Novel bicyclic borate beta-lactamase inhibitor and application thereof

Abstract

The invention provides a novel bicyclic borate beta lactamase inhibitor and application thereof. A series of beta-lactamase inhibitors with novel structures are obtained through design, some problems existing in clinical application of existing beta-lactamase inhibitors are solved, and the beta-lactamase inhibitors can be used as broad-spectrum inhibitors of SBL and MBL, particularly can be used as efficient MBL inhibitors, and have important significance in clinic.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the priority of the prior application filed with the China National Intellectual Property Office on June 12, 2024, with the patent application number 2024107523899, and the title of "A novel bicyclic boronate β-lactamase inhibitor and its and application". The entire contents of the prior application are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of medicine, and specifically relates to a novel bicyclic boronate β-lactamase inhibitor and its and application. BACKGROUND

[0004] β-lactam antibiotics are the most widely used antibiotics in the clinic for the treatment of bacterial infections due to their excellent antibacterial activity and selectivity. However, due to the frequent use of antibiotics, bacteria have acquired some resistance mechanisms to these drugs. The emergence and widespread dissemination of bacterial resistance have undermined their efficacy and pose a significant threat to public health. The production of β-lactamase is the most common mechanism of bacterial resistance, which destroys the antibiotic before it reaches the penicillin-binding protein (PBPs) target. Other resistance mechanisms include modification of PBPs, changes in outer membrane permeability, and upregulation of endogenous efflux mechanisms. To date, according to the β-lactamase database, nearly 7000 β-lactamases have been reported, and this number continues to grow. β-lactamases inactivate β-lactam antibiotics by hydrolyzing the β-lactam ring. According to the diversity of their sequences and structures, β-lactamases can be divided into classes A, B, C, and D. Classes A, C, and D are nucleophilic serine β-lactamases (SBLs), and class B is a metallo-β-lactamase (MBL).

[0005] The first generation of inhibitors, clavulanic acid, sulbactam, and tazobactam, which have been widely used in the clinic, are mainly active against class A SBLs, with a relatively limited range of action. However, in contrast to the successful clinical application of SBL inhibitors, there are currently no available high-efficiency MBL inhibitors in the clinic. For example, the currently synthesized compounds VNRX-5133, VNRX-5236, and QPX7728, etc. can be used as broad-spectrum inhibitors of SBLs and MBLs, but they are all in the clinical trial or preclinical stage. With the increasing demand for pharmaceuticals, it is therefore of great significance to develop a pan-spectrum inhibitor that can inhibit all types of β-lactamases, which can greatly promote the use of β-lactam antibiotics.

[0006] SUMMARY

[0007] The application aims to provide a novel bicyclic borate beta lactamase inhibitor and its application.

[0008] The application overcomes some problems existing in the clinical application of the prior art beta lactamase inhibitor by designing a series of beta lactamase inhibitors with novel structures, and the beta lactamase inhibitor can be used as a broad-spectrum inhibitor of SBL and MBL, and especially can be used as a high-efficiency MBL inhibitor, which has important significance in the clinic.

[0009] The application aims to provide a compound shown in structural formula (I), a racemate, a stereoisomer, a pharmaceutically acceptable salt,

[0010]

[0011] M is (CH2) m ; m is 0-4,

[0012] R is selected from hydrogen, amino, aryl, heterocyclic aryl, heterocyclic alkyl, cycloalkyl, and these groups can be substituted;

[0013] R 1 , R 2 , R 3 are independently selected from hydrogen, halogen, hydroxyl, C 1-6 alkyl, C 1-6 alkoxy.

[0014] According to the compound of claim 1, when R is selected from aryl, it can be phenyl, and the substituent group can be C 1-6 alkylamino, C 1-6 alkyl, halogen.

[0015] According to the embodiment of the application, when R is selected from heterocyclic aryl, it can be thiophene, furan, pyrrole, pyridine.

[0016] According to the embodiment of the application, when R is selected from heterocyclic alkyl, it can be piperidine, morpholine, piperazine, pyrrolidine.

[0017] According to the embodiment of the application, when R is selected from cycloalkyl, the substituent group can be C 1-6 alkylamino, C 1-6 alkyl, halogen.

[0018] According to the embodiment of the application, R 1 , R 2 , R 3 are independently selected from hydrogen, fluorine, methoxy.

[0019] According to the embodiment of the application, the compound shown in structural formula (I) is selected from the following compounds:

[0020]

[0021] The second object of the present application is to provide a pharmaceutical composition comprising a compound as shown in structural formula (I), its racemate, stereoisomer, pharmaceutically acceptable salt, and pharmaceutically acceptable excipient.

[0022] According to an embodiment of the present application, the pharmaceutical composition further comprises a beta-lactam antibiotic.

[0023] According to an embodiment of the present application, wherein the beta-lactam antibiotic is penicillin, cephalosporin, carbapenem, monobactam, bridged monobactam or a combination thereof.

[0024] According to an embodiment of the present application, wherein the cephalosporin is cefepime, ceftriaxone, cefiniazid, cefotaxime, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin or a combination thereof.

[0025] According to an embodiment of the present application, wherein the carbapenem is imipenem, biapenem, doripenem, meropenem and ertapenem or a combination thereof.

