A 3,4-dihydroquinolinone derivative and its application

Abstract

This invention discloses a 3,4-dihydroquinolineone derivative and its applications, belonging to the field of biomedical technology. Based on the structures of quinolineones such as Buchapine, Semecarpifoline, Peniprequinolone, and Penigequinolone B, which possess antibacterial activity, and combined with computer-aided design using the DprE1 enzyme, this invention simplifies the structure of natural products containing quinolineone cores, synthesizes and discovers compounds with anti-tuberculosis activity possessing 3,4-dihydroquinolineone structures. The obtained quinolineone derivatives exhibit good inhibitory activity against Mycobacterium tuberculosis and also show good inhibitory activity against clinically drug-resistant Mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a 3,4-dihydroquinolinone derivative and its applications. Background Technology

[0002] Tuberculosis (TB) is an infectious disease that has plagued humanity for thousands of years, ranking among the top ten causes of death worldwide and being the leading cause of death among single infectious diseases. TB is a serious infectious disease primarily caused by Mycobacterium tuberculosis. Most of the drugs currently used to treat TB were discovered between the 1940s and 1970s and have been in use for over half a century. Common anti-TB treatments include first-line drugs such as isoniazid (INH), rifampin (RFP), pyrazinamide (PZA), ethambutol (EMB), and streptomycin (SM); second-line drugs such as para-aminosalicylic acid (PAS), prothionamide (PTH), amikacin (AMK), capreomycin (CPM), ofloxacin (OXF), ethionamide (ETO), moxifloxacin (MFX), rifabutin (RFB), rifapentine (RPT), and cycloserine (CS), as well as bedaquiline, the first new mechanism anti-TB drug in nearly 50 years. However, tuberculosis treatment typically requires a combination of four or more first-line drugs, with a treatment course exceeding six months to achieve a cure. Second- or third-line drugs are used as adjuncts when first-line drugs prove ineffective. In recent years, due to the long-term use of traditional drugs and non-standard treatment practices, drug resistance has become increasingly serious in clinical practice. The emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and total drug-resistant (TDR) tuberculosis strains has rendered existing drugs and treatment regimens ineffective, even threatening a situation where no cure is available. The consequences of widespread transmission of these drug-resistant strains would be unimaginable. Therefore, the development of novel anti-tuberculosis drugs has become an urgent task for all of humanity.

[0003] The prevention and treatment of tuberculosis and the development of new drugs first require the discovery and elucidation of the molecular mechanisms of key physiological activities of its pathogens. Among these, the cell wall synthesis and energy metabolism processes of Mycobacterium tuberculosis are considered ideal entry points, as many anti-tuberculosis drugs and investigational drugs target these important physiological pathways. For example, first-line drugs isoniazid and ethambutol, and the clinical investigational drug SQ109, all exert their anti-tuberculosis effects by inhibiting cell wall synthesis. The marketed drug bedaquilin and the clinical investigational drug Q203 achieve their effect of killing Mycobacterium tuberculosis by inhibiting the respiratory chain of the energy metabolism pathway. Therefore, key proteins in the cell wall synthesis pathway are widely recognized as excellent drug targets. DprE1 enzyme, as a key synthase of arabinose, an important component of the cell wall, is an important target for anti-tuberculosis drugs. It can block the synthesis of arabinose, an essential component of the Mycobacterium tuberculosis cell wall, thereby killing the bacteria. This target is specific and highly effective. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a 3,4-dihydroquinolinone derivative and its application to prepare new drugs for the diagnosis and prevention of tuberculosis.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide a 3,4-dihydroquinolineone derivative with the structural formula shown in Formula I or II.

[0006]

[0007] Where X is C or N; X1 is H or F;

[0008] R1 is H or -OH or an ether bond or a long-chain ester group with 5 to 18 carbon atoms;

[0009] R2 is H or -OR4, R4 is a long-chain alkyl group with 5 to 18 carbon atoms, or a long-chain ester group with 5 to 18 carbon atoms, or an amino acid residue, or any one of Y1 to Y9.

[0010]

[0011] R5 is an alkyl group with 1 to 16 carbon atoms, or -PH(=O)OH; R6 is an alkyl group with 1 to 16 carbon atoms, or an acyl group with 1 to 5 carbon atoms; n is an integer from 0 to 10;

[0012] R3 is

[0013] A is a substituted aryl group, or a substituted heterocyclic group, or any one of pyridyl, thienyl, quinolinyl, isoquinolinyl, benzothienyl, quinoxolinyl, benzofuranyl, benzodioxacyclopenteneyl, benzoxazolyl, or benzimidazolyl; X2 is O, S, or N.

[0014] Based on the above technical solution, the present invention can be further improved as follows.

[0015] Furthermore, the 3,4-dihydroquinolinone derivative is any one of A1 to A13:

[0016]

[0017]

[0018] The present invention also discloses the use of the above-mentioned 3,4-dihydroquinolinone derivative in the preparation of medicaments for the diagnosis, prevention and / or treatment of tuberculosis.

[0019] The beneficial effects of this invention are:

[0020] In the screening of Mycobacterium tuberculosis inhibitors, this invention simplifies the structure of natural products containing quinoline ketone cores by combining computer-aided design with DprE1 enzyme, based on the structures of quinoline ketones such as Buchapine, Semecarpifoline, Peniprequinolone, and Penigequinolone B, which have antibacterial activity. This leads to the synthesis and discovery of compounds with anti-tuberculosis activity and 3,4-dihydroquinoline ketone structures.

