Triazine dicarboxylic acid compounds and their antibacterial uses

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to triazine dicarboxylic acid compounds, their preparation methods, and their use as antibacterial drugs. The compounds are shown in formula (1), wherein M1 is independently selected from hydrogen, C… 1‑8 Alkyl, C 1‑8 Alkoxy, methylthio, phenyl; L1 is independently selected from aryl or heteroaryl; L2 is independently selected from hydrogen, C 1‑8 Alkyl, phenyl, and substituted phenyl compounds. The molecules involved in this invention have nitrogen-substituted triazine linkers connecting both ends, representing a novel structural type of compound. This series of compounds exhibits strong inhibitory activity against Acinetobacter baumannii and warrants further research and development.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial drug technology, specifically relating to triazine dicarboxylic acid compounds, their preparation methods, and their uses as antibacterial drugs. Background Technology

[0002] In recent years, the emergence of drug-resistant bacteria and pathogens such as mycoplasma has led to a gradual decrease in the availability of effective antibiotics. On the other hand, antimicrobial drug development has been relatively slow: the number of approved antibiotics is small, and most are still of known structural types, making them prone to cross-resistance. Statistics show that in the past 20 years, only three new structural types of antimicrobial drugs have been approved globally, and these only target Gram-positive bacteria such as Staphylococcus aureus, with no new structural types targeting Gram-negative bacteria such as Acinetobacter baumannii. Therefore, antimicrobial drugs with new structural types or new mechanisms of action address significant clinical needs.

[0003] The compound involved in this patent belongs to the triazine dicarboxylic acid class of compounds. Due to the nitrogen substitution of the triazine linker connecting both ends of the molecule, it is a novel compound structure. The compound involved in this patent exhibits strong anti-Acinetobacter baumannii activity. Compound TN11 has a minimum inhibitory concentration (MIC) of 0.25 μg / mL against Acinetobacter baumannii ATCC19606, which is comparable to the activity of the marketed drug levofloxacin (MIC: 0.25 μg / mL); with the addition of 1% mouse serum, the MIC is 0.5 μg / mL, demonstrating good in vitro antibacterial activity. Therefore, the compound involved in this patent has the potential for further development into a clinical antibacterial drug. Summary of the Invention

[0004] The technical problem addressed by this patent is to overcome the deficiency of existing anti-drug drugs for resistant bacteria by providing compounds with novel structural types or their pharmaceutically acceptable salts, which exhibit strong antibacterial activity. This patent provides triazine dicarboxylic acid compounds, their preparation methods, and their use as antibacterial drugs.

[0005] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0006] Firstly, a triazine dicarboxylic acid compound is provided, which is a compound as shown in formula (1):

[0007]

[0008] Wherein: M1 is independently selected from hydrogen, C 1-8 Alkyl, C 1-8 Alkoxy, methylthio, phenyl;

[0009] L1 is independently selected from aryl or heteroaryl;

[0010] L2 is independently selected from hydrogen and C.1-8 Alkyl, phenyl, substituted phenyl.

[0011] The C 1-8 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, straight-chain or branched pentyl, straight-chain or branched hexyl, straight-chain or branched heptyl, and straight-chain or branched octyl.

[0012] The aryl group is selected from phenyl, naphthyl, anthraceneyl, phenanthryl, or pyrene;

[0013] The heteroaryl group is selected from furan, thiophene, oxazole, thiazole, isoxazole, oxadiazole, thiadiazole, pyrrole, pyrazole, imidazole, pyridine, pyrimidine, pyrazine, pyridazine, phthalazine, quinoline, isoquinoline, pteridine, purine, indole, isoindole, benzofuranyl, benzothiophenyl, benzopyridyl, benzopyrimidinyl, benzopyrazinyl, benzoimidazolyl, or benzophthalazinyl.

[0014] The substituted phenyl group is characterized in that,

[0015] R1, R2, R3, R4, and R5 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, nitro, cyano, hydroxyl, amino, and aminomethyl.

[0016] As a preferred option, the triazine dicarboxylic acid compound shown in Formula 1 is:

[0017] M1 is independently selected from hydrogen, methyl, methoxy, methylthio, and phenyl;

[0018] L1 is independently selected from phenyl and thiophene;

[0019] L2 is independently selected from n-butyl, phenyl, 4-chlorophenyl, 4-fluorophenyl, 3-fluorophenyl, and 3,4-difluorophenyl.

[0020] More preferably, the triazine dicarboxylic acid compound provided by the present invention is:

[0021]

[0022] Secondly, the present invention provides a method for preparing triazine dicarboxylic acid compounds, which includes the following steps:

[0023]

[0024] (1) The intermediate (f) is converted into the compound (1) of claims 1-4 under alkaline conditions;

[0025] The alkaline conditions are provided by sodium hydroxide, potassium hydroxide, or lithium hydroxide.

[0026] The definitions of L1, L2 and M1 are the same as in claim 1.

[0027]

[0028] (2) Intermediate (d) and intermediate (e) are reacted in an organic solvent to prepare intermediate (f);

[0029] The organic solvent is selected from acetone and acetonitrile.

[0030] The definitions of L1, L2 and M1 are the same as in claim 1.

[0031]

[0032] (3) The intermediate (c) is converted into the intermediate (d) by Pd / C catalysis under hydrogen conditions and in an organic solvent;

[0033] The organic solvent is selected from tetrahydrofuran or a dichloromethane-methanol mixture.

[0034] The definitions of L1 and L2 are the same as in claim 1.

[0035]

[0036] (4) Intermediate (a) and intermediate (b) were reacted under alkaline conditions in an organic solvent to prepare intermediate (c);

[0037] The organic solvent is selected from tetrahydrofuran and N,N-dimethylformamide, and the alkaline conditions are selected from sodium hydrogen.

[0038] The definitions of L1 and L2 are the same as in claim 1.

