Substituted thiophene imide ethyl ester compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a substituted thiophene imide ethyl ester compound as well as a preparation method and application thereof. Specifically, the present invention relates to a compound of formula (I), a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the compound of the present invention, in which R1, R2 and Ar are as described in the specification. The invention aims to prepare a novel DprE1 inhibitor which has good inhibitory activity on both a DprE1 wild type and a DprE1 mutant type, has anti-mycobacterium tuberculosis activity, particularly has a better inhibitory effect on drug-resistant mycobacterium tuberculosis, has an obviously weakened inhibitory effect on liver drug enzyme CYP 2C9, is low in cytotoxicity, and can be used for preparing a novel DprE1 inhibitor. The compound has excellent anti-tuberculosis activity in macrophages, can be used for treatment or prophylactic treatment of infectious diseases caused by bacteria, especially tuberculosis (TB) diseases caused by mycobacterium tuberculosis, and can also be used for overcoming the problems of mycobacterium tuberculosis DprE1 target protein mutation and drug-resistant mycobacterium tuberculosis infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine. In particular, it relates to substituted thienyl imine ethyl ester compounds represented by general formula (I), a preparation method thereof, an application thereof in the preparation of DprE1 inhibitors, a pharmaceutical composition taking the compound as an active ingredient, and an application thereof in the treatment and / or prevention of infectious diseases caused by Mycobacterium tuberculosis. BACKGROUND

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). The lung is the most common site of infection, and clinically manifests as pulmonary tuberculosis, accounting for about 60% to 80% of cases. In addition, Mycobacterium tuberculosis can also infect lymph nodes, pleura, bones and joints, ears and other sites, forming extrapulmonary tuberculosis. According to the WHO report, tuberculosis is one of the top ten deadly diseases in the world, and is the single infectious disease with the highest mortality rate, seriously threatening human health. About one-fourth of the world's population has latent infection of Mycobacterium tuberculosis. In 2022, 7.5 million people worldwide were newly diagnosed with tuberculosis and officially reported as tuberculosis cases, the highest number in a single year since the WHO began global tuberculosis surveillance in the mid-1990s. In 2022, a total of 1.3 million people died of tuberculosis worldwide, almost returning to the level in 2019 (Global tuberculosis report 2023). According to the WHO's strategy to end tuberculosis based on the Sustainable Development Goals, the global tuberculosis burden remains heavy.

[0003] In 1993, the World Health Organization launched the DOTS (directly observed therapy short course) program to overcome the tuberculosis crisis. The program prescribed first-line anti-tuberculosis drugs consisting of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB). Although the number of cases has decreased significantly, the development of drug-resistant bacteria has become increasingly serious due to poor patient compliance, resulting in multi-drug resistant tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB) and totally drug-resistant tuberculosis (TDR-TB), making treatment more difficult. In the past 40 years, only three drugs, Bedaquiline, Delamanid and PA-824 (Pretomanid), have been approved for marketing for the treatment of drug-resistant tuberculosis, and other drugs are still in the clinical research stage. Therefore, it is urgent to develop anti-tuberculosis drugs with independent intellectual property rights, new skeletons and novel mechanisms of action to treat and control tuberculosis, especially drug-resistant tuberculosis.

[0004] The cell wall is the outermost structure of Mtb, which has the functions of stabilizing cell structure, protecting cells from mechanical damage and osmotic pressure, maintaining its virulence and pathogenicity, etc. Therefore, drugs targeting the cell wall are the hotspots of anti-tuberculosis drug research and development. Most anti-tuberculosis drugs target the assembly of Mycobacterium tuberculosis cell wall, including mycolic acid (MA) biosynthesis (isoniazid / ethionamide) and arabinogalactan (AG) / lipoarabinomannan (LAM) biosynthesis (ethambutol). Decaprenylphosphoryl-beta-D-arabinofuranose (DPA) is an important component of the cell wall of Mycobacterium, which is the only substrate of arabinosyltransferase and plays an important role in the synthesis of Mycobacterium cell wall polysaccharides (the synthesis of AG and LAM), while DprE1 is a key enzyme involved in DPA biosynthesis. DprE1 is decaprenylphosphoryl-beta-D-ribose-2'-epimerase, which is involved in the biosynthesis of DPA. DprE1 can oxidize decaprenylphosphoryl-D-ribose (DPR) to decaprenylphosphoryl-2-ketose (DPX), and DprE2 can reduce DPX to DPA. Through the synergistic action of DprE1 and DprE2, the epimerization of DPR to DPA is catalyzed, and the inhibition of the activity of DprE1 can hinder the synthesis of cell wall and ultimately kill Mycobacterium tuberculosis (DprE1 Inhibitors: Enduring Aspirations for Future Antituberculosis Drug Discovery. ChemMedChem 2023, e202300099).

[0005] DprE1 is located in the periplasmic space of Mycobacterium tuberculosis, and drugs targeting this enzyme do not need to enter the cell to exert anti-tuberculosis effect, which makes DprE1 susceptible to drug interference and an ideal drug target. In addition, DprE1 only exists in prokaryotes and does not exist in humans, indicating that DprE1 inhibitors may have less toxic side effects on humans, which is particularly important for patients infected with drug-resistant bacteria who need long-term drug treatment. Therefore, the development of targeted inhibitors against DprE1 has important application prospects in the research of new anti-tuberculosis drugs.

[0006] Currently, there is no marketed drug for DprE1 inhibitors, which can be divided into two categories: covalent binding inhibitors and non-covalent binding inhibitors. Non-covalent binding inhibitors have diverse structures, with OPC167832, TBA7371 and TCA1 as representatives.

[0007] TCA1 is a non-covalent binding type DprE1 inhibitor obtained by high-throughput screening (Identification of a small molecule with activity against drug-resistant and persistent tuberculosis. PNAS 2013, 110(27), E2510-E2517), which has strong anti-tuberculosis activity in vivo and in vitro, but the compound has the defects of metabolic instability and strong inhibition of liver enzyme CYP 2C9. CYP 2C9 is one of the main drug metabolizing enzymes in the liver, accounting for 20% of the total amount of liver microsomal P450 protein. About 16% of the drugs used clinically are metabolized by it, such as the hypoglycemic drug tolbutamide, the antihypertensive drug losartan, and the antiepileptic drug phenytoin. Due to the strong CYP 2C9 inhibition, it will affect the in vivo metabolism of these drugs, and further cause serious drug-drug interaction problems.

[0008] CN 110759889 A published on February 7, 2020 discloses a compound shown in formula (V) for treating and / or preventing infectious diseases caused by Mycobacterium tuberculosis, and its application in treating Mycobacterium tuberculosis is described in The Journal of Medicinal Chemistry journal in 2021, volume 64, pages 6141-6261. It is based on TCA1 for structure-based drug design, has effective anti-Mycobacterium tuberculosis activity and low cytotoxicity, and shows good hepatocyte stability, but it still has strong inhibition on liver enzyme CYP 2C9, which is not conducive to the combined drug treatment of tuberculosis, so the development of new anti-tuberculosis drugs with strong anti-tuberculosis activity, strong target inhibition and improved liver enzyme inhibition has important application value.

[0009]

[0010] The compound TCA1 is synthesized according to the method in the literature (CN 105473578 A), the compound V is synthesized according to the synthetic route in the literature (CN 110759889 A), and in vitro anti-tuberculosis activity, DprE1 enzyme inhibition activity, intracellular anti-tuberculosis activity of macrophages, cytotoxicity, and CYP 2C9 inhibition are tested, so as to compare the compound of the present application with the positive control compound. SUMMARY

[0011] The technical problem to be solved by the present invention is to provide a substituted thiophene ethyl imide compound with novel structure and strong anti-Mycobacterium tuberculosis activity. The present invention finds that the substituted thiophene ethyl imide compound has excellent anti-Mycobacterium tuberculosis activity and also has excellent inhibitory effect on drug-resistant Mycobacterium tuberculosis. It has excellent DprE1 target enzyme inhibition, showing good inhibitory effect against both DprE1 WT type and DprE1 C387S and DprE1 Y314C mutants. In addition, the compound has low cytotoxicity and significantly improved CYP 2C9 inhibition. At the same time, it has strong intracellular anti-tuberculosis activity and can be used for the treatment or preventive treatment of infectious diseases caused by bacteria, particularly pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis. It can also be used to overcome problems associated with Mycobacterium tuberculosis DprE1 target protein mutations and drug-resistant Mycobacterium tuberculosis infection. The present invention was completed based on the above findings. SUMMARY OF THE INVENTION

[0013] To this end, the present invention provides, in a first aspect, a compound represented by general formula (I) or a pharmaceutically acceptable salt thereof,

[0014]

[0015] in,

[0016] R1 is selected from H, substituted or unsubstituted C1-C3 alkyl, F, Cl, Br, NO2;

[0017] R2 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted morpholinyl;

[0018] Ar is selected from substituted or unsubstituted phenyl, pyridyl, pyrimidinyl;

[0019] The substituents of the substituted C1-C3 alkyl in R1, the substituted C3-C7 cycloalkyl, the substituted morpholinyl, and the substituted phenyl in R2 are each independently selected from the following groups: F, Cl, Br, nitro, cyano, and C1-C3 alkyl.

[0020] In a preferred embodiment, the compound is represented by the general formula (II):

[0021]

[0022] Wherein, R1 and R2 are defined as described in the first aspect of the present invention.

[0023] R3 is independently selected from F, Cl, Br, CN, NO2, NO, NH2;

[0024] In another preferred embodiment, the compound is represented by the general formula (III):

[0025]

[0026] wherein,

[0027] R1is selected from H, F, Cl, Br;

[0028] R2is selected from substituted or unsubstituted C4-C6cycloalkyl;

[0029] The substituents of the C4-C6cycloalkyl group in R2may be optionally selected from F, Cl, Br, nitro, cyano, C1-C3alkyl.

[0030] In another preferred embodiment, the compound is represented by the general formula (IV):

[0031]

[0032] wherein,

[0033] R2is selected from substituted or unsubstituted cyclopentyl;

[0034] The substituents of the cyclopentyl group in R2may be optionally selected from F, Cl, Br, nitro, cyano, C1-C3alkyl.

[0035] A compound according to any one of the first aspect of the application, which is a compound of the application as prepared in the Examples (represented by a structural formula or described by a systematic name) or a pharmaceutically acceptable salt thereof.

[0036] A compound according to any one of the first aspect of the application, which is a compound selected from:

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] A second aspect of the application provides a process for preparing a compound according to any one of the first aspect of the application, comprising the steps of:

[0044]

[0045] Compound B is reacted with amine compound A in a suitable solvent (e.g. DMF, dichloromethane, preferably DMF) in the presence of condensing reagent HATU and base Et3N under air at -10°C to 50°C for 1-24 hours, preferably at room temperature for 8-15 hours, to give a compound of formula I;

[0046] wherein R1, R2 and Ar are as defined in the first aspect of the application.

[0047] Compound A in the present application can be readily prepared according to the methods known in the prior art publications, for example (CN105473578A).

[0048] The third aspect of the present application provides a pharmaceutical composition comprising a therapeutically and / or prophylactically effective amount of a compound of any one of the first aspect of the application or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients.

[0049] The fourth aspect of the present application provides the use of a compound of any one of the first aspect of the application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of the third aspect of the present application, in the manufacture of a deoxy-scyllo-inositol phosphoryl β-D-ribo-2'-epimerase (DprE1) inhibitor.

[0050] The fifth aspect of the present application provides the use of a compound of any one of the first aspect of the present application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of the third aspect of the present application, in the manufacture of a medicament for the treatment and / or prevention of an infectious disease caused by Mycobacterium tuberculosis.

[0051] The foregoing outlines certain aspects of the application, but is not limited to these aspects. These aspects and other aspects will become more fully apparent from the following description. DETAILED DESCRIPTION

[0053] The various aspects and features of the present application are further described below.

[0054] All documents cited in the present application, in total, are incorporated by reference into the present application, and in the event of a conflict between the present application and the incorporated documents, the present application controls. In addition, various terms and phrases used in the present application have their ordinary meanings as understood by one of ordinary skill in the art, unless otherwise defined in the present application. However, the present application still wishes to provide more detailed explanations and interpretations of these terms and phrases, and in the event of a conflict between the ordinary meanings and the present application, the present application controls. Below are the definitions of various terms used in the present application, which are applicable to the terms used throughout the present specification, unless otherwise specified in the specific context.

[0055] In general, the term "substituted or unsubstituted" means that one or more hydrogen atoms in the given structure are replaced by a particular substituent. Unless otherwise indicated, an optional substituent group can be substituted at any available position on the group. When the given structure can be substituted at more than one position with one or more substituents selected from a particular group, the substituents can be the same or different at each position.

[0056] C i -C j represents a moiety having an integer "i" (inclusive) to an integer "j" (inclusive) number of carbon atoms. Thus, for example, C1-C3 alkyl refers to an alkyl group having from 1 to 3 (inclusive) carbon atoms.

[0057] As described herein, the term "alkyl" refers to an alkyl group having the indicated number of carbon atoms, which is straight-chained or branched, and which can include subgroups thereof, e.g., where "C1-C3 alkyl" is mentioned, it can also include groups in the sub-range represented by C1-C2 alkyl, as well as specific groups such as methyl, ethyl, n-propyl, isopropyl.

[0058] As described herein, the term "cycloalkyl" refers to a cyclic alkyl group having the indicated number of ring carbon atoms, and which can include subgroups thereof, e.g., where "C3-C7 cycloalkyl" is mentioned, it can also include groups in the sub-ranges represented by C3-C5 cycloalkyl, C4-C6 cycloalkyl, C5-C7 cycloalkyl, etc., as well as specific groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl.

[0059] "Room temperature" in the present application means a temperature from 10 °C to 40 °C. In some embodiments, "room temperature" means a temperature from 20 °C to 30 °C; in other embodiments, room temperature means 25 °C.

[0060] As described herein, the term "effective amount" means an amount of a drug that can achieve a desired treatment of a disease or condition described herein in a subject.

[0061] As described herein, the term "pharmaceutically acceptable" such as in the description of "pharmaceutically acceptable salts" means that the salt is not only physiologically acceptable to the subject, but also that the salt is a synthetic material that is of use in pharmacy.

[0062] As described herein, the term "pharmaceutical composition", which can also be referred to as "composition", can be used to achieve a desired treatment of a disease or condition described herein in a subject, particularly a mammal.

[0063] "Treating" a disease includes:

[0064] (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a mammal that is exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease,

[0065] (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms,

[0066] (3) relieving the disease, i.e., causing the regression of the disease or its clinical symptoms.

[0067] A "therapeutically effective amount" means the amount of a compound that, when administered to a mammal for treatment of a disease, is sufficient to effect treatment for that disease. The therapeutically effective amount will vary depending on the compound, the disease to be treated, and its severity, as well as the age, body weight, gender, and general health of the mammal. A therapeutically effective amount can also refer to any amount of a compound that is sufficient to achieve a desired beneficial effect, including preventing, inhibiting, or relieving a disease as described in (1)-(3) above. For example, the amount of a compound can be between 0.1-250 mg / kg, or preferably, 0.5-100 mg / kg, or more preferably, 1-50 mg / kg, or even more preferably, 2-20 mg / kg. Preferably, the amount of the compound is administered to the mammal twice a day. More preferably, the amount of the compound is administered to the mammal once a day.

[0068] As described herein, the term "disease and / or disorder" refers to a physical state of the subject that is associated with the disease and / or disorder described herein. For example, the disease and / or disorder described herein refers to a Mycobacterium tuberculosis infectious disease.

[0069] As described herein, the term "subject" can refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., that receives a compound of Formula I or a pharmaceutical composition thereof of the present application for treatment of a disease or disorder described herein.