[0021]

[0022] The 3,4-dihydroquinolinone synthesized in this invention differs significantly from known quinolinones (such as those disclosed in CN201580045974 and WO2016 / 031255A) in that it possesses a linker piperidine ring in the middle of the compound, and its A ring portion adopts a benzothiazine-type aromatic heterocyclic structure with stronger binding affinity to DprE1 enzymes. In this invention, the linker ring employs spirocyclic, fused, and halopiperidine structures. The benzene ring portion of the parent 3,4-quinolinone retains the substitution characteristics of the natural products Buchapine, Semecarpifoline, Peniprequinolone, and Penigequinolone B. The resulting quinolinone derivative exhibits good inhibitory activity against Mycobacterium tuberculosis and also shows good inhibitory activity against clinically drug-resistant Mycobacterium tuberculosis. Detailed Implementation

[0023] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0024] Example 1

[0025] A 3,4-dihydroquinolinone derivative, namely 5-{[1-(4-chloro-2,6-difluorophenyl)-4-hydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinolin-2(1H)-one (A1), has the following structural formula:

[0026]

[0027] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0028] (1) Preparation of 8-fluoro-5-hydroxy-2(1H)-quinolinone (a1), the structural formula of which is as follows:

[0029]

[0030] Under nitrogen protection, 3,3-dimethoxypropionic acid (9.5 g, 63 mmol) was added to a three-necked round-bottom flask containing reagent-grade toluene (95 mL), followed by N,N-diisopropylethylamine (14 mL, 1.25 eq, 79 mmol). The reaction mixture was then cooled to 0 °C, and trimethylacetyl chloride (9.7 mL, 1.25 eq, 79 mmol) was added dropwise over 5 minutes. After the addition was complete, the reaction mixture became turbid and a precipitate formed. The reaction mixture was then heated to room temperature and stirred for 4 hours. 3-Amino-4-fluorophenol (8.9 g, 63 mmol, 1 eq) solid was rapidly added to the reaction mixture at room temperature, and the heterogeneous reaction mixture was stirred overnight. HPLC analysis showed that N-(2-fluoro-5-hydroxyphenyl)-3,3-dimethoxypropionamide had formed. The reaction mixture was cooled to 0 °C again, and concentrated sulfuric acid (50 mL, 15 eq, 950 mmol) was added dropwise over 45 minutes via a feeding funnel. After the addition was complete, the reaction was brought back to room temperature and stirred for 45 minutes, during which the formation of two layers was clearly observed. The reaction mixture was then carefully transferred to a 500 mL Erlenmeyer flask containing cold water (~30V, 300 mL) and placed in an ice bath, resulting in precipitation. Additional water (10V, 100 mL) was added dropwise to the remaining viscous and oily sulfuric acid layer in the round-bottom flask under cooling conditions, and the resulting precipitate was mixed with the material in the Erlenmeyer flask. The mixture was stirred at room temperature for 30 minutes, the precipitate was filtered, and washed successively with water and toluene (5V each), then with MTBE (5V) and dried under vacuum at room temperature for 30 minutes. The funnel was placed in a beaker and dried in a vacuum oven at 60°C for 15 hours to give 12.8 g of a pale yellow solid with a purity of 66% and a yield of 75%. This crude product was added to a 9% sodium bicarbonate aqueous solution (130 mL, 10V) and stirred at room temperature for 1 hour. After the gas has completely escaped (about 40 minutes), the solid was collected by filtration and washed with water (2 × 20 mL). It was then dried in a vacuum oven at 60 °C under a gentle nitrogen flow for 15 hours to obtain 7.5 g of beige solid, with a total yield of 62%; melting point: 192–195 °C. 1 H NMR(400MHz,DMSO-d6)δ8.01(dd,J=9.8Hz,0.9Hz,1H),7.19(dd,J=10.9,8.8Hz,1H ), 6.52 (dd, J=8.8, 3.3Hz, 1H), 6.45 (d, J=9.8Hz, 1H); ESI-MS: m / z=180.0432[M+H] + .

[0031] (2) Preparation of 8-fluoro-5-acetoxy-2(1H)-quinolinone (a2), the structural formula of which is as follows:

[0032]

[0033] 12 mL of acetic anhydride and 2 g (10.4 mmol, 93% purity) of 8-fluoro-5-hydroxy-2(1H)-quinolinone were added to a 20 mL reaction flask and heated to 120 °C for 2 h. The reaction mixture was cooled to room temperature (precipitation occurred upon cooling) and poured into ice water at 10 °C. The mixture was stirred at this temperature for 1 h. The precipitate was collected by filtration and washed with water (3 °C) to give 2.1 g of a beige solid, 90% yield; melting point: 241–244 °C; ESI-MS: m / z = 224.0389 [M+H] + .

[0034] (3) Preparation of 8-fluoro-5-acetoxy-3,4-dihydro-2(1H)-quinolinone (a3), the structural formula of which is as follows:

[0035]

[0036] In a small high-pressure reactor, 10% Pd / C (20 mg, 10% purity) was added to 2 mL of acetic acid (10V) containing 0.2 g (0.832 mmol) of 8-fluoro-5-acetoxy-2(1H)-quinolinone. The vessel was purged with nitrogen and backfilled, and the mixture was then heated at 75°C for 8 hours under a hydrogen atmosphere at 60 psi (4 atm). The vessel was cooled to 40°C, the autoclave was purged, and the nitrogen atmosphere was replaced. The residue was filtered through 2% (w / w) diatomaceous earth. The filtrate was concentrated under reduced pressure to give 214 mg of white solid, 97% yield; melting point: 176–179°C; ESI-MS: m / z = 224.0389 [M+H] + .

