WO2021088753A1

WO2021088753A1 - Benzothiazole compounds, and preparation method therefor and use thereof

Info

Publication number: WO2021088753A1
Application number: PCT/CN2020/125774
Authority: WO
Inventors: 刘吉元; 张雅林; 代焕琴
Original assignee: 西北农林科技大学
Priority date: 2019-11-07
Filing date: 2020-11-02
Publication date: 2021-05-14
Also published as: CN112778297A; CN112778297B

Abstract

Disclosed are benzothiazole compounds, and a preparation method therefor and the anti-tuberculosis use thereof. The benzothiazole compounds are as shown below, and are highly effective, have a low toxicity and are stable anti-tuberculosis drugs. (I)

Description

Benzothiazole compounds and preparation method and application thereof

Technical field

The invention belongs to the field of medicine, and specifically relates to a benzothiazole compound, a preparation method thereof, and an anti-tuberculosis application.

Background technique

Tuberculosis (Tuberculosis, TB) is the disease with the highest mortality rate among infectious diseases caused by a single cause in the world. The World Health Organization has listed tuberculosis as a disease that seriously endangers global public health. The current first-line anti-tuberculosis drugs used in clinical practice generally have the defects of long treatment cycle, large toxic side effects, and extensive drug resistance, which can no longer meet the demand for cure. Therefore, it is very urgent to develop new mechanisms of action and anti-tuberculosis drugs with novel frameworks.

TB is caused by Mycobacterium tuberculosis (MTB). The cell wall plays a vital role in the survival and reproduction of the bacteria, and hindering the formation of MTB cell wall has become the main idea of current anti-tuberculosis drug development. Anti-tuberculosis drugs such as isoniazid, ethambutol, cycloserine are synthesized against the cell wall. Developed for related channels. In MTB, arabinose is a synthetic raw material for arabinogalactan, an important component of cell wall. As an important precursor for arabinose synthesis, the synthesis of DPA (Decaprenyl-Phosphoryl-D-Arabinose) requires the participation of decisopentenyl phosphoryl β-D-2'-epimerase 1 (DprE1). After the activity of DprE1 is inhibited, it can effectively block the synthesis of DPA, thereby blocking the synthesis of arabinose, so that the cell wall of MTB cannot be synthesized normally, achieving the purpose of killing MTB. DprE1 enzyme only exists in the pathogen MTB, and there is no related homologous protein in human host cells. It has become a new anti-tuberculosis target.

According to the mechanism of action, inhibitors targeting DprE1 can be roughly divided into two categories: covalent binding and non-covalent binding. Most covalent binding inhibitors covalently bind to the Cys387 active site in DprE1, resulting in irreversible inactivation of DprE1. However, a series of mutations (Cys387Ser, Cys387Gly, Cys387Ala, etc.) of DprE1 at the Cys387 site have greatly increased the resistance of MTB to covalent inhibitors. The emergence of DprE1 non-covalent binding inhibitors solves the disadvantages of covalent binding inhibitors represented by benzothiazinones (BTZs). In May 2013, the first inhibitor TCA1(ethyl(2-(benzo[d]thiazole-2-carboxamido)thiophene-3-carbonyl)carbamate that non-covalently binds to DprE1, CAS No.: 864941-32- 2) It was found that the chemical structure of TCA1 can be found in patent WO2014/190199Al (Table A, Cpd ID 1). This compound shows good bactericidal activity against replicating, non-replicating and drug-resistant MTB, which is not available in BTZs of. The excellent activity of TCA1 against various types of MTB strains and the unique mechanism of action make it an excellent anti-tuberculosis lead compound. However, there are few studies on the modification of the structure of TCA1. In addition, the current clinical use of first-line anti-tuberculosis treatment The drug rifampin generally has the defects of long treatment cycle, large side effects and extensive drug resistance. Therefore, there is still a need for high-efficiency, low-toxicity, and stable anti-tuberculosis drugs.

Summary of the invention

The emergence of TCA1, a non-covalent binding inhibitor targeting DprE1, provides a new direction for the treatment of drug-resistant MTB and the development of DprE1 inhibitors. The present invention proposes a class of benzothiazole TCA1 derivatives, which have superior performance on DprE1 activity inhibition, drug resistance and non-drug resistance MTB strain inhibition, and cytotoxicity, and can be used for anti-tuberculosis drugs. The present invention is a more efficient, low-toxic and stable anti-tuberculosis drug unexpectedly discovered in the reasonable optimization of TCA1.

Therefore, an object of the present invention is to provide a benzothiazole compound of the following general formula (I) or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for preparing the benzothiazole compound of the following general formula (I) or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide the use of the benzothiazole compound of the following general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of medicines.

The present invention provides a benzothiazole compound of the following general formula (I) or a pharmaceutically acceptable salt thereof,

Among them, R1 can be:

Preferably, R1 can be:

More preferably, R1 can be:

In a specific embodiment, the benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof is the following compound or a pharmaceutically acceptable salt thereof:

In the most preferred embodiment, the benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof is the following compound or a pharmaceutically acceptable salt thereof:

The present invention provides a preparation method of a benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof, which comprises the following steps:

(1) Synthesis of compound B: Add cyanoacetic acid, R1-substituted methyl carbamate compound A and acetic anhydride into the reaction vessel, heat up the oil bath, and protect with Ar. After the reaction is complete, pour the reaction solution into ice water Extract with ethyl acetate, wash with saturated sodium bicarbonate solution once, dry and concentrate to obtain compound B;