[0026] The third object of the present application is to provide a use of a pharmaceutical composition comprising a compound as shown in structural formula (I), its racemate, stereoisomer, pharmaceutically acceptable salt, and pharmaceutically acceptable excipient in the manufacture of a medicament for treating bacterial infection in a subject.

[0027] According to an embodiment of the present application, wherein the infection comprises bacteria selected from the group consisting of Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Acinetobacter baumannii, Escherichia coli.

[0028] Compared with the prior art, the present application has the beneficial effects in that:

[0029] (1) The present application provides a novel bicyclic borate beta-lactamase inhibitor and its preparation method and application. The borate beta-lactamase inhibitor can be used as a broad-spectrum inhibitor of SBL and MBL. The compound has excellent biological activity test data, and can be used with various antibiotics, especially as an efficient MBL inhibitor, which has important significance in clinical application.

[0030] (2) The present application designs a novel bicyclic borate beta-lactam inhibitor with a novel structure. A new synthetic route is designed by completing route exploration and optimizing the conditions of key steps. The synthetic route has mild reaction conditions, readily available raw materials, and good yield. The new route has very important innovation for the synthesis of borate beta-lactam inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The hydrogen spectrum of the compound 1 of the present application.

[0032] Figure 2 The hydrogen spectrum of the compound 2 of the present application.

[0033] Figure 3 The hydrogen spectrum of the compound 3 of the present application.

[0034] Figure 4 The hydrogen spectrum of the compound 5 of the present application.

[0035] Figure 5 The hydrogen spectrum of the compound 6 of the present application.

[0036] Figure 6 The hydrogen spectrum of the compound 7 of the present application. DETAILED DESCRIPTION

[0037] The present application is further described in detail by the following specific examples and drawings. The following examples are only descriptive and not limiting, and the protection scope of the present application cannot be limited by them. If not otherwise specified, the raw materials used can be obtained by market or self-made.

[0038] Example 1: 4-(N-methylcarbamoyl)-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid

[0039]

[0040] Step 1: Synthesis of compound 1b (methyl 2-methoxy-3-methylbenzoate)

[0041] Into a three-necked flask, 10.0 g (165.7 mmol) of compound 1a and 125 mL of dry acetone were added under nitrogen atmosphere, followed by 26.4 g (190.6 mmol) of anhydrous potassium carbonate, 24.0 g (190.6 mmol) of dimethyl sulfate, and the mixture was stirred under reflux for 20 h. After the reaction was completed, it was cooled to room temperature, and the solid residue was removed by filtration. The filtrate was concentrated under reduced pressure, and the residue was diluted with 200 mL of water, stirred for 15 min, and extracted with ethyl acetate (3 x 90 mL). The organic phase was washed with 0.5 N sodium hydroxide solution (100 mL x 2), and finally with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 10:1, v / v) to obtain 10.422 g of colorless oil with a yield of 88%. 1HNMR (400 MHz, CDC13) δ 7.65 (d, J = 7.4 Hz, 1H), 7.36 (d, J = 7.4 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 3.93 (s, 3H), 3.85 (s, 3H), 2.34 (s, 3H).

[0042] Step 2: Synthesis of compound lc (2-methoxy-3-methylbenzoic acid)

[0043] In a round bottom flask, 2.6 g (14.43 mmol) of compound lb and 30 mL of methanol were added, followed by the addition of 20 mL of 15% sodium hydroxide aqueous solution, and the reaction was stirred at room temperature for 2 h. After the reaction was completed, the reaction system was concentrated under reduced pressure, the crude product was diluted with 30 mL of water, 5N HC1 was added to adjust the pH to 4, and ethyl acetate was used for extraction (3 x 50 mL), the organic phase was combined, washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA:AcOH = 100:10:1, v / v) to obtain 2.086 g of white solid with a yield of 87.0%. 1 HNMR (400 MHz, CDC13) δ 7.98 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 3.95 (s, 3H), 2.40 (s, 3H).

[0044] Step 3: Synthesis of compound Id (tert-butyl 2-methoxy-3-methylbenzoate)

[0045] Under a nitrogen atmosphere, 2.0 g (12.04 mmol) of compound lc, 20 mL of anhydrous dichloromethane, and 0.25 mL of anhydrous DMF were added to a three-necked flask, followed by the slow dropwise addition of 1.54 mL (17.9 mmol) of oxalyl chloride. The reaction was stirred at room temperature for 90 min, the reaction system was concentrated under reduced pressure, followed by the addition of 30 mL of anhydrous tert-butyl alcohol, and the reaction was stirred at 40°C for 20 h. After the reaction was completed, it was cooled to room temperature, the reaction system was concentrated under reduced pressure, the crude product was diluted with 30 mL of water, dichloromethane was used for extraction (3 x 50 mL), the organic phase was combined, washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 40:1, v / v) to obtain 1.81 g of colorless oil with a yield of 67%. 1 HNMR (400 MHz, CDC13) δ 7.57 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 3.85 (s, 3H), 2.33 (s, 3H), 1.63 (s, 9H).

