Compound for recovering sensitivity of beta-lactam antibiotics, composition and application

Abstract

The invention discloses a compound for recovering sensitivity of beta-lactam antibiotics, a composition and application, and belongs to the field of chemical medicines. The invention provides a boric acid compound with a brand new structure as shown in a formula (I) and a pharmaceutically acceptable salt thereof, on one hand, the boric acid compound has better metabolic stability and biological activity, and the administration time interval is expanded; on the other hand, the method can be better used for interfering a beta-lactam antibiotic reaction pathway, so that MSSA and MRSA bacteria have better sensitivity to lactam drugs.

Description

Technical Field

[0001] This invention belongs to the field of chemical medicine, specifically relating to a compound, composition, and use for restoring the sensitivity of β-lactam antibiotics. Background Technology

[0002] Staphylococcus aureus is a Gram-positive bacterium and one of the most common pathogens causing hospital-acquired and community-acquired infections. It easily leads to skin and soft tissue infections, pneumonia, endocarditis, pseudomembranous colitis, osteomyelitis, and other conditions. When it enters the bloodstream, it can even cause systemic infections such as sepsis and septicemia, posing a serious threat to human health.

[0003] Although penicillin has been clinically effective in treating infectious diseases caused by Staphylococcus aureus since its advent, the increased frequency and scope of penicillin use have led some Staphylococcus aureus strains to develop penicillinase, which hydrolyzes penicillin containing a β-lactam ring, resulting in penicillin resistance. With advancements in science and technology, a new type of semi-synthetic penicillin resistant to penicillinase hydrolysis, methicillin, has been developed. While methicillin effectively controlled infections caused by penicillinase-producing *S. aureus* in the short term, its detection rate has been steadily increasing since the first discovery of methicillin-resistant *S. aureus* (MRSA) in the 1960s. In recent years, due to frequent invasive procedures, the establishment of multiple intravenous access sites, and the widespread use of antibiotics, the resistance of *S. aureus* has been on the rise. MRSA exhibits multidrug resistance and is characterized by wide transmission routes, high pathogenicity, and high mortality, thus gradually attracting clinical attention.

[0004] Historically, β-lactam antibiotics have been commonly used to treat Staphylococcus aureus infections. However, the efficacy of these antibiotics is significantly reduced in MRSA mediated by the Staphylococcus aureus resistance determinant (mec) and β-lactamase regulatory gene (bla). When β-lactam antibiotics act on MRSA, they are recognized by the BlaR and / or MecR sensor domains, which transmit signals to the cytoplasm, causing desuppression of the antibiotic resistance gene and leading to resistance. Therefore, inhibiting the sensor domain will shut down this response system, thereby restoring the sensitivity of both MSSA and MRSA bacteria to β-lactam antibiotics.

[0005] The structures of currently reported high-activity small molecules are as follows:

[0006] The metabolic stability and bioactivity of this compound need further improvement. Summary of the Invention

[0007] [Technical Issues]

[0008] This invention provides a novel class of boric acid compounds and their pharmaceutically usable salts. Compared with existing compounds, these compounds have better metabolic stability and biological activity, and can be better used to interfere with the β-lactam antibiotic response pathway, thereby making MSSA and MRSA bacteria more sensitive to β-lactam drugs.

[0009] [Technical Solution]

[0010] This invention provides borate compounds with the structure shown in general formula (I), or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, or prodrugs thereof:

[0011]

[0012] In the formula,

[0013] X is independently selected from O, S, and Se;

[0014] Y1, Y2, Y3, Y4, Y5, and Y6 are independently selected from CH and N;

[0015] R1 and R2 are independently selected from H and D, respectively;

[0016] When X is selected from S and R1 and R2 are selected from H, at least one of Y1, Y2, Y3, Y4, Y5, and Y6 is N;

[0017] R3, R4, and R5 are independently selected from H, halogens (F, Cl, Br, I), alkyl halogens, SF5, and CN, respectively.

[0018] In one embodiment of the present invention, the haloalkyl group may specifically be CF3.

