by silica gel column (petroleum ether/ethyl acetate = <NUM>/<NUM>) to obtain compound 65c (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H]+.

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of 65c (<NUM>, <NUM> mmol) in methanol (<NUM>). After <NUM> minutes, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added and reacted for <NUM>. Inorganic salts were removed by filtration, and ethyl acetate was added for extraction. The organic phase was washed with saturated brine, dried over anhydrous magnesium sulfate and spin-dried to obtain the crude product, which was purified on a silica gel column (eluent: dichloromethane/methanol=<NUM>/<NUM>-<NUM>/<NUM>) to obtain compound 65d (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H]+.

Under an ice bath, to a solution of 65d (<NUM>, <NUM> mmol) in DMF (<NUM>), <NUM>-ethyl-<NUM>-methyl-<NUM>-pyrazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added. After reacting for <NUM>, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then heated to room temperature to react for <NUM>. The reaction solution was slowly poured into water, and a yellow solid precipitated out. The solid was filtered and dried to obtain 65e (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H]+.

Under an ice bath, TFA (<NUM>) was added to 65e (<NUM>, <NUM> mmol) in DCM (<NUM>), and then heated to room temperature for <NUM>. The solvent was spin-dried under reduced pressure, ethyl acetate was added and then free TFA was removed by spin-drying under reduced pressure to obtain compound 65f (<NUM>, containing trifluoroacetic acid). MS(ESI)m/z =<NUM>[M+H]+.

65f (<NUM>, <NUM> mmol, <NUM>% purity) was dispersed in n-butanol (<NUM>), and <NUM>-chloro-<NUM>-(<NUM>-morpholinopropoxy)-<NUM>-nitrobenzamide (<NUM>, <NUM> mmol) and DIPEA (<NUM>, <NUM> mmol) were added. The reaction solution was heated to <NUM> and stirred for <NUM>. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated brine respectively, dried over anhydrous sodium sulfate and spin-dried, and purified by silica gel column (eluent: dichloromethane/methanol=<NUM>/<NUM>) to obtain <NUM> (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>.

Under an ice bath, ammonium hydroxide (<NUM>) was added to a solution of <NUM> (<NUM>, <NUM> mmol) in methanol (<NUM>). <NUM> minutes later, an aqueous solution of sodium dithionite (<NUM>, <NUM> mmol) was added, and the mixture was slowly heated to react at room temperature for <NUM>. The inorganic salt was removed by filtration, the filtrate was concentrated to obtain the crude product, and then purified by reversed-phase column chromatography (eluent: acetonitrile/water=<NUM>/<NUM>) to obtain compound <NUM> (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H]+.

Under an ice bath, <NUM>-ethyl-<NUM>-methylthiazole-<NUM>-carbonyl isothiocyanate (<NUM>, <NUM> mmol) was added to a solution of <NUM> (<NUM>, <NUM> mmol) in DMF (<NUM>). After <NUM> of reaction, DIPEA (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, and then the mixture was heated to room temperature for <NUM>. The reaction solution was purified by preparative HPLC to obtain <NUM> (<NUM>, yield <NUM>%). MS(ESI)m/z = <NUM>[M+H]+.

The following experimental examples are used to illustrate the beneficial effects of the present invention:.

The protein thermal transfer test (TSA) was used to determine the binding affinity of the compound to Sting protein. The 100ug/ml Sting protein was mixed with different concentrations of the compound and 5X SYPRO Orange dye in a buffer of <NUM> Hepes, <NUM> NaCl, <NUM> MgCl<NUM>, <NUM> DTT, pH=<NUM>. The protein dissolution curve was measured on a qPCR instrument, the Tm value was fitted with Protein Thermal Shift Software <NUM> software, and the Tm difference of the protein was calculated when different concentrations of compound and no compound were added. The dissociation constant Kd was fitted and obtained according to the change of ΔTm with the concentration of the compound. The compound <NUM> reported in the article (<NPL>) was used as a positive reference compound. The test results are shown in Table <NUM> below. The Kd value of each compound is classified according to the following instructions:.