[0046] Step 4: Synthesis of compound 1e (tert-butyl 3-bromomethyl-2-methoxybenzoate)

[0047] In a round-bottom flask, 1.69 g (7.6 mmol) of compound 1d and 25 mL of carbon tetrachloride were added, followed by 1.55 g (8.74 mmol) of N-bromosuccinimide and 0.36 g (1.52 mmol) of dibenzoyl peroxide, and the reaction was stirred at reflux for 5 h. After the reaction was completed, it was cooled to room temperature, and the solid residue was removed by filtration. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE:EA = 100:1, v / v) to obtain 1.56 g of a colorless oil with a yield of 68%. 1 H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.7 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.13 (t, J = 7.7 Hz, 1H), 4.60 (s, 2H), 3.99 (s, 3H), 1.63 (s, 9H).

[0048] Step 5: Synthesis of compound 1f (tert-butyl 3-allyl-2-methoxybenzoate)

[0049] In a three-necked flask, 143 mg (0.75 mmol) of cuprous iodide and 30 mL of anhydrous tetrahydrofuran were added under a nitrogen atmosphere, and the temperature was lowered to -50°C. A 10 mL (10 mmol) solution of vinylmagnesium bromide (1 mol / L in THF) was added dropwise over 5 min, and after 10 min, a solution of 750 mg (2.5 mmol) of compound 1e in 10 mL of anhydrous tetrahydrofuran was added dropwise over 5 min. The reaction was maintained for 30 min, quenched with saturated ammonium chloride solution, diluted with 100 mL of water, and extracted with ethyl acetate (3 x 30 mL). The organic phases were combined, washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 80:1, v / v) to obtain 373 mg of a colorless oil with a yield of 60.1%. 1 H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 6.10-5.99 (m, 1H), 5.17-5.11 (m, 2H), 3.82 (s, 3H), 3.48 (d, J = 6.6 Hz, 2H), 1.63 (s, 9H).

[0050] Step 6: Synthesis of compound 1g (2-(3-(tert-butoxycarbonyl)-2-methoxyphenyl)acetic acid)

[0051] A round-bottom flask was charged with 3.07 g (12.38 mmol) of compound 1f and 100 mL of acetonitrile, 3.42 g (24.76 mmol) of potassium carbonate in water (100 mL) and 10.59 g (49.52 mmol) of sodium periodate in water (200 mL), 0.98 g (6.19 mmol) of potassium permanganate, 50 mL of water. The reaction was stirred at room temperature for 4 h, after the reaction was completed, 5N AcOH was added to adjust the pH to 5, extracted with ethyl acetate (3 x 30 mL), the organic phase was combined, washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (EA: PE: AcOH = 20:90:1, v / v) to give 2.58 g of a yellow oil with a yield of 78.4%. 1 HNMR (400 MHz, DMSO-d6) δ 7.53 (d, J = 7.7 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.13 (t, J = 7.6 Hz, 1H), 3.72 (s, 3H), 3.61 (s, 2H), 1.55 (s, 9H).

[0052] Step 7: Synthesis of compound 1h (tert-butyl 2-methoxy-3-(2-oxo-2- phenoxyethyl)benzoate)

[0053] A Schlenk flask was evacuated and refilled with nitrogen three times, and then 1.54 g (5.79 mmol) of compound 1g and 1.53 g (6.07 mmol) of 2-chloro-1-methylpyridine iodide were added under a nitrogen atmosphere, 573 mg (6.07 mmol) of phenol and 14 mL of dimethyl carbonate were added, and the reaction was stirred at room temperature for 10 min. Then 1.48 g (13.89 mmol) of 2,6-dimethylpyridine was added, and the reaction was stirred at 60°C. The progress of the reaction was monitored by TLC. After the reaction was completed, the solid residue was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 20:1, v / v) to give 1.43 g of a yellow oil with a yield of 72.1%. 1 H NMR (400 MHz, DMSO-d6) δ 7.60-7.55 (m, 2H), 7.45-7.41 (m, 2H), 7.29-7.24 (m, 1H), 7.19-7.17 (m, 1H), 7.16-7.12 (m, 2H), 3.99 (s, 2H), 3.80 (s, 3H), 1.56 (s, 9H).

[0054] Step 8: Synthesis of compound 1i (tert-butyl 2-methyl-3-(1-oxo-1- phenoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)prop-2-yl)-2- methoxybenzoate)

[0055] The Schlenk flask was evacuated and refilled with nitrogen three times, 20 mL of anhydrous tetrahydrofuran was added under nitrogen atmosphere, the temperature was reduced to -78 °C, and 4.2 mL (8.4 mmol) of lithium diisopropylamide solution (2 M in THF) was slowly added to the reaction flask.