[0037] (4) Preparation of 8-fluoro-5-hydroxy-3,4-dihydro-2(1H)-quinolinone (a4), the structural formula of which is as follows:

[0038]

[0039] 214 mg (0.95 mmol) of 8-fluoro-5-acetoxy-3,4-dihydro-2(1H)-quinolinone was added to 1 mL of MeOH (5 V) and 1 mL of concentrated hydrochloric acid (5 V) was added. The reaction mixture was heated at 100 °C for 1 hour. It was then cooled to 40 °C and water (2 mL) was added (precipitation occurred), and the mixture was stirred at 30 °C for 1 hour. The reaction mixture was then cooled to 0 °C and stirred for another 1 hour. The precipitate was collected by filtration and dried to give 113 mg of white solid, yield 79%; melting point: 192–194 °C. 1H NMR (400MHz, DMSO-d6) δ9.89 (s, 1H), 9.45 (s, 1H), 6.85 (t, J = 10.4Hz, 1H), 6.41 (dd, J = 8.9, 4.0Hz, 1H), 2.80 (t, J=7.8Hz, 2H), 2.44 (dd, J=7.8, 6.3Hz, 2H); ESI-MS: m / z=182.0330[M+H] + .

[0040] (5) Preparation of 8-(4-chloro-2,6-difluorophenyl)-1,4-dioxa-8-azaspiro[4.5]decane (a5), the structural formula of which is as follows:

[0041]

[0042] 1-Bromo-2,6-difluoro-4-chlorobenzene (940 mg, 4.13 mmol), sodium tert-butoxide (473 mg, 4.9 mmol), tris(diphenylmethyleneacetone)dipalladium (38 mg, 0.041 mmol), 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (77 mg, 0.12 mol), 1,4-dioxa-8-azaspiro[4.5]decane (590 μL, 4.6 mmol), and toluene (3 mL) were added to a microwave reaction tube. The tube was sealed and then irradiated with microwave at 130 °C for 1 hour. After the reaction was complete, water and ethyl acetate were added to the mixture and the layers were separated. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 10) to give 800 mg of light yellow crystals, yield 67%; melting point 72–75 °C. 1 H NMR (400MHz, CDCl3) δ6.91-6.84(m,2H),4.01(s,4H),3.24(t,J=5.6Hz,4H),1.84(t,J=5.6Hz,4H); ESI-MS: m / z=290.0317[M+H] + .

[0043] (6) Preparation of 1-(4-chloro-2,6-difluorophenyl)piperidin-4-one (a6), the structural formula of which is as follows:

[0044]

[0045] A solution of 8-(4-chloro-2,6-difluorophenyl)-1,4-dioxa-8-azaspiro[4.5]decane (800 mg, 2.76 mol) in acetone (20 mL) was added to 10 mL of 5 N hydrochloric acid, and the reaction mixture was heated under reflux for 3 hours. The acetone was removed by concentration under reduced pressure, and the residue was neutralized with 5 N sodium hydroxide, extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 10) to give 521 mg of a yellow solid, yield 76%; melting point 53–55 °C. 1 H NMR (400MHz, CDCl3) δ6.97–6.89(m,2H),3.47(t,J=6.0Hz,4H),2.59(t,J=6.0Hz,4H); ESI-MS: m / z=246.0310[M+H] + .

[0046] (7) Preparation of 6-(4-chloro-2,6-difluorophenyl)-1-oxa-6-azaspiro[2.5]octane (a7), the structural formula of which is as follows:

[0047]

[0048] Under nitrogen protection, dimethyl sulfoxide (6.8 mL) was added to a reaction flask containing trimethyl sulfoxide (550 mg, 2.49 mmol) and sodium tert-butoxide (240 mg, 2.49 mmol), and the reaction mixture was stirred at room temperature for 30 minutes. A solution of 1-(4-chloro-2,6-difluorophenyl)piperidin-4-one (516 mg, 2.1 mmol) in dimethyl sulfoxide (3 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 2.5 hours. Water was added to the reaction solution under ice cooling, followed by extraction with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 10) to give 415 mg of a yellow solid, 76% yield; melting point 55–58 °C. 1 H NMR(400MHz, CDCl3)δ6.93–6.85(m,2H),3.42–3.34(m,2H),3.25–3.19(m,2H), 2.72(s,2H),1.99–1.91(m,2H),1.66–1.60(m,2H); ESI-MS: m / z=260.0263[M+H] + .

[0049] (8) Preparation of 5-{[1-(4-chloro-2,6-difluorophenyl)-4-hydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinoline-2(1H)-one (A1)

[0050] Under nitrogen protection, 8-fluoro-5-hydroxy-3,4-dihydro-2(1H)-quinolinone (453 mg, 2.5 mmol), 6-(4-chloro-2,6-difluorophenyl)-1-oxa-6-azaspiro[2.5]octane (650 mg, 2.5 mmol), and potassium phosphate heptahydrate (846 mg, 2.5 mmol) were added to a 5 mL solution of N,N-dimethylformamide / isopropanol (1:1). After complete dissolution, the mixture was stirred overnight at 70 °C. After the reaction was complete, the mixture was cooled to room temperature, water was added to the reaction solution (precipitation occurred), and the mixture was stirred for another 10 minutes. The precipitate was collected by filtration and dried to give 710 mg of a white solid, yield 64%; melting point (ethyl acetate / methanol): 205–208 °C. 1 H NMR (400MHz, DMSO-d6) δ10.00(brs,1H),7.29–7.21(m,2H),7.01(t,J=9.7Hz,1H),6.58(dd,J=9.1,3.7Hz,1H),4.70(brs,1H),3.77(s,2H),3.40 –3.35(m,2H),2.99–2.96(m,2H),2.93(t,J=7.6Hz,2H),2.47(t,J=7.6Hz ,2H),1.81–1.78(m,2H),1.65–1.62(m,2H); ESI-MS: m / z=441.1204[M+H] + .