The above experimental results show that the compound of the present invention has a good ability to bind to the Sting protein, and has a similar in vitro affinity with the reference compound, so the compound of the present invention can be used as an effective STING protein regulator.

In this experiment, the function of sting agonist was evaluated by detecting the changes of IFN-β and CXCL10 (IP10) cytokines produced by human peripheral blood mononuclear cell line THP1 cells (Shanghai Cell Bank) stimulated by the compounds. On the first day of the experiment, the ELISA plate was coated according to the IFN-β (R&D, #DY814-<NUM>) and IP10 (BD, #<NUM>) ELISA test kit instructions. The compound was dissolved into DMSO stock solution and diluted with culture medium to a 2X working concentration, added to a <NUM>-well plate, <NUM>µL per well. The THP1 cells in the logarithmic growth phase were taken to count, and were diluted to a concentration of <NUM>* <NUM><NUM>/mL, added to the above-mentioned <NUM>-well plate containing the compounds, with 100µL per well, mixed well, and incubated in a <NUM>, <NUM>% CO<NUM> incubator for <NUM> hours. On the second day, the above cell culture supernatant was taken, 100µL per well, and tested according to IFN-β and IP10 ELISA test kits respectively. The OD450 value was read, and converted into IFN-β and IP10 concentration according to the standard curve, and fitted with GraphPad <NUM> to calculate the EC<NUM> value of the dose-efficiency curve. EC<NUM> is the half-maximal effect concentration (concentration for <NUM>% of maximal effect, EC<NUM>), which refers to the concentration of the drug that can cause <NUM>% of the individual to be effective.

The compound <NUM> reported in the article (<NPL>) was used as a positive reference compound.

The experimental results are shown in Table <NUM> below, and the EC<NUM> values of each compound are classified according to the following instructions:.

In Table <NUM>, "ND" means that the test has not been performed yet.

The above experimental data show that the compounds of the present invention have good stimulating activity of IFN-β and CXCL10 (IP10) cytokines produced by THP1 cells, and have a good STING protein agonistic function. Especially for compounds <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, etc., compared with the reference compound, the compounds of the present invention have comparable or even higher activity of stimulating the immune cells to produce cytokines.

Balb/C mice were inoculated with <NUM>×<NUM><NUM> CT26 cells subcutaneously on the back. After inoculation, tumor-bearing mice with tumor volume in the range of <NUM>-<NUM><NUM> were selected for the experiment. According to the random method of tumor size and segment, they were divided into <NUM> groups with <NUM> mice in each group. Different doses of drugs (compounds of the present invention) were given via tail vein injection, and the control group was given a control vehicle (<NUM>% DMSO, <NUM>% PEG400 formulated in physiological saline). Three hours after the administration, blood was taken from the mouse orbital vein, and EDTA•<NUM> was used as an anticoagulant. The collected mouse whole blood was centrifuged at <NUM> for <NUM> minutes and then the upper plasma was taken for testing. Three hours after administration, mouse tumor tissues were taken, weighed and homogenized by adding <NUM> times volume of PBS. After centrifuging the homogenate at <NUM>,<NUM> rpm for <NUM> minutes, the supernatant was collected for testing. The detection was performed according to the IFN-β detection kit (R&D, #DY814-<NUM>), the OD450 value was read, which was converted to the IFN-β concentration according to the standard curve.

Experimental results show that the tail vein administration of mice can induce the release of IFN-β downstream of the STING pathway in blood and tumors. After administration of the compound of the present invention, the intratumor IFN-β level was more than <NUM> times that of the plasma IFN-β level, indicating that cytokines were relatively enriched in tumor tissues, and blood cytokines were low, suggesting a higher treatment window.