[0056] A mixture of 1.428 g (4.17 mmol) of compound 1h and 7.472 g (41.7 mmol) of hexamethylphosphoric triamide was treated with anhydrous acetonitrile to remove water and then dissolved in 15 mL of anhydrous tetrahydrofuran. The resulting solution was slowly added dropwise to the reaction flask, and after one hour of reaction, 1.787 g (6.67 mmol) of iodomethylboronic acid pinacol ester was added, and the reaction progress was monitored by TLC. After the reaction was completed, it was quenched with saturated ammonium chloride solution, diluted with 50 mL of water, extracted with ethyl acetate (3 x 50 mL), the organic phases were combined, washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA = 10:1, v / v) to give 1.631 g of a light yellow oil with a yield of 81%. 1 H NMR (400 MHz, CDC13) δ 7.67 (d, J = 7.7 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.35 (t, J = 7.9 Hz, 2H), 7.22 - 7.13 (m, 2H), 7.06 - 7.01 (m, 2H), 4.54 - 4.50 (m, 1H), 3.95 (s, 3H), 1.64 (s, 9H), 1.29 (d, J = 9.7 Hz, 2H), 1.23 (d, J = 2.4 Hz, 12H).

[0057] Step 9: Synthesis of compound 1j (tert-butyl 2-methoxy-3-(1-(methylamino)-1-oxo-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-prop-2-yl)benzoate)

[0058] The Schlenk flask was evacuated and refilled with nitrogen three times, 242 mg (0.5 mmol) of compound 1i and 5 mL of anhydrous THF were added under nitrogen atmosphere, 168 mg (2.5 mmol) of methylamine hydrochloride, 2.5 mmol of TEA were added, and the reaction was carried out at 40 °C for 16 h. The reaction progress was monitored by TLC, after the reaction was completed, it was diluted with water, extracted with dichloromethane (4 x 30 mL), washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 77 mg of a light yellow oil with a yield of 37%. 1H NMR (400 MHz, CDC13) δ 7.63 (d, J = 7.7 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.12 (t, J = 7.7 Hz, 1H), 4.17 - 4.12 (m, 1H), 3.92 (s, 3H), 2.70 (d, J = 4.8 Hz, 3H), 2.05 (s, 1H), 1.62 (s, 9H), 1.55 (dd, J = 15.9, 8.6 Hz, 1H), 1.36 (dd, J = 15.9, 8.3 Hz, 1H), 1.14 (d, J = 8.3 Hz, 12H).

[0059] Step 10: Synthesis of compound 1 (4-(N-methylcarbamoyl)-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid)

[0060] Schlenk flask was evacuated and refilled with nitrogen three times, 210 mg (0.5 mmol) of compound 28-1 treated with anhydrous acetonitrile and 8 mL of anhydrous DCM were added, then the temperature was reduced to -78°C, 2.5 mL of BC13 solution in DCM (2.5 mmol, 1M in DCM) was slowly added dropwise within 20 min. TLC was used to monitor the progress of the reaction, after the reaction was completed, 1 mL of water was added to quench, freeze-dried, the solid was dissolved in 5 mL of methanol, filtered, and the filtrate was purified by large silica gel plate to obtain 40 mg of white solid, with a yield of 32%. 1 H NMR (400 MHz, DMSO-d6) δ 7.43 - 7.53 (m, 1H), 6.86 - 7.07 (m, 1H), 6.41 - 6.48 (m, 1H), 4.09 - 4.27 (m, 1H), 2.89 (s, 1.5H), 2.73 (s, 1.5H), 1.26 (s, 1H), 0.84 - 0.92 (m, 1H). ESI-MS: m / z [M+H]+ 250.1.

[0061] Example 2: 2-hydroxy-4-((thiophen-2-ylmethyl)carbamoyl)-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0062]

[0063] Compound 2 was prepared similarly as described in example 1 (step 1-10) by replacing the methylamine hydrochloride in step 9 with 2-thiophenemethylamine. White solid 61 mg was obtained with a yield of 37%. 1HNMR (400 MHz, CD3OD) δ 7.65-7.74 (m, 1H), 7.32-7.33 (m, 1H), 7.08-7.12 (m, 3H), 6.96-6.99 (m, 2H), 6.70-6.77 (m, 1H), 4.59-4.67 (m, 1H), 3.63-3.69 (m, 1H), 1.12-1.20 (m, 1H), 0.94 (s, 1H). ESI-MS: m / z [M+H]+ 332.1.

[0064] Example 3: 4-((4-Fluorophenylethyl)carbamoyl)-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0065]

[0066] Compound 3 was prepared similarly as described in Example 1 (steps 1-10) by replacing methylamine hydrochloride in step 9 with p-fluorophenethylamine. White solid 66 mg, yield 37%. 1 HNMR (400 MHz, CD3OD) δ 7.65-7.74 (m, 1H), 7.32-7.33 (m, 1H), 7.08-7.12 (m, 3H), 6.96-6.99 (m, 2H), 6.70-6.77 (m, 1H), 4.59-4.67 (m, 1H), 3.63-3.69 (m, 1H), 1.12-1.20 (m, 1H), 0.94 (s, 1H). ESI-MS: m / z [M+H]+ 332.1.

[0067] Example 4: 4-((4-Aminobutyl)carbamoyl)-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0068]

[0069] Compound 4 was prepared similarly as described in Example 1 (steps 1-10) by replacing methylamine hydrochloride in step 9 with 1,4-butanediamine. White solid 43 mg, yield 28%. 1 HNMR (400 MHz, CD3OD) δ 7.65-7.74 (m, 1H), 7.32-7.33 (m, 1H), 7.08-7.12 (m, 3H), 6.96-6.99 (m, 2H), 6.70-6.77 (m, 1H), 4.59-4.67 (m, 1H), 3.63-3.69 (m, 1H), 1.12-1.20 (m, 1H), 0.94 (s, 1H). ESI-MS: m / z [M+H]+ 332.1.