[0070] In yet another aspect, the present application also relates to pharmaceutical compositions comprising a compound of the present application as an active ingredient. The pharmaceutical compositions can be prepared according to methods known in the art. The compounds of the present application or pharmaceutical compositions containing them can be administered in unit dosage form, in which case the pharmaceutical composition is divided into unit dosages and the active ingredient of the present application or a pharmaceutical composition containing it is present in an amount of such dosages.

[0071] The compounds of the present application or pharmaceutical compositions containing them can be administered in unit dosage form, in which case the pharmaceutical composition is divided into unit dosages and the active ingredient of the present application or a pharmaceutical composition containing it is present in an amount of such dosages.

[0072] The administration form can be a liquid form, a solid form or a semi-solid form. The liquid form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including water injection, powder injection and infusion), eye drops, nose drops, lotion and liniment, etc.; the solid form can be a tablet (including ordinary tablet, enteric-coated tablet, chewable tablet, dispersible tablet, effervescent tablet, oral disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pellet, a drop, a suppository, a film, a patch, an aerosol (powder) mist, a spray, etc.; the semi-solid form can be an ointment, a gel, a paste, etc.

[0073] The compound of the present application can be prepared into a common preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation and various micro-particle administration systems.

[0074] In order to prepare the tablet of the compound of the present application, various excipients known in the art can be widely used, including diluents, wetting agents, binding agents, disintegrating agents, lubricants, solubilizers. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binding agents can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrating agents can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; the lubricants and solubilizers can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0075] The tablet can be further prepared into a coated tablet, such as a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, or a double-layer tablet and a multi-layer tablet.

[0076] In order to prepare the capsule of the administration unit, the effective component, the compound of the present application, can be mixed with diluents and solubilizers, and the mixture can be directly placed in a hard capsule or a soft capsule. The effective component, the compound of the present application, can also be mixed with diluents, binding agents and disintegrating agents to prepare granules or pellets, and then placed in a hard capsule or a soft capsule. The various diluents, wetting agents, binding agents, disintegrating agents and solubilizers used for preparing the tablet of the compound of the present application can also be used for preparing the capsule of the compound of the present application.

[0077] For injection, the compounds of the present application can be dissolved in water, ethanol, isopropyl alcohol, propylene glycol or a mixture thereof, and an appropriate amount of solubilizers, co-solubilizers, pH adjusters, osmotic pressure adjusters commonly used in the art can be added. The solubilizers or co-solubilizers can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjusters can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjusters can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a supporting agent.

[0078] In addition, if necessary, coloring agents, preservatives, flavors, odorants or other additives can also be added to the pharmaceutical preparation.

[0079] For the purpose of medication and enhancement of therapeutic effect, the pharmaceutical or pharmaceutical composition of the present application can be administered by any known administration method.

[0080] The compound or composition of the present application can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present application has a synergistic effect with other therapeutic drugs, the dosage thereof should be adjusted according to the actual situation.

[0081] Advantageous technical effects

[0082] The inventors determined the minimum inhibitory concentration (MIC) of the synthesized compounds by MABA (Microplate alamar blue assay) method, and the results showed that the compounds generally had strong anti-mycobacterium tuberculosis activity. Among the tested compounds, the MIC of 19 compounds was <0.5 μg / mL, and the MIC of 9 compounds was <0.016 g / mL, which was significantly stronger than the anti-tuberculosis activity of TCA1 and compound V. In addition, some of the compounds of the present application showed better inhibitory effect than the positive control compounds TCA1 and compound V in terms of resistance to drug-resistant mycobacterium tuberculosis (drug-resistant strains 13946 and strain 14862). Some of the compounds of the present application had good DprE1 enzyme inhibitory activity (including DprE1 wild type and DprE1 Y314C mutant), and had stronger enzyme inhibitory activity than the positive control compound TCA1 for DprE1 C387S mutant. The results of the intracellular anti-tuberculosis activity of mycobacterium tuberculosis macrophages showed that compounds 43 and 44 showed strong intracellular anti-tuberculosis activity, which was better than compound TCA1. The cytotoxicity results showed that the compounds of the present application had lower cytotoxicity than TCA1. The CYP2C9 inhibition showed that compounds 11, 13, 38 and 39 had low inhibition effect on CYP 2C9 liver enzyme, which was significantly improved compared with compound V. The present application provides a class of compounds with novel structure, strong DprE1 enzyme wild type and mutant inhibitory activity, strong in vitro anti-sensitive mycobacterium tuberculosis and drug-resistant mycobacterium tuberculosis activity, strong intracellular anti-tuberculosis activity, low cytotoxicity, and significant improvement in liver enzyme CYP2C9 inhibition, which can be used for the treatment or prophylactic treatment of infectious diseases caused by bacteria, especially tuberculosis caused by mycobacterium tuberculosis, and can also be used to overcome problems related to mycobacterium tuberculosis DprE1 target protein mutation and drug-resistant mycobacterium tuberculosis infection. DETAILED DESCRIPTION

[0083] The present application can be described in detail by the following examples, but it does not mean any unfavorable limitation to the present application. The present application has been described in detail herein, and specific examples thereof have also been disclosed. It is obvious to those skilled in the art to make various changes and improvements to the specific embodiments of the present application without departing from the spirit and scope of the present application.

[0084] For all the following examples, standard procedures and purification methods known to those skilled in the art can be used. Unless otherwise specified, all temperatures are expressed in °C (degrees Celsius). The structure of the compound is determined by nuclear magnetic resonance spectrum (NMR) and / or mass spectrum (MS).

[0085] Preparation Examples section

[0086] The structures of the compounds were determined by H NMR spectroscopy ( 1 H NMR spectra were determined using H NMR. Shifts (δ) for H and C NMR spectra are given in parts per million (ppm). Coupling constants (J) are in Hertz (Hz). NMR spectra were measured using a JEOL-400 or QOne-500 NMR spectrometer, using deuterated chloroform (CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6) as solvents and tetramethylsilane (TMS) as the internal standard.

[0087] The liquid chromatography-mass spectrometer was Agilent 1100 series LC / MSD trap mass spectrometer (ESI-MS).

[0088] The electronic balance used was the Japanese Yanaco LY-300 electronic balance.

[0089] The rapid preparative liquid chromatograph was a Biotage isolera one preparative chromatograph using a prepacked silica gel column (12 / 25 / 40 g, 40-60 μm, ) were purchased from Beijing Chengda Instrument Co., Ltd.

[0090] All anhydrous solvents were treated by standard methods, and all other reagents were commercially available of analytical grade.

[0091] The present invention uses the following abbreviations:

[0092] (Boc)2O is di-tert-butyl dicarbonate.

[0093] (CH3CO)2O is acetic anhydride.

[0094] DCM is dichloromethane.

[0095] DMAP is 4-dimethylaminopyridine.

[0096] DMF is N,N-dimethylformamide.

[0097] DMSO is dimethyl sulfoxide.

[0098] EA is ethyl acetate.

[0099] Et3N is triethylamine.

[0100] HATU is 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0101] NCS is N-chlorosuccinimide.

[0102] Oxone is a potassium monopersulfate complex salt.

[0103] PE is petroleum ether.

[0104] TFA is trifluoroacetic acid.

[0105] THF is tetrahydrofuran.

[0106] Preparation Example

[0107] Preparation Example 1

[0108] Preparation of (2-aminothiophene-3-carbonyl)carbamate ethyl ester (Intermediate A-1)

[0109]

[0110] First Step: Preparation of (2-cyanoacetyl)carbamate ethyl ester 2a

[0111] Under Ar protection, compound cyanoacetic acid 1a (15.0 g, 176.35 mmol), 140.6 mL of toluene, carbamate ethyl ester (15.7 g, 176.35 mmol), 9.4 mL of DMF were sequentially placed in a 250 mL round-bottom flask, and POCl3(8.22 mL, 88.18 mmol) was slowly added dropwise at 0 °C. After the addition was complete, the reaction temperature was raised to 50 °C, and the reaction was allowed to proceed for 4 h. TLC detection showed that the reaction was complete. The reaction liquid was poured out, and the remaining brownish solid was added to 500 mL of water, slurried, and suction filtered. The product was dried under infrared light to obtain intermediate 2a, a white solid, 15.61 g, in a yield of 56.8%. 1 H NMR (400 MHz, DMSO-d6) δ: 10.99 (br s, 1H), 4.13 (q, J = 7.2 Hz, 2H), 4.10 (s, 2H), 1.21 (t, J = 7.2 Hz, 3H).

[0112] Second Step: Preparation of 2-(2-aminothiophene-3-carbonyl)carbamate ethyl ester A-1

[0113] At 0 °C, compound 2a (15.61 g, 100.0 mmol), 150 mL of methanol, 1,4-dithane-2,5-diol (7.61 g, 50.0 mmol), and Et3N (15.3 mL, 110.0 mmol) were sequentially placed in a 250 mL round-bottom flask. After the addition was complete, the reaction temperature was raised to 50 °C, and the reaction was allowed to proceed for 4 h. TLC detection showed that the reaction was complete. Concentration under reduced pressure yielded a yellow solid. 100 mL of DCM was added, the mixture was slurried, suction filtered, and sequentially washed with saturated aqueous NH4Cl and water to obtain A-1, a white solid, 13.69 g, in a yield of 63.9%. 1H NMR (400 MHz, DMSO-d6) δ: 10.02 (s, 1H), 7.64 (s, 2H), 7.24 (d, J = 6.0 Hz, 1H), 6.23 (d, J = 6.0 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 1.24 (t, J = 7.2 Hz, 3H).

[0114] Preparation Example 2

[0115] Preparation of (2-amino-5-nitrothiophene-3-carbonyl)carbamic acid ethyl ester (Intermediate A-3)

[0116]

[0117] First Step: Preparation of (2-amino-5-methylthiophene-3-carbonyl)carbamic acid ethyl ester A-2

[0118] Compound 2a (5.00 g, 32.05 mmol) and propionaldehyde (2.88 mL, 32.05 mmol) were added to a solution of S (1.03 g, 32.05 mmol) and morpholine (1.56 mL, 17.95 mmol) in MeOH (50 mL), and the mixture was stirred at 50 °C for 3 h. TLC detection showed that the starting material was completely reacted. The mixture was cooled to room temperature, extracted with dichloromethane (30 mL x 3), and the combined organic phases were washed with deionized water, saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was separated by silica gel column chromatography using a mixture of petroleum ether-ethyl acetate (V:V = 70:30) as the eluent to give a yellow solid 3.316 g in a yield of 45.4%. 1 H NMR (400 MHz, DMSO-d6) δ: 9.85 (s, 1H), 7.53 (s, 2H), 6.90 (s, 1H), 4.11 (q, J = 6.8 Hz, 2H), 2.16 (s, 3H), 1.23 (t, J = 6.8 Hz, 3H).

[0119] Preparation Example 3

[0120] Preparation of (2-amino-5-nitrothiophene-3-carbonyl)carbamic acid ethyl ester (Intermediate A-3)

[0121]

[0122] First Step: Preparation of (3-((ethoxycarbonyl)aminocarbonyl)thiophen-2-yl)carbamic acid tert-butyl ester 3a

[0123] Compound A-1 (2.00 g, 9.34 mmol) was added to a solution of DCM (40 mL), DMAP (0.11 g, 0.93 mmol) was added, Et3N (2.60 ml, 18.68 mmol) was added under ice bath, (Boc)20 (2.15 ml, 9.34 mmol) was added slowly dropwise under ice bath, then the ice bath was removed, and the reaction was carried out at room temperature for 12 h. TLC detection showed that the reaction was complete. The reaction was quenched by adding 40 ml of water, and the solution was separated. The aqueous phase was extracted with DCM (30 mL x 3), and the organic phase was washed with 3M HCl (20 mL x 2), saturated sodium chloride aqueous solution, and anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the obtained crude product was separated by silica gel column chromatography using a mixture of petroleum ether and ethyl acetate (V:V = 80:20) as the eluent to obtain 1.264 g of white solid with a yield of 43.1%. 1 H NMR (400 MHz, DMSO-d6) δ: 10.73 (s, 1H), 10.62 (s, 1H), 7.60 (d, J = 6.0 Hz, 1H), 6.94 (d, J = 6.0 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 1.50 (s, 9H), 1.26 (t, J = 7.2 Hz, 3H).

[0124] Second step: preparation of tert-butyl (3-((ethoxycarbonyl)aminocarbonyl)-5- nitrothiophen-2-yl)carbamate 4a

[0125] Compound 3a (1.00 g, 3.18 mmol) was added to a solution of (CH3CO)20 (25 mL), 70% HNO3 (1.33 ml, 7.95 mmol) was added under ice bath, then the ice bath was removed, and the reaction was carried out at room temperature for 10 min. TLC detection showed that the reaction was complete. 70 ml of water was added, and the solution was slurried under ice bath. The filtrate was dried by infrared drying to obtain 1.005 g of brown-red solid with a yield of 88.0%. 1 H NMR (400 MHz, DMSO-d6) δ: 11.16 (s, 1H), 11.01 (s, 1H), 8.87 (s, 1H), 4.19 (q, J = 7.2 Hz, 2H), 1.52 (s, 9H), 1.27 (t, J = 7.2 Hz, 3H).

[0126] Third step: preparation of ethyl (2-amino-5-nitrothiophene-3-carbonyl)carbamate A-3

[0127] Compound 4a (1.00 g, 2.78 mmol) was added to a solution of DCM (15 mL), TFA (5.11 ml, 66.72 mmol) was added under ice bath, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 50 min. TLC detection showed that the reaction was complete. 60 ml of water was added, and the mixture was filtered, and dried by infrared lamp to obtain a yellow solid 0.645 g, with a yield of 89.5%. 1 H NMR (500 MHz, DMSO-d6) δ: 10.61 (s, 1H), 8.99 (s, 2H), 8.69 (s, 1H), 4.15 (q, J = 7.0 Hz, 2H), 1.25 (t, J = 7.0 Hz, 3H).

[0128] Example

[0129] Example 1

[0130]

[0131] 2-(4-(benzamido)benzamido)thiophene-3-carbonylcarbamic acid ethyl ester (compound 1)

[0132] Synthetic route:

[0133]

[0134] First step: preparation of methyl 4-(benzamido)benzoate 2b

[0135] Methyl 4-aminobenzoate 1b (1.00 g, 6.62 mmol) and pyridine (1.1 mL, 13.24 mmol) were dissolved in anhydrous dichloromethane (15 mL), and then benzoyl chloride (0.763 mL, 6.62 mmol) was slowly added to the above solution through a constant pressure dropping funnel. After the addition was completed, the reaction was stirred at room temperature for 3 h. TLC detection showed that the reaction was complete. The reaction solution was washed with deionized water, 3M hydrochloric acid aqueous solution, and saturated brine in turn, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a white solid. The white solid was dried under infrared lamp to obtain a white solid 1.444 g with a yield of 85.8%. 1 H NMR (400 MHz, DMSO-d6) δ: 10.56 (s, 1H), 7.99-7.97 (m, 1H), 7.96 (s, 5H), 7.65-7.59 (m, 1H), 7.58-7.52 (m, 2H), 3.84 (s, 3H).

[0136] Second step: preparation of 4-(benzamido)benzoic acid B-1

[0137] Dissolve 2b (1.00 g, 3.92 mmol) in 15 mL MeOH, add aqueous solution of LiOH-H2O (0.82 g, 19.60 mmol) in 10 mL, continue to stir at 40 °C for 1 h, after TLC detection of the reaction is complete, cool to room temperature, add 1 M aqueous HC1 solution under ice bath conditions, adjust pH = 1-3, a large amount of white solid is precipitated, reduce pressure and filter, dry under infrared lamp, obtain white solid 0.941 g, yield 99.5%. 1 H NMR (500 MHz, DMSO-d6) δ: 12.74 (s, 1H), 10.55 (s, 1H), 8.00-7.90 (m, 6H), 7.65-7.58 (m, 1H), 7.58-7.50 (m, 2H).