[0051] Example 2

[0052] A 3,4-dihydroquinolinone derivative, namely 5-{[1-(4-chloro-2,6-difluorophenyl)-1,2,3,6-tetrahydropyridin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinolin-2(1H)-one (A2), has the following structural formula:

[0053]

[0054] Under nitrogen protection, Burgess reagent (597 mg, 2.5 mmol) was added to a tetrahydrofuran (8 mL) solution of 5-{[1-(4-chloro-2,6-difluorophenyl)-4-hydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinoline-2(1H)-one (A1) (441 mg, 1 mmol). The reaction mixture was stirred at room temperature for 16.5 h, followed by stirring at 60 °C for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and water was added to the reaction solution (precipitation occurred). The precipitate was collected by filtration and dried to give 350 mg of a white solid, yield 82%; melting point: 218–220 °C.1 H NMR(400MHz,DMSO-d6)δ10.02(s,1H),7.34–7.26(m,3H),7.07–7.00(m,1H),5.88(brs,1H),4.48(s,2H),3.64(brs ,2H),3.29–3.24(m,2H),3.22–3.03(m,2H),2.89(t,J=7.6Hz,2H),2.27–2.23(m,2H); ESI-MS:m / z=423.0367[M+H] + .

[0055] Example 3

[0056] A 3,4-dihydroquinolinone derivative, namely (3R,4R)-1-(4-chloro-2,6-difluorophenyl)-4-{[(8-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}-4-hydroxypiperidin-3-yl-L-valine (A3), has the following structural formula:

[0057]

[0058] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0059] (1) Preparation of 5-{[(3R,4R)-1-(4-chloro-2,6-difluorophenyl)-3,4-dihydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinoline-2(1H)-one (b1), the structural formula of which is as follows:

[0060]

[0061] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0062] At room temperature, a solution of bis(dihydroquinolinidyl)phthalazine ((DHQD)2PHAL) (34.05 mg, 0.043 mmol) and potassium osmium(VI) dihydrate (4.03 mg, 0.011 mmol) in acetone-water (2:1) (20 mL) was stirred for 15 minutes. Under ice bath conditions, a solution of 4.8 M N-methylmorpholine-N-oxide (228 μL, 1.09 mmol) and 5-{[1-(4-chloro-2,6-difluorophenyl)-1,2,3,6-tetrahydropyridin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinolin-2(1H)-one (A2) (308 mg, 0.728 mmol) in acetone-water (2:1) was added. This mixture was stirred at room temperature for 1.5 days. After the reaction was complete, a saturated aqueous sodium sulfite solution was added to the reaction mixture, and the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate) and recrystallized from the residue with acetic acid / water. The precipitate was collected by filtration and dried under reduced pressure (60°C) to give 140 mg of a white solid (>99% ee), yield 42%; melting point: 193–195°C. 1 H NMR (400MHz, DMSO-d6) δ10.01(s,1H),7.30–7.22(m,2H),7.02(t,J=9.2Hz,1H),6.59(dd,J=9.2,3.7 Hz,1H),4.87(d,J=6.4Hz,1H),4.52(s,1H),4.03(d,J=8.8Hz,1H),3.76–3.70(m,1H),3.69(d,J=8.8 Hz,1H),3.34–3.30(m,1H),3.22(t,J=10.7Hz,1H),2.98(dd,J=10.7,4.5Hz,1H),2.93–2.86(m,3H), 2.50–2.45(m,2H),1.91(dt,J=13.4,4.7Hz,1H),1.70(d,J=13.4Hz,1H); ESI-MS:m / z=457.0349[M+H] + .

[0063] (2) Preparation of (3R,4R)-1-(4-chloro-2,6-difluorophenyl)-4-{[(8-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}-4-hydroxypiperidin-3-yl{[(9H-fluoren-9-yl)methoxy]carbonyl}-L-valine (b2), the structural formula of which is as follows:

[0064]

[0065] Fmoc-L-valine (30 mg, 0.088 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21 mg, 0.11 mmol), and 4-dimethylaminopyridine (2.5 mg, 0.0088 mmol) were added to a DMF / DCM (1:2, 1 mL) solution and stirred at room temperature for 10 minutes. Then, 5-{[(3R,4R)-1-(4-chloro-2,6-difluorophenyl)-3,4-dihydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinoline-2(1H)-one (b1) (40 mg, 0.088 mmol) were added to the reaction solution and reacted overnight at room temperature. After the reaction was completed, water and dichloromethane were added to the reaction solution and the layers were separated. The organic layer was washed successively with 5% hydrochloric acid, saturated sodium bicarbonate and brine, dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 2:1) to give 60 mg of white solid, yield 87%; melting point: 87-91℃.

[0066] (3) Preparation of (3R,4R)-1-(4-chloro-2,6-difluorophenyl)-4-{[(8-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}-4-hydroxypiperidin-3-yl-L-valine (A3)

[0067] (3R,4R)-1-(4-chloro-2,6-difluorophenyl)-4-{[(8-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}-4-hydroxypiperidin-3-yl{[(9H-fluorene-9-yl)methoxy]carbonyl}-L-valine (b2) (60 mg, 0.077 mmol) was added to a 20% solution of 4-methylpiperidin / dichloromethane. The mixture was stirred at room temperature for 40 minutes. The reaction solution was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol / triethylamine = 30:1:0.1%). The residue was washed with isopropyl ether, and the precipitate was collected by filtration to give 35 mg of white solid, yield 81%; melting point: 185–188 °C. 1H NMR (400MHz, CDCl3) δ7.91(s,1H),6.94–6.86(m,3H),6.44(dd,J=9.1,3.8Hz,1H),5.26(dd,J=9.9,5.0H z,1H),3.93–3.84(m,2H),3.48(t,J=10.8Hz,2H),3.35(d,J=4.9Hz,1H),3.26(dd,J=11.1,4.8Hz,1H),3. 09–2.99(m,3H),2.65(t,J=7.6Hz,2H),2.13(td,J=12.1,4.8Hz,1H),2.01(dd,J=12.1,6.7Hz,1H),1.97 –1.91(m,1H),1.71(brs,2H),0.95(d,J=6.8Hz,3H),0.87(d,J=6.8Hz,3H); ESI-MS:m / z=578.2639[M+Na] + .