[0070] Example 5: 2-Hydroxy-4-((piperidin-2-ylmethyl)carbamoyl)-3,4-dihydro-2H- benzo[e][1,2]oxaborinine-8-carboxylic acid

[0071]

[0072] Compound 5 was prepared in a similar manner as described in Example 1 (steps 1-10) by replacing methylamine hydrochloride in step 9 with N-BOC-piperidin-2- methylamine. White solid 63 mg, yield 38%. 1 HNMR (400 MHz, DMSO-d6) δ 7.42-7.67 (m, 1H), 7.15-7.28 (m, 1H), 6.51-6.62 (m, 1H), 4.11-4.31 (m, 1H), 3.69-3.80 (m, 4H), 1.23-1.72 (m, 7H), 0.83-0.91 (m, 1H). ESI-MS: m / z [M+H]+ 333.2.

[0073] Example 6: 4-((4-(Aminomethyl)benzyl)carbamoyl)-2-hydroxy-3,4-dihydro-2H- benzo[e][1,2]oxaborinine-8-carboxylic acid

[0074]

[0075] Compound 6 was prepared in a similar manner as described in Example 1 (steps 1-10) by replacing methylamine hydrochloride in step 9 with (N-BOC-aminomethyl)benzylamine. White solid 71 mg, yield 40%. 1 HNMR (400 MHz, DMSO-d6) δ 7.12-7.68 (m, 6H), 6.61-6.83 (m, 1H), 3.89-4.29 (m, 5H), 1.24 (s, 1H), 0.86-0.92 (m, 1H). ESI-MS: m / z [M+H]+ 354.1.

[0076] Example 7: 4-(((4-((2-Aminoethyl)amino)cyclohexyl)methyl)carbamoyl)-2-hydroxy-3,4- dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic acid

[0077]

[0078]

[0079] Step A: Synthesis of tert-butyl ((4-((2-((benzyloxy)carbonyl)amino)ethyl)amino)cyclohexyl)methyl)carbamate (Compound m)

[0080] Schlenk flask was evacuated and refilled with nitrogen three times, 500 mg (2.57 mmol) N-benzyl-2-oxoethylamine and 15 mL methanol were added, and the mixture was cooled to 0 °C, then 500 mg (2.20 mmol) l was added. The reaction was stirred for 20 min, and 130 mg (3.43 mmol) sodium borohydride was added in portions over 15 min. The reaction progress was monitored by TLC. After the reaction was completed, the reaction system was concentrated under reduced pressure. The crude product was diluted with a small amount of water, extracted with dichloromethane (4 x 50 mL), and the organic phases were combined. The organic phase was washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA:Et3N = 30:60:0.5, v / v / v) to obtain 504 mg of a light yellow solid with a yield of 56.6%. ESI-MS: m / z [M+H]+406.

[0081] Schlenk flask was evacuated and refilled with nitrogen three times, 500 mg (2.57 mmol) N-benzyl-2-oxoethylamine and 15 mL methanol were added, and the mixture was cooled to 0 °C, then 500 mg (2.20 mmol) l was added. The reaction was stirred for 20 min, and 130 mg (3.43 mmol) sodium borohydride was added in portions over 15 min. The reaction progress was monitored by TLC. After the reaction was completed, the reaction system was concentrated under reduced pressure. The crude product was diluted with a small amount of water, extracted with dichloromethane (4 x 50 mL), and the organic phases were combined. The organic phase was washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA:Et3N = 30:60:0.5, v / v / v) to obtain 504 mg of a light yellow solid with a yield of 56.6%. ESI-MS: m / z [M+H]+406.

[0082] Step B: Synthesis of benzyl (2-((4-(aminomethyl)cyclohexyl)amino)ethyl)carbamate

[0083] Schlenk flask was evacuated and refilled with nitrogen three times, 500 mg (2.57 mmol) N-benzyl-2-oxoethylamine and 15 mL methanol were added, and the mixture was cooled to 0 °C, then 500 mg (2.20 mmol) l was added. The reaction was stirred for 20 min, and 130 mg (3.43 mmol) sodium borohydride was added in portions over 15 min. The reaction progress was monitored by TLC. After the reaction was completed, the reaction system was concentrated under reduced pressure. The crude product was diluted with a small amount of water, extracted with dichloromethane (4 x 50 mL), and the organic phases were combined. The organic phase was washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE:EA:Et3N = 30:60:0.5, v / v / v) to obtain 504 mg of a light yellow solid with a yield of 56.6%. ESI-MS: m / z [M+H]+406. 1 HNMR (400 MHz, DMSO-d6) δ 7.39 - 7.29 (m, 5H), 5.01 (s, 2H), 3.06 - 3.00 (m, 3H), 2.57 (t, J = 6.6 Hz, 2H), 2.37 (d, J = 6.4 Hz, 2H), 2.30 - 2.21 (m, 1H), 1.88 - 1.80 (m, 2H), 1.74 - 1.71 (m, 2H), 1.16 - 1.13 (m, 1H), 0.95 - 0.80 (m, 4H). ESI-MS: m / z [M+H]+306.