[0019] In one embodiment of the present invention, the above-mentioned boric acid compound may be selected from:

[0020]

[0021] In one embodiment of the present invention, the pharmaceutically acceptable salt is an inorganic salt or an organic salt, wherein the inorganic salt is selected from sodium salt, calcium salt, potassium salt, magnesium salt, silver salt, and lithium salt; and the organic salt is selected from meglumine salt, tromethamine salt, diethylamine salt, lysine salt, choline salt, arginine salt, tert-butylamine salt, and N,N-dibenzylethylenediamine salt.

[0022] The present invention also provides a pharmaceutical composition comprising the compound described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and pharmaceutical excipients.

[0023] In one embodiment of the present invention, the pharmaceutical excipient includes a pharmaceutically acceptable diluent, excipient, or carrier.

[0024] In one embodiment of the invention, a therapeutically effective amount of the compound of formula (I) is administered in combination with other drugs.

[0025] In one embodiment of the present invention, the other drug may be any one or more β-lactam antibiotics.

[0026] In one embodiment of the present invention, the dosage form of the pharmaceutical composition includes injection, lyophilized powder for injection, suspension, implant, embolization, capsule, tablet, pill and oral liquid.

[0027] The compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated into solid dosage forms for oral administration, including, but not limited to, capsules, tablets, pills, powders, granules, etc. In these solid dosage forms, the compound of general formula (I) of the present invention is mixed as the active ingredient with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate. Or it may be mixed with the following components: (1) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol and silica; (2) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, gum arabic; (3) humectants, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginic acid, certain silicates and sodium carbonate; (5) slowing agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate; (8) adsorbents, such as kaolin; (9) lubricants, such as talc, calcium stearate, solid polyethylene glycol, sodium dodecyl sulfate, etc., or mixtures thereof. Buffers may also be included in capsules, tablets and pills. The solid dosage forms, such as tablets, sugar pills, capsules, pellets, and granules, can be coated or microencapsulated with coating and shell materials such as enteric coatings and other materials known in the art. They may contain opaque agents, and the release of the active ingredient from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active ingredient may also be formed into microcapsules with one or more of the excipients described above.

[0028] The compounds of this invention, or pharmaceutically acceptable salts thereof, can be formulated into liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, etc. In addition to the compound of general formula (I) or its pharmaceutically acceptable salt as the active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water and other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil, etc., or mixtures thereof. Besides these inert diluents, the liquid dosage forms of this invention may also include conventional adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. The suspending agents include, for example, ethoxylated octadecyl alcohol, polyoxyethylene sorbitol, and dehydrated sorbitol, microcrystalline cellulose, agar, etc., or mixtures thereof.

[0029] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, and excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0030] The compounds of this invention or pharmaceutically acceptable salts thereof can be formulated into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays, and inhalers. The compounds of general formula (I) of this invention or pharmaceutically acceptable salts thereof, as active ingredients, are mixed under sterile conditions with a physiologically acceptable carrier and optionally with preservatives, buffers, and propellants, if necessary.

[0031] The pharmaceutical compositions of the present invention comprise a compound of general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, excipient, and diluent. In preparing the pharmaceutical compositions, the compound of general formula (I) or a pharmaceutically acceptable salt thereof is typically mixed with a pharmaceutically acceptable carrier, excipient, or diluent. The content of the compound of general formula (I) or a pharmaceutically acceptable salt thereof can be 0.01-1000 mg, for example 0.05-800 mg, 0.1-500 mg, 0.01-300 mg, 0.01-200 mg, 0.05-150 mg, 0.05-50 mg, etc.

[0032] According to one embodiment of the invention, a therapeutically effective amount of the compound of formula (I) is administered in combination with other drugs.

[0033] In one embodiment of the invention, the pharmaceutical composition, when formulated, can be administered systematically or sequentially, and can be delivered to the tumor site by any effective mode of administration. The mode of administration includes, but is not limited to, oral administration of the compound or composition, local, percutaneous, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal injection, intranasal instillation, intracavitary or intravesical instillation, intraocular, intra-arterial, intralesional, or via application to mucous membranes such as nasal, laryngeal, and bronchial mucosal tubes.

[0034] This invention also provides the use of the above-mentioned boric acid compounds or their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels, and prodrugs in the preparation of medicaments for treating bacterial infections.