[0068] Example 4

[0069] A 3,4-dihydroquinolinone derivative, namely (3R,4R)-1-(4-chloro-2,6-difluorophenyl)-4-{[(8-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}-4-hydroxypiperidin-3-yl stearate (A4), has the following structural formula:

[0070]

[0071] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0072] Stearic acid (0.088 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21 mg, 0.11 mmol), and 4-dimethylaminopyridine (2.5 mg, 0.0088 mmol) were added to a DMF / DCM (1:2, 1 mL) solution and stirred at room temperature for 10 minutes. Then, 5-{[(3R,4R)-1-(4-chloro-2,6-difluorophenyl)-3,4-dihydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinoline-2(1H)-one (b1) (40 mg, 0.088 mmol) were added to the reaction solution and reacted overnight at room temperature. After the reaction was completed, water and dichloromethane were added to the reaction solution and the layers were separated. The organic layer was washed successively with 5% hydrochloric acid, saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 2:1) to give 69 mg of white solid, yield 86%; melting point: 65-67℃. 1H NMR(400MHz, CDCl3)δ8.15(s,1H),6.92(d,J=9.1Hz,1H),6.90–6.85(m,2H),6.45(dd,J=9.1,3.9Hz,1H), 5.22(dd,J=10.0,5.1Hz,1H),3.91–3.82(m,2H),3.52–3.40(m,2H),3.26(dd,J=11.1,4.9Hz,1H),3.09–2. 99(m,3H),2.66(t,J=7.7Hz,2H),2.32(t,J=7.2Hz,2H),2.13(td,J=12.2,4.9Hz,1H),1.92(dt,J=13.8,2 .4Hz,1H),1.58(t,J=7.0Hz,2H),1.39–1.13(m,28H),0.89(t,J=6.8Hz,3H); ESI-MS:m / z=707.2571[M-17] + .

[0073] Example 5

[0074] A 3,4-dihydroquinolinone derivative, namely 1-(4-chloro-2,6-difluorophenyl)-4-{[(8-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}piperidin-4-yl stearate (A5), has the following structural formula:

[0075]

[0076] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0077] Stearic acid (85.2 mg, 0.3 mmol) was added to thionyl chloride (0.3 mL), and the mixture was refluxed for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove thionyl chloride, yielding an acyl chloride intermediate for later use. Under ice bath conditions, 60% sodium hydride (12 mg, 0.3 mmol) was added to a solution of 5-{[1-(4-chloro-2,6-difluorophenyl)-4-hydroxypiperidin-4-yl]methoxy}-8-fluoro-3,4-dihydroquinoline-2(1H)-one (A1) (110 mg, 0.25 mmol) in N,N-dimethylformamide (0.5 mL), and then the mixture was brought to room temperature and reacted for half an hour. Then, under ice bath conditions, a solution of the above acyl chloride intermediate in dichloromethane (0.5 mL) was slowly added dropwise to the above N,N-dimethylformamide reaction solution. After the addition was complete, the mixture was brought to room temperature and reacted for 4 hours. After the reaction was completed, water and dichloromethane were added to the reaction solution and the layers were separated. The organic layer was washed with brine, dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3:1) to give 120 mg of white solid, yield 67%; melting point: 75-78℃. 1 H NMR (400MHz, CDCl3) δ6.98(t,J=9.1,1H),6.94–6.87(m,2H),6.73(dd,J=9.1,3.7Hz,1H),3.88(s,2H),3.49(t,J=11.1Hz,2H),3 .12–3.05(m,4H),3.01–2.96(m,2H),2.69(dd,J=7.5,6.1Hz,2H),1.91–1.76(m,6H),1.31–1,24(m,28H),0.90(t,J=6.8Hz,3H).

[0078] Example 6

[0079] A 3,4-dihydroquinolinone derivative, namely 5-((1-(1-methyl-1H-pyrazole-4-carbonyl)-1,2,3,6-tetrahydropyridin-4-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (A6), has the following structural formula:

[0080]

[0081] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0082] (1) Preparation of tert-butyl 4-hydroxy-4-{[(2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}piperidine-1-carboxylic acid (c1), the structural formula of which is as follows:

[0083]

[0084] The preparation method is the same as A1, and 1.45 g of white solid was finally obtained, with a yield of 83%; melting point: 166-168℃; 1 H NMR (400MHz, CDCl3) δ8.46(brs,1H),7.13(t,J=8.1Hz,1H),6.57(d,J=8.3Hz,1H),6.48(d,J=7.9Hz,1H),3.87–4.04(m,2H),3.85(s,2H),3.1 8–3.31(m,2H),2.98(t,J=7.7Hz,2H),2.64(t,J=7.7Hz,2H),2.20–2.23(m,1H),1.66–1.78(m,4H),1.49(s,9H); ESI-MS: m / z=277.1137[M-99] + .

[0085] (2) Preparation of 4-{[(2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (c2), the structural formula of which is as follows:

[0086]

[0087] The synthesis method was the same as that for compound A2, yielding 2.2 g of a beige solid in 82% yield; melting point: 186–189 °C; ESI-MS: m / z = 360.1589 [M+H] + .

[0088] (3) Preparation of 5-[(1,2,3,6-tetrahydropyridin-4-yl)methoxy]-3,4-dihydroquinoline-2(1H)-one (c3), the structural formula of which is as follows:

[0089]

[0090] Compound 4-{[(2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (c2) (675 mg, 1.88 mmol) was added to 14 mL of hydrochloric acid / methanol (1:2) solution. The mixture was stirred at room temperature for 2 hours, and the solution became clear. After the reaction was complete, the mixture was concentrated under reduced pressure to give a yellow solid. This crude product was used directly in the next reaction without purification. ESI-MS: m / z = 259.1475 [M+H] + .