[0039] Thirdly, the present invention provides the antibacterial use of triazine dicarboxylic acid compounds or their use in the preparation of antibacterial drugs.

[0040] Preferably, the antibacterial use is for the treatment and prevention of infectious diseases in humans or animals caused by pathogenic microorganisms such as bacteria, mycoplasma, chlamydia, rickettsia, spirochetes, and fungi.

[0041] More preferably, the bacteria are selected from Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Clostridium difficile, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, etc.; the mycoplasma is selected from Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium, etc.; and the chlamydia is selected from Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, and Chlamydia bovis, etc.

[0042] More preferably, the Acinetobacter baumannii is selected from drug-sensitive Acinetobacter baumannii, multidrug-resistant Acinetobacter baumannii, pan-drug-resistant Acinetobacter baumannii, carbapenem-resistant Acinetobacter baumannii, and pan-drug-resistant Acinetobacter baumannii; the Staphylococcus aureus is selected from methicillin-sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus; the Staphylococcus epidermidis is selected from methicillin-sensitive Staphylococcus epidermidis and methicillin-resistant Staphylococcus epidermidis; the Enterococcus faecalis is selected from vancomycin-sensitive Enterococcus faecalis and vancomycin-resistant Enterococcus faecalis; the Enterococcus faecium is selected from vancomycin-sensitive Enterococcus faecium and vancomycin-resistant Enterococcus faecium; and the Mycoplasma is selected from drug-sensitive Mycoplasma pneumoniae or drug-resistant Mycoplasma pneumoniae.

[0043] Fourthly, the present invention provides an antibacterial pharmaceutical composition comprising the above-mentioned triazine dicarboxylic acid compound or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers.

[0044] Furthermore, the carrier is selected from conventional pharmaceutical excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, lubricants, etc.

[0045] Preferably, the dosage form of the drug is selected from injections, tablets, pills, capsules, suspensions, emulsions or ointments, and the route of administration is selected from intravenous or intramuscular injection, oral administration, transdermal administration, mucosal administration, rectal administration, vaginal administration, etc.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The triazine dicarboxylic acid compounds of this invention are novel antibacterial drugs. Experiments have confirmed that these compounds have a strong inhibitory effect on Acinetobacter baumannii. Since this structural type is different from clinically used antibacterial drugs, it is expected to solve the problem of bacterial resistance and has the value for in-depth research and the potential for clinical application. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments, but the embodiments are only for illustrating the present invention and not for limiting the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; unless otherwise specified, the materials and reagents used are commercially available. I. Preparation and Structural Confirmation of New Compounds

[0049] Example 1: Preparation and structural confirmation of compound TN1

[0050]

[0051] 1.96 g (10 mmol, 1.0 eq) of 2-amino-5-nitrobenzoate was added to a 100 mL round-bottom flask and dissolved in 30 mL of tetrahydrofuran (THF). Then, sodium hydroxide (306 mg, 7.65 mmol, 3.0 eq) was added and the mixture was stirred in an ice bath for 30 minutes. 4-Butylbenzenesulfonyl chloride (3.48 g, 15 mmol, 1.5 eq) was dissolved in 10 mL of THF and added dropwise in an ice bath. After the addition was complete, stirring was continued for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 20 mL of saturated ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain methyl 2-((4-butylphenyl)sulfonamido)-5-nitrobenzoate (3.5 g, yield: 89.3%).

[0052] 3.5 g of methyl 2-((4-butylphenyl)sulfonamido)-5-nitrobenzoate was dissolved in 25 mL of THF, and 350 mg of dry palladium on carbon (palladium content: 10%) was added. The mixture was then placed in a hydrogenation reactor (H2: 0.4 MPa) and stirred for 4 h. The mixture was filtered through diatomaceous earth, the filtrate was evaporated to dryness, and the residue was separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 5-amino-2-((4-butylphenyl)sulfonamido)benzoate (2.0 g, yield: 61.9%).

[0053] Add methyl 5-amino-2-((4-butylphenyl)sulfonamide)benzoate (200 mg, 0.55 mmol, 2.2 eq), 2,4-dichloro-6-methyl-1,3,5-triazine (41 mg, 0.25 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain 5,5'-((6-methyl-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-((4-butylphenyl)sulfonamido)benzoate). The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5,5'-((6-methyl-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(2-((4-butylphenyl)sulfonamido)benzoic acid) (TN1, 60 mg, yield: 30.5%).

[0054]

[0055] White solid. 1 H NMR (400MHz, DMSO-d6) δ9.40(s,2H),8.16(s,2H),7.67(s,6H),7.30(t,J=11.0Hz,6H),2.56(d,J= 7.8Hz,3H),2.19(s,3H),1.49(p,J=7.5Hz,4H),1.24(h,J=7.8Hz,5H),0.84(t,J=7.4Hz,6H).HRMS calcd for C 38 H 41 N7O8S2[M+H] + ,788.2531; found,788.2544.

[0056] Example 2: Preparation and detection of compound TN2

[0057]

[0058] Add methyl 5-amino-2-((4-butylphenyl)sulfonamide)benzoate (200 mg, 0.55 mmol, 2.2 eq), 2,4-dichloro-6-methoxy-1,3,5-triazine (43 mg, 0.25 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain 5,5'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-((4-butylphenyl)sulfonamido)benzoate). The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to give the target compound 5,5'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(2-((4-butylphenyl)sulfonamido)benzoic acid) (TN2,48 mg, yield: 24%).

[0059] White solid. 1H NMR (400MHz, DMSO-d6) δ9.63(s,2H),8.15(s,2H),7.88(d,J=55.9Hz,2H),7.66(d,J=7.9Hz,4H),7.41(d,J=9.0Hz,2H),7.33( d,J=7.9Hz,4H),3.84(s,3H),2.58(t,J=7.7Hz,3H),1.49(p,J=7.6Hz,4H),1.23(q,J=7.5Hz,5H),0.84(t,J=7.3Hz,6H).HRMS calcd for C 38 H 41 N7O9S2[M+H] + ,804.2480; found,804.2488.