(2) Synthesis of compound C: Add compound B, 2,5-dihydroxy-1,4-dithiane, methanol, and morpholine to the reaction vessel, and the reaction is complete in 2 hours in an oil bath, directly pass through the column, and eluate. To obtain compound C;

(3) Synthesis of compound (I): add 2-formic acid benzothiazole and compound C to the reaction vessel, add pyridine and stir to dissolve it, add phosphorus oxychloride dropwise, and add dichloromethane to the system after fully stirring Mix with water, quickly stir to separate the organic layer, wash once with saturated sodium bicarbonate solution, directly pass through a short silica gel column, and elute to obtain compound (I); alternatively, add 2-formic acid benzothiazole and dichloride to the reaction vessel Methane, cool to 0°C, add oxalyl chloride dropwise, react for several hours, add pyridine to the system, stir for several minutes, add compound C, react for several hours, add methanol to the system to quench the reaction, wash once with saturated sodium bicarbonate solution, The sample is directly mixed through a silica gel column and eluted to obtain compound (I).

In addition, the present invention provides the use of the benzothiazole compound of the general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of anti-tuberculosis drugs.

Preferably, it is the use of a benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an anti-tuberculosis drug as a DprE1 inhibitor.

Preferably, it is the use of a benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an anti-tuberculosis drug as an H37Rv inhibitor.

Preferably, it is the use of a benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an anti-tuberculosis drug as a rifampin-resistant H37Rv inhibitor.

Preferably, it is the use of a benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an anti-tuberculosis drug as a BCG inhibitor.

Preferably, it is the use of a benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an anti-tuberculosis drug targeting DprE1.

Preferably, it is the use of a benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of an anti-tuberculosis drug with low toxicity.

Description of the drawings

^{Figure 1 is a 1} H-NMR spectrum of the compound TCA1 in an experimental example of the present invention dissolved in deuterated DMSO.

Figure 2 is a mass spectrum of the compound TCA1 involved in an experimental example of the present invention.

Figure 3 is a graph showing the inhibition of the activity of the compounds of the present invention LZDT1 and TCA1 on Mycobacterium tuberculosis DprE1.

Figure 4 is a graph showing the bactericidal curve of the compounds of the present invention LZDT1 and TCA1 against Mycobacterium tuberculosis H37Rv.

Figure 5 is a graph showing the bactericidal curve of the compounds of the present invention LZDT1, TCA1 and rifampicin against Mycobacterium tuberculosis H37Rv.

Figure 6 is a graph showing the sterilization curves of compounds LZDT1, TCA1 and rifampicin of the present invention against rifampicin-resistant H37Rv strains.

Figure 7 is a graph showing the bactericidal curves of the compounds LZDT1, TCA1 and rifampicin against BCG strains of the present invention.

Figure 8 is a graph showing the bactericidal curve of the compounds LZDT1, TCA1 and rifampicin against BCG (DprE1 overexpression) strains.

Figure 9 is the toxicity curve of the compound LZDT1 of the present invention on human liver cancer cells HepG2.

Figure 10 shows the toxicity curve of TCA1 on human liver cancer cells HepG2.

Figure 11 shows the toxicity curve of TCA1 on human neuroblastoma cells SH-SY5Y.

Figure 12 is the toxicity curve of compound LZDT1 of the present invention on human neuroblastoma cell SH-SY5Y.

Figure 13 is the toxicity curve of compound LZDT2 of the present invention on human neuroblastoma cell SH-SY5Y.

Figure 14 shows the toxicity curve of TCA1 on human embryonic kidney cells HEK293.

Figure 15 is the toxicity curve of the compound LZDT1 of the present invention on human embryonic kidney cells HEK293.

Figure 16 is the toxicity curve of the compound LZDT2 of the present invention on human embryonic kidney cells HEK293.

Detailed ways

The present invention will be explained by the following examples, but the present invention is not limited thereto. The experimental methods shown in the following examples are conventional methods unless otherwise specified. The reagents and materials shown are all commercially available products.

Preparation Example 1 Preparation of compound LZDT1

Add 2.3g of cyanoacetic acid, 3g of compound A1 (cyclopropylmethyl carbamate) and 5mL of acetic anhydride into a 25mL single-necked flask. The oil bath is heated to 85-90℃, protected by Ar, and the reaction is complete for 3-4h. The reaction solution was poured into ice water for extraction with ethyl acetate, washed once with saturated sodium bicarbonate solution, dried and concentrated to obtain 1.5 g of red-brown solid compound B1.

Add compound B1 1g, 2,5-dihydroxy-1,4-dithiane 0.42g, methanol 12mL, and morpholine 0.53g to a 100mL single-necked flask, oil bath 65℃, 2h reaction is complete, directly pass the column, EA: PE=1:2 eluted to obtain 210 mg of solid compound C1.

Add 166mg of 2-formic acid benzothiazole and 8mL of dichloromethane to a 25mL two-necked flask, cool at 0°C, add 129mg of oxalyl chloride dropwise, and react for 2h. Add 175mg of pyridine to the system, stir for 10min and then add compound C1 212mg, react for 3h 2mL methanol was added to the system to quench the reaction, washed once with 10mL saturated sodium bicarbonate solution, and directly mixed through the column, EA:PE=1:2 eluted to obtain 35mg of pure solid compound LZDT1, the yield was 9.9%.

Alternatively, add 60 mg of 2-formic acid benzothiazole and compound C1 80.5 mg to a 25 mL two-necked flask, add 1 mL of pyridine and stir to clear, add 0.05 mL of phosphorus oxychloride dropwise at -5°C, stir for 1 hour and add dichloromethane to the system 5mL and 1mL of water, quickly stir to separate the organic layer and wash once with saturated sodium bicarbonate solution, dry and concentrate directly to mix the sample through a silica gel column, EA: PE=1: 2 elution to obtain 15mg of pure solid compound LZDT1, the yield is 15 %.