[0138] Third step: preparation of 2-(4-(benzamide)benzamide)thiophene-3-carbonyl ethyl carbamate (compound 1)

[0139] Dissolve B-1 (0.29 g, 1.22 mmol) in DMF (2 mL), then add A-1 (0.20 g, 0.93 mmol) and HATU (0.71 g, 1.87 mmol), Et3N (0.30 mL, 2.16 mmol), DMAP (11 mg, 0.09 mmol), stir at room temperature for 12 h. TLC detection of the reaction is complete, add deionized water (30 mL), extract with dichloromethane (30 mL x 3), combine the organic phase, wash with 1 M aqueous HC1, saturated sodium chloride solution, dry over anhydrous sodium sulfate, filter, reduce pressure and concentrate, the obtained crude product is separated by silica gel column chromatography, dichloromethane-methanol (V:V = 100:1) mixture as eluent, obtain white solid 0.022 g, yield 4.1%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.59 (s, 1H), 10.80 (s, 1H), 10.65 (s, 1H), 8.10-8.03 (m, 2H), 8.03-7.94 (m, 4H), 7.73 (d, J = 6.0 Hz, 1H), 7.67-7.59 (m, 1H), 7.59-7.53 (m, 2H), 7.07 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 22 H 20 N3O5S: 438.1118; found: 438.1115. Example 2

[0140]

[0141] 2-(4-(cyclopropanecarboxamido)benzamido)thiophene-3-carbonylcarbamic acid ethyl ester (Compound 2)

[0142] Synthetic route:

[0143]

[0144] First step: Preparation of methyl 4-(cyclopropanecarboxamido)benzoate 3b

[0145] Starting from 1b (1.70 g, 15.11 mmol) and cyclopropanecarbonyl chloride (0.80 mL, 9.62 mmol), using similar procedure as the first step in Example 1, intermediate 3b was obtained as a white solid 1.822 g, 86.7% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.53 (s, 1H), 7.96-7.85 (m, 2H), 7.78-7.68 (m, 2H), 3.81 (s, 3H), 1.86-1.74 (m, 1H), 0.85-0.80 (m, 4H).

[0146] Second step: Preparation of 4-(cyclopropanecarboxamido)benzoic acid B-2

[0147] Starting from 3b (1.00 g, 4.57 mmol), using similar procedure as the second step in Example 1, intermediate B-2 was obtained as a white solid 0.936 g, 99.2% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.56 (s, 1H), 7.90-7.84 (m, 2H), 7.75-7.67 (m, 2H), 1.91-1.76 (m, 1H), 0.86-0.78 (m, 4H).

[0148] Third step: Preparation of 2-(4-(cyclopropanecarboxamido)benzamido)thiophene-3-carbonylcarbamic acid ethyl ester (Compound 2)

[0149] Starting from B-2 (0.20 g, 0.98 mmol) and A-1 (0.25 g, 1.18 mmol), using similar procedure as the third step in Example 1, Compound 2 was obtained as a white solid 0.139 g, 35.3% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.78 (s, 1H), 10.62 (s, 1H), 7.90 (d, J = 6.8 Hz, 2H), 7.83 (d, J = 6.8 Hz, 2H), 7.71 (d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 1.88-1.79 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H), 0.89-0.79 (m, 4H). HRMS (ESI): m / z [M+H] + C 19 H 20 N3O5S: 402.1118; found: 402.1108.

[0150] Example 3

[0151]

[0152] 2-(4-(cyclobutanecarboxamido)benzamido)thiophene-3-carbonylaminomethylate (Compound 3)

[0153] Synthetic route:

[0154]

[0155] First step: Preparation of methyl 4-(cyclobutanecarboxamido)benzoate 4b

[0156] Using 1b (1.50 g, 10.08 mmol) and cyclobutanecarbonyl chloride (0.96 mL, 8.47 mmol) as raw materials, the similar operation steps in the first step of Example 1 were adopted to obtain the intermediate 4b, white solid 1.944 g, yield 99.3%. 1 H NMR (400 MHz, DMSO-d6) δ: 10.06 (s, 1H), 7.99-7.86 (m, 2H), 7.81-7.71 (m, 2H), 3.81 (s, 3H), 3.32-3.19 (m, 1H), 2.30-2.05 (m, 4H), 2.02-1.75 (m, 2H).

[0157] Second step: Preparation of 4-(cyclobutanecarboxamido)benzoic acid B-3

[0158] Using 4b (1.80 g, 7.72 mmol) as raw materials, the similar operation steps in the second step of Example 1 were adopted to obtain the intermediate B-3, white solid 1.642 g, yield 97.4%. 1H NMR (400 MHz, DMSO-d6) δ: 12.67 (s, 1H), 10.03 (s, 1H), 7.90-7.83 (m, 2H), 7.76-7.68 (m, 2H), 3.30-3.21 (m, 1H), 2.29-2.06 (m, 4H), 2.01-1.75 (m, 2H).

[0159] Third Step: Preparation of 2-(4-(cyclopentanecarboxamido)benzamido)thiophene-3- carboxylic acid ethyl ester (Compound 4)

[0160] Using B-3 (0.20 g, 0.91 mmol) and A-1 (0.24 g, 1.09 mmol) as starting material, the similar procedure as described in Example 1, Step 3 was used to give Compound 4 as a white solid 0.064 g, yield 16.9%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.79 (s, 1H), 10.15 (s, 1H), 7.91-7.84 (m, 4H), 7.71 (d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.32-3.26 (m, 1H), 2.29-2.20 (m, 2H), 2.17-2.05 (m, 2H), 2.02-1.90 (m, 1H), 1.86-1.81 (m, 1H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 20 H 22 N3O5S: 416.1275; found: 416.1278.

[0161] Example 4

[0162]

[0163] 2-(4-(cyclopentanecarboxamido)benzamido)thiophene-3-carboxylic acid ethyl ester (Compound 4)

[0164] Synthetic Route:

[0165]

[0166] First Step: Preparation of methyl 4-(cyclopentanecarboxamido)benzoate 5b

[0167] Starting from 1b (2.00 g, 13.24 mmol) and cyclopentanoic acid (1.70 mL, 15.89 mmol) in dry dichloromethane (15 mL), followed by the addition of EDCI (4.57 g, 23.84 mmol), DMAP (0.97 g, 7.95 mmol), stirring at room temperature for 6 h. TLC detection of the complete reaction of the starting material, the reaction liquid was washed with deionized water, 3M hydrochloric acid aqueous solution, saturated brine, dried over anhydrous sodium sulfate, concentrated to give white solid, dried under infrared lamp, white solid 3.189 g, yield 97.7%. 1 H NMR (400 MHz, DMSO-d6) δ: 10.20 (s, 1H), 7.94-7.86 (m, 2H), 7.78-7.71 (m, 2H), 3.81 (s, 3H), 2.88-2.74 (m, 1H), 1.92-1.79 (m, 2H), 1.78-1.62 (m, 4H), 1.61-1.49 (m, 2H).

[0168] Second step: preparation of 4-(cyclopentanecarboxamido)benzoic acid B-4

[0169] Starting from 5b (3.20 g, 12.96 mmol), using similar operation steps in the second step of Example 1, to obtain intermediate B-4, white solid 3.024 g, yield 99.9%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.66 (s, 1H), 10.18 (s, 1H), 7.88-7.84 (m, 2H), 7.76-7.68 (m, 2H), 2.89-2.73 (m, 1H), 1.92-1.78 (m, 2H), 1.77-1.61 (m, 4H), 1.61-1.47 (m, 2H).

[0170] Third step: preparation of 2-(4-(cyclopentanecarboxamido)benzamido)thiophene-3-carbonyl amino methyl carbonate (compound 4)

[0171] Starting from B-4 (0.20 g, 0.86 mmol) and A-1 (0.22 g, 1.03 mmol), using similar operation steps in the third step of Example 1, to obtain compound 4, white solid 0.113 g, yield 30.6%. 1H NMR (400 MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.79 (s, 1H), 10.29 (s, 1H), 7.91-7.84 (m, 4H), 7.72 (d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.87-2.70 (m, 1H), 1.94-1.52 (m, 8H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 21 H 24 N3O5S: 430.1431 ; found: 430.1438.

[0172] Example 5

[0173]

[0174] 2-(4-(cyclohexanecarboxamido)benzamido)thiophene-3-carboxylic acid ethyl ester (Compound 5)

[0175] Synthetic route:

[0176]

[0177] First step: Preparation of methyl 4-(cyclohexanecarboxamido)benzoate 6b

[0178] Using 1b (2.00 g, 13.24 mmol) and cyclohexanoic acid (1.70 g, 17.54 mmol) as starting materials, similar procedure as the first step in Example 4, the intermediate 6b was obtained as a yellow solid 3.421 g in 74.7% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.18 (s, 1H), 7.91-7.86 (m, 2H), 7.77-7.72 (m, 2H), 3.81 (s, 3H), 2.43-2.30 (m, 1H), 1.86-1.70 (m, 4H), 1.46-1.34 (m, 2H), 1.32-1.15 (m, 4H).

[0179] Second step: Preparation of 4-(cyclohexanecarboxamido)benzoic acid B-5

[0180] Using 6b (3.30 g, 12.64 mmol) as starting material, similar procedure as the second step in Example 1, the intermediate B-5 was obtained as a white solid 3.087 g in 99.1% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.65 (s, 1H), 10.14 (s, 1H), 7.90-7.83 (m, 2H), 7.76-7.65 (m, 2H), 2.42-2.30 (m, 1H), 1.85-1.69 (m, 4H), 1.68-1.62 (m, 1H), 1.48-1.34 (m, 2H), 1.33-1.12 (m, 3H).

[0181] Third Step: Preparation of Ethyl 2-(4-(cyclohexanecarboxamido)benzamido)thiophene-3- carboxylate (Compound 5)

[0182] Using B-5 (0.10 g, 0.41 mmol) and A-1 (0.09 g, 0.41 mmol) as starting material, the similar procedure as described in Example 1, Step 3 was used to obtain Compound 5 as a white solid 0.036 g, yield 20.1%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.65 (s, 1H), 10.14 (s, 1H), 7.90-7.83 (m, 2H), 7.76-7.65 (m, 2H), 2.42-2.30 (m, 1H), 1.85-1.69 (m, 4H), 1.68-1.62 (m, 1H), 1.48-1.34 (m, 2H), 1.33-1.12 (m, 3H). + C 22 H 26 N3O5S: 444.1588; Found: 444.1585. Example 6

[0183]

[0184] Ethyl 2-(4-(cycloheptanecarboxamido)benzamido)thiophene-3-carboxylate (Compound 6)

[0185] Synthetic Route:

[0186]

[0187] First Step: Preparation of Methyl 4-(cycloheptanecarboxamido)benzoate 7b

[0188] Starting from 1b (0.96 g, 8.41 mmol) and cycloheptanecarboxylic acid (0.97 mL, 7.01 mmol), using similar procedure as in Example 4, Step 1, intermediate 7b was obtained as a yellow solid 1.412 g, 73.4% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.13 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 3.81 (s, 3H), 2.56-2.52 (m, 1H), 1.91-1.79 (m, 2H), 1.78-1.38 (m, 10H).

[0189] Second Step: Preparation of 4-(cycloheptanecarboxamido)benzoic acid B-6

[0190] Starting from 7b (1.20 g, 4.36 mmol), using similar procedure as in Example 1, Step 2, intermediate B-6 was obtained as a white solid 1.126 g, 98.1% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.65 (s, 1H), 10.22 (s, 1H), 7.87-7.84 (m, 2H), 7.74-7.70 (m, 2H), 2.60-2.54 (m, 1H), 1.88-1.80 (m, 2H), 1.76-1.38 (m, 10H).

[0191] Third Step: Preparation of 2-(4-(cycloheptanecarboxamido)benzamido)thiophene-3- carboxylic acid ethyl ester (Compound 6)

[0192] Starting from B-6 (0.20 g, 0.77 mmol) and A-1 (0.20 g, 0.92 mmol), using similar procedure as in Example 1, Step 3, Compound 6 was obtained as a white solid 0.100 g, 28.4% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.78 (s, 1H), 10.21 (s, 1H), 7.93-7.80 (m, 4H), 7.71 (d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.55-2.52 (m, 1H), 1.91-1.81 (m, 2H), 1.77-1.67 (m, 2H), 1.66-1.58 (m, 2H), 1.58-1.42 (m, 6H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 23H 28 N3O5S: 458.1744; found: 458.1746.

[0193] Example 7

[0194]

[0195] 2-(4-(tetrahydro-2H-pyran-4-carboxamido)benzamido)thiophene-3-carboxylic acid ethyl ester (Compound 7)

[0196] Synthetic route:

[0197]

[0198] First step: Preparation of methyl 4-(tetrahydro-2H-pyran-4-carboxamido)benzoate 8b

[0199] Using 1b (1.05 g, 9.24 mmol) and tetrahydro-2H-pyran-4-carboxylic acid (1.00 g, 7.69 mmol) as starting materials, the similar procedure as the first step in Example 4 was used to obtain intermediate 8b as a white solid 1.759 g, 86.7% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.23 (s, 1H), 7.95-7.85 (m, 2H), 7.78-7.71 (m, 2H), 3.95-3.87 (m, 2H), 3.81 (s, 3H), 3.42-3.28 (m, 2H), 2.70-2.56 (m, 1H), 1.77-1.57 (m, 4H).

[0200] Second step: Preparation of 4-(tetrahydro-2H-pyran-4-carboxamido)benzoic acid B-7

[0201] Using 8b (1.40 g, 5.32 mmol) as starting material, the similar procedure as the second step in Example 1 was used to obtain intermediate B-7 as a white solid 1.270 g, 96.3% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.66 (s, 1H), 10.21 (s, 1H), 7.98-7.74 (m, 2H), 7.78-7.63 (m, 2H), 3.97-3.83 (m, 2H), 3.40-3.32 (m, 2H), 2.70-2.58 (m, 1H), 1.77-1.57 (m, 4H).

[0202] Third step: Preparation of 2-(4-(tetrahydro-2H-pyran-4-carboxamido)benzamido)thiophene-3-carboxylic acid ethyl ester (Compound 7)

[0203] Using B-7 (0.20 g, 0.80 mmol) and A-1 (0.20 g, 0.92 mmol) as the starting material, the similar procedure as in Example 1, Step 3 was used to obtain compound 7, white solid 0.083 g, yield 23.2%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.55 (br s, 1H), 10.79 (br s, 1H), 10.32 (br s, 1H), 7.91-7.84 (m, 4H), 7.72 (d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.94-3.90 (m, 2H), 3.39-3.33 (m, 2H), 2.71-2.57 (m, 1H), 1.77-1.60 (m, 4H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 21 H 24 N3O6S: 446.1380; found: 446.1386.

[0204] Example 8

[0205]

[0206] Synthesis route of 2-(4-(4-methylcyclohexane-1-carboxamido)benzamido)thiophene-3-carboxylate (Compound 8):

[0207]

[0208] First Step: Preparation of methyl 4-(4-methylcyclohexane-1-carboxamido)benzoate 9b

[0209] Using 1b (0.80 g, 7.02 mmol) and p-methylcyclohexanoic acid (1.00 g, 7.02 mmol) as the starting material, the similar procedure as in Example 4, Step 1 was used to obtain intermediate 9b, white solid 1.672 g, yield 86.7%. 1 H NMR (400 MHz, DMSO-d6) δ: 10.17-10.10 (m, 1H), 7.89 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H), 2.48-2.44 (m, 1H), 1.87-1.66 (m, 3H), 1.61-1.47 (m, 3H), 1.47-1.38 (m, 2H), 0.93 (d, J = 6.4 Hz, 3H), 0.89-0.87 (m, 1H).