[0060] Example 3: Preparation and detection of compound TN3

[0061]

[0062] Add methyl 5-amino-2-((4-butylphenyl)sulfonamide)benzoate (200 mg, 0.55 mmol, 2.2 eq), 2,4-dichloro-1,3,5-triazine (39 mg, 0.25 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain 5,5'-((1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-((4-butylphenyl)sulfonamido)benzoate). The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h and monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5,5'-((1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(2-((4-butylphenyl)sulfonamido)benzoic acid) (TN 3.63 mg, yield: 33%).

[0063] White solid. 1H NMR (400MHz, DMSO-d6) δ10.77(s,2H),9.84(s,2H),8.33(s,1H),8.00(s,3H),7.67(d,J=8.0Hz,4H),7.47(d,J=9.0Hz,2H), 7.35(d,J=8.0Hz,4H),2.59(t,J=7.8Hz,4H),1.49(p,J=7.6Hz,4H),1.22(p,J=7.2Hz,4H),0.84(t,J=7.3Hz,6H).HRMScalcd for C 37 H 41 N7O8S2[M+H] + ,774.2374;found,774.2398.

[0064] Example 4: Preparation and detection of compound TN4

[0065]

[0066] Add methyl 5-amino-2-((4-butylphenyl)sulfonamide)benzoate (200 mg, 0.55 mmol, 2.2 eq), 2,4-dichloro-6-phenyl-1,3,5-triazine (56 mg, 0.25 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain 5,5'-((6-phenyl-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-((4-butylphenyl)sulfonamido)benzoate). The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5,5'-((6-phenyl-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(2-((4-butylphenyl)sulfonamido)benzoic acid) (TN4,88 mg, yield: 44%).

[0067] White solid. 1H NMR(500MHz,DMSO-d6)δ9.85(s,2H),8.39(s,3H),8.18–7.76(m,2H),7.69(d,J=7.9Hz,4H),7.55(dt,J=37.8,8.6Hz,5H) ,7.47–7.23(m,5H),2.58(d,J=8.4Hz,4H),1.48(p,J=8.0Hz,4H),1.22(q,J=7.9Hz,4H),0.82(dt,J=9.4,4.5Hz,6H).HRMS calcd for C 40 H 46 N4O9S2[M+H] + ,791.2779;found,791.2779.

[0068] Example 5: Preparation and detection of compound TN5

[0069]

[0070] Add methyl 5-amino-2-((4-butylphenyl)sulfonamide)benzoate (200 mg, 0.55 mmol, 2.2 eq), 2,4-dichloro-6-methylthio-1,3,5-triazine (49 mg, 0.25 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain 5,5'-((6-methylthio-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-((4-butylphenyl)sulfonamido)benzoate). The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5,5'-((6-methylthio-1,3,5-triazine-2,4-diyl)bis(azanediyl))bis(2-((4-butylphenyl)sulfonamido)benzoic acid) (TN5,80 mg, yield: 39%).

[0071] White solid. 1H NMR(500MHz,DMSO-d6)δ9.76(s,2H),8.54–8.12(m,1H),8.10–7.70(m,2H),7.67(d,J=7.9Hz,4H),7.55–7.20 (m,7H),2.61–2.56(m,4H),2.44(s,3H),1.49(p,J=7.6Hz,4H),1.23(q,J=7.5Hz,4H),0.86–0.82(m,6H).HRMS calcd for C 38 H 41 N7O8S3[M+H] + ,820.2252;found,820.2276.

[0072] Example 6: Preparation and detection of compound TN6

[0073]

[0074] 2-Amino-5-nitrobenzene methyl ester (500 mg, 2.55 mmol, 1.0 eq) was added to a 100 mL round-bottom flask and dissolved in 15 mL of tetrahydrofuran (THF). Sodium hydroxide (306 mg, 7.65 mmol, 3.0 eq) was then added and stirred in an ice bath for 30 minutes. [1,1'-biphenyl]-4-sulfonyl chloride (965 mg, 3.83 mmol, 1.5 eq) was dissolved in 10 mL of THF and added dropwise in an ice bath. After the addition was complete, stirring was continued for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 10 mL of saturated ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 2-([1,1'-biphenyl]-4-sulfonamido)-5-nitrobenzene methyl ester (820 mg, yield: 78.4%).

[0075] Methyl 2-([1,1'-biphenyl]-4-sulfonamido)-5-nitrobenzoate (820 mg, 2 mmol, 1.0 eq) was dissolved in 25 mL of THF, and dry palladium on carbon (205 mg, palladium content: 10%) was added. The mixture was then placed in a hydrogenation reactor (H2: 0.4 MPa) and stirred for 4 h. The mixture was filtered through diatomaceous earth, the filtrate was evaporated to dryness, and the residue was separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-([1,1'-biphenyl]-4-sulfonamido)-5-aminobenzoate (630 mg, yield: 82.5%).

[0076] 2-([1,1'-biphenyl]-4-sulfonamido)-5-aminobenzoate methyl ester (630 mg, 1.65 mmol, 2.2 eq), 2,4-dichloro-6-methoxy-1,3,5-triazine (134 mg, 0.75 mmol, 1.0 eq), and acetone (40 mL) were added to a 50 mL round-bottom flask. The reaction mixture was heated at 60 °C for 2 h and monitored by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain 5,5'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-([1,1'-biphenyl]-4-sulfonamido)benzoate). The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5,5'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-([1,1'-biphenyl]-4-sulfonamido)benzoic acid) (TN6,280 mg, yield: 44%).