¹ H NMR (400MHz, DMSO-d ₆ ) δ 13.10 (s, 1H), 10.93 (s, 1H), 8.32 (d, J = 7.9 Hz, 2H), 7.81 (d, J = 5.9 Hz, 1H) ,7.68(dt,J＝19.1,7.3Hz,2H),7.21(d,J＝5.8Hz,1H),4.03(d,J＝7.3Hz,2H),1.27–1.14(m,1H),0.59( h,J＝4.5Hz,2H),0.38(t,J＝4.8Hz,2H).MS(ESI)cacld.for C ₁₈ H ₁₅ N ₃ O ₄ S ₂ 401.97[M+1] ⁺ .

Preparation Example 2 Preparation of compound LZDT2

Add 2.7 g of cyanoacetic acid, 3.9 g of compound A2 (cyclobutyl methyl carbamate), and 8 mL of acetic anhydride into a 50 mL single-necked flask. The oil bath is heated to 85-90°C, protected by Ar, and the reaction is complete for 3-4 hours. The liquid was poured into ice water and extracted with ethyl acetate, washed once with saturated sodium bicarbonate solution, dried and concentrated to obtain 2.2 g of red-brown solid compound B2.

Add compound B2 1g, 2,5-dihydroxy-1,4-dithiane 0.39g, methanol 10mL, and morpholine 0.49g to a 100mL single-necked flask, oil bath 65℃, 2h reaction is complete, directly pass the column, EA: PE=1:2 eluted to obtain 0.4 g of solid compound C2.

Add 74 mg of 2-formic acid benzothiazole and 105 mg of compound C2 to a 25 mL two-neck flask, add 1 mL of pyridine and stir to clear, add 0.05 mL of phosphorus oxychloride dropwise at -5°C, stir for 1 hour, add 5 mL of dichloromethane and 1 mL of water to the system , Quickly agitate and separate the organic layer, wash it once with saturated sodium bicarbonate solution, and directly pass through a short silica gel column, eluting with EA:PE=1:3 to obtain 5 mg of pure solid compound LZDT2, with a yield of 4.16%.

¹ H NMR (400MHz, Chloroform-d) δ 13.03 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.94 (s, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.13 (d, J = 5.9 Hz, 1H), 6.96 (d, J = 5.8 Hz, 1H), 4.26 ( d,J=7.0Hz,2H),2.71(p,J=7.4Hz,1H), 2.15–2.08(m,2H),2.02–1.9(m,2H),1.88–1.78(m,2H).MS (ESI)cacld.for C ₁₉ H ₁₇ N ₃ O ₄ S ₂ 416.1[M+1] ⁺ .

Preparation Example 3 Preparation of compound LZDT3

Add 0.62 g of cyanoacetic acid, 1 g of compound A3 (cyclopentyl methyl carbamate) and 8 mL of acetic anhydride to a 50 mL single-necked flask. The oil bath is heated to 85-90°C, protected by Ar, and the reaction is complete for 3-4 hours. The reaction solution was poured into ice water for extraction with ethyl acetate, washed once with saturated sodium bicarbonate, dried and concentrated to obtain 1.5 g of off-white solid compound B3.

Add compound B3 1.34g, 2,5-dihydroxy-1,4-dithiane 0.485g, methanol 13mL and morpholine 0.61g to a 100mL single-necked flask, oil bath 65℃, 2h reaction complete, directly pass the column, EA : PE=1:2 eluted to obtain 0.85 g of solid compound C3.

Add 16.7 mg of 2-formic acid benzothiazole and compound C3 25 mg to a 25 mL two-neck flask, add 1 mL of pyridine and stir to clear, add 0.05 mL of phosphorus oxychloride dropwise at -5°C, stir for 1 hour, add 5 mL of dichloromethane and water to the system 1 mL, quickly stirred and separated the organic layer, washed once with saturated sodium bicarbonate solution, directly passed through a short silica gel column, eluted with EA:PE=1:3 to obtain 3 mg of pure solid compound LZDT3, with a yield of 7.5%.

¹ H NMR (400MHz, Chloroform-d) δ 13.03 (s, 1H), 8.27 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.65–7.51 (m, 2H), 7.11 (d, J = 6.0 Hz, 1H), 6.96 (d, J = 5.9 Hz, 1H), 4.17 (d, J = 7.2 Hz, 2H), 2.30 (p, J ＝7.7Hz,1H),1.80(m,2H)1.64(dd,J＝12.8,7.3Hz,3H),1.26(m,3H).MS(ESI)cacld.for C ₂₀ H ₁₉ N ₃ O ₄ S ₂ 430.1[M+1] ⁺ .

Preparation Example 4 Preparation of compound LZDT4

Add 1.39 g of cyanoacetic acid, 2.48 g of compound A4 (cyclohexyl methyl carbamate) and 8 mL of acetic anhydride to a 50 mL single-necked flask. The oil bath is heated to 85-90°C, protected by Ar, and the reaction is complete for 3 to 4 hours. The reaction solution was poured into ice water and extracted with ethyl acetate, washed once with saturated sodium bicarbonate, dried and concentrated to obtain 1.2 g of red-brown solid compound B4.

Add 0.957g of compound B4, 0.325g of 2,5-dihydroxy-1,4-dithiane, 10mL of methanol, and 0.41g of morpholine to a 100mL single-necked flask. The reaction is complete in an oil bath at 65°C for 2h, and then directly pass the column, EA : PE=1:2 eluted to obtain 0.3 g of solid compound C4.