[0210] Second Step: Preparation of 4-(4-methylcyclohexane-1-carboxamido)benzoic acid B-8

[0211] Using 9b (1.60 g, 5.82 mmol) as the raw material, the similar operation steps in the second step of Example 1 were adopted to obtain the intermediate B-8, white solid 1.416 g, yield 93.4%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.64 (s, 1H), 10.18-1.11 (m, 1H), 7.87-7.84 (m, 2H), 7.74-7.70 (m, 2H), 2.48-2.45 (m, 1H), 1.85-1.65 (m, 3H), 1.61-1.35 (m, 5H), 1.00-0.91 (m, 3H), 0.89-0.87 (m, 1H).

[0212] Third Step: Preparation of 2-(4-(4-methylcyclohexane-1-carboxamido)benzamido)thiophene-3-carboxylic acid ethyl ester (Compound 8)

[0213] Using B-8 (0.20 g, 0.77 mmol) and A-1 (0.20 g, 0.92 mmol) as the raw materials, the similar operation steps in the third step of Example 1 were adopted to obtain Compound 8, yellow solid 0.099 g, yield 28.1%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.78 (s, 1H), 10.25 (s, 1H), 7.96-7.79 (m, 4H), 7.71 (d, J = 6.0 Hz, 1H), 7.05 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.35-2.25 (m, 1H), 1.88-1.79 (m, 2H), 1.76-1.72 (m, 2H), 1.52-1.31 (m, 3H), 1.29 (t, J = 7.2 Hz, 3H), 1.02-0.91 (m, 2H), 0.89 (d, J = 6.4 Hz, 3H). HRMS (ESI): m / z [M+H] + C 23 H 28 N3O5S: 458.1744; found: 458.1738.

[0214] Example 9

[0215]

[0216] 2-(4-(cyclobutanecarboxamido)-3-fluorobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 9) synthesis route:

[0217]

[0218] First step: preparation of methyl 4-(cyclobutanecarboxamido)-3-fluorobenzoate 11b

[0219] Using methyl 4-amino-3-fluorobenzoate 10b (2.00 g, 11.83 mmol) and cyclobutanecarbonyl chloride (1.40 mL, 11.83 mmol) as starting materials, the similar procedure as the first step in Example 1 was adopted to obtain intermediate 11b as a yellow solid 2.158 g in 72.7% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 9.81 (s, 1H), 8.23 (t, J = 8.0 Hz, 1H), 7.81-7.68 (m, 2H), 3.84 (s, 3H), 3.51-3.38 (m, 1H), 2.30-2.05 (m, 4H), 2.03-1.75 (m, 2H).

[0220] Second step: preparation of 4-(cyclobutanecarboxamido)-3-fluorobenzoic acid B-9

[0221] Using 11b (2.00 g, 7.97 mmol) as starting material, the similar procedure as the second step in Example 1 was adopted to obtain intermediate B-9 as a white solid 1.097 g in 58.5% yield. 1 H NMR (400 MHz, CDCl3) δ: 8.57 (t, J = 8.0 Hz, 1H), 7.96-7.86 (m, 1H), 7.82-7.79 (m, 1H), 7.41 (s, 1H), 3.34-3.16 (m, 1H), 2.49-2.35 (m, 2H), 2.34-2.23 (m, 2H), 2.12-2.03 (m, 1H), 2.02-1.91 (m, 1H).

[0222] Third step: preparation of 2-(4-(cyclobutanecarboxamido)-3-fluorobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 9)

[0223] Using B-9 (0.20 g, 0.84 mmol) and A-1 (0.20 g, 0.92 mmol) as starting materials, the similar procedure as the third step in Example 1 was adopted to obtain Compound 9 as a white solid 0.219 g in 60.2% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.54 (s, 1H), 10.81 (s, 1H), 9.88 (s, 1H), 8.33-8.29 (m, 1H), 7.79-7.69 (m, 3H), 7.09 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.50-3.40 (m, 1H), 2.30-2.08 (m, 4H), 2.02-1.78 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H]+calcd for C 20 H 21 FN3O5S: 434.1180; found: 434.1169. Example 10

[0224]

[0225] Synthetic route of 2-(4-(cyclopentanecarboxamido)-3-fluorobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 10):

[0226]

[0227] First step: Preparation of methyl 4-(cyclopentanecarboxamido)-3-fluorobenzoate 12b

[0228] Using 10b (2.00 g, 11.83 mmol) and cyclopentanecarbonyl chloride (1.60 mL, 14.20 mmol) as starting materials, the similar procedure as the first step in Example 1 was adopted to obtain the intermediate 12b, yellow solid 2.967 g, yield 93.3%. 1 H NMR (400 MHz, DMSO-d6) δ: 9.92 (s, 1H), 8.21 (t, J = 8.0 Hz, 1H), 7.79-7.68 (m, 2H), 3.84 (s, 3H), 3.05-3.00 (m, 1H), 1.79-1.43 (m, 8H).

[0229] Second step: Preparation of 4-(cyclopentanecarboxamido)-3-fluorobenzoic acid B-10

[0230] Using 12b (1.80 g, 6.79 mmol) as starting material, the similar procedure as the second step in Example 1 was adopted to obtain the intermediate B-10, white solid 1.654 g, yield 97.0%. 1H NMR (400 MHz, DMSO-d6) δ: 13.04 (s, 1H), 9.89 (s, 1H), 8.16 (t, J = 8.0 Hz, 1H), 7.80-7.63 (m, 2H), 3.11-2.86 (m, 1H), 1.98-1.45 (m, 8H).

[0231] Third Step: Preparation of 2-(4-(cyclopentanecarboxamido)-3- fluorobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 10)

[0232] Using B-10 (0.20 g, 0.80 mmol) and A-1 (0.21 g, 0.96 mmol) as starting material, the similar procedure as described in Example 1, Step 3 was used to give Compound 10 as a white solid 0.156 g, 43.6% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.54 (br s, 1H), 10.81 (br s, 1H), 9.99 (br s, 1H), 8.29 (t, J = 8.0 Hz, 1H), 7.79-7.70 (m, 3H), 7.09 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.08-2.97 (m, 1H), 1.93-1.83 (m, 2H), 1.79-1.64 (m, 4H), 1.58-1.54 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 23 FN3O5S: 448.1337; found: 448.1333.

[0233] Example 11

[0234]

[0235] Synthetic route of 2-(4-(cyclopentanecarboxamido)-3-nitrobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 11):

[0236]

[0237] First Step: Preparation of methyl 4-(cyclopentanecarboxamido)-3-nitrobenzoate 14b

[0238] Using methyl 4-amino-3-nitrobenzoate 13b (1.00 g, 5.10 mmol) and cyclopentanecarbonyl chloride (0.92 mL, 7.65 mmol) as starting materials, the similar procedure as the first step in Example 1 was used to obtain intermediate 14b as a yellow solid 1.051 g in 70.5% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.52 (s, 1H), 8.39 (d, J = 2.0 Hz, 1H), 8.21 (dd, J = 8.4, 2.0 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 3.89 (s, 3H), 2.92-2.85 (m, 1H), 1.92-1.80 (m, 2H), 1.79-1.51 (m, 6H).

[0239] Second Step: Preparation of 4-(cyclopentanecarboxamido)-3-nitrobenzoic acid B-11

[0240] Using 14b (0.50 g, 1.71 mmol) as starting material, the similar procedure as the second step in Example 1 was used to obtain intermediate B-11 as a yellow solid 0.248 g in 52.1% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 13.41 (s, 1H), 10.49 (s, 1H), 8.37 (d, J = 2.0 Hz, 1H), 8.18 (dd, J = 8.4, 2.0 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 2.94-2.81 (m, 1H), 1.92-1.80 (m, 2H), 1.79-1.48 (m, 6H).

[0241] Third Step: Preparation of 2-(4-(cyclopentanecarboxamido)-3-nitrobenzamido)thiophene-3-carbonylcarbamate (Compound 11)

[0242] Using B-11 (0.20 g, 0.72 mmol) and A-1 (0.15 g, 0.72 mmol) as starting materials, the similar procedure as the third step in Example 1 was used to obtain Compound 11 as a yellow solid 0.166 g in 48.7% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.58 (s, 1H), 10.82 (s, 1H), 10.58 (s, 1H), 8.43 (d, J = 2.0 Hz, 1H), 8.19 (dd, J = 8.4, 2.0 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 6.0 Hz, 1H), 7.11 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.96-2.84 (m, 1H), 1.88-1.82 (m, 2H), 1.80-1.51 (m, 6H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 23 N4O7S: 475.1282; found: 475.1271.

[0243] Example 12

[0244]

[0245] Synthesis route of 2-(4-(cyclohexanecarboxamido)-3-nitrophenyl)benzamide (Compound 12):

[0246]

[0247] First step: preparation of methyl 4-(cyclohexanecarboxamido)-3-nitrobenzoate 15b

[0248] Using 13b (1.00 g, 5.10 mmol) and cyclohexanecarbonyl chloride (1.02 mL, 7.65 mmol) as starting materials, the similar procedure as the first step in Example 1 was adopted to obtain the intermediate 15b, yellow solid 0.798 g, yield 51%. 1 H NMR (400 MHz, CDCl3) δ: 10.65 (s, 1H), 8.96 (d, J = 8.8 Hz, 1H), 8.90 (d, J = 2.0 Hz, 1H), 8.26 (dd, J = 8.8, 2.0 Hz, 1H), 3.96 (s, 3H), 2.44-2.35 (m, 1H), 2.07-2.02 (m, 2H), 1.89-1.85 (m, 2H), 1.61-1.22 (m, 6H).

[0249] Second step: preparation of 4-(cyclohexanecarboxamido)-3-nitrobenzoic acid B-12

[0250] Using 15b (0.50 g, 1.63 mmol) as the starting material, a similar procedure as in Example 1, Step 2 was used to obtain intermediate B-12 as a yellow solid 0.301 g, 63.2% yield. 1 H NMR (400 MHz, CDC13) δ: 10.70 (s, 1H), 9.01 (d, J = 8.8 Hz, 1H), 8.97 (d, J = 2.0 Hz, 1H), 8.32 (dd, J = 8.8, 2.0 Hz, 1H), 2.47-2.35 (m, 1H), 2.11-2.01 (m, 2H), 1.90-1.85 (m, 2H), 1.62-1.22 (m, 6H).

[0251] Preparation of 2-(4-(cyclohexanecarboxamido)-3-nitrobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 12)

[0252] Using B-12 (0.15 g, 0.51 mmol) and A-1 (0.11 g, 0.51 mmol) as the starting materials, a similar procedure as in Example 1, Step 3 was used to obtain Compound 12 as a yellow solid 0.088 g, 35.3% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.58 (s, 1H), 10.83 (s, 1H), 10.54 (s, 1H), 8.43 (d, J = 2.0 Hz, 1H), 8.19 (dd, J = 8.4, 2.0 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 6.0 Hz, 1H), 7.11 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.45-2.39 (m, 1H), 1.87-1.63 (m, 5H), 1.46-1.15 (m, 8H). HRMS (ESI): m / z [M+H] + C 22 H 25 C7N4S: 489.1438; found: 489.1459.

[0253] Example 13

[0254]

[0255] Synthetic route of 2-(4-(cyclopentanecarboxamido)-3-cyanobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 13):

[0256]

[0257] First Step: Preparation of methyl 4-(cyclopentanecarboxamido)-3- cyano benzoate 17b

[0258] Using methyl 4-amino-3-cyano benzoate 16b (0.50 g, 2.84 mmol) and cyclopentanecarbonyl chloride (0.53 mL, 4.26 mmol) as starting materials, the similar procedure as the first step in Example 1 was followed to give the intermediate 17b as a white solid 0.585 g in 75.6% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.33 (s, 1H), 8.27 (d, J = 2.0 Hz, 1H), 8.19 (dd, J = 8.8, 2.0 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 3.87 (s, 3H), 2.94-2.90 (m, 1H), 1.89-1.56 (m, 8H).

[0259] Second Step: Preparation of 4-(cyclopentanecarboxamido)-3-cyano benzoic acid B-13

[0260] Using 17b (0.20 g, 0.73 mmol) as starting material, the similar procedure as the second step in Example 1 was followed to give the intermediate B-13 as a white solid 0.186 g in 98.1% yield. 1 H NMR (500 MHz, DMSO-d6) δ: 13.27 (br s, 1H), 10.31 (s, 1H), 8.23 (d, J = 2.0 Hz, 1H), 8.17 (dd, J = 8.5, 2.0 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 2.95-2.87 (m, 1H), 1.89-1.74 (m, 4H), 1.69-1.56 (m, 4H).

[0261] Third Step: Preparation of ethyl 2-(4-(cyclopentanecarboxamido)-3-cyano benzamido)thiophene-3-carboxylic acid amide (Compound 13)

[0262] Using B-13 (0.18 g, 0.68 mmol) and A-1 (0.15 g, 0.68 mmol) as starting materials, the similar procedure as the third step in Example 1 was followed to give Compound 13 as a white solid 0.125 g in 40.3% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.52 (s, 1H), 10.82 (s, 1H), 10.39 (s, 1H), 8.29 (d, J = 2.0 Hz, 1H), 8.17 (dd, J = 8.8, 2.0 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.10 (d, J = 6.0 Hz, 1H), 4.21 (t, J = 7.2 Hz, 2H), 3.00-2.88 (m, 1H), 1.97-1.51 (m, 8H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 22 H 23 O5N4S:455.1384;found:455.1375.

[0263] Example 14

[0264]

[0265] Synthesis route of 2-(4-(cyclohexanecarboxamido)-3-cyanobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 14):

[0266]

[0267] First step: Preparation of methyl 4-(cyclohexanecarboxamido)-3-cyanobenzoate 18b

[0268] Using 16b (0.40 g, 2.27 mmol) and cyclohexanecarbonyl chloride (0.61 mL, 4.54 mmol) as starting materials, the similar procedure as the first step in Example 1 was adopted to obtain the intermediate 18b as a white solid 0.530 g, yield 81.5%. 1 H NMR (400 MHz, CDCl3) δ: 8.62 (d, J = 8.8 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.22 (dd, J = 8.8, 2.0 Hz, 1H), 7.83 (s, 1H), 3.93 (s, 3H), 2.40-2.32 (m, 1H), 2.09-1.82 (m, 4H), 1.58-1.23 (m, 6H).

[0269] Second step: Preparation of 4-(cyclohexanecarboxamido)-3-cyanobenzoic acid B-14

[0270] Using 18b (0.20 g, 0.70 mmol) as starting material, similar procedures to the second step in Example 1 were adopted to obtain intermediate B-14 as a white solid (0.169 g) with a yield of 88.9%. 1 H NMR(400MHz,DMSO-d6)δ:13.22(br s,1H),10.24(s,1H),8.23(d,J=2.0Hz,1H),8.16(dd,J=8.8,2.0Hz,1H),7.7 5(d,J=8.8Hz,1H),2.48-2.44(m,1H),1.89-1.73(m,4H),1.48-1.17(m,6H).

[0271] Step 3: Preparation of ethyl 2-(4-(cyclohexanecarboxamido)-3-cyanobenzamido)thiophene-3-carbonylcarbamate (Compound 14)

[0272] Using B-14 (0.15 g, 0.55 mmol) and A-1 (0.12 g, 0.55 mmol) as starting materials, similar procedures to the third step in Example 1 were adopted to obtain compound 14 as a white solid (0.108 g). The yield was 41.9%. 1 H NMR(400MHz,DMSO-d6)δ:12.51(s,1H),10.83(s,1H),10.34(s,1H),8.29(d, J=2.0Hz,1H),8.17(dd,J=8.8,2.0Hz,1H),7.86(d,J=8.8Hz,1H),7.71(d,J=6 .0Hz,1H),7.11(d,J=6.0Hz,1H),4.22(q,J=7.2Hz,2H),2.49-2.46(m,1H),1 .92-1.60(m,5H),1.46-1.40(m,2H),1.36-1.15(m,6H).HRMS(ESI):m / z[M+H] + calcd for C 23 H 25 O5N4S:469.1540; found:469.1533.