[0077]

[0078] White solid. 1 H NMR(500MHz,DMSO-d6)δ10.91(s,2H),9.77(s,2H),8.13(s,1H),7.84(s,9H),7.68(d,J=7.4H z,5H),7.52(d,J=8.9Hz,2H),7.47(t,J=7.4Hz,4H),7.41(t,J=7.3Hz,3H),3.85(s,3H).HRMS calcd for C 42 H 33 N7O9S2[M+H] + ,844.1854;found,844.1866.

[0079] Example 7: Preparation and detection of compound TN7

[0080]

[0081] 2-Amino-5-nitrobenzene methyl ester (500 mg, 2.55 mmol, 1.0 eq) was added to a 100 mL round-bottom flask and dissolved in 15 mL of tetrahydrofuran (THF). Sodium hydride (306 mg, 7.65 mmol, 3.0 eq) was then added and stirred in an ice bath for 30 minutes. 4'-Chloro-[1,1'-biphenyl]-4-sulfonyl chloride (1.095 g, 3.83 mmol, 1.5 eq) was dissolved in 10 mL of THF and added dropwise in an ice bath. After the addition was complete, stirring was continued for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 10 mL of saturated ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 2-((4'-chloro-[1,1'-biphenyl])-4-sulfonamido)-5-nitrobenzene methyl ester (900 mg, yield: 79%).

[0082] Methyl 2-((4'-chloro-[1,1'-biphenyl])-4-sulfonamido)-5-nitrobenzoate (900 mg, 2.02 mmol, 1.0 eq) was dissolved in 25 mL of THF, and dry palladium on carbon (240 mg, palladium content: 10%) was added. The mixture was then placed in a hydrogenation reactor (H2: 0.4 MPa) and stirred for 4 h. The mixture was filtered through diatomaceous earth, the filtrate was evaporated to dryness, and the residue was separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 5-amino-2-((4'-chloro-[1,1'-biphenyl])-4-sulfonamido)benzoate (780 mg, yield: 93%).

[0083] Add methyl 5-amino-2-((4'-chloro-[1,1'-biphenyl])-4-sulfonamido)benzoate (780 mg, 1.875 mmol, 2.2 eq), 2,4-dichloro-6-methoxy-1,3,5-triazine (153 mg, 0.85 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Heat the reaction mixture at 60 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain 5,5'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-((4'-chloro-[1,1'-biphenyl])-4-sulfonamido)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5,5'-((6-methoxy-1,3,5-triazine-2,4-diyl)bis(azadiyl))bis(2-((4'-chloro-[1,1'-biphenyl])-4-sulfonamido)benzoic acid) (TN 7,320 mg, yield: 41%).

[0084]

[0085] White solid. 1 H NMR(500MHz,DMSO-d6)δ10.83(s,2H),9.83(s,2H),7.84(s,10H),7.72(d,J=8.1 Hz,4H),7.66(d,J=7.7Hz,1H),7.51(t,J=7.4Hz,7H),3.84(d,J=2.1Hz,3H).HRMS calcd for C 42 H 31 Cl2N7O9S2[M+H] + ,912.1074; found,912.1089. Example 8: Preparation and detection of compound TN8

[0086]

[0087] 2-amino-5-nitrobenzoate methyl ester (2 g, 10.2 mmol, 1.0 eq) was added to a 100 mL round-bottom flask and dissolved in 30 mL of tetrahydrofuran (THF). Sodium hydroxide (1.8 mg, 45 mmol, 4.4 eq) was then added and stirred in an ice bath for 30 minutes. 5-(4-chlorophenyl)thiophene-2-sulfonyl chloride (4.3 g, 1.47 mmol, 1.44 eq) was dissolved in 10 mL of THF and added dropwise in an ice bath. After the addition was complete, stirring was continued for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 20 mL of saturated ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain 2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)-5-nitrobenzoate methyl ester (4.2 g, yield: 91.1%).

[0088] Methyl 2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)-5-nitrobenzoate (4.2 g, 9.3 mmol, 1.0 eq) was dissolved in 25 mL of THF, and dry palladium on carbon (840 mg, palladium content: 10%) was added. The mixture was then placed in a hydrogenation reactor (H2: 0.4 MPa) and stirred for 4 h. The mixture was filtered through diatomaceous earth, the filtrate was evaporated to dryness, and the residue was separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 5-amino-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)benzoate (3.8 g, yield: 96.8%).

[0089] Add methyl 5-amino-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)benzoate (200 mg, 0.47 mmol, 2.2 eq), 2,4-dichloro-6-methyl-1,3,5-triazine (35 mg, 0.22 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)-5-(4-((4-(5-(4-chlorophenyl)phenylthiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methyl-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, 3 mL of methanol, and 2.5 mL of saturated lithium hydroxide solution. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5-((4-((3-carboxy-4-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-6-methyl-1,3,5-triazin-2-yl)amino)-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)benzoic acid (TN8, 56 mg, yield: 27%).

[0090] White solid. 1 H NMR (400MHz, DMSO-d6) δ9.79(s,2H),8.21(d,J=5.1Hz,1H),8.04(s,3H),7.71–7.62(m,4H),7.50(dt,J=22.9,11.4Hz,10H),2.26(s,3H).HRMS calcd forC 38 H 27 Cl2N7O8S4[M+H] + ,930.0073; found,930.0046.

[0091] Example 9: Preparation and detection of compound TN9

[0092] The synthesis method was the same as that for the preparation of intermediates in Example 8, using methyl 5-amino-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamide)benzoate as the starting material.

[0093]

[0094] Add methyl 5-amino-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)benzoate (200 mg, 0.47 mmol, 2.2 eq), 2,4-dichloro-1,3,5-triazine (33 mg, 0.22 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)-5-(4-((4-(5-(4-chlorophenyl)phenylthiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5-((4-((3-carboxy-4-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-1,3,5-triazin-2-yl)amino)-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)benzoic acid (TN 9.54 mg, yield: 27%).