Add 68 mg of 2-formic acid benzothiazole and 100 mg of compound C4 to a 25 mL two-neck flask, add 1 mL of pyridine and stir to clear, add 0.05 mL of phosphorus oxychloride dropwise at -5°C, stir for 1 hour, add 5 mL of dichloromethane and 1 mL of water to the system The organic layer was separated by rapid stirring, washed once with saturated sodium bicarbonate solution, and directly passed through a short silica gel column, eluted with EA:PE=1:3 to obtain 7 mg of pure solid compound LZDT4, with a yield of 4.45%.

¹ H NMR (400MHz, Chloroform-d) δ 13.03 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.89 (s, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 5.9 Hz, 1H), 6.96 (d, J = 5.9 Hz, 1H), 4.10 ( d, J=6.4 Hz, 2H), 1.84-1.73 (m, 9H), 1.25 (m, 2H). MS(ESI)cacld.for C ₂₁ H ₂₁ N ₃ O ₄ S ₂ 444.0[M+1] ⁺ .

Experimental Example 1 Inhibition experiment of the compound of the present invention on Mycobacterium tuberculosis (MTB) DprE1

Experiment: Dissolve DprE1, flavin adenine dinucleotide (FAD), horseradish peroxidase (HRP), and Amplex Red in 50mM glycine buffer (pH 8.0, containing 200mM potassium glutamate, 0.002% Brij-35, 1% DMSO) to make the final concentrations 300 nM, 1 μM, 0.2 μM, and 50 μM, respectively. Mix evenly, transfer an appropriate volume into a 384-well blackboard (Corning, Item No. 3573). Compound TCA1 was purchased from Life Chemicals Inc. (ethyl(2-(benzo[d]thiazole-2-carboxamido)thiophene-3-carbonyl)carbamate, CAS No.: 864941-32-2), and its ¹ H-NMR spectrum The graphs and mass spectra are shown in Fig. 1 and Fig. 2, which are the same in the following experimental examples. ^{Figure 1 is a 1} H-NMR spectrum of compound TCA1 dissolved in deuterated DMSO. Figure 2 is a mass spectrum of compound TCA1. Add 0-50μM (including the compound LZDT1 of the present invention and the positive control drug TCA1) of the test compound (including the compound of the present invention LZDT1 and the positive control drug TCA1) in each well, and each concentration is repeated twice, and the well with an equal volume of DMSO is used as a negative control. After incubating at 30°C for 10 min, farnesyl-phosphoryl-β-D-ribofuranose (FPR) with a final concentration of 300 μM was added to maintain the final volume of the reaction solution at 30 μL. After mixing uniformly, use the dynamic mode to perform fluorescence detection on the full-wavelength multifunctional microplate reader Tecan M200, the excitation light wavelength λex=530nm, and the emission light wavelength λem=595nm. By fitting the initial reaction rate and the corresponding concentration of the test compound (including the compound of the present invention LZDT1 and the positive control drug TCA1), the inhibitory concentration (IC ₅₀ ) of TCA1 and the compound of the present invention LZDT1 on DprE1 activity is calculated.

Results: The experimental results are shown in Figure 3. Figure 3 is a graph showing the inhibition of the activity of the compounds of the present invention LZDT1 and TCA1 on Mycobacterium tuberculosis DprE1. IC LZDT1 compounds of the present invention against Mycobacterium tuberculosis (MTB) DprE1 ₅₀ is 0.0158 ± 0.0028μM, significantly less than the positive control IC TCA1 Mycobacterium tuberculosis (MTB) DprE1 of ₅₀ (0.0583 ± _0.0099μM).

Conclusion: The experimental results show that the present invention obtains a compound LZDT1 with higher DprE1 inhibitory activity against Mycobacterium tuberculosis (MTB).

Experimental Example 2 Inhibition experiment of the compound of the present invention on Mycobacterium tuberculosis H37Rv

Experiment: Prepare 7H9 medium for culturing Mycobacterium tuberculosis H37Rv strain. Weigh 0.94g 7H9 medium powder, add 0.4mL glycerol, 0.1mL Tween80, fully dissolve with double distilled water and dilute to 180mL, and sterilize at 121°C for 10min. Before use, add 20mL filter-sterilized bacteria-enhancing agent OADC (0.255g NaCl, 1.5g BSA-V, 0.6g glucose, 0.016g sodium oleate, 0.0012g catalase, double distilled water to make the volume to 30mL) .

The H37Rv strain was inoculated into a 250 mL Erlenmeyer flask containing 40 mL of 7H9 medium (the final concentration of kanamycin was 20 μg/mL), and cultured in a constant temperature shaker at 37° C. at 110 rpm for 10 days to mid-log growth period to obtain the H37Rv bacterial solution. Dilute it with fresh 7H9 medium (with OADC added) to OD ₆₀₀ =0.05～0.06 (corresponding to ~10 ⁶ CFU/mL bacterial concentration at this time). Use a multichannel pipette to pipette 100 μL of the bacterial suspension into each well of a 96-well microtiter plate. Add 2 μL of the compound LZDT1 (4 mg/mL) of the present invention dissolved in DMSO to each well, and perform a two-fold gradient dilution in sequence. DMSO was used as a negative control, rifampicin and TCA1 were used as a positive control, and then each test plate was sealed with Parafilm parafilm, and incubated in a 37°C constant temperature incubator for 7 days. Add 30μL of resazurin to each well, continue to incubate at 37°C for 24 hours, observe the growth of the bacteria under a microscope, and use a multifunctional microplate reader to detect the absorbance at 570nm. The final concentration of the compound of the present invention corresponding to the wells where the growth of the H37Rv strain is inhibited by more than 90% (the fluorescence value is lower than 90% of the negative control group) is defined as the minimum inhibitory concentration (MIC) of the compound against the H37Rv strain.