[0273] Example 15

[0274]

[0275] Synthesis route of ethyl 2-(3-amino-4-(cyclopentanecarboxamido)benzamido)thiophene-3-carbonylcarbamate (Compound 15):

[0276]

[0277] 2-(3-amino-4-(cyclopentanecarboxamido)benzamido)thiophene-3-carboxylic acid ethyl ester (Compound 15)

[0278] Compound 11 (0.15 g, 0.32 mmol) was dissolved in 10 ml THF, 180 mg 10% Pd / C was added, followed by H2 replacement for three times, the reaction was carried out under H2 atmosphere for 4 h, TLC detection showed that the raw material was completely reacted. The filter paper and diatomite were padded and filtered, the filter residue was washed with THF (10 ml x 3), the filtrate was collected and concentrated under reduced pressure to obtain Compound 15, white solid 122 mg, yield 86.9%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.45 (s, 1H), 10.77 (s, 1H), 9.27 (s, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 2.0 Hz, 1H), 7.11 (dd, J = 8.4, 2.0 Hz, 1H), 7.04 (d, J = 6.0 Hz, 1H), 5.36 (s, 2H), 4.21 (q, J = 7.2 Hz, 2H), 2.92-2.88 (m, 1H), 1.93-1.49 (m, 8H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 25 N4O5S: 445.1540; found: 445.1545. Example 16 Example 17

[0279]

[0280] 2-(4-(cyclopentanecarboxamido)-3-nitrosobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 16)

[0281] Synthetic route:

[0282]

[0283] 2-(4-(cyclopentanecarboxamido)-3-nitrosobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 16)

[0284] Compound 15 (0.10 g, 0.23 mmol) was dissolved in 20 ml DCM, and Oxone (0.21 g, 0.34 mmol) was dissolved in 20 ml H2O and added to the above solution. After 10 h of reaction at room temperature, TLC detection showed that the reaction had not proceeded. After adding 20 ml DCM, the solution was separated, and the aqueous phase was extracted with DCM (20 ml x 2). The organic phase was washed with 1 M aqueous HCl (20 ml x 1), saturated aqueous NaHCO3, and saturated aqueous NaCl. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product was separated by silica gel column chromatography using a mixture of dichloromethane-methanol (V:V = 99.8:0.2) as the eluent to obtain 0.012 g of a yellow solid, with a yield of 11.7%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 11.74 (s, 1H), 10.82 (s, 1H), 8.78 (d, J = 8.8 Hz, 1H), 8.37 (dd, J = 8.8, 2.0 Hz, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.09 (d, J = 6.0 Hz, 1H), 6.89 (d, J = 2.0 Hz, 1H), 4.24 (q, J = 7.2 Hz, 2H), 3.27-3.20 (m, 1H), 2.01-1.97 (m, 2H), 1.88-1.58 (m, 6H), 1.31 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 23 N4O6S: 459.1333; found: 459.1341.

[0285] Example 18

[0286]

[0287] Synthetic route of 2-(4-(cyclobutanecarboxamido)-2-fluorobenzamido)thiophene-3- carbonylaminomethyl dihydrogen phosphate (Compound 17):

[0288]

[0289] First step: Preparation of methyl 4-(cyclobutanecarboxamido)-2-fluorobenzoate 20b

[0290] Using methyl 4-amino-2-fluorobenzoate 19b (2.00 g, 11.82 mmol) and cyclobutanecarbonyl chloride (1.60 mL, 14.18 mmol) as starting materials, the similar procedure as the first step in Example 1 was used to obtain intermediate 20b as a white solid 2.817 g in 95.0% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.24 (s, 1H), 7.84 (t, J = 8.8 Hz, 1H), 7.75-7.71 (m, 1H), 7.41-7.38 (m, 1H), 3.81 (s, 3H), 3.30-3.19 (m, 1H), 2.29-2.07 (m, 4H), 2.01-1.88 (m, 1H), 1.87-1.75 (m, 1H).

[0291] Second Step: Preparation of 4-(cyclobutanecarboxamido)-2-fluorobenzoic acid B-17

[0292] Using 20b (2.70 g, 10.7 mmol) as starting material, the similar procedure as the second step in Example 1 was used to obtain intermediate B-17 as a white solid 2.158 g in 84.9% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.92 (s, 1H), 10.23 (s, 1H), 7.81 (t, J = 8.8 Hz, 1H), 7.72-7.68 (m, 1H), 7.39-7.36 (m, 1H), 3.30-3.21 (m, 1H), 2.28-2.07 (m, 4H), 2.02-1.87 (m, 1H), 1.87-1.75 (m, 1H).

[0293] Third Step: Preparation of 2-(4-(cyclobutanecarboxamido)-2-fluorobenzamido)thiophene-3-carbonylcarbamate (Compound 17)

[0294] Using B-17 (0.20 g, 0.84 mmol) and A-1 (0.20 g, 0.92 mmol) as starting materials, the similar procedure as the third step in Example 1 was used to obtain Compound 17 as a yellow solid 0.133 g in 36.5% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.63 (d, J = 11.2 Hz, 1H), 10.73 (s, 1H), 10.33 (s, 1H), 8.01 (t, J = 8.8 Hz, 1H), 7.87-7.83 (m, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.53-7.50 (m, 1H), 7.07 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.29-3.23 (m, 1H), 2.32-2.19 (m, 2H), 2.18-2.10 (m, 2H), 2.03-1.89 (m, 1H), 1.89-1.76 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 20 H 21 FN3O5S: 434.1180; found: 434.1191. Example 19

[0295]

[0296] 2-(4-(cyclopentanecarboxamido)-2-fluorobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 18) synthesis route:

[0297]

[0298] First Step: Preparation of methyl 4-(cyclopentanecarboxamido)-2-fluorobenzoate 21b

[0299] Using 19b (2.00 g, 11.82 mmol) and cyclopentanecarbonyl chloride (1.50 mL, 14.18 mmol) as starting material, similar procedure as the first step in Example 1 was taken to give the intermediate 21b, yellow solid 2.952 g, 94.3% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.38 (s, 1H), 7.84 (t, J = 8.8 Hz, 1H), 7.74-7.70 (m, 1H), 7.41-7.38 (m, 1H), 3.81 (s, 3H), 2.85-2.74 (m, 1H), 1.91-1.80 (m, 2H), 1.77-1.62 (m, 4H), 1.62-1.50 (m, 2H).

[0300] Second Step: Preparation of 4-(cyclopentanecarboxamido)-2-fluorobenzoic acid B-18

[0301] Using 21b (2.80 g, 10.57 mmol) as the starting material, a similar procedure as the second step in Example 1 was used to obtain intermediate B-18 as a white solid 2.547 g, 95.6% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.92 (s, 1H), 10.34 (s, 1H), 7.81 (t, J = 8.8 Hz, 1H), 7.71-7.67 (m, 1H), 7.38-7.36 (m, 1H), 2.84-2.76 (m, 1H), 1.91-1.80 (m, 2H), 1.77-1.63 (m, 4H), 1.62-1.50 (m, 2H).

[0302] Step 3: Preparation of 2-(4-(cyclopentanecarboxamido)-2- fluorobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 18)

[0303] Using B-18 (0.20 g, 0.80 mmol) and A-1 (0.19 g, 0.88 mmol) as the starting materials, a similar procedure as the third step in Example 1 was used to obtain Compound 18 as a yellow solid 0.101 g, 28.1% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.63 (d, J = 11.2 Hz, 1H), 10.73 (s, 1H), 10.47 (s, 1H), 8.02 (t, J = 8.8 Hz, 1H), 7.87-7.83 (m, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.52-7.70 (m, 1H), 7.07 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.89-2.74 (m, 1H), 1.94-1.81 (m, 2H), 1.81-1.63 (m, 4H), 1.63-1.51 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 23 FN3O5S: 448.1337; found: 448.1339.

[0304] Example 20

[0305]

[0306] Synthetic route of 2-(4-(cyclopentanecarboxamido)-2-nitrobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 19):

[0307]

[0308] First Step: Preparation of methyl 4-(cyclopentanecarboxamido)-2- nitrobenzoate 23b

[0309] Using methyl 4-amino-2-nitrobenzoate 22b (0.40 g, 2.04 mmol) and cyclopentanecarbonyl chloride (0.31 mL, 2.55 mmol) as starting materials, the similar procedure as the first step in Example 1 was used to obtain intermediate 23b as a yellow solid 0.585 g in 98.2% yield. 1 H NMR (400 MHz, CDC13) δ: 8.11 (s, 1H), 7.76 (br s, 2H), 7.67 (br s, 1H), 3.88 (s, 3H), 2.76-2.68 (m, 1H), 2.00-1.86 (m, 4H), 1.81-1.77 (m, 2H), 1.65-1.61 (m, 2H).

[0310] Second Step: Preparation of 4-(cyclopentanecarboxamido)-2-nitrobenzoic acid B-19

[0311] Using 23b (0.58 g, 1.98 mmol) as starting material, the similar procedure as the second step in Example 1 was used to obtain intermediate B-19 as a pink solid 0.476 g in 86.2% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 13.62 (s, 1H), 10.54 (s, 1H), 8.23 (d, J = 2.0 Hz, 1H), 7.85 (d, J = 6.8 Hz, 1H), 7.80 (dd, J = 6.8, 2.0 Hz, 1H), 2.84-2.78 (m, 1H), 1.91-1.80 (m, 2H), 1.78-1.62 (m, 4H), 1.58-1.55 (m, 2H).

[0312] Third Step: Preparation of ethyl 2-(4-(cyclopentanecarboxamido)-2-nitrobenzamido)thiophene-3-carboxylate (Compound 19)

[0313] Using B-19 (0.30 g, 1.08 mmol) and A-1 (0.23 g, 1.08 mmol) as starting materials, the similar procedure as the third step in Example 1 was used to obtain Compound 19 as a yellow solid 0.203 g in 39.7% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.06 (s, 1H), 10.79 (s, 1H), 10.59 (s, 1H), 8.49 (s, 1H), 7.95 (d, J = 6.8 Hz, 1H), 7.82 (d, J = 6.8 Hz, 1H), 7.67 (d, J = 6.0 Hz, 1H), 7.12 (d, J = 6.0 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H), 2.85-2.79 (m, 1H), 1.90-1.83 (m, 2H), 1.81-1.65 (m, 4H), 1.64-1.54 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 23 N4O7S: 475.1282; found: 475.1270.

[0314] Example 21

[0315]

[0316] Synthesis route of 2-(4-(cyclohexanecarboxamido)-2-nitrobenzamido)thiophene-3- carboxylate (Compound 20):

[0317]

[0318] First step: Preparation of methyl 4-(cyclohexanecarboxamido)-2-nitrobenzoate 24b

[0319] Using 22b (0.50 g, 2.55 mmol) and cyclohexanecarbonyl chloride (0.43 mL, 3.19 mmol) as starting material, similar procedure as the first step in Example 1 was applied to give the intermediate 24b as a yellow solid 0.777 g in 99.5% yield. 1 H NMR (400 MHz, CDCl3) δ: 8.11 (d, J = 2.0 Hz, 1H), 7.78-7.75 (m, 2H), 3.88 (s, 3H), 2.32-2.24 (m, 1H), 1.99-1.91 (m, 2H), 1.87-1.81 (m, 2H), 1.58-1.48 (m, 2H), 1.36-1.20 (m, 4H).

[0320] Second step: Preparation of 4-(cyclohexanecarboxamido)-2-nitrobenzoic acid B-20

[0321] Using 24b (0.60 g, 1.96 mmol) as the starting material, a similar procedure to the second step in Example 1 was used to obtain intermediate B-20 as a white solid 0.472 g, 82.4% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 13.59 (s, 1H), 10.49 (s, 1H), 8.23 (d, J = 2.0 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.80 (dd, J = 8.4, 2.0 Hz, 1H), 2.39-2.32 (m, 1H), 1.87-1.71 (m, 4H), 1.66-1.63 (m, 1H), 1.45-1.15 (m, 5H).

[0322] Preparation of 2-(4-(cyclohexanecarboxamido)-2-nitrobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 20)

[0323] Using B-20 (0.30 g, 1.03 mmol) and A-1 (0.22 g, 1.03 mmol) as the starting materials, a similar procedure to the third step in Example 1 was used to obtain Compound 20 as a yellow solid 0.180 g, 35.9% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.06 (s, 1H), 10.79 (s, 1H), 10.54 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 7.94 (dd, J = 8.4, 2.0 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 6.0 Hz, 1H), 7.12 (d, J = 6.0 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 2.43-2.31 (m, 1H), 1.86-1.75 (m, 4H), 1.67-1.64 (m, 1H), 1.47-1.37 (m, 2H), 1.33-1.19 (m, 6H). HRMS (ESI): m / z [M+H] + calcd for C 22 H 25 O7N4S: 489.1438; found: 489.1439.

[0324] Example 22

[0325]

[0326] Synthesis route of 2-(4-(cyclobutanecarboxamido)benzamido)-5-methylthiophene-3- carboxylic acid ethyl ester (Compound 21):

[0327]

[0328] Preparation of ethyl 2-(4-(cyclobutanecarboxamido)benzamido)-5-methylthiophene-3-carbonylcarbamate (Compound 21)

[0329] Using B-3 (0.20 g, 0.91 mmol) and A-2 (0.25 g, 1.09 mmol) as raw materials, the similar operation steps as the third step in Example 1 were adopted to obtain compound 21 as a yellow solid (0.119 g) with a yield of 30.5%. 1 H NMR(400MHz,DMSO-d6)δ:12.46(br s,1H),10.64(br s,1H),10.14(br s,1H),7.94-7.75(m,4H),7.41(s,1H),4.24-4.16(m,2H),3.30-3.24(m,1H),2.38(s,3H),2.29-2.1 9(m,2H),2.18-2.07(m,2H),2.05-1.89(m,1H),1.82(s,1H),1.31-1.24(m,3H).HRMS(ESI):m / z[M+H] + C 21 H 24 N3O5S:430.1431; found:430.1430. Example 23

[0330]

[0331] Synthesis route of ethyl 2-(4-(cyclohexanecarboxamido)benzamido)-5-methylthiophene-3-carbonylcarbamate (Compound 22):

[0332]

[0333] Preparation of ethyl 2-(4-(cyclohexanecarboxamido)benzamido)-5-methylthiophene-3-carbonylcarbamate (Compound 22)

[0334] Using B-5 (0.20 g, 0.81 mmol) and A-2 (0.22 g, 0.97 mmol) as raw materials, the similar operation steps as the third step in Example 1 were adopted to obtain compound 22 as a yellow solid (0.114 g) with a yield of 30.8%. 1H NMR (400 MHz, DMSO-d6) δ: 12.46 (br s, 1H), 10.63 (br s, 1H), 10.23 (br s, 1H), 7.96-7.78 (m, 4H), 7.41 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.85-1.73 (m, 4H), 1.67-1.64 (m, 1H), 1.47-1.36 (m, 2H), 1.33-1.14 (m, 7H). HRMS (ESI): m / z [M+H] + C 23 H 28 N3O5S: 458.1744; found: 458.1744.