[0084]

[0085] The above step is similar to the procedure described in Example 1 (steps 1-10) using the compound n described above to replace the methylamine hydrochloride in step 9 to produce compound 7k as a light yellow solid. The compound 7k, 0.5 mmol, was dissolved in 5.0 mL of methanol and transferred to a micro hydrogenation reactor. 30 mg of 20% palladium hydroxide on carbon was added and the reactor was purged with hydrogen three times. The hydrogen pressure was controlled at 30 atm and the reaction was allowed to proceed at room temperature for 6 h. The reaction mixture was filtered and the filtrate was concentrated and purified by preparative TLC to give 51 mg of a white solid as a 26% yield. 1 H NMR (400 MHz, CD3OD) δ 7.61-7.62 (m, 1H), 7.00-7.02 (m, 1H), 7.67-7.70 (m, 1H), 3.61-3.65 (m, 1H), 3.05-3.29 (m, 4H), 2.44-2.75 (m, 3H), 1.28-1.59 (m, 8H), 1.02-1.16 (m, 2H), 0.83-0.88 (m, 1H). ESI-MS: m / z [M+H]+ 390.3.

[0086] Example 8: 7-Fluoro-4-(N-methylcarbamoyl)-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0087]

[0088] The above step is similar to the procedure described in Example 1 (steps 1-10) using 6-fluoro-2-hydroxy-3-methylbenzoic acid to replace 3-methylsalicylic acid in step 1 to produce compound 8 as a white solid. 52 mg, 35% yield.

[0089] Example 9: 7-Methoxy-4-(N-methylcarbamoyl)-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0090]

[0091] The above step is similar to the procedure described in Example 1 (steps 1-10) using 2-hydroxy-6-methoxy-3-methylbenzoic acid to replace 3-methylsalicylic acid in step 1 to produce compound 9 as a white solid. 45 mg, 33% yield.

[0092] Example 10: 7-Fluoro-2-hydroxy-4-((thiophen-2-ylmethyl)carbamoyl)-3,4- dihydro-2H-benzo[e][l,2]oxaborinine-8-carboxylic acid

[0093]

[0094] Compound 10 was prepared in analogy to the procedure described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 6-fluoro-2-hydroxy-3- methylbenzoic acid. White solid 51 mg, yield 36%.

[0095] Example 11 : 2-Hydroxy-7-methoxy-4-((thiophen-2-ylmethyl)carbamoyl)-3,4- dihydro-2H-benzo[e][l,2]oxaborinine-8-carboxylic acid

[0096]

[0097] Compound 11 was prepared in analogy to the procedure described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 2-hydroxy-6-methoxy-3- methylbenzoic acid. White solid 43 mg, yield 34%.

[0098] Example 12: 7-Fluoro-4-((4-fluorophenylethyl)carbamoyl)-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0099]

[0100] Compound 12 was prepared in analogy to the procedure described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 6-fluoro-2-hydroxy-3-methylbenzoic acid. White solid 55 mg, yield 40%.

[0101] Example 13: 4-((4-Fluorophenylethyl)carbamoyl)-2-hydroxy-7-methoxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0102]

[0103] Compound 13 was prepared in analogy to the procedure described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 2-hydroxy-6-methoxy-3-methylbenzoic acid. White solid 56 mg, yield 40%.

[0104] Example 14: 4-((4-Aminobutyl)carbamoyl)-7-fluoro-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0105]

[0106] Compound 14 was prepared in analogy to the procedures described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 6-fluoro-2-hydroxy-3- methylbenzoic acid. White solid 53 mg, yield 29%.

[0107] Example 15: 4-((4-aminobutyl)carbamoyl)-2-hydroxy-7-methoxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0108]

[0109] Compound 15 was prepared in analogy to the procedures described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 2-hydroxy-6-methoxy-3- methylbenzoic acid. White solid 54 mg, yield 28%.

[0110] Example 16: 7-fluoro-2-hydroxy-4-((piperidin-2-ylmethyl)carbamoyl)-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0111]

[0112] Compound 16 was prepared in analogy to the procedures described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 6-fluoro-2-hydroxy-3-methylbenzoic acid. White solid 58 mg, yield 35%.

[0113] Example 17: 2-hydroxy-7-methoxy-4-((piperidin-2-ylmethyl)carbamoyl)-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0114]

[0115] Compound 17 was prepared in analogy to the procedures described in Example 1 (steps 1-10) by replacing 3-methylsalicylic acid in step 1 with 2-hydroxy-6-methoxy-3-methylbenzoic acid. White solid 58 mg, yield 38%.

[0116] Example 18: 4-((4-(aminomethyl)benzyl)carbamoyl)-7-fluoro-2-hydroxy-3,4-dihydro-2H- benzo[e][l,2]oxaborinine-8-carboxylic acid

[0117]

[0118] The procedure described in Example 1 (steps 1-10) was followed using 6-fluoro-2- hydroxy-3-methylbenzoic acid in place of 3-methylsalicylic acid in step 1 to prepare compound 18. White solid 56 mg, 39% yield.