Results: According to the absorbance changes corresponding to different test concentrations, the lowest inhibitory concentration (MIC ₉₀ ) of the three compounds of the present invention LZDT1, rifampicin and TCA1 against the H37Rv strain was calculated. The results are shown in Table 1. The MIC of the compound LZDT1 of the present invention against the H37Rv strain is the same as that of rifampicin, and is much higher than the MIC _{90 of} TCA1 against the H37Rv strain.

Table 1 The lowest inhibitory concentration of the compounds of the present invention LZDT1, rifampicin and TCA1 against H37Rv strain

Conclusion: The experimental results show that the compound LZDT1 of the present invention has higher antibacterial activity against the H37Rv strain, and the antibacterial activity of the compound LZDT1 of the present invention against the H37Rv strain is equivalent to that of the first-line anti-tuberculosis drug rifampicin.

Experimental Example 3 Detection of the bactericidal activity of the compound of the present invention against Mycobacterium tuberculosis H37Rv

Experiment: Configure 7H10-ADC medium plate, treat Mycobacterium tuberculosis with compounds corresponding to MIC concentration for 7 days, take 100μl of bacterial solution and coat 7H10-ADC plate, cultivate at 37℃ for 4 weeks, CFU count, CFU reduced by 90% compound The concentration is the minimum bactericidal concentration. According to the inhibitory concentration and bactericidal concentration of the compound, the influence of the compound on the growth curve of the strain is determined.

Add Mycobacterium tuberculosis with an OD value of 0.1 to a 15ml centrifuge tube, and then add 5μg/mL compound to culture at 37°C on a 80rpm shaker, and take them at 0 days, 3 days, 7 days, 14 days, and 21 days. Bacteria solution was coated with 7H10-ADC plate, cultured at 37°C for 4 weeks for CFU count, and sterilization curve was drawn. At the same time, 2μg/mL rifampicin and 5μg/mL TCA1 and DMSO were used as controls.

Results: After LZDT1 was treated at a concentration of 5μg/mL, CFU began to decrease after 3 days, and the bacterial concentration decreased by 4 log values after 21 days. The experimental results are shown in Figure 4. Figure 4 is a graph showing the bactericidal curve of the compounds of the present invention LZDT1 and TCA1 against Mycobacterium tuberculosis H37Rv.

Conclusion: The experimental results show that the bactericidal effect of the compound LZDT1 of the present invention is better than that of TCA1 in the first 7 days, and the bactericidal effect of 2 μg/mL is equivalent to that of the positive control rifampicin (rifamycin).

_{Experimental Example 4 Determination of MIC 99} of the compound of the present invention against several Mycobacterium tuberculosis

Experiment: Prepare 7H9 medium for culturing Mycobacterium tuberculosis. Weigh 0.94g 7H9 medium powder, add 0.4mL glycerol, 0.1mL Tween80, fully dissolve with double distilled water and dilute to 180mL, and sterilize at 121°C for 10min. Before use, add 20mL filter-sterilized bacteria-enhancing agent OADC (0.255g NaCl, 1.5g BSA-V, 0.6g glucose, 0.016g sodium oleate, 0.0012g catalase, double distilled water to make the volume to 30mL) .

Mycobacterium bovine tuberculosis BCG (BCG), DprE1 overexpressed Mycobacterium tuberculosis BCG (BCG (DprE1 overexpression)), H37Rv, rifampin-resistant H37Rv, Mycobacterium smegmatis and DprE1 mutant (DprE1C387S) tuberculosis 6 strains of mycobacteria were inoculated into a 250mL Erlenmeyer flask containing 40mL 7H9 medium (final concentration of kanamycin is 20μg/mL), cultured in a constant temperature shaker at 37°C at 110rpm for 10 days to mid-logarithmic growth. BCG, BCG (DprE1 overexpression), H37Rv, rifampicin-resistant H37Rv, Mycobacterium smegmatis and DprE1 mutant (DprE1C387S) Mycobacterium tuberculosis broth. Dilute it with fresh 7H9 medium (with OADC added) to OD ₆₀₀ =0.05～0.06 (corresponding to ~10 ⁶ CFU/mL bacterial concentration at this time). Use a multichannel pipette to pipette 100 μL of the bacterial suspension into each well of a 96-well microtiter plate. Add 2 μL of the compound LZDT1 (4 mg/mL) of the present invention dissolved in DMSO to each well, and perform a two-fold gradient dilution in sequence. DMSO was used as a negative control, rifampicin and TCA1 were used as a positive control, and then each test plate was sealed with Parafilm parafilm, and incubated in a 37°C constant temperature incubator for 7 days. Add 30μL of resazurin to each well, continue to incubate at 37°C for 24 hours, observe the growth of the bacteria under a microscope, and use a multifunctional microplate reader to detect the absorbance at 570nm. BCG, BCG (DprE1 overexpression), H37Rv, rifampicin-resistant H37Rv, Mycobacterium smegmatis and DprE1 mutant (DprE1C387S) Mycobacterium tuberculosis strain growth is inhibited by more than 99% (fluorescence value is lower than 99% of the negative control group) The final concentration of the compound of the present invention corresponding to the hole above) is defined as the compound's resistance to BCG, BCG (DprE1 overexpression), H37Rv, rifampicin-resistant H37Rv, Mycobacterium smegmatis and DprE1 mutant (DprE1C387S) smudge The minimum inhibitory concentration of mycobacterium strain (MIC ₉₉ ) The same method was used to determine the minimum inhibitory effect of the compound LZDT2 of the present invention on BCG, BCG (DprE1 overexpression), H37Rv, rifampicin-resistant H37Rv, Mycobacterium smegmatis and DprE1 mutant (DprE1C387S) Mycobacterium tuberculosis and other 6 strains Concentration (MIC ₉₉ ).