[0035] The present invention also provides a method for treating bacterial infection, comprising administering a therapeutically effective amount of the above-described compound or compound composition to a subject infected with bacteria, thereby treating the bacterial infection.

[0036] In addition, the present invention provides a method for treating methicillin-resistant Staphylococcus aureus (MRSA) infection, comprising administering a therapeutically effective amount of antibiotic and the above-described compound or compound combination to a subject infected with MRSA, thereby treating the bacterial infection.

[0037] The present invention also provides a method for restoring the sensitivity of β-lactam antibiotic-resistant bacteria to bacteria susceptible to β-lactam antibiotics, comprising administering an effective amount of the above-mentioned boric acid compound to a patient infected with β-lactam antibiotic-resistant bacteria, wherein the administration of the compound restores the sensitivity of the bacteria to β-lactam antibiotics.

[0038] Beneficial effects:

[0039] This invention provides a class of boronic acid compounds with novel structures as shown in formula (I) and their pharmaceutically acceptable salts, which, on the one hand, have better metabolic stability and biological activity than the reference compound (R4), and extend the dosing interval;

[0040] On the other hand, it can also be used more effectively to interfere with the β-lactam antibiotic response pathway, thereby making MSSA and MRSA bacteria more sensitive to β-lactam drugs. Detailed Implementation

[0041] The following examples are illustrative and not limiting of the synthesis of compounds of general formula (I). All temperatures are in degrees Celsius. Unless otherwise stated, all evaporations were performed under reduced pressure. Unless otherwise stated, reagents were purchased from commercial suppliers and used without further purification. The structures of the final products, intermediates, and starting materials were confirmed by standard analytical methods, such as elemental analysis and spectroscopic characterization, such as MS and NMR. Abbreviations used are conventional abbreviations in the art.

[0042] Example 1: ((1-(2,4-bis(trifluoromethyl)phenyl)methyl-d2)-6-cyano-1H-benzo[d]imidazol-2-yl)thio)methyl)boronic acid (compound 1)

[0043]

[0044] Step 1: Preparation of (2,4-bis(trifluoromethyl)phenyl)methane-d2-ol (intermediate a-2)

[0045] Ethyl 2,4-bis(trifluoromethyl)benzoate (intermediate a-1, 1.43 g, 5.0 mmol) was dissolved in anhydrous THF (15 mL). Lithium aluminum deuteride (210 mg, 5.0 mmol) was slowly added under ice bath conditions. After the addition was complete, the reaction was carried out at 0 °C for 3 hours. After the reaction was completed, water was slowly added to quench the reaction. The mixture was extracted with ethyl acetate (50 mL x 2), and the organic phases were combined. The mixture was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to give intermediate a-2 (0.75 g, yield: 61%). MS-ESI (m / z): 247.10 [M+1] + .

[0046] Step 2: Preparation of 1-(chloromethyl-d2)-2,4-bis(trifluoromethyl)benzene (intermediate a-3)

[0047] Under nitrogen atmosphere, acetonitrile (10 ml) and (2,4-bis(trifluoromethyl)phenyl)methane-d2-ol (272 mg, 1.1 mmol) were added to the reactor. The mixture was heated to 45 ± 5 °C. Thionyl chloride (1.0 ml) was added over approximately 10 min while maintaining the temperature at 45 ± 5 °C. The mixture was stirred until the reaction was complete. Water (10 ml) was slowly added over approximately 30 min while maintaining the temperature at 45 ± 5 °C, followed by mixing for approximately 1 h. Over several hours, the suspension was cooled to 5 ± 5 °C and then mixed for approximately 1 h. The product was collected and washed with water (5 ml) and acetonitrile (5 ml). The product was dried under vacuum at 40–60 °C to give intermediate a-3 (213 mg, yield: 73%). MS-ESI (m / z): 264.98 [M+1] +

[0048] Step 3: Preparation of 4-amino-3-((2,4-bis(trifluoromethyl)phenyl)methyl-d2)amino)benzonitrile (intermediate a-4)