[0335] Example 24

[0336]

[0337] 5-Methyl-2-(4-(4-methylcyclohexane-1-carboxamido)benzamido)thiophene-3- carbonylcarbamic acid ethyl ester (Compound 23)

[0338] Synthetic route:

[0339]

[0340] Preparation of 5-Methyl-2-(4-(4-methylcyclohexane-1-carboxamido)benzamido)thiophene-3- carbonylcarbamic acid ethyl ester (Compound 23)

[0341] Using B-8 (0.20 g, 0.77 mmol) and A-2 (0.21 g, 0.92 mmol) as the starting material, the similar procedure as in Example 1, Step 3 was used to obtain Compound 23, yellow solid 0.135 g, yield 37.2%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.46 (br s, 1H), 10.63 (br s, 1H), 10.23 (br s, 1H), 7.96-7.78 (m, 4H), 7.41 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 2.37 (s, 3H), 1.85-1.73 (m, 4H), 1.67-1.64 (m, 1H), 1.47-1.36 (m, 2H), 1.33-1.14 (m, 7H). HRMS (ESI): m / z [M+H] + C 24 H 30N3O5S: 472.1901; found: 472.1902. Example 25

[0342]

[0343] Synthesis route of 5-chloro-2-(4-(cyclopropanecarboxamido)benzamido)thiophene-3- carboxylic acid ethyl ester (Compound 24):

[0344]

[0345] Preparation of 5-chloro-2-(4-(cyclopropanecarboxamido)benzamido)thiophene-3- carboxylic acid ethyl ester (Compound 24)

[0346] Compound 2 (0.05 g, 0.13 mmol) was dissolved in anhydrous dichloromethane (8 mL), then NCS (0.019 g, 0.14 mmol) and FeCl3(2 mg, 0.01 mmol) were added, Ar protection, heated to reflux stirring for 16 h. TLC detection of raw materials completely, add deionized water (5 mL), extracted with dichloromethane (30 mL x 3), the combined organic phase was washed with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the obtained crude product was separated by silica gel column chromatography, dichloromethane-ethyl acetate (V:V = 100:3) mixture as eluent, 0.032 g of white solid was obtained, yield 58.2%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.76 (s, 1H), 10.64 (s, 1H), 7.91-7.81 (m, 4H), 7.80 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 1.91-1.78 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.88-0.82 (m, 4H). HRMS (ESI): m / z [M+H] + C 19 H 19 ClN3O5S: 436.0728; found: 436.0714.

[0347] Example 26

[0348]

[0349] Synthesis route of 5-chloro-2-(4-(cyclobutanecarboxamido)benzamido)thiophene-3- carboxylic acid ethyl ester (Compound 25):

[0350]

[0351] Preparation of 5-chloro-2-(4-(cyclobutanecarboxamido)benzamido)thiophene-3-carbonylcarbamate (Compound 25)

[0352] Compound 25 was obtained using compound 3 (0.05 g, 0.12 mmol) as starting material by similar procedures to those in Example 24. The obtained compound 25 was obtained as a white solid (0.045 g). The yield was 83.3%. 1 H NMR (400MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.75 (s, 1H), 10.16 (s, 1H), 7.91-7.82 (m, 4H), 7.79 (s, 1H), 4.21 (q, J = 7.2Hz, 2H), 3.32-3. 22(m,1H),2.31-2.18(m,2H),2.18-2.08(m,2H),2.00-1.92(m,1H),1.88-1.76(m,1H),1.28(t,J=7.2Hz,3H).HRMS(ESI):m / z[M+H] + C 20 H 21 ClN3O5S:450.0885; found:450.0886.

[0353] Example 27

[0354]

[0355] Synthesis route of ethyl 5-chloro-2-(4-(cyclopentanecarboxamido)benzamido)thiophene-3-carbonylcarbamate (Compound 26):

[0356]

[0357] Preparation of 5-chloro-2-(4-(cyclopentanecarboxamido)benzamido)thiophene-3-carbonylcarbamate (Compound 26)

[0358] Compound 4 (0.10 g, 0.23 mmol) was used as starting material and the similar operation steps as in Example 24 were adopted to obtain compound 26 as a white solid (0.052 g). The yield was 48.6%. 1H NMR (400 MHz, DMSO-d6) δ: 12.56 (br s, 1H), 10.76 (br s, 1H), 10.31 (br s, 1H), 7.90-7.84 (m, 4H), 7.80 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.85-2.79 (m, 1H), 1.89-1.86 (m, 2H), 1.80-1.63 (m, 4H), 1.62-1.51 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 21 H 23 ClN3O5S: 464.1041; found: 464.1036.

[0359] Example 28

[0360]

[0361] Synthesis route of 5-chloro-2-(4-(cyclohexanecarboxamido)benzamido)thiophene-3-carbonylcarbamic acid ethyl ester (Compound 27):

[0362]

[0363] Preparation of 5-chloro-2-(4-(cyclohexanecarboxamido)benzamido)thiophene-3-carbonylcarbamic acid ethyl ester (Compound 27)

[0364] Using Compound 5 (0.05 g, 0.12 mmol) as the raw material, the similar operation steps in Example 24 were adopted to obtain Compound 27, yellow solid 0.027 g, yield 47.3%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.55 (s, 1H), 10.75 (s, 1H), 10.26 (s, 1H), 7.89-7.83 (m, 4H), 7.79 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.40-2.34 (m, 1H), 1.84-1.74 (m, 4H), 1.67-1.64 (m, 1H), 1.46-1.37 (m, 2H), 1.33-1.19 (m, 6H). HRMS (ESI): m / z [M+H] + C 22 H 25 ClN3O5S: 478.1198; found: 478.1180.

[0365] Example 29

[0366]

[0367] 5-chloro-2-(4-(cycloheptanecarboxamido)benzamido)thiophene-3-carboxylic acid ethyl ester (Compound 28) synthesis route:

[0368]

[0369] Preparation of 5-chloro-2-(4-(cycloheptanecarboxamido)benzamido)thiophene-3- carboxylic acid ethyl ester (Compound 28)

[0370] Using compound 6 (0.10 g, 0.22 mmol) as the starting material, using similar procedure steps in Example 24, compound 28 was obtained as a white solid 0.062 g, yield 57.4%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.55 (br s, 1H), 10.76 (br s, 1H), 10.23 (br s, 1H), 7.90-7.82 (m, 4H), 7.80 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 1.91-1.82 (m, 2H), 1.78-1.40 (m, 11H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H]+C 23 H 27 ClN3O5S: 492.1354; found: 492.1336. Example 30

[0371]

[0372] 5-chloro-2-(4-(cyclobutanecarboxamido)-3-fluorobenzamido)thiophene-3-carboxylic acid ethyl ester (Compound 29)

[0373] Synthesis route:

[0374]

[0375] Preparation of 5-chloro-2-(4-(cyclobutanecarboxamido)-3-fluorobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 29)

[0376] Compound 9 (0.10 g, 0.23 mmol) was dissolved in anhydrous dichloromethane (5 mL), and sulfuryl chloride (0.02 mL, 0.25 mmol) was added under ice bath condition, Ar protection, 5 min stirring at 0 °C. TLC detection of the complete reaction of the raw material, the addition of deionized water (5 mL), extraction with dichloromethane (10 mL x 3), the combined organic phase was washed with saturated aqueous sodium chloride solution, anhydrous sodium sulfate drying, filtration, reduced pressure concentration, the obtained crude product was separated by silica gel column chromatography, dichloromethane-ethyl acetate (V:V = 100:3) mixture as eluent, 0.086 g of white solid was obtained, the yield was 79.6%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.53 (s, 1H), 10.78 (s, 1H), 9.89 (s, 1H), 8.34-8.30 (m, 1H), 7.79 (s, 1H), 7.79-7.72 (m, 2H), 4.21 (q, J = 7.2 Hz, 2H), 3.52-3.40 (m, 1H), 2.30-2.08 (m, 4H), 2.02-1.78 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 20 H 20 FClN3O5S: 468.0791; found: 468.0789.

[0377] Example 31

[0378]

[0379] 5-chloro-2-(4-(cyclopentanecarboxamido)-3-fluorobenzamido)thiophene-3- carbonylaminomethvate (Compound 30)

[0380] Synthetic route:

[0381]

[0382] Preparation of 5-chloro-2-(4-(cyclopentanecarboxamido)-3-fluorobenzamido)thiophene-3- carbonylaminomethvate (Compound 30)

[0383] Compound 30 was obtained by using similar operation procedure in Example 29, with compound 10 (0.10 g, 0.22 mmol) as raw material, white solid 0.058 g, yield 54.7%. 1H NMR (400 MHz, DMSO-d6) δ: 12.53 (s, 1H), 10.78 (s, 1H), 10.00 (s, 1H), 8.32-8.28 (m, 1H), 7.79 (s, 1H), 7.77-7.70 (m, 2H), 4.21 (q, J = 7.2 Hz, 2H), 3.04-3.00 (m, 1H), 1.92-1.82 (m, 2H), 1.78-1.65 (m, 4H), 1.60-1.53 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 21 H 22 FClN3O5S: 482.0947; found: 482.0931.

[0384] Example 32

[0385]

[0386] 5-Chloro-2-(4-(cyclopentanecarboxamido)-3-nitrobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 31)

[0387] Synthetic route:

[0388]

[0389] Preparation of 5-Chloro-2-(4-(cyclopentanecarboxamido)-3-nitrobenzamido)thiophene-3- carboxylic acid ethyl ester (Compound 31)

[0390] Starting from compound 11 (0.05 g, 0.11 mmol), using similar procedure as in Example 29, compound 31 was obtained as yellow solid 0.029 g in 54.3% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.53 (s, 1H), 10.78 (s, 1H), 10.00 (s, 1H), 8.32-8.28 (m, 1H), 7.79 (s, 1H), 7.77-7.70 (m, 2H), 4.21 (q, J = 7.2 Hz, 2H), 3.04-3.00 (m, 1H), 1.92-1.82 (m, 2H), 1.78-1.65 (m, 4H), 1.60-1.53 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 21 H 22 ClN4O7S: 509.0892; found: 509.0907.

[0391] Example 33

[0392]

[0393] 5-chloro-2-(4-(cyclohexanecarboxamido)-3-nitrobenzamido)thiophene-3- carbonylcarbamate (Compound 32)

[0394] Synthetic route:

[0395]

[0396] Preparation of 5-chloro-2-(4-(cyclohexanecarboxamido)-3-nitrobenzamido)thiophene- 3-carbonylcarbamate (Compound 32)

[0397] Using similar procedure as in Example 29, starting from Compound 12 (0.05 g, 0.10 mmol), Compound 32 was obtained as a yellow solid 0.037 g in 69.8% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.57 (s, 1H), 10.81 (s, 1H), 10.54 (s, 1H), 8.42 (d, J = 2.0 Hz, 1H), 8.18 (dd, J = 8.4, 2.0 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.79 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.48-2.37 (m, 1H), 1.90-1.72 (m, 4H), 1.69-1.60 (m, 1H), 1.46-1.14 (m, 8H). HRMS (ESI): m / z [M+H] + C 22 H 24 ClN4O7S: 523.1049; found: 523.1073.

[0398] Example 34

[0399]

[0400] 5-chloro-2-(4-(cyclohexanecarboxamido)-3-nitrobenzamido)thiophene-3- carbonylcarbamate (Compound 32)

[0401] Synthetic route:

[0402]

[0403] 5-chloro-2-(4-(cyclopentanecarboxamido)-3-cyanobenzamido)thiophene-3- carbonylcarbamate (Compound 33)

[0404] Using similar procedure as in Example 29, starting from compound 13 (0.06 g, 0.13 mmol), compound 33 was obtained as a white solid 0.050 g, yield 77.5%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 10.81 (s, 1H), 10.41 (s, 1H), 8.29 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.98-2.90 (m, 1H), 1.96-1.84 (m, 2H), 1.83-1.53 (m, 6H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 22 H 22 ClN4O5S: 489.0994; found: 489.0991.

[0405] Example 35

[0406]

[0407] 5-chloro-2-(4-(cyclohexanecarboxamido)-3-cyanobenzamido)thiophene-3- carbonylcarbamate (Compound 34)

[0408] Synthetic route:

[0409]

[0410] 5-chloro-2-(4-(cyclohexanecarboxamido)-3-cyanobenzamido)thiophene-3- carbonylcarbamate (Compound 34)

[0411] Using similar procedure as in Example 29, starting from compound 14 (0.05 g, 0.11 mmol), compound 34 was obtained as a white solid 0.051 g, yield 94.8%. 1H NMR (400 MHz, DMSO-d6) δ: 12.50 (s, 1H), 10.80 (s, 1H), 10.35 (s, 1H), 8.28 (s, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.78 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.49-2.45 (m, 1H), 1.90-1.75 (m, 4H), 1.70-1.61 (m, 1H), 1.51-1.37 (m, 2H), 1.35-1.21 (m, 6H). HRMS (ESI): m / z [M+H] + C 23 H 24 ClN4O5S: 503.1150; found: 503.1144.

[0412] Example 36

[0413]

[0414] 5-Chloro-2-(4-(cyclopentanecarboxamido)-2-nitrobenzamido)thiophene-3- carbonylaminomethvate (Compound 35)

[0415] Synthetic route:

[0416]

[0417] Preparation of 5-Chloro-2-(4-(cyclopentanecarboxamido)-2-nitrobenzamido)thiophene-3- carbonylaminomethvate (Compound 35)

[0418] Using Compound 19 (0.08 g, 0.16 mmol) as the starting material, Compound 35 was obtained as a yellow solid 0.066 g in 80.5% yield by using similar procedure as in Example 29. 1 H NMR (400 MHz, DMSO-d6) δ: 12.50 (s, 1H), 10.80 (s, 1H), 10.35 (s, 1H), 8.28 (s, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.78 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.49-2.45 (m, 1H), 1.90-1.75 (m, 4H), 1.70-1.61 (m, 1H), 1.51-1.37 (m, 2H), 1.35-1.21 (m, 6H). HRMS (ESI): m / z [M+H] + C 21 H22 CIN4O7S: 509.0892; found: 509.0881.

[0419] Example 37

[0420]

[0421] 2-(4-(cyclopentanecarboxamido)-3-fluorobenzamido)-5-nitrothiophene-3- carboxylic acid ethyl ester (Compound 36)

[0422] Synthetic route:

[0423]

[0424] Preparation of 2-(4-(cyclopentanecarboxamido)-3-fluorobenzamido)-5-nitrothiophene- 3-carboxylic acid ethyl ester (Compound 36)

[0425] Using B-10 (0.10 g, 0.41 mmol) and A-3 (0.10 g, 0.39 mmol) as the starting material, the similar procedure as in Example 1, Step 3 was used to obtain Compound 36, yellow solid 0.078 g, yield 40.6%. 1 H NMR (500 MHz, DMSO-d6) δ: 12.85 (s, 1H), 11.19 (s, 1H), 10.05 (s, 1H), 8.97 (s, 1H), 8.32 (s, 1H), 7.82 (s, 2H), 4.22 (q, J = 7.0 Hz, 2H), 3.06-2.99 (m, 1H), 1.95-1.79 (m, 2H), 1.76-1.63 (m, 4H), 1.60-1.51 (m, 2H), 1.30 (t, J = 7.0 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 22 FN4O7S: 493.1188; found: 493.1175.

[0426] Example 38

[0427]

[0428] 2-(6-(cyclobutanecarboxamido)nicotinamido)thiophene-3-carboxylic acid ethyl ester (Compound 37)

[0429] Synthetic route:

[0430]

[0431] First Step: Preparation of methyl 6-(cyclobutanecarboxamido)nicotinate 26b

[0432] Using methyl 6-aminonicotinate 25b (1.00 g, 6.57 mmol) and cyclobutanecarbonyl chloride (0.90 mL, 7.88 mmol) as starting materials, the similar procedure as the first step in Example 1 was used to obtain intermediate 26b as a white solid 1.131 g in 63.1% yield. 1 HNMR (400 MHz, DMSO-d6) δ: 10.72 (s, 1H), 8.82 (m, 1H), 8.32-8.19 (m, 2H), 3.85 (s, 3H), 3.50-3.34 (m, 1H), 2.34-2.03 (m, 4H), 2.02-1.71 (m, 2H).