Results: According to the changes in absorbance corresponding to different test concentrations, the four compounds of the present invention, LZDT1, LZDT2, rifampicin and TCA1, were calculated to affect BCG, BCG (DprE1 overexpression), H37Rv, rifampicin-resistant H37Rv, smegma Table 2 shows _{the minimum inhibitory concentration (MIC 99} ) of Mycobacterium and DprE1 mutant (DprE1C387S) Mycobacterium tuberculosis and other strains. _{The MIC 99 of the} compound LZDT1 of the present invention to each strain is higher than that of TCA1.

_{Table 2 The MIC 99 of the} compounds of the present invention LZDT1, LZDT2, rifampicin and TCA1 against several Mycobacterium tuberculosis strains

Conclusion: The experimental results show that the compound LZDT1 of the present invention has higher antibacterial activity against the H37Rv strain and the rifampin-resistant H37Rv strain, and the compound LZDT1 of the present invention has better antibacterial activity against the rifampin-resistant H37Rv strain than the first-line antibody The tuberculosis drug rifampicin.

Experimental Example 5 Detection of the bactericidal activity of the compound of the present invention against Mycobacterium tuberculosis H37Rv

Experiment: Configure the 7H10-ADC medium plate, _{treat Mycobacterium tuberculosis with the compound corresponding to the concentration of 20×MIC 99 for} 7 days, take 100 μL of the bacterial solution to coat the 7H10-ADC plate, culture at 37°C for 4 weeks, CFU count, CFU decrease by 99 % Compound concentration is the minimum bactericidal concentration. According to the inhibitory concentration and bactericidal concentration of the compound, the influence of the compound on the growth curve of the strain is determined.

Add Mycobacterium tuberculosis with an OD of 0.1 to a 15ml centrifuge tube, and then add 6.2μg/mL compound to culture at 37°C and 80rpm on a shaker for 0 days, 3 days, 7 days, 14 days, and 21 days. The bacteria solution was coated on a 7H10-ADC plate, and incubated at 37°C for 4 weeks to count CFU and draw a sterilization curve. At the same time, 2μg/mL rifampicin and 12.6μg/mL TCA1 and DMSO were used as controls.

Results: When LZDT1 was treated at a concentration of 6.2μg/mL, CFU began to decrease after 3 days, and the bacterial concentration decreased by 3.5 log after 21 days. The experimental results are shown in Figure 5. Figure 5 is a graph showing the bactericidal curve of the compounds of the present invention LZDT1, TCA1 and rifampicin against Mycobacterium tuberculosis H37Rv.

Conclusion: The experimental results show that the bactericidal effect of the compound LZDT1 of the present invention is better than that of TCA1 and rifampicin in the first 7 days, and the bactericidal effect of 2 μg/mL rifampicin (rifamycin) is equivalent after 21 days.

Experimental Example 6 Detection of the bactericidal activity of the compound of the present invention against rifampicin-resistant H37Rv strain

Experiment: Configure 7H10-ADC medium plate, _{treat rifampicin-resistant H37Rv strain with the compound corresponding to 20×MIC 99 for} 7 days, take 100μL of bacterial solution and coat 7H10-ADC plate, culture at 37℃ for 4 weeks, CFU count, CFU The compound concentration reduced by 99% is the minimum bactericidal concentration. According to the inhibitory concentration and bactericidal concentration of the compound, the influence of the compound on the growth curve of the strain is determined.

Add the rifampicin-resistant H37Rv strain with an OD value of 0.1 to a 15mL centrifuge tube, and then add 12.5μg/mL of the compound of the present invention LZDT1 to culture in a shaker at 37°C and 80rpm, respectively on day 0, day 3, day 7 On the 14th and 21st days, the bacteria solution was applied to the 7H10-ADC plate and incubated at 37°C for 4 weeks to count the CFU and draw the sterilization curve. At the same time, 20μg/mL rifampicin and 25μg/mL TCA1 and DMSO were used as controls.

Results: After LZDT1 was treated at a concentration of 12.5μg/mL, CFU began to decrease after 3 days, and the bacterial concentration decreased by 3.5 log after 21 days. The experimental results are shown in Figure 6. Figure 6 is a graph showing the sterilization curves of compounds LZDT1, TCA1 and rifampicin of the present invention against rifampicin-resistant H37Rv strains.

Conclusion: The experimental results show that the bactericidal effect of the compounds of the present invention LZDT1 and TCA1 on rifampicin-resistant H37Rv is better than that of rifampicin after 21 days, and the bactericidal effect of the compound LZDT1 of the present invention is better than that of TCA1 in the first 7 days.

Experimental Example 7 Detection of the bactericidal activity of the compound of the present invention against BCG strains

Experiment: Configure the 7H10-ADC medium plate, _{treat the BCG strain with the compound of the corresponding concentration of 20×MIC 99 for} 7 days, take 100μL of bacterial solution to coat the 7H10-ADC plate, incubate at 37℃ for 4 weeks, CFU count, CFU reduced by 99% The compound concentration is the minimum bactericidal concentration. According to the inhibitory concentration and bactericidal concentration of the compound, the influence of the compound on the growth curve of the strain is determined.