[0049] In a 100 mL three-necked round-bottom flask equipped with a magnetic stir bar, 3,4-diaminobenzonitrile (intermediate b-1, 60 mg, 0.45 mmol) and potassium carbonate (K₂CO₃) (375 mg, 0.2 mmol) were added, followed by 5 mL of anhydrous DMF to obtain a suspension. 1-(chloromethyl-d₂)-2,4-bis(trifluoromethyl)benzene (intermediate a-3) (26.4 mg, 0.1 mmol) was added dropwise. The mixture was stirred at room temperature and reacted under a nitrogen atmosphere for 24 hours. The reaction mixture was diluted with EtOAc (5 mL) and washed with water (3 × 2 mL) and brine (2 mL). The organic layer was dried over Na₂SO₄ and filtered. The filtrate was evaporated to give an oil. The target compound intermediate a-4 (213 mg, yield: 33%) was purified by silica gel chromatography. MS-ESI (m / z): 362.08 [M+1] +

[0050] Step 4: Preparation of 3-(2,4-bis(trifluoromethyl)phenyl)methyl-d2)-2-thio-2,3-dihydro-1H-benzo[d]imidazolium-5-nitrile (intermediate a-5)

[0051] In a 50 mL round-bottom flask equipped with a magnetic stir bar, intermediate a-4 (253 mg, 0.7 mmol) and 1,1'-thiocarbonyldiimidazole (TCDI, 196 mg, 1.1 mmol) were dissolved in anhydrous DMF (5.0 mL). The reaction mixture was refluxed for 3 hours and then cooled to room temperature. The reaction mixture was poured into a beaker containing 10 mL of ice water and stirred for 30 minutes to give a grayish-white precipitate. The precipitate was filtered and washed with deionized water (5 mL) and n-pentane (5 mL). The product was dried under vacuum overnight to give solid intermediate a-5 (133 mg, yield: 47%). MS-ESI (m / z): 404.09 [M+1] +

[0052] Step 5: Preparation of 1-((2,4-bis(trifluoromethyl)phenyl)methyl-d2)-2-((6-methyl-4,8-dioxo-1,3,6,2-diazaborane-2-yl)methyl)thio)-1H-benzo[d]imidazolium-6-nitrile (intermediate a-6)

[0053] At room temperature, diisopropyl-azodicarboxylate (130 mg, 0.64 mmol) was slowly added to an anhydrous THF solution (10 mL) of intermediate b-2 (100 mg, 0.54 mmol), polymer-bound triphenylphosphine PPh3 (0.50 g, 0.80 mmol), and intermediate a-5 (324 mg, 0.80 mmol) fitted with a magnetic stir bar. The reaction mixture was stirred for 6 hours. The triphenylphosphine resin was filtered and washed with ethyl acetate (10 mL × 3). The filtrate was concentrated to an oil under reduced pressure. The oil was suspended in t-BuOMe (20 mL), resulting in a white precipitate. The product was collected by filtration, and the filtrate was discarded. The product precipitate was resuspended in t-BuOMe (20 mL) three times. The target product, white borate intermediate a-6 (86 mg, yield: 27%), was obtained by silica gel chromatography. MS-ESI (m / z): 573.19 [M+1] + .

[0054] Step 6: Preparation of ((1-(2,4-bis(trifluoromethyl)phenyl)methyl-d2)-6-cyano-1H-benzo[d]imidazol-2-yl)thio)methyl)boronic acid (compound 1)

[0055] At room temperature, the borate ester of intermediate a-6 (241 mg, 0.42 mmol) and lithium hydroxide monohydrate (68 mg, 1.68 mmol) were dissolved in a 25 mL THF / H2O (95:5 v / v, 10 mL) flask equipped with a magnetic stirrer. The reaction mixture was stirred for 6 hours, and then the reaction mixture was concentrated under reduced pressure to give a colorless oil. The colorless oil was dissolved in t-BuOMe (10 mL) and washed with saturated NaHCO3 solution (10 mL). The aqueous layer was washed with t-BuOMe (10 mL x 3). The organic layer was collected and dried with Na2SO4. The target compound 1 (86 mg, yield: 27%) was obtained by concentrating the organic layer and drying under vacuum overnight. MS-ESI (m / z): 462.09 [M+1] + .