[0095] White solid. 1 H NMR(400MHz,DMSO-d6)δ10.96(s,2H),9.93(s,2H),8.35(s,1H),8.09(s,3H),7.68(d, J=8.1Hz,4H),7.59(d,J=3.9Hz,2H),7.55(d,J=8.9Hz,4H),7.47(d,J=8.2Hz,5H).HRMS calcd for C 37 H 25 Cl2N7O8S4[M+Na] + ,915.9917;found,915.9917.

[0096] Example 10: Preparation and detection of compound TN10

[0097]

[0098] 1.5 g (7.69 mmol, 1.0 eq) of 2-amino-5-nitrobenzoate was added to a 100 mL round-bottom flask and dissolved in 30 mL of tetrahydrofuran (THF). Sodium hydroxide (923 mg, 23 mmol, 3.0 eq) was then added and stirred in an ice bath for 30 minutes. 5-Bromothiophene-2-sulfonyl chloride (3 g, 11.54 mmol, 1.5 eq) was dissolved in 10 mL of THF and added dropwise in an ice bath. After the addition was complete, stirring was continued for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 20 mL of saturated ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain methyl 2-((5-bromothiophene)-2-sulfonamido)-5-nitrobenzoate (3.22 g, yield: 99.7%).

[0099] To a 100 mL round-bottom flask, add methyl 2-((5-bromothiophene)-2-sulfonamido)-5-nitrobenzoate (3.22 g, 7.67 mmol, 1.0 eq), iron powder (2.13 g, 38 mmol, 5.0 eq), tetrahydrofuran, methanol, and 5 mL of saturated ammonium chloride solution. Heat the reaction mixture at 85 °C for 2 h, monitoring the reaction by TLC. After the reaction is complete, filter the diatomaceous earth solution while hot, concentrate the filtrate by rotary evaporation, add water, extract with ethyl acetate, combine the organic phases, dry to dryness with anhydrous sodium sulfate, and perform column chromatography to obtain methyl 2-((5-bromothiophene)-2-sulfonamido)-5-aminobenzoate (2.8 g, yield: 93.6%).

[0100] Add methyl 2-((5-bromothiophene)-2-sulfonamido)-5-aminobenzoate (2.8 g, 7.2 mmol, 2.2 eq), 2,4-dichloro-6-methoxy-1,3,5-triazine (590 mg, 3.3 mmol, 1.0 eq), and acetone (40 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 40 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to give methyl 2-((5-bromothiophene)-2-sulfonamido)-5-((4-(4-(((5-bromothiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate (2.6 g, yield: 88.8%).

[0101] Add 4-fluorophenylboronic acid (50 mg, 0.36 mmol, 3.2 eq), methyl 2-((5-bromothiophene)-2-sulfonamido)-5-((4-(4-((5-bromothiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate (100 mg, 0.11 mmol, 1.0 eq), potassium carbonate (50 mg, 0.36 mmol, 3.2 eq), 1,4-dioxane (10 mL), and water (2 mL) to a 100 mL round-bottom flask, and purge with nitrogen three times. Add tetra(triphenylphosphine)palladium (28 mg, 0.024 mmol, 0.22 eq), and purge with nitrogen three times. The reaction solution was heated under reflux at 130 °C for 4 h, the solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate and water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(4-fluorophenyl)thiophene)-2-sulfonamido)-5-(4-((4-(5-(-4-fluorophenyl)phenyl)-2-sulfonamide)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction solution was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5-(4-((3-carboxy-4-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)-2-((5-(4-fluorophenyl)thiophene)-2-sulfonamido)benzoic acid (40 mg, yield: 40.8%).

[0102] White solid. 1 H NMR(500MHz, DMSO-d6)δ9.51(s,2H),7.89(d,J=45.7Hz,1H),7.68(d,J=7.1Hz,5H),7.51–7.35(m,6H),7.24(t,J=8.6Hz,6H),3.84(s,3H).HRMS calcd forC 38 H 27 F2N7O9S4[M+H] + ,892.0794; found,892.0795.

[0103] Example 11: Preparation and detection of compound TN11

[0104] Methyl benzoate 2-((5-bromothiophene)-2-sulfonamido)-5-((4-(4-(((5-bromothiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazine-2-yl)amino)benzoate was used as the starting material and the synthesis method was the same as that for the preparation of intermediates in Example 10.

[0105]

[0106] Add 3,4-difluorophenylboronic acid (55 mg, 0.36 mmol, 3.2 eq), methyl 2-((5-bromothiophene)-2-sulfonamido)-5-((4-(4-((5-bromothiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate (100 mg, 0.11 mmol, 1.0 eq), potassium carbonate (50 mg, 0.36 mmol, 3.2 eq), 1,4-dioxane (10 mL), and water (5 mL) to a 100 mL round-bottom flask, and purge with nitrogen three times. Add tetra(triphenylphosphine)palladium (28 mg, 0.024 mmol, 0.22 eq), and purge with nitrogen three times. The reaction solution was heated under reflux at 130 °C for 4 h, the solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate and water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)-5-(4-((4-(5-(-4-fluorophenyl)phenyl)-2-sulfonamide)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction solution was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, followed by extraction with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Separation was performed by silica gel column chromatography, and the solvent was removed by rotary evaporation to obtain the target compound 5-(4-((3-carboxy-4-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)-2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)benzoic acid (55 mg, yield: 54%).

[0107] White solid. 1H NMR(500MHz,DMSO-d6)δ9.62–9.39(m,2H),8.24(s,1H),7.79(q,J=15.9,13.0Hz,4H),7.56–7.30(m,11H),3.84(s,3H).HRMS calcd for C 38 H 25 F4N7O9S4[M+H] + ,928.0605;found,928.0598.