Add the BCG strain with an OD value of 0.1 to a 15mL centrifuge tube, and then add 25μg/mL of the compound of the present invention LZDT1 to culture at 37°C on a 80rpm shaker, respectively at 0 days, 3 days, 7 days, 14 days, and 21 days. The bacteria solution was coated on a 7H10-ADC plate, and incubated at 37°C for 4 weeks to count CFU and draw a sterilization curve. At the same time, 6.2μg/mL rifampicin and 100μg/mL TCA1 and DMSO were used as controls.

Results: After LZDT1 was treated at a concentration of 25μg/mL, CFU began to decrease after 3 days, and the bacterial concentration decreased by 3 log values after 21 days. The experimental results are shown in Figure 7. Figure 7 is a graph showing the bactericidal curves of the compounds LZDT1, TCA1 and rifampicin against BCG strains of the present invention.

Conclusion: The experimental results show that the bactericidal effect of the compound LZDT1 of the present invention on BCG after 21 days is equivalent to that of TCA1 and rifampicin, but the bactericidal effect of the compound LZDT1 of the present invention is better than that of TCA1 and rifampicin in the first 7 days.

Experimental Example 8 Detection of the bactericidal activity of the compound of the present invention against BCG (DprE1 overexpression) strains

Experiment: Configure the 7H10-ADC medium plate, _{treat the BCG (DprE1 overexpression) strain with the compound corresponding to the concentration of 20×MIC 99 for} 7 days, take 100 μL of the bacterial solution and coat the 7H10-ADC plate, incubate at 37°C for 4 weeks, and count the CFU. The concentration of the compound that reduces CFU by 99% is the minimum bactericidal concentration. According to the inhibitory concentration and bactericidal concentration of the compound, the influence of the compound on the growth curve of the strain is determined.

Add the BCG (DprE1 overexpression) strain with an OD value of 0.1 to a 15mL centrifuge tube, and then add 100μg/mL compound to culture in a shaker at 37°C and 80rpm. After 21 days, the bacteria solution was applied to 7H10-ADC plate, and the CFU was counted by culturing at 37°C for 4 weeks, and the sterilization curve was drawn. At the same time, 6.2μg/mL rifampicin and 500μg/mL TCA1 and DMSO were used as controls.

Results: When LZDT1 was treated at a concentration of 100 μg/mL, the CFU of the BCG (DprE1 overexpression) strain was basically unchanged. The CFU of the BCG (DprE1 overexpression) strain treated with TCA1 and rifampicin has a larger range. The experimental results are shown in Figure 8. Figure 8 is a graph showing the bactericidal curve of the compounds LZDT1, TCA1 and rifampicin against BCG (DprE1 overexpression) strains.

Conclusion: The experimental results show that the target of the compound LZDT1 of the present invention is DprE1, and the targeting of DprE1 is better than TCA1.

Experimental Example 9 Detection of the toxicity of the compound of the present invention on human liver cancer cells HepG2

Experiment: Amplify and culture human liver cancer cells HepG2. When the cells grow to the logarithmic growth phase, adjust the cell density to 3×10 ⁴ cells/mL and inoculate them into 96-well cell culture plates with 100 μL per well, with each concentration gradient 3 multiple holes. TCA1 and LZDT1 were configured with complete medium to be 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μg/mL and 8 concentration gradients were applied to the cells. After culturing for 24h, 48h and 72h, 10μL of CCK-8 was added to each well and incubated in a 37°C, 5% CO ₂ incubator in the dark for 2h. The microplate reader measured the OD value at the same time point at the wavelength of 492nm, and the measured OD value was used to analyze the influence of cell proliferation.

Inhibition rate (%)=(control group OD value-experimental group OD value)/(control group OD value-blank group OD value)×100%

Results: 72h and the compound of the present invention inhibit the TCA1 LZDT1 proliferation of HepG2 cells in concentrations (IC ₅₀₎ were 62.57μg / mL and 82.78μg / mL. At 24h and 48h, neither TCA1 nor the compound LZDT1 of the present invention inhibited the proliferation of human liver cancer cells HepG2 by 50%. The experimental results are shown in Table 3, Figure 9 and Figure 10. Figure 9 is the toxicity curve of the compound LZDT1 of the present invention on human liver cancer cells HepG2. Figure 10 shows the toxicity curve of TCA1 on human liver cancer cells HepG2.

Table 3 The inhibitory concentration of the compounds of the present invention LZDT1 and TCA1 on the proliferation of human liver cancer cells HepG2

Conclusion: The toxicity of the compound LZDT1 of the present invention to human liver cancer cells HepG2 is lower than that of TCA1, indicating that the present invention reduces its toxicity to cells after modification of TCA1.

Experimental Example 10 Toxicity detection of the compound of the present invention on human neuroblastoma cell SH-SY5Y

Experiment: Expand and culture human neuroblastoma cells SH-SY5Y. When the cells grow to the logarithmic growth phase, adjust the cell density to 3×10 ⁴ cells/mL and inoculate them into 96-well cell culture plates with 100 μL per well. , 3 replicate holes per concentration gradient. TCA1, LZDT1 and LZDT2 were configured with complete medium to be 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μg/mL, respectively, with 8 concentration gradients to act on the cells. After culturing for 24h, 48h and 72h, 10μL of CCK-8 was added to each well and incubated in a 37°C, 5% CO ₂ incubator in the dark for 2h. The microplate reader measured the OD value at the same time point at the wavelength of 492nm, and the measured OD value was used to analyze the influence of cell proliferation.