[0056] 1 H NMR (400MHz, DMSO-d6) ppmδ: 8.19 (br, 2H), 8.01 (s, 1H), 7.90 (m, 1H), 7.76 (d, J = 8.0Hz, lH), 7.63 (m, lH), 6.90 (m, 2H), 3.91 (s, 2H).

[0057] Example 2: ((1-((2,4-bis(trifluoromethyl)phenyl)methyl-d2)-1H-benzo[d]imidazol-2-yl)thio)methyl)boronic acid (compound 2)

[0058]

[0059] Compound 2 was synthesized using the same method as compound 1. MS-ESI (m / z): 437.05 [M+1] + .

[0060] 1H NMR(400MHz,DMSO-d6)ppmδ:8.35(br,2H),8.11(s,1H),7.73(m,lH),7.27(d ,J=8.0Hz,1H),7.25(m,lH),7.22(m,2H),6.57(d,J=8.0Hz,1H),3.92(s,2H).

[0061] Example 3: ((1-((2,4-bis(trifluoromethyl)phenyl)methyl-d2)-6-(trifluoromethane)-1H-benzo[d]imidazol-2-yl)thio)methyl)boronic acid (compound 3)

[0062]

[0063] Compound 3 was synthesized using the same method as compound 1. MS-ESI (m / z): 505.08 [M+1] + .

[0064] 1 H NMR (400MHz, DMSO-d6) ppmδ: 8.39 (br, 2H), 8.04 (s, 1H), 7.87 (m, lH), 7.73 (d, J = 8.2Hz, 1H), 7.69 (m, lH), 7.09 (m, 2H), 3.91 (s, 2H).

[0065] Example 4: ((1-(2,4-bis(trifluoromethyl)benzyl)-6-cyano-1H-benzo[d]imidazol-2-yl)seleno)methyl)boronic acid (compound 4)

[0066]

[0067] Step 1: Synthesis of 4-amino-3-(2,4-bis(trifluoromethyl)benzyl)amino)benzonitrile (intermediate c-1)

[0068] Intermediate c-1 was synthesized using the same method as intermediate a-4. MS-ESI (m / z): 360.05 [M+1] + .

[0069] Step 2: Synthesis of 3-(2,4-bis(trifluoromethyl)benzyl)-2-seleno-2,3-dihydro-1H-benzo[d]imidazol-5-nitrile (intermediate c-2)

[0070] Under a nitrogen atmosphere and at room temperature, diamine intermediate c-1 (324 mg, 0.9 mmol), tBuOK (453.6 mg, 4.05 mmol), Se (213.2 mg, 2.7 mmol), and iPrOH / 1,4-dioxane (1.5 / 1.5 mL) were added to a Schlenk reaction tube. CHCl3 (0.72 mL, 9 mmol) was added dropwise below 0 °C. The mixture was stirred in a heating mantle at 50 °C for 16 hours. After cooling to room temperature, saturated NH4Cl aqueous solution (5 mL) was added. The mixture was extracted with ethyl acetate (3 x 10 mL). The organic layer was dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1) to give the target product intermediate c-2 (153 mg, yield: 38%). MS-ESI (m / z): 450.03 [M+1] + .

[0071] Step 3: Synthesis of 1-(2,4-bis(trifluoromethyl)benzyl)-2-(((6-methyl-4,8-dioxo-1,3,6,2-diazaborane-2-yl)seleno)methyl)-1H-benzo[d]imidazolium-6-onitrile (intermediate c-3)

[0072] Intermediate c-3 was synthesized using the same method as intermediate a-6. MS-ESI (m / z): 619.05 [M+1] + .

[0073] Step 4: Synthesis of ((1-(2,4-bis(trifluoromethyl)benzyl)-6-cyano-1H-benzo[d]imidazol-2-yl)seleno)methyl)boronic acid (compound 2)

[0074] Compound 4 was synthesized using the same method as compound 1. MS-ESI (m / z): 508.02 [M+1]+.