[0108] Example 12: Preparation and detection of compound TN12

[0109] Methyl benzoate 2-((5-bromothiophene)-2-sulfonamido)-5-((4-(4-(((5-bromothiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazine-2-yl)amino)benzoate was used as the starting material and the synthesis method was the same as that for the preparation of intermediates in Example 10.

[0110]

[0111] Add 3-fluorophenylboronic acid (50 mg, 0.36 mmol, 3.2 eq), methyl 2-((5-bromothiophene)-2-sulfonamido)-5-((4-(4-((5-bromothiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate (100 mg, 0.11 mmol, 1.0 eq), potassium carbonate (50 mg, 0.36 mmol, 3.2 eq), 1,4-dioxane (10 mL), and water (2 mL) to a 100 mL round-bottom flask, and purge with nitrogen three times. Add tetra(triphenylphosphine)palladium (28 mg, 0.024 mmol, 0.22 eq), and purge with nitrogen three times. The reaction solution was heated under reflux at 130 °C for 4 h, the solvent was removed by rotary evaporation, and the mixture was extracted with ethyl acetate and water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(3-fluorophenyl)thiophene)-2-sulfonamido)-5-(4-((4-(5-(-4-fluorophenyl)phenyl)-2-sulfonamide)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction solution was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, followed by extraction with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Separation was performed by silica gel column chromatography, followed by further solvent removal by rotary evaporation to obtain the target compound 5-(4-((3-carboxy-4-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)-2-((5-(3-fluorophenyl)thiophene)-2-sulfonamido)benzoic acid. (59 mg, yield: 58%)

[0112] White solid. 1 H NMR(500MHz,DMSO-d6)δ9.59–9.32(m,2H),8.29–7.59(m,2H),7.58–7.31(m,14H),7.21–7.16(m,2H),3.84(s,3H).HRMS calcd for C 38 H 27 F2N7O9S4[M+H] + ,914.0613;found,914.0596.

[0113] Example 13: Preparation and detection of compound TN13

[0114]

[0115] Add 3,4-difluorophenylboronic acid (6.3 g, 39.9 mmol, 1.15 eq), 2-iodothiophene (7.3 g, 34.8 mmol, 1.0 eq), potassium carbonate (6.9 g, 50 mmol, 1.5 eq), 1,4-dioxane (100 mL), and water (20 mL) to a 250 mL round-bottom flask, and purge with nitrogen three times. Add tetra(triphenylphosphine)palladium (1.5 g, 1.3 mmol, 0.04 eq), and purge with nitrogen three times. Heat the reaction mixture under reflux at 130 °C for 4 h, remove the solvent by rotary evaporation, extract with ethyl acetate and water, wash the organic phase with saturated brine, dry to anhydrous sodium sulfate, separate by silica gel column chromatography, and remove the solvent by rotary evaporation to obtain 2-(3,4-difluorophenyl)thiophene (6.77 g, yield: 99.2%).

[0116] 2-(3,4-difluorophenyl)thiophene (6.77 g, 34.5 mmol, 1.0 eq), ethyl acetate (100 mL), and acetic anhydride (6.9 mL, 69 mmol, 2.0 eq) were added to a 250 mL round-bottom flask. Concentrated sulfuric acid (4.0 mL, 69 mmol, 2.0 eq) was slowly added dropwise under ice bath conditions. After the addition was complete, the mixture was moved to room temperature and stirred for 24 h. A white solid precipitated upon the addition of saturated brine (150 mL) and ethyl acetate (100 mL). The organic phase was filtered and dried to give 5-(3,4-difluorophenyl)thiophene-2-sulfonic acid (9.5 g, yield: 99.7%).

[0117] Add 9.5 g (34.4 mmol, 1.0 eq) of 5-(3,4-difluorophenyl)thiophene-2-sulfonic acid, 100 mL of thionyl chloride, and 1 drop of N,N-dimethylformamide to a 250 mL round-bottom flask. Heat the reaction mixture under reflux at 85 °C for 8 h, cool to room temperature, and slowly pour the mixture into crushed ice (300 g), generating a large amount of gas. Extract with dichloromethane, dry the organic phase with anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain 7.2 g (71.2% yield) of 5-(3,4-difluorophenyl)thiophene-2-sulfonyl chloride.

[0118] 1.96 g (10 mmol, 1.0 eq) of 2-amino-5-nitrobenzoate was added to a 100 mL round-bottom flask and dissolved in 30 mL of tetrahydrofuran (THF). Sodium hydroxide (1.2 mg, 30 mmol, 3.0 eq) was then added and stirred in an ice bath for 30 minutes. 5-(3,4-difluorophenyl)thiophene-2-sulfonyl chloride (4.41 g, 15 mmol, 1.5 eq) was dissolved in 10 mL of THF and added dropwise in an ice bath. After the addition was complete, stirring was continued for 2 h, and the reaction was monitored by TLC. After the reaction was complete, 20 mL of saturated ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain methyl 2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)-5-nitrobenzoate (4.1 g, yield: 90.3%).

[0119] Methyl 2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)-5-nitrobenzoate (4.1 g, 9.03 mmol, 1.0 eq) was dissolved in 25 mL of THF, and dry palladium on carbon (1 g, palladium content: 10%) was added. The mixture was stirred in a hydrogenation reactor (H2: 0.4 MPa) for 4 h. After diatomaceous earth filtration, the filtrate was evaporated to dryness, separated by silica gel column chromatography, and the solvent was removed by rotary evaporation to obtain methyl 5-amino-2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)benzoate (2.7 g, yield: 70.5%).