[0091] (3) Preparation of 5-((1-(1-methyl-1H-pyrazole-4-carbonyl)-1,2,3,6-tetrahydropyridin-4-yl)methoxy)-3,4-dihydroquinoline-2(1H)-one (A7)

[0092] 1-Methyl-4-pyrazolonic acid (126 mg, 1 mmol) was added to thionyl chloride (1 mL), and the mixture was refluxed and stirred for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove thionyl chloride, yielding an acyl chloride intermediate for later use. The acyl chloride intermediate was dissolved in dichloromethane (4 mL) and added dropwise under ice bath conditions to a solution of compound 5-[(1,2,3,6-tetrahydropyridin-4-yl)methoxy]-3,4-dihydroquinoline-2(1H)-one (c3) (258 mg, 1 mmol) and triethylamine (278 μL, 2 mmol) in dichloromethane (4.0 mL). The reaction solution was allowed to react overnight at room temperature. After the reaction was completed, dichloromethane and water were added to the reaction solution and the layers were separated. The organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane:methanol = 25:1) to give 194 mg of white solid, yield 53%; melting point: 213-215℃. 1 H NMR (400MHz, DMSO-d6) δ: 10.03 (s, 1H), 8.09 (s, 1H), 7.70 (s, 1H), 7.07 (t, J = 8 .1Hz,1H),6.62(d,J=8.3Hz,1H),6.50(d,J=7.9Hz,1H),5.84(brs,1H),4.48( s,2H),4.24–4.03(m,2H),3.86(s,3H),3.71(t,J=5.7Hz,2H),2.83(t,J=7.7H z,2H),2.42(t,J=7.7Hz,2H),2.29–2.18(m,2H); ESI-MS:m / z=367.1496[M+H] + .

[0093] Example 7

[0094] A 3,4-dihydroquinolinone derivative, namely 5-(((3R,4R)-3,4-dihydroxy-1-(1-methyl-1H-pyrazole-4-carbonyl)piperidin-4-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (A7), has the following structural formula:

[0095]

[0096] The synthesis method of the 3,4-dihydroquinolinone derivative in this embodiment is the same as that of compound b1; 50 mg of white solid was finally obtained, with a yield of 48%; melting point: 230-233 °C;1 H NMR (400MHz, DMSO-d6) δ: 10.02 (s, 1H), 8.06 (s, 1H), 7.67 (s, 1H), 7.08 (t, J = 8.1Hz, 1H), 6.58(d,J=8.3Hz,1H),6.49(d,J=7.9Hz,1H),5.07(d,J=5.1Hz,1H),4.70(s,1H),4.03(d, J=8.9Hz,1H),3.86(s,3H),3.72(d,J=8.9Hz,1H),3.62–3.56(m,1H),3.36–3.37(m,4H), 2.84–2.76(m,2H),2.41(t,J=7.7Hz,2H),1.76–1.67(m,2H); ESI-MS:m / z=401.1302[M+H] + .

[0097] Example 8

[0098] A 3,4-dihydroquinolinone derivative, namely 5-((1-(1H-pyrazole-4-carbonyl)-1,2,3,6-tetrahydropyridin-4-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (A8), has the following structural formula:

[0099]

[0100] The synthesis method of the 3,4-dihydroquinolinone derivative in this embodiment is the same as that of compound A6; 103 mg of white solid was finally obtained, with a yield of 40%; melting point: 236-240 °C; 1 H NMR (400MHz, DMSO-d6) δ: 13.20 (s, 1H), 10.02 (s, 1H), 8.11 (s, 1H), 7.76 (s, 1H) ),7.07(t,J=8.1Hz,1H),6.62(d,J=8.4Hz,1H),6.49(d,J=7.9Hz,1H),5.84(b rs,1H),4.48(s,2H),4.20–4.06(m,2H),3.72(t,J=5.7Hz,2H),2.83(t,J=7.7 Hz,2H),2.42(t,J=7.7Hz,2H),2.26–2.20(m,2H); ESI-MS:m / z=367.1496[M+H] + .

[0101] Example 9

[0102] A 3,4-dihydroquinolinone derivative, namely 5-((4-hydroxy-1-(1-methyl-1H-pyrazole-4-carbonyl)piperidin-4-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (A9), has the following structural formula:

[0103]

[0104] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0105] (1) Preparation of 1-(1-methyl-1H-pyrazole-4-carbonyl)piperidin-4-one (d1), the structural formula of which is as follows:

[0106]

[0107] The preparation method is as described in A6, and a white solid of 680 mg was finally obtained, with a yield of 54%; melting point: 122-124℃; 1 H NMR (400MHz, CDCl3) δ7.75 (s, 1H), 7.65 (s, 1H), 3.97 (t, J = 6.3Hz, 4H), 3.92 (s, 3H), 2.51 (t, J = 6.3Hz, 4H); ESI-MS: m / z = 208.0993 [M+H] + .

[0108] (2) Preparation of 1-methyl-1H-pyrazol-4-yl)(1-oxa-6-azaspiro[2.5]octane-6-yl) methyl ketone (d2), the structural formula of which is as follows:

[0109]

[0110] The preparation method is the same as described in a7. This crude product requires no purification and can be used directly in the next reaction. ESI-MS: m / z = 222.0870 [M+H] + .

[0111] (3) Preparation of 5-((4-hydroxy-1-(1-methyl-1H-pyrazole-4-carbonyl)piperidin-4-yl)methoxy)-3,4-dihydroquinoline-2(1H)-one (A10)

[0112] The preparation method is as described in A1, and 250 mg of white solid was finally obtained, with a yield of 65%; melting point: 206-208℃; 1H NMR (400MHz, DMSO-d6) δ10.02(s,1H),8.04(s,1H),7.66(s,1H),7.07(t,J=8.1Hz,1H),6.58(d,J=8.2Hz,1H),6.49(d,J=7.9Hz,1H), 4.86(s,1H),3.86(s,3H),3.78(s,2H),3.39–3.36(m,4H),2.86(t,J=7.7Hz,2H),2.42(t,J=7.7Hz,2H); ESI-MS:m / z=385.1887[M+H] + .