[0119] Example 19: 4-((4-(Aminomethyl)benzyl)carbamoyl)-2-hydroxy-7-methoxy-3,4- dihydro-2H-benzo[e][l,2]oxaborinine-8-carboxylic acid

[0120]

[0121] The procedure described in Example 1 (steps 1-10) was followed using 2-hydroxy-6- methoxy-3-methylbenzoic acid in place of 3-methylsalicylic acid in step 1 to prepare compound 19. White solid 54 mg, 41% yield.

[0122] Example 20: 4-(((4-((2-Aminopropyl)amino)cyclohexyl)methyl)carbamoyl)-7-fluoro-2- hydroxy-3,4-dihydro-2H-benzo[e][l,2]oxaborinine-8-carboxylic acid

[0123]

[0124] The procedure described in Example 7 was followed using 6-fluoro-2-hydroxy-3- methylbenzoic acid in place of 3-methylsalicylic acid in step 1 to prepare compound 20. White solid 52 mg, 46% yield.

[0125] Example 21: 4-(((4-((2-Aminopropyl)amino)cyclohexyl)methyl)carbamoyl)-2-hydroxy-7- methoxy-3,4-dihydro-2H-benzo[e][l,2]oxaborinine-8-carboxylic acid

[0126]

[0127] The procedure described in Example 7 was followed using 2-hydroxy-6-methoxy-3- methylbenzoic acid in place of 3-methylsalicylic acid in step 1 to prepare compound 21. White solid 61 mg, 42% yield.

[0128] Example 22: Compounds inhibit multiple beta-lactamases

[0129] The experimental scheme refers to the latest version of the American Clinical Laboratory Standardization Committee CLSI drug sensitivity experiment standard, according to the CLSI M-100 antimicrobial drug sensitivity test execution standard 35th edition, with Micrococcus luteus as the strain, cefepime and ceftriaxone are selected for combination with the test compound, and avibactin sodium is used as a control to determine the enzyme inhibition activity (bacteriostatic ring diameter (mm)) of target compounds 1-21. The results are shown in Table 1.

[0130] Experimental content:

[0131] Preparation of bacterial solution and bacterial plate: Take Micrococcus luteus CMCC28001 strain slant, cultivate at 37℃ overnight, take 2mL sterile saline in the slant test tube, elute the bacterial lawn, take all the eluate and add 2.5mL sterile saline to mix, then take 0.5mL mixed solution and add to 200mL MH agar medium to mix to prepare a bacterial plate, after condensation, use a puncher to dig out the agar in the hole.

[0132] Solution preparation: weigh 98% cefepime 12.5mg, add 19.14mL sterile water to dissolve, prepare cefepime mother liquor with a concentration of 640μg / ml; weigh 93.5% ceftriaxone 12.5mg, add 18.26mL sterile water to dissolve, prepare 640μg / mL ceftriaxone mother liquor; add a certain amount of inhibitor compound and dimethyl sulfoxide respectively to prepare 640μg / mL inhibitor mother liquor.

[0133] Experimental steps: take 0.5mL cefepime / ceftriaxone mother liquor and test compound mother liquor for 1:1 mixing, get 320μg / mL compound solution.

[0134] Take 0.5mL cefepime / ceftriaxone mother liquor and 0.5mL sterile water for mixing, get 320μg / mL single solution.

[0135] Add 100μL of prepared enzyme dilution solution ① and ② to 1mL of cefepime single and compound solution (1:1) (the final concentration of cefepime in the substrate is 291μg / mL), and take avibactin as the inhibitor control.

[0136] Add 50μL of prepared enzyme dilution solution ① and ② to 1.0mL of ceftriaxone single and ceftriaxone and each test compound compound (1:1) (the final concentration of ceftriaxone in the substrate is 305μg / mL), and take avibactin as the inhibitor control.

[0137] Mix immediately and place in a 37℃ water bath for 15min. Mix 0.1mL of the incubated solution with 1.9mL sterile water, take 50μL of the above mixture and add it to a large plate, place it in a 37℃ incubator for 45min.

[0138] Table 1 Inhibition of various β-lactamases by compounds of the application (zone diameter, mm)

[0139]

[0140]

[0141] As can be seen from Table 1, cefepime and ceftriaxone alone are hydrolyzed by β-lactamases 1 and 2, and lose antibacterial activity. After cefepime and ceftriaxone are each mixed with the target compound at a ratio of 1:1, the test compound can inhibit β-lactamases 1 and 2, so that cefepime and ceftriaxone can exert antibacterial effects, and the inhibitory effect on β-lactamases 1 and 2 is equivalent to that of avibactam sodium.

[0142] Example 23: In vitro antibacterial experiment for β-lactamase inhibition

[0143] The difference in inhibitory effect on various β-lactamase bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae) between cefepime / test compound (1-21) and cefepime / avibactam was compared.

[0144] (1) The test strain (Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Acinetobacter baumannii) nutrient agar slant was inoculated into 2 mL of CAMHB broth medium, and incubated at 35-37°C for 6 h, and then subjected to MBCOD turbidity with a No. 0.5 McFarland turbidity tube, appropriately diluted, and adjusted to a concentration of about 10 7 CFU / mL for standby.