[0075] 1 H NMR (400MHz, DMSO-d6) ppmδ: 8.07 (br, 2H), 7.94 (s, 1H), 7.85 (m, 1H), 7.72 (d, J = 8.0Hz, 1H), 7.60 (m, lH), 6.85 (m, 2H), 5.69 (s, 2H), 2.08 (s, 2H).

[0076] Synthesis of intermediate d-3: 3-(chloromethyl)-2,6-bis(trifluoromethyl)pyridine

[0077]

[0078] Step 1: Synthesis of 2,6-bis(trifluoromethyl)pyridin-3-yl)methanol (intermediate d-2)

[0079] Intermediate d-1 (144 mg, 0.5 mmol) of 2,6-bis(trifluoromethyl)nicotinic acid ethyl ester was dissolved in anhydrous THF (10 mL), and lithium aluminum hydride (19 mg, 0.5 mmol) was slowly added under ice bath conditions. After the addition was complete, the reaction was carried out at 0 °C for 3 hours. After the reaction was completed, water was slowly added to quench the reaction, and the mixture was extracted with ethyl acetate (10 mL x 2). The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate d-2 (85 mg, yield: 69%).

[0080] MS-ESI(m / z): 246.09 [M+1] + .

[0081] Step 2: Synthesis of 3-(chloromethyl)-2,6-bis(trifluoromethyl)pyridine (intermediate d-3)

[0082] Intermediate d-3 was synthesized using the same method as intermediate a-3. MS-ESI (m / z): 263.94 [M+1] + .

[0083] Synthesis of intermediate d-6: (5-(chloromethyl)-2,4-bis(trifluoromethyl)pyrimidine)

[0084]

[0085] Intermediate d-6 was synthesized using the same method as intermediate d-3. MS-ESI (m / z): 265.04 [M+1] + .

[0086] Example 5: ((1-((2,6-bis(trifluoromethyl)pyridin-3-yl)methyl)-6-cyano-1H-benzo[d]imidazol-2-yl)thio)methyl)boronic acid (compound 5)

[0087]

[0088] Compound 5 was synthesized using the same method as compound 1. MS-ESI (m / z): 461.07 [M+1]+.

[0089] 1 H NMR (400MHz, DMSO-d6) ppmδ: 8.24 (br, 2H), 7.95 (m, 1H), 7.84 (d, J = 8.2Hz, lH), 7.63 (m, lH), 6.95 (m, 2H), 5.72 (s, 2H), 3.95 (s, 2H).

[0090] Example 6: ((1-((2,4-bis(trifluoromethyl)pyrimidin-5-yl)methyl)-6-cyano-1H-benzo[d]imidazol-2-yl)thio)methyl)boronic acid (compound 6)

[0091]

[0092] Compound 6 was synthesized using the same method as compound 1. MS-ESI (m / z): 462.01 [M+1]+.

[0093] 1 H NMR (400MHz, DMSO-d6)ppmδ:8.44(br,2H), 8.19(s,lH),7.67(m,lH),7.13(m,2H),5.75(s,2H),3.97(s,2H).

[0094] Example 7: ((1-(2,4-bis(trifluoromethyl)benzyl)-6-cyano-1H-imidazo[4,5-b]pyridin-2-yl)thio)methyl)boronic acid (compound 7)

[0095]

[0096] Compound 7 was synthesized using the same method as compound 1. MS-ESI (m / z): 461.11 [M+1]+.

[0097] 1 H NMR (400MHz, DMSO-d6)ppmδ:8.89(s,1H),8.45(s,1H),8.38(br,2H),7.97(s,lH),7.23(m,2H),5.79(s,2H),3.91(s,2H).

[0098] Example 8: Evaluation of the stability of the compound in human liver microsomes:

[0099] The liver microsomal stability of compounds 1-7 in Examples was compared with that of the reference compound.

[0100] Assay System: The metabolic stability of the test compounds (example compounds and reference compounds) was tested using liver microsomes from mixed male and female samples with 1 mM NADPH. Samples were analyzed using mass spectrometry. HRMS was used to determine the peak area response ratio (corresponding to the peak area of ​​the test compound or control divided by the peak area of ​​the analytical internal standard) without running a standard curve. HRMS scans were performed within an appropriate m / z range to detect all possible metabolites.

