Novel boric acid compound crystal form and preparation method thereof

Abstract

The invention discloses a novel crystal form of a boric acid compound and a preparation method thereof, and particularly discloses a compound as shown in a formula (I) and a preparation method and application of the crystal form of the compound. # imgabs0 #

Description

New crystal form of boric acid compound and preparation method thereof Technical Field

[0001] The present invention discloses a new crystal form of a boric acid compound and a preparation method thereof, and specifically discloses a compound of formula (I) and a preparation method and application of the crystal form thereof. Background Art

[0002] Since the discovery of penicillin by British scientist Fleming in 1928, β-lactam antibiotics have saved countless lives. However, with the emergence of various β-lactamases, β-lactam antibiotics are increasingly losing their activity. Serine hydrolases (ESBLs), KPCs, and metallo-β-lactamases (MBLs) are increasingly prevalent in various bacteria, exacerbating the safety risks posed by these bacteria to humans. Compounds such as phenylboronic acid have shown significant inhibitory effects against metallo-β-lactamases (MBLs).

[0003] The launch of antibiotic-boronic acid β-lactamase inhibitor combination drugs, represented by meropenem / vaborbactam, has sparked hope for the development of boronic acid β-lactamase inhibitors. Rempex Pharmaceuticals (WO2018 / 005662A1) reported a class of boronic acid β-lactamase inhibitors obtained by modifying phenylboronic acid. Optimization of this series of compounds led to the development of QPX7728, a compound currently in Phase I clinical development and currently being advanced into clinical trials by Qpex Biopharma. Another boronic acid β-lactamase inhibitor currently under development is VNRX-5133 (WO2014 / 089365) developed by VenatoRx Pharmaceuticals, currently in Phase III clinical trials.

[0004] In view of the severe drug resistance situation, it is urgent to develop a new generation of broad-spectrum β-lactamase inhibitors to solve the current drug resistance problem. The boronic acid molecules in the present invention have corresponding potential.

[0005] Summary of the Invention

[0006] The present invention provides a compound of formula (I), the structural formula of which is as follows:

[0007] The present invention also provides a crystalline form A of the compound of formula (I), which has an X-ray powder diffraction pattern (XRPD) having characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, 23.800±0.200° and 29.861±0.200°;

[0008] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, 20.660±0.200°, 23.800±0.200°, 29.300±0.200°, 29.861±0.200°, 35.001±0.200° and 36.002±0.200°.

[0009] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, 18.642±0.200°, 20.660±0.200°, 21.258±0.200°, 22.518±0.200°, 23.800±0.200°, 29.300±0.200°, 29.861±0.200°, 34.061±0.200°, 35.001±0.200° and 36.002±0.200°.

[0010] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, 14.542±0.200°, 16.538±0.200°, 17.820±0.200°, 18.642±0.200°, 20.660±0.200°, 21.258±0.200°, 22.518±0.200°, 23.800±0.200°, 24.912±0.200°, 25.800±0.200°, 26.912±0.200°, 27.912±0.200°, 28. 5.499±0.200°, 27.415±0.200°, 27.844±0.200°, 29.300±0.200°, 29.861±0.200°, 30.478±0.200°, 31.314±0.200°, 32.061±0.200°, 33.504±0.200°, 34.061±0.200°, 35.001±0.200°, 36.002±0.200° and 38.960±0.200°.

[0011] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 5.959°, 11.860°, 14.542°, 16.538°, 17.820°, 18.642°, 20.660°, 21.258°, 22.518°, 23.800°, 25.499°, 27.415°, 27.844°, 29.300°, 29.861°, 30.478°, 31.314°, 32.061°, 33.504°, 34.061°, 35.001°, 36.002° and 38.960°.