[0145] (2) An appropriate amount of sterile water and dimethyl sulfoxide were taken to dissolve the test compound into a stock solution, and then serially diluted with physiological saline. 1280 μg / mL of cefepime and 640 μg / mL of enzyme inhibitor compound 1 were mixed at a ratio of 1:1 to obtain a solution with a concentration of cefepime / enzyme inhibitor of 640 / 320 μg / mL. Serially diluted with physiological saline at a ratio of 1:2 to obtain solutions with concentrations of 320 / 160, 160 / 80, 80 / 40, 40 / 20, 20 / 10 and 10 / 5 μg / mL, respectively. For cefepime alone, serially diluted to obtain solutions with drug concentrations of 1280, 640, 320, 160, 80, 40, 20 and 10 μg / mL, respectively.

[0146] (3) After the MHA medium was autoclaved (121°C, 15 min), the temperature was reduced to about 40°C, and then mixed and poured into a flat plate for solidification. The specific operation is as follows:

[0147] For cefepime alone, 1 mL of the sample solution (concentrations are 1280, 640, 320, 160, 80, 40, 20 and 10 μg / mL, respectively) was taken and added into a sterile flat plate, and 9 mL of 40°C MHA medium was added immediately, and mixed well to obtain the sample flat plate with final concentrations of 128, 64, 32, 16, 8, 4, 2 and 1 μg / mL, respectively.

[0148] For the compound, the flat plate with a test concentration of 128 / 64 μg / mL was obtained by adding 8 mL of MHA to 2 mL of a solution with a concentration of 640 / 320 μg / mL. For the rest of the test concentrations, 1 mL of a solution with a corresponding concentration was added to 9 mL of MHA to obtain the sample flat plate with final concentrations of 64 / 32, 32 / 16, 16 / 8, 8 / 4, 4 / 2, 2 / 1 and 1 / 0.5 μg / mL, respectively.

[0149] (4) Multi-point inoculator was used for inoculation 4 CFU / point bacteria were inoculated on the sample flat plates from low concentration to high concentration. Meanwhile, three flat plates without the sample were inoculated as controls, and were placed at 37°C for 16-20 h. The observation results were taken, and the growth conditions were recorded. The lowest concentration at which the visible growth of bacteria was inhibited was recorded as the MIC value of the compound.

[0150] Using the above method, the compounds of the embodiments of the present application can be evaluated according to their ability to inhibit the β-lactamase-producing bacteria in the presence of β-lactam antibiotics. The results of these experiments are shown in Table 2, in terms of antibiotic concentration, in which A represents MIC > 128 μg / mL, B represents MIC between 4-128 μg / mL, C represents MIC < 4 μg / mL, and NT = not tested.

[0151] Table 2 Broad-spectrum inhibition of bacterial growth

[0152]

[0153]

[0154] As can be seen from Table 2, the MIC value of cefepime / compound is significantly lower than that of cefepime alone, and the bacteriostatic effect is equivalent to that of commercially available cefepime / avibactam. The compounds of the present application have an inhibitory effect on various β-lactamases, and the biological activity test data all show excellent performance, and can be used as broad-spectrum inhibitors of SBL and MBL, especially have a high inhibitory effect on class B β-lactamase, which has important significance in the clinic.

Claims

1. A compound of structural formula (I), racemates, stereoisomers, pharmaceutically acceptable salts thereof, M is (CH2) m ; m is 0-4, R is selected from the group consisting of hydrogen, amino, aryl, heteroaryl, heterocycloalkyl, cycloalkyl, which can be substituted; R 1 , R 2 , R 3 are independently of one another selected from hydrogen, halogen, hydroxyl, C 1-6 alkyl, C 1-6 alkoxy.

2. The compound of claim 1, R is selected from aryl, which can be phenyl, and the substituent group can be C 1-6 alkylamino, C 1-6 alkyl, halogen.

3. The compound of claim 1, when R is selected from heteroaryl, it can be thienyl, furanyl, pyrrolyl, pyridyl.

4. The compound of claim 1, when R is selected from heterocycloalkyl, it can be piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl.

5. The compound of claim 1, wherein when R is selected from cycloalkyl, the substituent group can be C 1-6 alkylamino, C 1-6 alkyl, halogen.

6. The compound according to any one of claims 1 to 5, R 1 , R 2 , R 3 are independently from each other selected from hydrogen, fluoro, methoxy.

7. The compound of claim 6, wherein, The compound of structural formula (I) is selected from the group consisting of:

8. A pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition of claim 8, further comprising a beta-lactam antibiotic; wherein the beta-lactam antibiotic is a penicillin, a cephalosporin, a carbapenem, a monobactam, a bridged monobactam, or a combination thereof, wherein, the cephalosporin is cefepime, ceftriaxone, cefonicid, cefoxitin, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin or a combination thereof, wherein the carbapenem is imipenem, biapenem, doripenem, meropenem and ertapenem or a combination thereof.

10. Use of a pharmaceutical composition according to any one of claims 8 in the manufacture of a medicament for treating a bacterial infection in a subject; wherein the infection comprises bacteria selected from the group consisting of Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Acinetobacter baumannii, Escherichia coli.

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