[0113] Example 10

[0114] A 3,4-dihydroquinolinone derivative, namely 5-((4-(benzyloxy)-1-(1H-pyrazol-4-carbonyl)piperidin-4-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (A10), has the following structural formula:

[0115]

[0116] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0117] (1) Preparation of tert-butyl 4-(benzyloxy)-4-(((2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy)methyl)piperidine-1-carboxylic acid (e1), the structural formula of which is as follows:

[0118]

[0119] Under ice bath conditions, 50% sodium hydride (195 mg, 4.07 mmol) was added to a solution of 4-hydroxy-4-{[(2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy]methyl}piperidine-1-carboxylic acid tert-butyl ester (c1) (1.4 g, 3.71 mmol) in N,N-dimethylformamide (8 mL) and stirred for 15 min. Benzyl bromide (500 μL, 4.2 mmol) was added to the mixture, and the reaction mixture was then brought to room temperature and stirred for 1 h. Under ice cooling, a saturated aqueous solution of ammonium chloride was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 10–1 / 1) to give 1.36 g of a yellow oil, 78% yield; ESI-MS: m / z = 411.1974 [M-55] + .

[0120] (2) Preparation of 5-((4-(benzyloxy)piperidin-4-yl)methoxy)-3,4-dihydroquinoline-2(1H)-one (e2), the structural formula of which is as follows:

[0121]

[0122] Trifluoroacetic acid (1.5 mL, 19.7 mmol) was added to a solution of tert-butyl piperidine-1-carboxylate (e1) (920 mg, 1.97 mmol) in dichloromethane (4 mL). The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, the reaction mixture was slowly added to a saturated sodium carbonate solution and the layers were separated. The aqueous layer was extracted with dichloromethane, and the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 630 mg of white solid, yield 87%; melting point: 176–180 °C. 1 H NMR (400MHz, DMSO-d6) δ7.32–7.28(m,2H),7.23–7.18(m,3H),7.07(t,J=8.3Hz,1H) ,6.67(d,J=8.3Hz,1H),6.57(d,J=8.3Hz,1H),5.21(s,1H),5.14(s,2H),3.80(s,2H ),3.24–3.20(m,2H),3.16–3.10(m,2H),2.96(t,J=7.5Hz,2H),2.67(t,J=7.5Hz,2H ),1.95(td,J=13.9,4.7Hz,2H),1.72(d,J=13.6Hz,2H); ESI-MS:m / z=367.1336[M+H] + .

[0123] (3) Preparation of 5-((4-(benzyloxy)-1-(1H-pyrazole-4-carbonyl)piperidin-4-yl)methoxy)-3,4-dihydroquinoline-2(1H)-one (A10)

[0124] The preparation method is as described in A6, and 40 mg of white solid was finally obtained, with a yield of 50%; melting point: 210-212℃; 1H NMR (400MHz, DMSO-d6) δ13.16(s,1H),8.04(s,1H),7.76(s,1H),7.30(t,J=7.4Hz, 2H),7.21(t,J=7.5Hz,3H),7.06(t,J=8.3Hz,1H),6.66(d,J=8.3Hz,1H),6.56(d,J =8.3Hz,1H),5.13(s,2H),4.89(s,1H),3.79(s,2H),3.41–3.39(m,4H),2.94(t,J= 7.5Hz, 2H), 2.66 (t, J=7.5Hz, 2H), 1.69–1.63 (m, 4H); ESI-MS: m / z=461.2200[M+H] + .

[0125] Example 11

[0126] A 3,4-dihydroquinolinone derivative, namely 8-nitro-2-(4-(((2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy)methyl)-3,6-dihydropyridin-1(2H)-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (A11), has the following structural formula:

[0127]

[0128] The 3,4-dihydroquinolinone derivative in this embodiment was prepared through the following steps:

[0129] (1) Preparation of 2-chloro-3-nitro-5-trifluoromethylbenzoic acid (f1), the structural formula of which is as follows:

[0130]

[0131] 2-Chloro-5-trifluoromethylbenzoic acid (1 g, 4.45 mmol) was added to 50 mL of concentrated sulfuric acid, followed by the addition of potassium nitrate (900 mg, 8.9 mmol) at 0 °C. The mixture was stirred at 90 °C for 3 hours. After the reaction was complete, the mixture was cooled to room temperature and poured into ice water, resulting in the precipitation of a large amount of solid. The solid was filtered and washed with ice water to give 1.1 g of a white solid, yield 91%; melting point: 168–170 °C. 1 H NMR (400MHz, DMSO-d6) δ8.70 (s, 1H), 8.39 (s, 1H).

[0132] (2) Preparation of a 3,4-dihydroquinolinone derivative, which is 8-nitro-2-(4-(((2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy)methyl)-3,6-dihydropyridin-1(2H)-yl)-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (A11).