[0012] In some embodiments of the present invention, the X-ray powder diffraction pattern of the above-mentioned crystal form A has characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, and / or 14.542±0.200°, and / or 16.538±0.200°, and / or 17.820±0.200°, and / or 18.642±0.200°, and / or 20.660±0.200°, and / or 21.258±0.200°, and / or 22.518±0.200°, and / or 23.800±0.200°, and / or 25.49 9±0.200°, and / or 27.415±0.200°, and / or 27.844±0.200°, and / or 29.300±0.200°, and / or 29.861±0.200°, and / or 30.478±0.200°, and / or 31.314±0.200°, and / or 32.061±0.200°, and / or 33.504±0.200°, and / or 34.061±0.200°, and / or 35.001±0.200°, and / or 36.002±0.200°, and / or 38.960±0.200°.

[0013] In some embodiments of the present invention, the XRPD pattern of the above-mentioned crystal form A is basically as shown in Figure 1.

[0014] In some embodiments of the present invention, the XRPD pattern analysis data of the above-mentioned Form A is shown in Table 1.

[0015] Table 1 XRPD pattern analysis data of the crystal form of the compound of formula (I)

[0016] In some embodiments of the present invention, the above-mentioned crystal form A has a differential scanning calorimetry curve with endothermic peaks at 105.93±3°C and 118.45±3°C.

[0017] In some embodiments of the present invention, the DSC spectrum of the above-mentioned crystal form A is shown in Figure 2.

[0018] In some embodiments of the present invention, the thermogravimetric analysis curve of the above-mentioned crystal form A shows a weight loss of 15.146% at 150.0±3°C.

[0019] In some embodiments of the present invention, the TGA spectrum of the above-mentioned crystal form A is shown in Figure 3.

[0020] The present invention also provides a method for preparing the crystalline form A of compound of formula (I), comprising:

[0021] 1) adding the compound of formula (I) to an alcohol solvent, acetone, ethyl acetate, tert-butyl methyl ether, tetrahydrofuran, dimethyltetrahydrofuran, 1,4-dioxane, acetonitrile, water, dichloromethane, chloroform, toluene, n-heptane, dimethyl sulfoxide, N,N-dimethylacetamide, N-methylpyrrolidone, or a mixed solvent of any two of the above solvents;

[0022] 2) stirring at -20 to 50° C. for 1 to 192 hours;

[0023] 3) filtering, collecting the filter cake, and drying the filter cake at 30-40° C. for 10-12 hours, preferably at 35° C. for 10-12 hours;

[0024] Wherein, the compound of formula (I) is

[0025] In some embodiments of the present invention, the mixed solvent is selected from an alcohol solvent, a mixed solvent of acetone or acetonitrile and water, and a mixed solvent of an alcohol solvent and methyl tert-butyl ether.

[0026] In some embodiments of the present invention, the volume ratio of the above-mentioned alcohol solvent, acetone, acetonitrile and water is selected from 1:50 to 1:4.

[0027] In some embodiments of the present invention, the mixed solvent is selected from a mixed solvent of an alcohol solvent and methyl tert-butyl ether.

[0028] In some embodiments of the present invention, the volume ratio of the alcohol solvent to methyl tert-butyl ether is selected from 10:1 to 1:1.

[0029] In some embodiments of the present invention, the alcohol solvent is selected from methanol, ethanol and isopropanol.

[0030] In some embodiments of the present invention, the mass volume ratio of the compound of formula (I) to the solvent is selected from 1:1 to 200.

[0031] The present invention also provides the use of the compound of formula (I), the crystal form A of the compound of formula (I), or the preparation method of the crystal form A of the compound of formula (I) in the preparation of drugs for treating diseases related to bacterial infections.

[0032] Technical Effects

[0033] The crystal form A of the compound of formula (I) of the present invention is easy to obtain, has good physical and chemical stability, and has high industrial application value and economic value. The present invention discloses a new class of broad-spectrum β-lactamase inhibitors containing a boronic acid structure, which can inhibit serine β-lactamase and metallo-β-lactamase. When used in combination with specific β-lactam antibiotics, its antibacterial activity can be significantly restored, especially against various super bacteria expressing β-lactamase, including carbapenem-resistant Enterobacteriaceae and carbapenem-resistant Acinetobacter baumannii. The compounds disclosed in the present invention have good water solubility; have good exposure regardless of oral or intravenous administration, excellent pharmacokinetic properties; good safety; can significantly restore the in vivo bactericidal activity of β-lactam antibiotics when used in combination with specific β-lactam antibiotics, have good efficacy, and will be used to treat diseases related to bacterial infections.