[0120] Add methyl 5-amino-2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)benzoate (424 mg, 1 mmol, 3.0 eq), 2,4-dichloro-1,3,5-triazine (50 mg, 0.335 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)-5-(4-(4-(5-(3,4-difluorophenyl,thiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, followed by extraction with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Separation was performed by silica gel column chromatography, and the solvent was removed by rotary evaporation again to obtain the target compound 5-(4-((3-carboxy-4-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-1,3,5-triazin-2-yl)amino)-2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)benzoic acid. (126 mg, yield: 42%)

[0121] White solid. 1 H NMR(500MHz,DMSO-d6)δ9.82(s,2H),8.32(s,1H),7.83(s,2H),7.64(s,1H),7.55(d,J=7.5Hz,4H),7.53–7.44(m,8H).HRMS calcd for C 37 H 23 F4N7O8S4[M+H] + ,898.0500; found,898.0521.

[0122] Example 14: Preparation and detection of compound TN14

[0123] Methyl 5-amino-2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamide)benzoate was used as the starting material, and the synthesis method was the same as that for the preparation of intermediates in Example 13.

[0124]

[0125] Add methyl 5-amino-2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)benzoate (390 mg, 0.92 mmol, 3.0 eq), 2,4-dichloro-6-methyl-1,3,5-triazine (50 mg, 0.307 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)-5-(4-(4-(5-(3,4-difluorophenyl,thiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methyl-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, followed by extraction with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. Separation was performed by silica gel column chromatography, followed by further solvent removal by rotary evaporation to obtain the target compound 5-(4-((3-carboxy-4-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-6-methyl-1,3,5-triazin-2-yl)amino)-2-((5-(3,4-difluorophenyl)thiophene)-2-sulfonamido)benzoic acid. (130 mg, yield: 46%)

[0126] White solid. 1 H NMR(500MHz,DMSO-d6)δ10.90(s,2H),8.10(d,J=69.0Hz,2H),7.81(s,2H),7.62–7.51(m,6H),7.47(s,6H),2.30(s,3H).HRMS calcd for C 38 H 25 F4N7O8S4[M+H] + ,921.0656;found,912.0631.

[0127] Example 15: Preparation and detection of compound TN15

[0128] The synthesis method was the same as that for the preparation of intermediates in Example 8, using methyl 5-amino-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamide)benzoate as the starting material.

[0129]

[0130] Add methyl 5-amino-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)benzoate (200 mg, 0.47 mmol, 2.2 eq), 2,4-dichloro-6-methoxy-1,3,5-triazine (39 mg, 0.22 mmol, 1.0 eq), and acetone (20 mL) to a 50 mL round-bottom flask. Reflux the reaction mixture at 70 °C for 2 h and monitor the reaction by TLC. After the reaction was complete, 20 mL of saturated sodium bicarbonate solution was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography. The solvent was removed by rotary evaporation to obtain methyl 2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)-5-(4-((4-(5-(4-chlorophenyl)phenylthiophene)-2-sulfonamido)-3-(methoxycarbonyl)phenyl)amino)-6-methoxy-1,3,5-triazin-2-yl)amino)benzoate. The solid was dissolved in 10 mL of THF, and 3 mL of methanol and 2.5 mL of saturated lithium hydroxide solution were added. The reaction mixture was heated at 60 °C for 2 h, and the reaction was monitored by TLC. After the reaction was complete, dilute hydrochloric acid (1 mol / L, 20 mL) was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then separated by silica gel column chromatography and the solvent was removed by rotary evaporation to obtain the target compound 5-((4-((3-carboxy-4-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)phenyl)amino)-6-methoxy-1,3,5-triazine-2-yl)amino)-2-((5-(4-chlorophenyl)thiophene)-2-sulfonamido)benzoic acid (TN 15, 54 mg, yield: 27%).

[0131] White solid. 1 H NMR (400MHz, DMSO-d6) δ9.75 (s, 2H), 7.96 (s, 3H), 7.68 (d, J = 8.0Hz, 4H), 7.57 (d ,J=4.0Hz,2H),7.55–7.50(m,4H),7.47(d,J=8.2Hz,5H),3.86(s,3H).HRMScalcd for C 38 H 27 Cl2N7O9S4[MH] - ,922.0057; found,922.0074.

[0132] II. Antibacterial Activity Assay

[0133] Example 1: Determination of the inhibitory activity of the compound against Acinetobacter baumannii ATCC 19606

[0134] 1. Preparation of test bacterial suspension

[0135] Acinetobacter baumannii standard strain ATCC 19606 was inoculated into 20 mL of tryptone soybean broth and incubated at 37°C with shaking at 200 rpm for 18 hours. When the clear medium became turbid, it indicated significant bacterial proliferation and vigorous growth. At this point, the bacterial suspension was diluted with fresh tryptone soybean broth to achieve an OD600 value between 0.3 and 0.5; it was then further diluted 10 times with fresh tryptone soybean broth. 3 The serum-free test bacterial solution was prepared by adding mouse serum and mixing thoroughly to prepare the test bacterial solution with 1% mouse serum.

[0136] 2. Determine the minimum inhibitory concentration.

[0137] Take a clean, sterile 96-well cell culture plate. Add 200 μL of the prepared serum-free test bacterial solution or the test bacterial solution with 1% mouse serum to each well in the first column. Add 100 μL of the serum-free test bacterial solution or the test bacterial solution with 1% mouse serum to each well in columns two through twelve. Add 4 μL of the pre-prepared 1.6 mg / mL DMSO solution of the test sample to each well in column one (three replicates per sample). Set up a positive control group (i.e., 4 μL of levofloxacin at the same concentration) and a blank control (i.e., no drug added). Starting from the wells in column one, use an 8-channel micropipette to sequentially add 100 μL of the sample from the previous column to each well in the next column for a 2-fold serial dilution, setting up 12 different concentrations of the compound solution: 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, and 0.015625 μg / mL. The 96-well cell culture plates were placed in an incubator at 37°C and cultured for 18 hours. The growth of Acinetobacter baumannii in each well was then observed. For each compound, the concentration of the compound in wells where no bacterial growth was observed was the minimum inhibitory concentration (MIC) of that compound. Levofloxacin was used as a positive control.