[0433] Second Step: Preparation of 6-(cyclobutanecarboxamido)nicotinic acid B-21

[0434] Using 26b (0.90 g, 3.80 mmol) as starting material, the similar procedure as the second step in Example 1 was used to obtain intermediate B-21 as a white solid 0.822 g in 98.3% yield. 1 HNMR (400 MHz, DMSO-d6) δ: 13.05 (s, 1H), 10.67 (s, 1H), 8.80-8.74 (m, 1H), 8.25-8.17 (m, 2H), 3.47-3.35 (m, 1H), 2.30-2.04 (m, 4H), 2.02-1.70 (m, 2H).

[0435] Third Step: Preparation of ethyl 2-(6-(cyclobutanecarboxamido)nicotinamido)thiophene-3- carboxylate (Compound 37)

[0436] Using B-21 (0.20 g, 0.90 mmol) and A-1 (0.23 g, 1.10 mmol) as starting materials, the similar procedure as the third step in Example 1 was used to obtain Compound 37 as a white solid 0.189 g in 50.4% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 10.80 (s, 1H), 10.78 (s, 1H), 8.92-8.65 (m, 1H), 8.46-8.18 (m, 2H), 7.71 (d, J = 6.0 Hz, 1H), 7.08 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.47-3.38 (m, 1H), 2.30-2.18 (m, 2H), 2.18-2.07 (m, 2H), 2.00-1.89 (m, 1H), 1.87-1.74 (m, 1H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H]+calcd for C 19 H 21 N4O5S: 417.1227; found: 417.1231.

[0437] Example 39

[0438]

[0439] 2-(6-(cyclopentanecarboxamido)nicotinamido)thiophene-3-carboxylate (Compound 38)

[0440] Synthetic route:

[0441]

[0442] First step: Preparation of methyl 6-(cyclopentanecarboxamido)nicotinate 27b

[0443] Using 25b (1.00 g, 6.57 mmol) and cyclopentanecarbonyl chloride (0.96 mL, 7.88 mmol) as starting material, similar procedure as the first step in Example 1 was adopted to give the intermediate 27b as a brown solid 1.131 g in 69.0% yield. 1 H NMR (500 MHz, DMSO-d6) δ: 10.84 (s, 1H), 8.87-8.79 (m, 1H), 8.29-8.19 (m, 2H), 3.85 (s, 3H), 3.07-2.89 (m, 1H), 1.91-1.80 (m, 2H), 1.75-1.62 (m, 4H), 1.60-1.48 (m, 2H).

[0444] Second step: Preparation of 6-(cyclopentanecarboxamido)nicotinic acid B-22

[0445] Using 27b (1.10 g, 4.40 mmol) as the starting material, the similar procedure as in Example 1, Step 2 was used to obtain intermediate B-22 as a white solid 0.624 g, in 60.6% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 10.91 (s, 1H), 10.81 (s, 1H), 8.90-8.80 (m, 1H), 8.35-8.22 (m, 2H), 7.71 (d, J = 6.0 Hz, 1H), 7.08 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.03-2.95 (m, 1H), 1.89-1.83 (m, 2H), 1.78-1.60 (m, 4H), 1.60-1.50 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H]

[0446] Step 3: Preparation of 2-(6-(cyclopentanecarboxamido) nicotinamido)thiophene-3- carboxylic acid ethyl ester (Compound 38)

[0447] Using B-22 (0.20 g, 0.78 mmol) and A-1 (0.17 g, 0.78 mmol) as the starting materials, the similar procedure as in Example 1, Step 3 was used to obtain Compound 38 as a white solid 0.204 g, in 59.3% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 10.91 (s, 1H), 10.81 (s, 1H), 8.90-8.80 (m, 1H), 8.35-8.22 (m, 2H), 7.71 (d, J = 6.0 Hz, 1H), 7.08 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.03-2.95 (m, 1H), 1.89-1.83 (m, 2H), 1.78-1.60 (m, 4H), 1.60-1.50 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 20 H 23 N4O5S: 431.1384; found: 431.1393.

[0448] Example 40

[0449]

[0450] 2-(6-(cyclohexanecarboxamido) nicotinamido)thiophene-3-carboxylic acid ethyl ester (Compound 39)

[0451] Synthetic route:

[0452]

[0453] First Step: Preparation of methyl 6-(cyclohexanecarboxamido)nicotinate 28b

[0454] Using 25b (1.00 g, 6.57 mmol) and cyclohexanecarbonyl chloride (1.06 mL, 7.88 mmol) as the starting material, the similar procedure as the first step in Example 1 was used to obtain the intermediate 28b as a white solid 1.701 g in 98.3% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.78 (s, 1H), 8.84-8.82 (m, 1H), 8.27-8.20 (m, 2H), 3.85 (s, 3H), 1.84-1.70 (m, 4H), 1.65-1.62 (m, 1H), 1.44-1.11 (m, 6H).

[0455] Second Step: Preparation of 6-(cyclohexanecarboxamido)nicotinic acid B-23

[0456] Using 28b (1.60 g, 6.10 mmol) as the starting material, the similar procedure as the second step in Example 1 was used to obtain the intermediate B-23 as a white solid 1.180 g in 77.9% yield. 1 H NMR (500 MHz, DMSO-d6) δ: 10.87 (s, 1H), 8.80 (s, 1H), 8.24 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 2.59-2.53 (m, 1H), 1.83-1.59 (m, 6H), 1.43-1.33 (m, 2H), 1.27-1.15 (m, 2H).

[0457] Third Step: Preparation of ethyl 2-(6-(cyclohexanecarboxamido)nicotinamido)thiophene-3-carboxylate (Compound 39)

[0458] Using B-23 (0.29 g, 0.81 mmol) and A-1 (0.21 g, 0.98 mmol) as the starting material, the similar procedure as the third step in Example 1 was used to obtain Compound 39 as a white solid 0.104 g in 28.8% yield. 1H NMR (400 MHz, DMSO-d6) δ: 12.52 (s, 1H), 10.84 (s, 1H), 10.81 (br s, 1H), 8.88-8.82 (m, 1H), 8.33-8.22 (m, 2H), 7.71 (d, J = 6.0 Hz, 1H), 7.08 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.62-2.52 (m, 1H), 1.87-1.71 (m, 4H), 1.66-1.63 (m, 1H), 1.47-1.34 (m, 2H), 1.31-1.20 (m, 6H). HRMS (ESI): m / z [M+H] + calcd for C 21 H 25 N4O5S: 445.1540; found: 445.1547.

[0459] Example 41

[0460]

[0461] Synthesis route of 2-(5-(cyclopentanecarboxamido)-2-pyridinoylamino)thiophene-3- carboxylic acid ethyl ester (Compound 40):

[0462]

[0463] First step: Preparation of methyl 5-(cyclopentanecarboxamido)-2-pyridinecarboxylate 30b

[0464] Using methyl 5-amino-2-pyridinecarboxylate 29b (1.00 g, 6.57 mmol) and cyclopentanecarbonyl chloride (0.96 mL, 7.92 mmol) as starting materials, the similar procedure as the first step in Example 1 was used to obtain the intermediate 30b as a white solid 1.059 g, yield 64.8%. 1 H NMR (400 MHz, DMSO-d6) δ: 10.43 (s, 1H), 8.84 (d, J = 2.4 Hz, 1H), 8.26 (dd, J = 8.8, 2.4 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 3.84 (s, 3H), 2.92-2.78 (m, 1H), 2.02-1.80 (m, 2H), 1.81-1.46 (m, 6H).

[0465] Second step: Preparation of 5-(cyclopentanecarboxamido)-2-pyridinecarboxylic acid B-24

[0466] Using 30b (1.00 g, 3.70 mmol) as the starting material, the similar procedure as in Example 1, Step 2 was used to obtain intermediate B-24 as a white solid 0.576 g, in 60.8% yield. 1 H NMR (500 MHz, DMSO-d6) δ: 12.89 (s, 1H), 10.40 (s, 1H), 8.85 (d, J = 2.5 Hz, 1H), 8.23 (dd, J = 8.5, 2.5 Hz, 1H), 8.01 (d, J = 8.5 Hz, 1H), 2.89-2.77 (m, 1H), 1.95-1.82 (m, 2H), 1.80-1.63 (m, 4H), 1.62-1.50 (m, 2H).

[0467] Step 3: Preparation of 2-(5-(cyclopentanecarboxamido)-2-pyridinamido)thiophene-3- carbonylaminomethylate (Compound 40)

[0468] Using B-24 (0.20 g, 0.78 mmol) and A-1 (0.20 g, 0.94 mmol) as the starting materials, the similar procedure as in Example 1, Step 3 was used to obtain Compound 40 as a yellow solid 0.325 g, in 96.7% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 13.17 (s, 1H), 10.73 (s, 1H), 10.55 (s, 1H), 8.88 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 8.8, 2.4 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.08 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.91-2.83 (m, 1H), 1.97-1.85 (m, 2H), 1.81-1.64 (m, 4H), 1.62-1.54 (m, 2H), 1.29 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 20 H 23 N4O5S: 431.1384; found: 431.1380. Example 42

[0469]

[0470] Synthetic route of 2-(5-(cyclohexanecarboxamido)-2-pyridinamido)thiophene-3- carbonylaminomethylate (Compound 41):

[0471]

[0472] First Step: Preparation of methyl 5-(cyclohexanecarboxamido)-2-pyridinecarboxylate 31b

[0473] Using 29b (1.00 g, 6.57 mmol) and cyclohexanecarbonyl chloride (0.85 mL, 6.57 mmol) as starting materials, the similar procedure as the first step in Example 1 was adopted to give the intermediate 31b as a white solid 1.312 g in 76.7% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 10.53 (s, 1H), 8.87 (d, J = 2.4 Hz, 1H), 8.32-8.23 (m, 1H), 8.03 (d, J = 8.8 Hz, 1H), 3.85 (s, 3H), 2.48-2.35 (m, 1H), 1.88-1.71 (m, 4H), 1.66-1.64 (m, 1H), 1.47-1.34 (m, 2H), 1.33-1.16 (m, 3H).

[0474] Second Step: Preparation of 5-(cyclohexanecarboxamido)-2-pyridinecarboxylic acid B-25

[0475] Using 31b (1.30 g, 4.90 mmol) as starting material, the similar procedure as the second step in Example 1 was adopted to give the intermediate B-25 as a white solid 0.850 g in 68.6% yield. 1 H NMR (500 MHz, DMSO-d6) δ: 10.55 (s, 1H), 8.91 (d, J = 2.5 Hz, 1H), 8.29 (dd, J = 8.5, 2.5 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 2.46-2.37 (m, 1H), 1.91-1.72 (m, 4H), 1.66-1.64 (m, 1H), 1.48-1.36 (m, 2H), 1.35-1.10 (m, 3H).

[0476] Third Step: Preparation of ethyl 2-(5-(cyclohexanecarboxamido)-2-pyridinecarbonylamino)thiophene-3-carboxylate (Compound 41)

[0477] Using B-25 (0.20 g, 0.78 mmol) and A-1 (0.20 g, 0.94 mmol) as starting materials, the similar procedure as the third step in Example 1 was adopted to give Compound 41 as a yellow solid 0.411 g in 94.8% yield. 1H NMR (400 MHz, DMSO-d6) δ: 13.16 (s, 1H), 10.73 (s, 1H), 10.50 (s, 1H), 8.88 (d, J = 2.4 Hz, 1H), 8.40 (dd, J = 8.8, 2.4 Hz, 1H), 8.15 (d, J = 8.8 Hz, 1H), 7.71 (d, J = 6.0 Hz, 1H), 7.08 (d, J = 6.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 2.45-2.38 (m, 1H), 1.90-1.83 (m, 2H), 1.80-1.73 (m, 2H), 1.68-1.65 (m, 1H), 1.52-1.36 (m, 2H), 1.36-1.23 (m, 6H). HRMS (ESI): m / z [M+H] + C 21 H 25 N4O5S: 445.1540; found: 445.1544. Example 43

[0478]

[0479] Synthesis route of 5-chloro-2-(6-(cyclobutanecarboxamido) nicotinamido)thiophene-3-carboxylic acid ethyl ester (Compound 42):

[0480]

[0481] Preparation of 5-chloro-2-(6-(cyclobutanecarboxamido) nicotinamido)thiophene-3-carboxylic acid ethyl ester (Compound 42)

[0482] Using Compound 37 (0.05 g, 0.12 mmol) as the raw material, the similar operation steps in Example 29 were adopted to obtain Compound 42, white solid 0.033 g, yield 61.1%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 10.80 (s, 1H), 10.77 (s, 1H), 8.82 (d, J = 2.4, 1H), 8.32 (d, J = 8.8 Hz, 1H), 8.25 (dd, J = 8.8, 2.4 Hz, 1H), 7.78 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.46-3.39 (m, 1H), 2.30-2.06 (m, 4H), 2.02-1.76 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 19 H 20CIN4OS: 451.0837; found: 451.0841.

[0483] Example 44

[0484]

[0485] Synthetic route of 5-chloro-2-(6-(cyclopentanecarboxamido) nicotinamido)thiophene-3-carbonylaminomethyl dihydrogen phosphate (Compound 43):

[0486]

[0487] Preparation of 5-chloro-2-(6-(cyclopentanecarboxamido) nicotinamido)thiophene-3-carbonylaminomethyl dihydrogen phosphate (Compound 43)

[0488] Using similar procedure as in Example 29, starting from Compound 38 (0.05 g, 0.11 mmol), Compound 43 was obtained as a yellow solid 0.033 g, yield 61.1%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 10.93 (s, 1H), 10.78 (s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 8.38-8.11 (m, 2H), 7.78 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.02-2.94 (m, 1H), 1.92-1.80 (m, 2H), 1.78-1.62 (m, 4H), 1.61-1.47 (m, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI): m / z [M+H] + C 20 H 22 CIN4OS: 451.0837; found: 451.0841.

[0489] Example 45

[0490]

[0491] Synthetic route of 5-chloro-2-(6-(cyclopentanecarboxamido) nicotinamido)thiophene-3-carbonylaminomethyl dihydrogen phosphate (Compound 43):

[0492]

[0493] Preparation of 5-chloro-2-(6-(cyclopentanecarboxamido) nicotinamido)thiophene-3-carbonylaminomethyl dihydrogen phosphate (Compound 43)

[0494] Using compound 39 (0.05 g, 0.11 mmol) as the starting material, the similar procedure as in Example 29 was used to obtain compound 44, white solid 0.037 g, yield 68.5%. 1 H NMR (400 MHz, DMSO-d6) δ: 12.51 (s, 1H), 10.86 (s, 1H), 10.78 (s, 1H), 8.84 (d, J = 2.4 Hz, 1H), 8.37-8.14 (m, 2H), 7.79 (s, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.61-2.54 (m, 1H), 1.88-1.70 (m, 4H), 1.66-1.63 (m, 1H), 1.45-1.35 (m, 2H), 1.31-1.17 (m, 6H). HRMS (ESI): m / z [M+H] + C 21 H 24 ClN4O5S: 479.1150; found: 479.1177.

[0495] Compound

[0496]

[0497] Synthesis route of 2-(2-(cyclopentanecarboxamido)pyrimidine-5- carboxamido)thiophene-3-carboxylic acid ethyl ester (compound 45):

[0498]

[0499] First step: Preparation of methyl 2-(cyclopentanecarboxamido)pyrimidine-5- carboxylate 33b

[0500] Using methyl 2-aminopyrimidine-5-carboxylate 32b (0.60 g, 3.92 mmol) and cyclopentanecarbonyl chloride (0.57 mL, 4.70 mmol) as the starting materials, the similar procedure as in Example 1, first step was used to obtain intermediate 33b, white solid 0.394 g, yield 40.4%. 1 H NMR (400 MHz, CDCl3) δ: 9.13 (s, 2H), 8.61 (s, 1H), 3.95 (s, 3H), 3.30-3.22 (m, 1H), 2.04-1.86 (m, 4H), 1.83-1.73 (m, 2H), 1.71-1.58 (m, 2H).