[0133] Oxaloyl chloride (212 μL, 2.5 mmol) and a catalytic amount of N,N-dimethylformamide were added sequentially to a suspension of 2-chloro-3-nitro-5-trifluoromethylbenzoic acid (f1) (269 mg, 1 mmol) in dichloromethane (3 mL). The reaction mixture was stirred at room temperature for 2 hours and the solvent was evaporated under vacuum. The residue was dissolved in dichloromethane (4 mL) and added dropwise to ammonium thiocyanate (153 mg, 2 mmol) with stirring. Then, a catalytic amount of polyethylene glycol 400 was added to the suspension and stirred at room temperature for 1.5 hours. After the starting material was consumed, 5-[(1,2,3,6-tetrahydropyridin-4-yl)methoxy]-3,4-dihydroquinoline-2(1H)-one (c3) (258 mg, 1 mmol) was added, and the mixture was stirred at room temperature for another 2 hours. The reaction mixture was then diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 1) to give 175 mg of a yellow solid, yield 33%; melting point: 249–252 °C. 1 H NMR (600MHz, DMSO-d6) δ10.03(s,1H),8.85(s,1H),8.80(s,1H),7.06(t,J=8.2Hz,1H),6.63(d,J=8.2Hz,1H),6.49(d,J=7.9Hz,1H),5.94(s,1H) ,4.53(s,2H),4.49–4.32(m,2H),4.17–3.97(m,2H),2.84(t,J=7.8Hz,2H ),2.42(t,J=7.8Hz,2H),2.40–2.29(m,2H); ESI-MS:m / z=533.1147[M+H] + .

[0134] Example 12

[0135] A 3,4-dihydroquinolinone derivative, namely 2-((3R,4R)-3,4-dihydroxy-4-(((2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (A12), has the following structural formula:

[0136]

[0137] The synthesis method was the same as that for compound b2; 8 mg of a yellow solid was finally obtained, with a yield of 45%; melting point: 172–175 °C; ESI-MS: m / z = 567.1199 [M+H] + .

[0138] Example 13

[0139] A 3,4-dihydroquinolinone derivative, namely 2-(4-(benzyloxy)-4-(((2-oxo-1,2,3,4-tetrahydroquinolin-5-yl)oxy)methyl)piperidin-1-yl)-8-nitro-6-(trifluoromethyl)-4H-benzo[e][1,3]thiazin-4-one (A13), has the following structural formula:

[0140]

[0141] The synthesis method is the same as that for compound A11; the final yield was 50 mg of yellow solid, with a yield of 40%. 1 H NMR (400MHz, DMSO-d6) δ8.85(d,J=1.9Hz,1H),8.80(d,J=1.9Hz,1H),7.29( t,J=7.4Hz,2H),7.20(t,J=7.7Hz,3H),7.06(t,J=8.3Hz,1H),6.67(d,J=8.3 Hz,1H),6.56(d,J=8.3Hz,1H),5.13(s,2H)5.12(s,1H),3.84(s,2H),3.35–3 .33(m,4H),2.94(t,J=7.5Hz,2H),2.64(t,J=7.5Hz,2H),1.91–1.74(m,4H). ESI-MS: m / z = 641.1674 [M+H] + .

[0142] Experimental Example

[0143] Example of anti-tuberculosis activity test: The experimental method used was microplate susceptibility testing, and the test strain was Mycobacterium tuberculosis H. 37 R v .

[0144] Experimental steps

[0145] 1. Dispensing medication:

[0146] The test compound was completely dissolved in DMSO to prepare a stock solution (1 mg / mL), filtered to remove bacteria, stored at -80°C, and aliquoted for use. It was then serially diluted with Mycobacterium tuberculosis Middlebrook 7H9 broth, and 100 μl / well was added to each well of a 96-well plate immediately before use. The final concentrations were: 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.0156 μg / mL.

[0147] Positive control drugs: Rifampin (R) and isoniazid (H) were provided by our laboratory. The final concentrations were: 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, and 0.0078 μg / mL, as shown in Table 1.

[0148] Table 1. Drug Concentration Table

[0149]

[0150] Mycobacterium tuberculosis:

[0151] Take H during the logarithmic growth phase 37 Rv, turbidity of ground bacteria to OD600 = 1.0 (approximately 5 × 10⁻⁶) 6 Take 200 μl of the test compound and dilute it in 10 ml of culture medium. Then, add 100 μl / well (about 104 CFU / well) to the culture plate using a continuous pipette. The control group is added with the same amount of bacteria as the test compound (D100), 1 / 10 of the amount of bacteria (D10), and 1 / 100 of the amount of bacteria (D1).

[0152] 2. Experimental Design

[0153] Taking a 96-well plate arrangement as an example:

[0154] compound 1 2 3 4 5 6 7 8 9 10 11 12 a3 A 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D100 a3 B 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D100 b3 C 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D10 b3 D 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D10 c3 E 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D10 c3 F 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D1 a1 G 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D1 a1 H 4 2 1 0.5 0.25 0.125 0.0625 0.03125 0.0156 0.0078 0.0039 D1

[0155] The remaining well plates were arranged similarly to the previous example, and the incubation period was 21 days.

[0156] 3. Results Observation

[0157] The culture was carried out at 37°C. Results were interpreted when obvious bacterial plaque growth was observed in the control wells with a 1% inoculum. Colony growth was observed in each group, and the lowest concentration of the drug group with no colony growth was taken as the MIC value of the test compound for that strain.

[0158] The experimental results were obtained by visually observing the growth of Mycobacterium tuberculosis in liquid culture medium in a 96-well plate for 21 days under the action of the drug, and comparing it with the control groups with the same inoculum amount of 100%, 10%, and 1%.

[0159] In this experiment, after 21 days of culture, the control strains with 100%, 10%, and 1% bacterial counts showed significant growth, with the 1% bacterial count showing the largest growth and a certain gradient in plaque size.

[0160] The results are shown in Table 2 below.

[0161] Table 2. In vitro anti-tuberculosis activity of compounds

[0162]

[0163]

[0164] Note: 1. 90% inhibition is defined as smaller plaques compared to the control group with an inoculum of 10%; 99% inhibition is defined as smaller plaques compared to the control group with an inoculum of 1%; 100% inhibition concentration is defined as the minimum concentration at which no plaques are visible to the naked eye.

[0165] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. The use of 3,4-dihydroquinolinone derivatives with the structural formula shown in Formula A3 in the preparation of drugs for the diagnosis, prevention and / or treatment of tuberculosis: 。

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