Results: 72h and the compound of the present invention inhibit the TCA1 LZDT2 LZDT1 and human neuroblastoma SH-SY5Y cell proliferation in the concentration (IC ₅₀₎ were 16.70μg / mL, 22.47μg / mL and 14.86μg / mL. At 24h and 48h, TCA1 and the compounds of the present invention LZDT1 and LZDT2 did not inhibit the proliferation of human neuroblastoma cells SH-SY5Y by 50%. The experimental results are shown in Table 4, Figure 11, Figure 12 and Figure 13. Figure 11 shows the toxicity curve of TCA1 on human neuroblastoma cells SH-SY5Y. Figure 12 is the toxicity curve of compound LZDT1 of the present invention on human neuroblastoma cell SH-SY5Y. Figure 13 is the toxicity curve of compound LZDT2 of the present invention on human neuroblastoma cell SH-SY5Y.

Table 4 The inhibitory concentration of the compounds of the present invention LZDT1, LZDT2 and TCA1 on the proliferation of human neuroblastoma cells SH-SY5Y

Conclusion: The toxicity of the compound LZDT1 of the present invention to human neuroblastoma cells SH-SY5Y is lower than that of TCA1, indicating that the modification of TCA1 by the present invention reduces its toxicity to cells.

Experimental Example 11 Toxicity detection of the compound of the present invention on human embryonic kidney cells HEK293

Experiment: Expand and culture human embryonic kidney cells HEK293. When the cells grow to the logarithmic growth phase, adjust the cell density to 3×10 ⁴ cells/mL and inoculate them into 96-well cell culture plates, 100 μL per well, each concentration Gradient 3 replicate holes. TCA1, LZDT1 and LZDT2 were configured with complete medium to be 0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μg/mL, respectively, with 8 concentration gradients to act on the cells. After culturing for 24h, 48h and 72h, 10μL of CCK-8 was added to each well and incubated in a 37°C, 5% CO ₂ incubator in the dark for 2h. The microplate reader measured the OD value at the same time point at the wavelength of 492nm, and the measured OD value was used to analyze the influence of cell proliferation.

_{Results: The 72h inhibitory concentration (IC 50} ) of TCA1 and the compounds of the present invention LZDT1 and LZDT2 on the proliferation of human embryonic kidney cells HEK293 were 25.11μg/mL, 34.52μg/mL and 28.28μg/mL, respectively. At 24h and 48h, TCA1 and the compounds of the present invention LZDT1 and LZDT2 did not inhibit the proliferation of human embryonic kidney cells HEK293 by 50%. The experimental results are shown in Table 5, Figure 14, Figure 15, and Figure 16. Figure 14 shows the toxicity curve of TCA1 on human embryonic kidney cells HEK293. Figure 15 is the toxicity curve of the compound LZDT1 of the present invention on human embryonic kidney cells HEK293. Figure 16 is the toxicity curve of the compound LZDT2 of the present invention on human embryonic kidney cells HEK293.

Table 5 The inhibitory concentration of the compounds of the present invention LZDT1, LZDT2 and TCA1 on the proliferation of human embryonic kidney cells HEK293

Conclusion: The toxicity of the compounds LZDT1 and LZDT2 of the present invention to human embryonic kidney cells HEK293 is lower than that of TCA1, indicating that the present invention reduces its toxicity to cells after modification of TCA1.

Based on the above description of the content of the invention, those skilled in the art can fully apply the invention, and all the same principles or similar modifications should be considered to be included in the scope of the invention.

Claims

The benzothiazole compound of the following general formula (I) or a pharmaceutically acceptable salt thereof,

Among them, R1 is:
The benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein R1 is:
The benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, which is the following compound or a pharmaceutically acceptable salt thereof:
The preparation method of the benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, comprising the following steps:

(1) Synthesis of compound B: Add cyanoacetic acid, R1-substituted methyl carbamate compound A and acetic anhydride into the reaction vessel, heat up the oil bath, and protect with Ar. After the reaction is complete, pour the reaction solution into ice water Extract with ethyl acetate, wash with saturated sodium bicarbonate solution once, dry and concentrate to obtain compound B;

(2) Synthesis of compound C: Add compound B, 2,5-dihydroxy-1,4-dithiane, methanol, and morpholine to the reaction vessel, and the reaction is complete in 2 hours in an oil bath, directly pass through the column, and eluate. To obtain compound C;

(3) Synthesis of compound (I): add 2-formic acid benzothiazole and compound C to the reaction vessel, add pyridine and stir to dissolve it, add phosphorus oxychloride dropwise, and add dichloromethane to the system after fully stirring Mix with water, quickly stir to separate the organic layer, wash once with saturated sodium bicarbonate solution, directly pass through a short silica gel column, and elute to obtain compound (I); alternatively, add 2-formic acid benzothiazole and dichloride to the reaction vessel Methane, cool to 0°C, add oxalyl chloride dropwise, react for several hours, add pyridine to the system, stir for several minutes, add compound C, react for several hours, add methanol to the system to quench the reaction, and wash once with saturated sodium bicarbonate solution. Mix the sample directly through a silica gel column and elute to obtain compound (I);

Here, the definition of R1 is the same as in claim 1.
The use of the benzothiazole compound of the general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-tuberculosis drug.
The benzothiazole compound of general formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof is prepared as DprE1 inhibitor, or as H37Rv inhibitor, or as rifampin-resistant H37Rv inhibitor, or as Use of BCG inhibitors in anti-tuberculosis drugs.
Use of the benzothiazole compound of general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-tuberculosis drug targeting DprE1.
The use of the benzothiazole compound of the general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-tuberculosis drug with low toxicity.