[0501] Second step: Preparation of 2-(cyclopentanecarboxamido)pyrimidine-5-carboxylic acid B-26

[0502] Using 33b (0.30 g, 1.20 mmol) as the starting material, a similar procedure to the second step in Example 1 was used to obtain intermediate B-26 as a white solid 0.200 g, 70.9% yield. 1 H NMR (400 MHz, DMSO-d6) δ: 13.44 (s, 1H), 10.92 (s, 1H), 9.03 (s, 2H), 3.13-3.01 (m, 1H), 1.88-1.82 (m, 2H), 1.77-1.59 (m, 4H), 1.59-1.48 (m, 2H).

[0503] Step 3: Preparation of ethyl 2-(2-(cyclopentanecarboxamido)pyrimidine-5- carboxamido)thiophene-3-carboxylate (Compound 45)

[0504] Using B-26 (0.19 g, 0.81 mmol) and A-1 (0.17 g, 0.81 mmol) as the starting materials, a similar procedure to the third step in Example 1 was used to obtain Compound 45 as a blue solid 0.052 g, 14.8% yield. 1 H NMR (500 MHz, DMSO-d6) δ: 12.45 (s, 1H), 11.05 (s, 1H), 10.85 (s, 1H), 9.08 (s, 2H), 7.70 (d, J = 6.0 Hz, 1H), 7.12 (d, J = 6.0 Hz, 1H), 4.21 (q, J = 7.0 Hz, 2H), 3.13-3.07 (m, 1H), 1.93-1.80 (m, 2H), 1.78-1.60 (m, 4H), 1.58-1.54 (m, 2H), 1.28 (t, J = 7.0 Hz, 3H). HRMS (ESI): m / z [M+H] + calcd for C 19 H 22 N5O5S: 432.1336; found: 432.1330.

[0505] 1. In vitro anti-Mycobacterium tuberculosis activity test

[0506] Method of determination: Microplate Alamar Blue Assay (MABA) method was used to determine the in vitro anti-tuberculosis activity. Principle of experiment: Alamar Blue added to the culture medium can be used as an oxidation-reduction indicator, and the color changes from blue to red, reflecting the consumption of oxygen molecules by the microorganism under study. The color change of Alamar Blue can be determined by a spectrophotometer, and the emission wavelength is 590 nm.

[0507] Method of experiment: The strain was Mycobacterium tuberculosis standard strain H 37Rv (ATCC 27294) and drug-resistant strains 13946 (resistant to INH, SM, RFP, EMB, RBT, PAS, OFLX.), drug-resistant strains 14862 (resistant to INH, SM, RFP, EMB, PAS, 1321, CPM.), and the synthesized target compound is set to 11 times of dilution concentration test gradient in the concentration range of 0.031-32 μg / mL. The Mycobacterium tuberculosis cultured to the logarithmic growth phase is centrifuged, washed and resuspended in a phosphate buffer, filtered and stored at -80℃. A 96-well sterile microplate is taken, 100 μL of the bacterial solution is added to each well, then 100 μL of the drug solution is added, and two blank growth control wells are set on each plate. Incubate at 37℃ for 7 days, add the premixed color developing solution to each well, incubate at 37℃ for 16-24 hours, observe the color change, and use an enzyme label instrument to measure the fluorescence value at 590 nm, and calculate the MIC 90 .

[0508] Table 1, in vitro anti-tubercle bacillus activity of some compounds

[0509] MIC (μg / mL) Compound MIC (μg / mL) Compound 4 Compound 32 0.425 Compound 9 0.104 Compound 33 0.124 Compound 10 <0.016 Compound 34 0.117 Compound 11 <0.016 Compound 37 0.005 Compound 12 0.252 Compound 38 <0.016 Compound 13 <0.016 Compound 39 0.007 Compound 14 0.031 Compound 42 0.004 Compound 16 0.023 Compound 43 0.289 Compound 29 <0.016 Compound 44 0.041 Compound 31 <0.016 Compound V 0.062 TCA1 0.020 ​ 0.476

[0510] It can be seen from the data in Table 1 that the compound in the application has good in vitro anti-tubercle bacillus activity, and the activity of compounds 11, 13 and 14 reaches ng / mL level, and the in vitro anti-tubercle activity is significantly better than that of the control compounds V and TCA1.

[0511] Table 2, in vitro anti-drug-resistant Mycobacterium tuberculosis activity of some compounds

[0512]

[0513]

[0514] a Resistant to isoniazid, streptomycin, rifampicin, ethambutol, rifabutin, p-aminosalicylic acid, ofloxacin. b Resistant to isoniazid, streptomycin, rifampicin, ethambutol, p-aminosalicylic acid, prothionamide, capreomycin.

[0515] It can be seen from the data in Table 2 that the compound in the application has good inhibitory effect on drug-resistant strains 13946 and strains 14862, and has better in vitro anti-drug-resistant Mycobacterium tuberculosis activity than the control compound TCA1.

[0516] 2, DprE1 enzyme inhibition IC 50 determination

[0517] Assay method: resazurin assay for DprE1 enzyme inhibition IC 50 .

[0518] Principle of the experiment: DprE1 oxidizes farnesyl phosphoyl-beta-D-ribose (FPR) to farnesyl phosphoyl-beta-D-2'-keto-erythro-pentofuranose (FPX) resulting in the formation of a two-electron reduced flavin intermediate (FADH2). To complete the catalytic cycle, FADH2 has to be re-oxidized to FAD. This can be achieved by resazurin, which upon reduction yields a highly fluorescent product, resorufin, and the reaction is monitored by following the increase in fluorescence intensity (λ ex = 530 nm, λ em = 595 nm) associated with resorufin formation.

[0519] Experimental method: Reactions in a total volume of 80 μL at 30°C were performed in 96-well plates containing different concentrations of FPR and DprE1 WT, C387S or Y314C mutants in a buffer containing 50 mM Hepes, pH 7.5, 100 mM NaCl, 1.5% (v / v) DMSO, 100 μM Tween-20, 2 μM FAD and 50 μM resazurin. The reactions were detected by detecting the increase in fluorescence intensity (λ ex = 530 nm, λ em = 595 nm) associated with resorufin formation. Steady-state kinetics studies were performed using a microplate reader Spectramax M5. For inhibition studies, DprE1 WT or mutants (1 μM) were measured in the presence of different inhibitor concentrations (0 to 50 μM) using the resazurin assay with 1 mM FPR. The positive control compound was TCA1. All inhibitors were dissolved in dimethyl sulfoxide (1% dimethyl sulfoxide) under all assay conditions. IC 50 values were obtained by plotting the inhibition effect with different inhibitor concentrations using graphpad Prism 9.0.

[0520] Table 3. DprE1 enzyme inhibition IC 50

[0521]

[0522] From the data in Table 3, it can be seen that the compounds of the present application have good DprE1 enzyme inhibition activity for DprE1 wild type and DprE1 Y314C mutant, and have stronger enzyme inhibition activity for DprE1 C387S mutant than the positive control compound TCA1, which is obviously one to two orders of magnitude better than TCA1.

[0523] 3. Intracellular anti-tuberculosis activity test of Mycobacterium tuberculosis in macrophage

[0524] The strain is Mycobacterium tuberculosis standard strain H 37 Rv (ATCC 27294), the macrophage is mouse monocyte macrophage J774A.1, and the positive control drugs are compounds TCA1 and RFP. The J774A.1 cells are cultured in 75 cm 2 square culture dishes with RPMI 1640 medium (containing 10% fetal bovine serum), and after cell growth and trypsin digestion, the cells are collected, suspended in 5 mL of medium, and then adjusted to a cell concentration of 4 x 10 5 cells / mL with the medium. 1 mL of the cell suspension is added to each well of a 24-well microplate, and the cells are incubated at 37°C in a 5% CO2 incubator for 16 hours to allow the cells to adhere and grow for Mycobacterium tuberculosis infection. Mycobacterium tuberculosis H 37 Rv is inoculated in 7H9 liquid medium (containing 10% OADC and 0.05% Tween-80), cultured at 37°C in a 5% CO2 environment for 21 days to the logarithmic growth phase, filtered with a microporous filter of 8 μm to obtain a single bacterial suspension, and the optical density (OD 570 value) of the single bacterial suspension is measured at 570 nm using a microplate reader. OD 570 0.1 corresponds to a bacterial concentration of 10 8 CFU / mL. The aforementioned adherent macrophages are infected with Mycobacterium tuberculosis at a multiplicity of infection (MOI) of 0.5:1, and after incubation at 37°C for 4 hours, the liquid medium is discarded, and the culture dishes are washed twice with sterile PBS to remove Mycobacterium tuberculosis that has not been infected and remains outside the macrophages. Then, 2 mL of freshly prepared RPMI 1640 medium (containing 10% fetal bovine serum) containing different concentrations of compounds is added to each well, and duplicate wells and negative control wells are set. The drugs are allowed to act for 3 days, the liquid medium is discarded, 200 μL of cell lysis solution (0.1% SDS) is added to each well, and after lysis at 37°C for 5 min, 800 μL of freshly prepared medium is added to neutralize the lysis, and 10-fold dilution is performed with normal saline. 100 μL of each of the different concentration dilutions is inoculated onto 7H10 solid culture plates, and the plates are incubated at 37°C in a 5% CO2 incubator for 3 weeks, and CFU counting is performed. The intracellular anti-tuberculosis activity of the compounds in mouse macrophages is evaluated according to the reduction in CFU in the experimental group relative to the negative control group.

[0525] Table 4. Intracellular anti-tuberculosis activity of some compounds in macrophages

[0526]

[0527] a Logarithmic growth of Mycobacterium tuberculosis (H 37 Rv) after infection in mouse J774A.1 macrophages10 CFU.

[0528] b Δlog 10 CFU=log 10 CFU (blank control group)-log 10 CFU (corresponding compound-treated groups).

[0529] As shown in Table 4, the compounds of the present invention exhibited strong intracellular anti-tuberculosis activity. Compound 43, compared with the blank control group, could reduce the expression of TB virus by 1.02 Log at 10 μg / mL. 10 CFU, stronger than the control compound TCA1 (reduced by 0.53 Log 10 CFU).

[0530] 4. Cytotoxicity test

[0531] Determination method: MTT assay. Experimental principle: Cell activity is determined by the reduction of oxidized 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (trade name: Thiazole Blue) / MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide] to an insoluble blue formazan compound through mitochondrial dehydrogenase (such as succinate dehydrogenase). The color development after dissolution with DMSO is used for determination. The conversion amount is positively correlated with the number of viable cells.

[0532] Experimental method: The prepared Vero cell suspension was inoculated in a 96-well plate at 50 μL / well. The highest test concentration of the test compound was 64 μg / mL. The test compound was serially diluted in a 96-well plate at a ratio of 1:3 with culture medium. The target compound was set at 6 concentrations, and 6 parallel wells were set at each concentration, 50 μL / well. At the same time, a cell control well without drug and a culture medium blank control well were set. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. MTT 10 μL / well was added and cultured for another 4 hours. The culture medium was discarded and 100 μL / well DMSO was added. After the formazan particles were completely dissolved, the optical density (OD) was measured at a wavelength of 570 nm. 570 ). Cell inhibition percentage (%) = [(cell control OD 570 Value - OD of the drug-dosing group 570 value) / (cell control OD 570 Value - Blank OD 570 The dose-response curve was fitted using Origin 7.0 software, and the concentration of the compound at which the cell inhibition rate was 50% (IC 50 ).

[0533] Table 5. Toxicity of some compounds to Vero cells

[0534]

[0535] As can be seen from the data in Table 5, the compounds of the present invention have low cytotoxicity and exhibit higher safety than the positive control compound TCA1.

[0536] 5. CYP 2C9 inhibition test

[0537] The test compound and the positive control compound V (0.05-50 μM) were mixed with human liver microsomes (0.1 mg / mL) and the CYP2C9 substrate diclofenac (Diclofenac) in a working solution and pre-incubated at 37 ° C for 5 minutes. Then, NADPH (1 mM) was added. After incubation for 10 minutes, the reaction was immediately terminated by adding a control solution containing an internal standard. After the sample was vortexed and centrifuged, the peak area ratio of the CYP 2C9 substrate product 4'-hydroxydiclofenac (4'-Hydroxydiclofenac) to the internal standard was detected by LC-MS / MS. The inhibition rate and IC at 0.5 μM were calculated based on the results. 50 .

[0538] Table 6. CYP 2C9 hepatic enzyme inhibition rate of some compounds at 0.5 μM

[0539]

[0540]

[0541] Table 7. CYP 2C9 hepatic enzyme inhibition IC values ​​of some compounds 50

[0542]

[0543] As can be seen from the data in Tables 6 and 7, the compounds of the present invention have low inhibitory effects on the CYP 2C9 hepatic enzyme. Among them, compounds 11, 13, 38, and 39 have significantly lower inhibitory effects on the CYP 2C9 hepatic enzyme than compound V, and their drugability is significantly improved.

[0544] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A compound represented by general formula (I) or a pharmaceutically acceptable salt thereof: in, R1 is selected from H, substituted or unsubstituted C1-C3 alkyl, F, Cl, Br, NO2; R2 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted morpholinyl; Ar is selected from substituted or unsubstituted phenyl, pyridyl, pyrimidinyl; The substituents of the substituted C1-C3 alkyl in R1, the substituted C3-C7 cycloalkyl, the substituted morpholinyl, and the substituted phenyl in R2 are each independently selected from the following groups: F, Cl, Br, nitro, cyano, and C1-C3 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the general formula (II): in, R1 is selected from H, substituted or unsubstituted C1-C3 alkyl, F, Cl, Br, NO2; R2 is selected from substituted or unsubstituted phenyl, substituted or unsubstituted C3-C7 cycloalkyl, substituted or unsubstituted morpholinyl; R3 is independently selected from F, Cl, Br, CN, NO2, NO, NH2; The substituents of the substituted C1-C3 alkyl in R1, the substituted C3-C7 cycloalkyl, the substituted morpholinyl, and the substituted phenyl in R2 are each independently selected from the following groups: F, Cl, Br, nitro, cyano, and C1-C3 alkyl.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the general formula (III): in, R1 is selected from H, F, Cl, Br; R2 is selected from substituted or unsubstituted C4-C6 cycloalkyl; The substituents of the C4-C6 cycloalkyl group in R2 may be selected from the following groups: F, Cl, Br, nitro, cyano, and C1-C3 alkyl.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by the general formula (IV): in, R2 is selected from substituted or unsubstituted cyclopentyl; The substituents of the cyclopentyl group in R2 may be selected from the following groups: F, Cl, Br, nitro, cyano, and C1-C3 alkyl.

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, which is selected from the following compounds:

6. A method for preparing the compound according to any one of claims 1 to 5, comprising the following steps: Compound A and compound B undergo a condensation reaction in the presence of a base and a condensing agent to obtain a compound represented by formula (I); Wherein, R1 and R2 are defined as any one of claims 1-5.

7. A pharmaceutical composition, characterized in that: The composition comprises a therapeutically and / or prophylactically effective amount of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and optionally one or more pharmaceutically acceptable excipients.

8. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 7, in the preparation of a decisopentenylphosphoyl β-D-ribose-2'-epimerase DprE1 inhibitor.

9. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 7, in the preparation of a medicament for treating and / or preventing infectious diseases caused by Mycobacterium tuberculosis.

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