[0034] Definition and Description

[0035] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0036] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0037] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0038] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0039] All solvents used in the present invention were commercially available and used without further purification.

[0040] The solvent used in the present invention is commercially available.

[0041] The present invention uses the following abbreviations: DCM represents dichloromethane; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOH represents ethanol; MeOH represents methanol; ACN (MeCN) represents acetonitrile; EtOAc represents ethyl acetate; THF represents tetrahydrofuran; H2O represents water; i-PrOH represents isopropyl alcohol; Acetone represents acetone; IPAc represents isopropyl acetate; THF represents tetrahydrofuran; 2-MeTHF represents 2-methyltetrahydrofuran; 1,4-Dioxane represents 1,4-dioxane; CHCl3 represents trichloromethane; Toluene represents toluene; n-Heptane represents n-heptane; DMAc represents N,N-dimethylacetamide; NMP represents N-methylpyrrolidone; MIC represents minimum inhibitory concentration

[0042] Compounds are manually or Software naming, commercially available compounds use supplier catalog names.

[0043] Instrument and analysis method of the present invention

[0044] 1.1 X-ray powder diffractometer (XRPD) method of the present invention

[0045] Testing instrument: X-ray diffractometer produced by Dandong Haoyuan Instrument Co., Ltd.

[0046] Test method: Approximately 10-20 mg of sample was used for XRPD detection.

[0047] Test conditions: See table.

[0048] Table 2 X-ray powder diffraction - test conditions

[0049] 1.2 Differential Scanning Calorimeter (DSC) Method of the Present Invention

[0050] Instrument model: DSC1 differential scanning calorimeter

[0051] Test method: Take an appropriate amount of sample and place it in a perforated aluminum crucible for testing. Heat the sample from 25℃ to 350℃ at a heating rate of 10℃ / min.

[0052] 1.3 Thermogravimetric Analysis (TGA) Method of the Present Invention

[0053] Instrument model: TGA550 thermogravimetric analyzer

[0054] Test method: Take an appropriate amount of sample and place it in an aluminum crucible, and then place it in a platinum hanging basket for testing. Under nitrogen (N2) conditions, at a gas flow rate of 40mL / min and a heating rate of 10℃ / min, heat the sample from room temperature to 500℃. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 is a Cu-Kα radiation XRPD spectrum of the crystalline form A of the compound of formula (I).

[0056] FIG2 is a DSC spectrum of the crystalline form A of compound of formula (I).

[0057] FIG3 is a TGA spectrum of the crystalline form A of compound of formula (I).

[0058] FIG4 is a single crystal X-ray diffraction (SXRD) ellipsoid diagram of the three-dimensional structure of the compound of formula (I). DETAILED DESCRIPTION

[0059] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the specific implementation methods are not intended to limit the content of the present invention.

[0060] Example 1: Preparation of compound of formula (I)

[0061] Step 1: Preparation of compound 1-2

[0062] Compound 1-1 (5.00 kg, 32.44 mol, 1.0 eq) was dissolved in ethylene glycol dimethyl ether (20 L). The reaction solution was cooled to 0-5°C. Acetone (2.17 kg, 37.31 mol, 2.74 L, 1.15 eq) and 4-dimethylaminopyridine (198.17 g, 1.62 mol, 0.05 eq) were added sequentially. Thionyl chloride (4.63 kg, 38.93 mol, 2.82 L, 1.2 eq) was then slowly added dropwise at 0-5°C. The reaction solution was then stirred at 0-5°C for 1 hour. The temperature was then slowly raised to room temperature (20-25°C) and stirring continued for 15 hours. The reaction solution was divided into two batches, half of the reaction solution was slowly added to water (15 L) and ethyl acetate (15 L) for quenching, the temperature was controlled at 5-15 ° C, the organic layer was separated and washed with saturated brine (10 L), saturated aqueous sodium bicarbonate solution (10 L), and saturated brine (10 L) once each. The separated organic layer was dried over anhydrous sodium sulfate (500.00 g), filtered and concentrated under reduced pressure. The other half was treated in the same way and combined to obtain compound 1-2.