[0101] Assay conditions: This assay was performed using a single incubation (N=1). The test compound was incubated at 37°C in a buffer containing 0.5 mg / mL liver microsomal protein. The reaction was initiated by adding a cofactor, and samples were taken at 0, 1, 2, 3, 4, and 5 hours. The positive control (5 μM serotonin) was incubated in parallel, and samples were taken at 0, 8, 16, 24, 32, and 48 hours.

[0102] Quality control assay: A parallel assay was performed using the control compound serotonin to confirm the enzymatic cleanliness of (liver) microsomes. After the final time point, NADPH was confirmed to have been added to the reaction mixture using fluorescence assay. The T1 / 2 of the control met acceptable internal standard requirements. Analytical method: Liquid chromatography column: Thermo BDS Hypersil C1830X 2.0 mm, 3 μm, with guard column MP.

[0103] Buffer solution: 25mM formic acid conjugated with buffer solution, pH 3.5;

[0104] Aqueous phase (A): 90% water, 10% buffer solution;

[0105] Organic phase (B): 90% acetonitrile, 10% buffer solution;

[0106] Flow rate: 300 microliters / minute;

[0107] Autosampler: Injection volume 10 μL.

[0108] Table 1. Gradient Procedure

[0109] Time (minutes) A％ B％ 0 100 0 1.5 0 100 2.0 0 100 2.2 100 0 3.5 100 0

[0110] The specific test results are shown in Table 2.

[0111] Table 2. Stability results of compounds in human liver microsomes

[0112]

[0113] The results showed that, compared with reference compound R4, compounds 1-4 of Examples had better metabolic stability and the potential to extend the dosing interval.

[0114] Example 9: Detection of the enhancing effect of the compound on the activity of β-lactam antibiotics

[0115] The minimum inhibitory concentrations (MICs) of the test compounds (compounds 1-7 in Examples and reference compound R4) with oxacillin (OXA), meropenem (MEM), cefoxitin (FOX), and ceftazidime (CPT) were determined and synergistic effects were investigated. Following literature methods, the MICs of the test compounds were determined by microdilution with Muller-Hinton broth.

[0116] Culture medium preparation: Muller-Hinton liquid medium: Accurately weigh 25g of Muller-Hinton liquid medium (Haibo Biotechnology). Dissolve in 1000mL of pure water, sterilize at 120℃, and store at 4℃ for later use. Muller-Hinton solid medium: Accurately weigh 35g of Muller-Hinton solid medium (Haibo Biotechnology), dissolve in 1000mL of pure water, and autoclave at 120℃. Prepare bacterial culture dishes, aspirate approximately 25mL of the above medium into each dish, and allow to solidify. Store in a refrigerator at 40℃ for subsequent experiments.

[0117] Preparation of bacterial suspension: Under sterile conditions, gently wipe the surface of the ampoule containing the bacterial strain with an alcohol swab, open the ampoule, add 500 μL of sterile physiological saline, and repeatedly pipette the bacterial strain until fully mixed. After complete homogeneity, aspirate 100 μL and spread it evenly onto a blood agar plate. Incubate at 37°C for 24 hours. Select a single colony, pick a loopful of the bacterial strain, and inoculate it into liquid culture medium. Incubate at 37°C on a shaker for 48 hours. The bacterial strain activation is then complete.

[0118] The test compound was first dissolved in dimethyl sulfoxide (DMSO) and then diluted for later use. Under aseptic conditions, Muller-Hinton medium was precisely pipetted into a 96-well plate (Thermo Fisher Scientific) at a rate of 96 μL / well. 4 μL of the stock solution was then precisely pipetted into the first column of wells. The solution in the first column was mixed thoroughly. The solution was then serially diluted to the next column, mixing thoroughly each time, until the 10th well was reached. 100 μL of a bacterial concentration of 5 × 10⁻⁶ was precisely pipetted into each well. 5 Add CFU / mL Muller-Hinton medium to columns 1 to 11, with column 12 containing 100 μL of sterile Muller-Hinton medium. Columns 1-10 are the experimental group, column 11 is the positive growth control, and column 12 is the negative control. Incubate at 37°C for 18 h and observe bacterial growth. Repeat the experiment 3 times.