[0138] 3. Data on the compound's activity against Acinetobacter baumannii (ATCC 19606) are shown in Table 1.

[0139] Table 1. Minimum inhibitory concentrations of compounds against Acinetobacter baumannii

[0140]

Claims

1. A compound of formula (1) or a pharmaceutically acceptable salt thereof, Wherein: M1 is independently selected from hydrogen, C 1-8 Alkyl, C 1-8 Alkoxy, methylthio, phenyl; L1 is independently selected from aryl or heteroaryl; L2 is independently selected from hydrogen and C. 1-8 Alkyl, phenyl, substituted phenyl. The C 1-8 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, straight-chain or branched pentyl, straight-chain or branched hexyl, straight-chain or branched heptyl, and straight-chain or branched octyl. The aryl group is selected from phenyl, naphthyl, anthraceneyl, phenanthryl, or pyrene; The heteroaryl group is selected from furan, thiophene, oxazole, thiazole, isoxazole, oxadiazole, thiadiazole, pyrrole, pyrazole, imidazole, pyridine, pyrimidine, pyrazine, pyridazine, phthalazine, quinoline, isoquinoline, pteridine, purine, indole, isoindole, benzofuranyl, benzothiophenyl, benzopyridyl, benzopyrimidinyl, benzopyrazinyl, benzoimidazolyl, or benzophthalazinyl. The substituted phenyl group, characterized in that , R1, R2, R3, R4, and R5 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, methoxy, nitro, cyano, hydroxyl, amino, and aminomethyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that... , M1 is independently selected from hydrogen, methyl, methoxy, methylthio, and phenyl; L1 is independently selected from phenyl and thiophene; L2 is independently selected from n-butyl, phenyl, 4-chlorophenyl, 4-fluorophenyl, 3-fluorophenyl, and 3,4-difluorophenyl.

3. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-2, characterized in that... The compounds are selected from the following group:

4. A method for preparing the compound according to any one of claims 1 to 3, characterized in that, Includes the following steps: The intermediate (f) is then converted into the compound (1) of claims 1-4 under alkaline conditions; The definitions of L1, L2 and M1 are the same as in claim 1.

5. The preparation method according to claim 4, wherein the alkaline conditions are provided by sodium hydroxide, potassium hydroxide or lithium hydroxide.

6. The preparation method according to claim 4, characterized in that, The intermediate (f) is prepared as follows: Intermediate (f) is prepared by reacting intermediate (d) and intermediate (e) in an organic solvent; The definitions of L1, L2 and M1 are the same as in claim 1.

7. The preparation method according to claim 6, wherein the organic solvent is selected from acetone and acetonitrile.

8. The preparation method according to claim 6, characterized in that, The intermediate (d) is prepared as follows: Intermediate (c) was converted into intermediate (d) by Pd / C catalysis under hydrogen conditions in an organic solvent; L1 and L2 are defined as in claim 1.

9. The preparation method according to claim 8, characterized in that... The organic solvent is selected from tetrahydrofuran or a dichloromethane-methanol mixture.

10. The preparation method according to claim 8, characterized in that, The intermediate (c) is prepared as follows: That is, intermediate (c) is prepared by reacting intermediate (a) and intermediate (b) under alkaline conditions in an organic solvent; The definitions of L1 and L2 are the same as in claim 1.

11. The preparation method according to claim 10, characterized in that, The organic solvent is selected from tetrahydrofuran and N,N-dimethylformamide, and the alkaline conditions are selected from sodium hydrogen.

12. A pharmaceutical composition, characterized in that... It includes the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier thereof.

13. The pharmaceutical composition according to claim 12, characterized in that... The carrier is selected from conventional pharmaceutical excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, lubricants, etc. The pharmaceutical composition is selected from injections, tablets, pills, capsules, suspensions, emulsions or ointments. The route of administration is selected from intravenous or intramuscular injection, oral administration, transdermal administration, mucosal administration, rectal administration, and vaginal administration.

14. Use of the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial drug.

15. The application according to claim 14, characterized in that... The application in the preparation of antibacterial drugs refers to the application in the preparation of drugs for the treatment or prevention of infectious diseases in humans or animals caused by pathogenic microorganisms such as bacteria, mycoplasma, chlamydia, rickettsia, spirochetes, and fungi.

16. The application according to claim 15, characterized in that... The bacteria are selected from Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Clostridium difficile, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae; the mycoplasma is selected from Mycoplasma pneumoniae, Ureaplasma urealyticum, Mycoplasma hominis, and Mycoplasma genitalium; and the chlamydia is selected from Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, and Chlamydia bovis.

17. The application according to claim 16, characterized in that... The *Acinetobacter baumannii* is selected from drug-sensitive *Acinetobacter baumannii*, multidrug-resistant *Acinetobacter baumannii*, pan-drug-resistant *Acinetobacter baumannii*, carbapenem-resistant *Acinetobacter baumannii*, and pan-drug-resistant *Acinetobacter baumannii*. The *Staphylococcus aureus* is selected from methicillin-sensitive *Staphylococcus aureus* and methicillin-resistant *Staphylococcus aureus*. The *Staphylococcus epidermidis* is selected from methicillin-sensitive *Staphylococcus epidermidis* and methicillin-resistant *Staphylococcus epidermidis*. The *Enterococcus faecalis* is selected from vancomycin-sensitive *Enterococcus faecalis* and vancomycin-resistant *Enterococcus faecalis*. The *Enterococcus faecium* is selected from vancomycin-sensitive *Enterococcus faecium* and vancomycin-resistant *Enterococcus faecium*. The *Mycoplasma* is selected from drug-sensitive *Mycoplasma pneumoniae* or drug-resistant *Mycoplasma pneumoniae*.

Images