[0063] Step 2: Preparation of compound 1-3

[0064] Compound 1-2 (4 kg, 20.60 mol, 1 eq) was dissolved in N,N-dimethylformamide (15 L) solution, and potassium carbonate (3.42 kg, 24.72 mol, 1.2 eq) and iodoethane (3.86 kg, 24.72 mol, 1.2 eq) were added. The mixture was stirred at 45-50°C for 5 hours. The reaction solution was cooled to room temperature (10-20°C), and water (30 L) was added to the reaction solution. A large amount of solid precipitated. The reaction solution was stirred at 10-20°C for 0.5 hours and filtered to obtain a crude product. The crude product was placed in ethyl acetate (4 L) and stirred at 10-15°C for 1 hour. After filtering, the filter cake was dried to obtain compound 1-3. 1 H NMR (400MHz, CDCl3) δppm 7.44(t,J=8.38Hz,1H),6.62(d,J=8.56Hz,1H),6.55(d,J=8.19Hz,1H),4.19(q, J=6.97Hz, 2H), 1.72 (s, 6H), 1.54 (t, J=7.03Hz, 3H). LCMS (ESI) m / z: 223.0 (M+1).

[0065] Step 3: Preparation of compound 1-4

[0066] Add anhydrous 2-methyltetrahydrofuran (18 L) to a 50 L high- and low-temperature reactor. Start the stirrer at 180 rpm and add compound 1-3 (4500.10 g, 20.25 mol, 1.0 eq) all at once. Heat the mixture in a circulating oil bath, maintaining the internal temperature at 40-45°C. Add dibromohydantoin (4052.81 g, 14.17 mol, 0.7 eq) in seven batches. After each addition, the internal temperature will rise to 50°C. Wait until the temperature drops to 40-45°C before adding the next batch. After all additions are complete, maintain the internal temperature at 45-50°C and stir for 30 minutes. Add an additional 200.02 g of dibromohydantoin, maintain the internal temperature at 45-50°C, and stir for 30 minutes. Cool the mixture to maintain the internal temperature at 25-30°C. The reaction mixture was concentrated under reduced pressure (45°C, pressure: <-0.08 MPa) to remove most of the solvent. 6.0 L of water and 6.0 L of ethyl acetate / n-heptane (volume ratio 1:3) were added to a rotary evaporator. The mixture was stirred at atmospheric pressure for 15 minutes, then filtered. The filter cake was rinsed once with 2.0 L of water and once with 1.0 L of n-heptane. The filter cake was an off-white solid. 8.0 L of water, 2.0 L of ethyl acetate, and 6.0 L of n-heptane were added to a 50 L high- and low-temperature reactor, and stirred at 25-30°C for 13 hours. The reaction mixture was filtered under reduced pressure, and the filter cake was rinsed once with 2.0 L of water and once with 2.0 L of n-heptane. The filter cake was vacuum dried (45°C, 22 hours, pressure: <-0.08 MPa) to obtain compound 1-4. 1H NMR (400MHz, CDCl3) δppm 7.65 (d, J=9.05Hz, 1H), 6.57 (d, J=9.05Hz, 1H), 4.18 (q, J=6.97Hz, 2H), 1.77 (s, 6H), 1.54 (t, J=7.03Hz, 3H). LCMS (ESI) m / z: 300.9 / 302.9 (M+1).