[0119] The experimental strains were purchased from the American Center for Type Culture Collection.

[0120] The comparative example selected was compound 4 of WO2024196958A2, which has the structure listed in Table 2.

[0121] The specific test results are shown in Tables 3-6.

[0122] Table 3. MIC results of OXA, MEM, FOX, and CPT alone and in combination with reference compound R4 (8 mg / L) against MSSA and MRSA strains.

[0123]

[0124] Table 4. MIC results of OXA, MEM, FOX, and CPT alone and in combination with compound 1 (8 mg / L) of Example on MSSA and MRSA strains.

[0125]

[0126] As shown in Tables 3 and 4, compound 1 of the examples showed better restoration of β-lactam antibiotic sensitivity compared to reference compound R4, with even better results when used in combination, especially against ATCC29213, USA300, N315, and ATCC43300. Table 5. MIC results of OXA, MEM, FOX, and CPT alone and in combination with compound 4 of the examples (8 mg / L) against MSSA and MRSA strains.

[0127]

[0128] As shown in Tables 3 and 5, compound 4 of the examples can also better restore the sensitivity of β-lactam antibiotics compared with the reference compound R4, and the combined use is even more effective, especially against ATCC29213, USA300, N315 and ATCC43300.

[0129] Table 6. MIC results of OXA alone and in combination with compounds 2, 3, 5, 6, 7 of Examples and reference compound R4 (8 mg / L) against strain N315.

[0130] compound N315, MIC (mg / L) OXA 128 2 16 2+OXA 4 3 16 3+OXA 4 5 16 5+OXA 1 6 16 6+OXA 0.5 7 16 7+OXA 1 R4 16 R4+OXA 2

[0131] As shown in Table 6, compounds 5, 6, and 7 in the examples also exhibited better inhibitory activity compared to the reference compound R4, demonstrating superior drug potential.

[0132] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. Boronic acid compounds having the structure shown in general formula (I) or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled substances, prodrugs: In the formula, X is independently selected from O, S, and Se; Y1, Y2, Y3, Y4, Y5, and Y6 are independently selected from CH and N; R1 and R2 are independently selected from H and D, respectively; When X is selected from S and R1 and R2 are selected from H, at least one of Y1, Y2, Y3, Y4, Y5, and Y6 is N; R3, R4, and R5 are independently selected from H, halogens, haloalkyl groups, SF5, and CN, respectively.

2. The boric acid compound or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug according to claim 1, characterized in that, The compounds specifically include:

3. The boric acid compound or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug according to claim 1 or 2, characterized in that, The pharmaceutically acceptable salt is an inorganic or organic salt. The inorganic salt is selected from sodium salts, calcium salts, potassium salts, magnesium salts, silver salts, and lithium salts. The organic salt is selected from meglumine salts, tromethamine salts, diethylamine salts, lysine salts, choline salts, arginine salts, tert-butylamine salts, and N,N-dibenzylethylenediamine salts.

4. A pharmaceutical composition comprising any one of the borate compounds of claims 1-3 or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, prodrug, and pharmaceutical excipient.

5. The pharmaceutical composition according to claim 4, characterized in that, Pharmaceutical excipients include pharmaceutically acceptable diluents, excipients, or carriers.

6. The pharmaceutical composition according to claim 4, characterized in that, The dosage forms of the pharmaceutical composition include injections, lyophilized powder for injection, suspensions, implants, embolic agents, capsules, tablets, pills, and oral liquids.

7. The pharmaceutical composition according to claim 4, comprising the use of an antibiotic in combination with any one of the borate compounds of claims 1-3 or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug, wherein the antibiotic is optionally a β-lactam antibiotic.

8. The use of any borate compound of claims 1-3 or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug in the preparation of a medicament for treating bacterial infections.

9. The application as described in claim 8, wherein the bacteria is Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, or methicillin-resistant Staphylococcus aureus.

10. The application as described in claim 8, characterized in that, The β-lactam antibiotic is selected for use in combination with any one of the borate compounds of claims 1-3 or their pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope labels, or prodrugs.

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