After Balb/c mice rested for one week, CT26 cells were inoculated subcutaneously on the back, and each mouse was inoculated with <NUM>µL cell suspension containing <NUM>×<NUM><NUM> cells. When the average tumor volume grew to about <NUM><NUM>, they were randomly divided into groups according to the size of the tumor, with <NUM> in each group. The mice in each group were administered intravenously through the tail vein on Day <NUM>, <NUM>, and <NUM> after grouping, and the control group was given a control vehicle (<NUM>% DMSO, <NUM>% PEG400 formulated in physiological saline). On the day of grouping, <NUM> times a week after the first administration, and before euthanasia, the length and short diameter of the tumor were measured and recorded with a vernier caliper, the tumor volume was calculated, and the tumor growth curve was drawn according to the tumor volume. The tumor volume was calculated according to the following formula: V=<NUM>/<NUM>×long diameter×short diameter×short diameter.

The experimental results are shown in <FIG>. After <NUM> days of administration, the compounds prepared in the examples of the present invention all effectively inhibited tumor growth and exhibited a dose-dependent tumor growth inhibitory effect.

Kunming rats were inoculated with H22 cells subcutaneously on their backs after a week of resting. Each mouse was inoculated with <NUM>µL of cell suspension containing <NUM>×<NUM><NUM> cells. When the average tumor volume grew to about <NUM><NUM>, they were randomly grouped according to the tumor size, with <NUM> in each group. On the Day <NUM>, <NUM> and <NUM> after grouping, the mice in the high, medium and low dose groups were administered intravenously via the tail vein (the compounds of the present invention), or on the Day <NUM>, <NUM>, and <NUM> the mice in the high dose group were administered intravenously via the tail vein. The control group was given a control vehicle (<NUM>% DMSO, <NUM>% PEG400 formulated in physiological saline). On the day of grouping, <NUM> times a week after the first administration, and before euthanasia, the length and short diameter of the tumor were measured and recorded with a vernier caliper, the tumor volume was calculated, and the tumor growth curve was drawn according to the tumor volume. The tumor volume was calculated according to the following formula: V=<NUM>/<NUM>×long diameter×short diameter×short diameter.

After <NUM> days of administration, the compounds of the present invention all effectively inhibited tumor growth, and exhibited a dose-dependent tumor growth inhibitory effect.

The tumor regression of each group of mice in the CT26 anti-tumor efficacy model of the above experimental example <NUM> was further observed. After <NUM> days of the last administration, CT26 cells were inoculated subcutaneously on the back. Each mouse was inoculated with <NUM>µL cell suspension containing <NUM>×<NUM><NUM> cells; mice that have not been treated with drugs were used as a control group to inoculate CT26 cells in the same way. The tumor was observed and the growth of the tumor was recorded.

The experimental results are shown in <FIG>. The CT26 tumors in the control group can grow after inoculation, but the tumors in the mice whose tumors disappeared completely after treatment with the compounds of the present invention cannot grow again after inoculation, indicating that these compounds activated the immune memory mechanism of the mice, and render mice had immunity to tumor cells that were re-inoculated.

Claim 1:
A compound, or the stereoisomer, or the pharmaceutically acceptable salt thereof, wherein the compound is represented by Formula II,
<CHM>
wherein X is selected from -O- and -S-, and X' is -O-;
A ring and A' ring are each independently selected from
<CHM>
optionally substituted with <NUM>-<NUM> Rc; and when X and X' are both -O-, at least one of A ring and A' ring is selected from
<CHM>
optionally substituted with <NUM>-<NUM> Rc;
R<NUM> is selected from C<NUM>-C<NUM> alkyl;
R<NUM>' is selected from
<CHM>
Re' is selected from C<NUM>-C<NUM> alkyl, <NUM>-<NUM> membered cycloalkyl, -C(O)Rf;
Rf is selected from -ORa', C<NUM>-C<NUM> alkyl, <NUM> to <NUM>-membered cycloalkyl, and Ra' is selected from C<NUM>-C<NUM> alkyl; and
wherein
the
<CHM>
optionally substituted with <NUM> to <NUM> Rc is
<CHM>
and
the
<CHM>
optionally substituted by <NUM>-<NUM> Rc is
<CHM>