[0067] Step 4: Preparation of compound 1-5

[0068] Add ethylene glycol dimethyl ether (23 L) to a 50 L high- and low-temperature reactor. Stirring was started at 180 rpm. The internal temperature was maintained at 25-30°C. A nitrogen flow was started and maintained. Compound 1-4 (2300.01 g, 7.64 mol, 1.0 eq), bis-naphthalene borate (2327.50 g, 9.17 mol, 1.2 eq), and potassium isooctanoate (2840.05 g, 15.58 mol, 2.0 eq) were added in a single addition. The atmosphere was flushed with nitrogen three times, maintaining a nitrogen flow. Tetrakistriphenylphosphine palladium (44.10 g, 38.21 mmol, 0.005 eq) was added in a single addition. The atmosphere was flushed with nitrogen five times, maintaining a nitrogen flow. The temperature was raised to an external temperature of 100°C and an internal temperature of 85-90°C. The reaction mixture was stirred at reflux for 16 h. The reaction solution was cooled to 20-25°C, 5.0L saturated brine was added to the reaction solution to quench, the internal temperature was controlled to be less than 30°C, and then 10.0L ethyl acetate and 5.0L saturated sodium bicarbonate aqueous solution were added and the liquid was separated. The organic phase was washed twice with saturated brine (5.0L×2) and the organic phase was separated. 500.20g of anhydrous sodium sulfate was added and dried for 30 minutes. After filtration and concentration, 4.0L n-heptane was added and stirred at 25-30°C for 20 minutes, filtered, and the filter cake was rinsed with 1.0L n-heptane. The filter cake was transferred to a tray and air-dried at room temperature to obtain 1912.20g of crude product. 19.0L ethyl acetate was added to a 50L high and low temperature reactor, stirring was started, the speed was 160 rpm, the internal temperature was controlled to 25-30°C, and the above crude product was added at one time under nitrogen flow protection and stirred to dissolve. Add 956.00 g of palladium-free silica gel (iMolbox-LMat-NH01) in one portion, control the internal temperature at 25-30°C, and stir for 19 hours. Filter, rinse the filter cake once with 1.0 L of ethyl acetate, and concentrate the filtrate on a rotary evaporator at 45°C under vacuum (vacuum <-0.08 MPa) to obtain a crude pale yellow solid. Add 2.0 L of n-heptane and stir at 25-30°C for 20 minutes. Filter, and rinse the filter cake with 0.5 L of n-heptane to obtain a white solid. Transfer to a tray and air-dry at room temperature to obtain compound 1-5. 1H NMR (400MHz, CDCl3) δppm 7.82 (d, J = 8.56Hz, 1H), 6.62 (d, J = 8.56Hz, 1H), 4.21 (q, J = 6.97Hz, 2H), 1.72 (s, 6H), 1.53 (t, J = 6.97Hz, 3H), 1.34 (s, 12H). LCMS(ESI)m / z:349.0(M+1).

[0069] Step 5: Preparation of compound 1-6

[0070] To a 50L high-low temperature reactor, add 7.2L of 2-methyltetrahydrofuran and 7.2L of water. Stirring was initiated at 160 rpm. Compound 1-5 (1800.00g, 5.17mol, 1.0eq) was added all at once. After stirring, the mixture was cooled to 10-15°C in a circulating oil bath. Sodium perborate (tetrahydrate) (875.02g, 5.69mol, 1.1eq) was added in four batches, maintaining the temperature between 10-20°C. After addition, the circulating oil bath was turned off. The mixture was slowly heated to 20-25°C and stirred for 1 hour. 8.0L of ethyl acetate was added to the reaction mixture; some white solid remained insoluble. The mixture was filtered to obtain a filtrate. The filtrate was transferred to a 50.0L separatory vessel and allowed to stand for separation. The aqueous phase was extracted once more with 4.0L of ethyl acetate, and the organic phases were combined. The organic phase was washed with 2.0 L saturated brine and 2.0 L saturated sodium thiosulfate solution respectively. The organic phase was tested with wet starch potassium iodide paper to ensure that it did not turn blue. The separated organic phase was washed once with 3.0 L saturated brine and dried over 500.05 g of anhydrous sodium sulfate for 15 h. The mixture was then filtered through a Buchner funnel and the filter cake was rinsed with 0.3 L ethyl acetate to obtain a light yellow filtrate. Most of the solvent was concentrated under reduced pressure, leaving about 1.5 L of solvent, with a large amount of solid precipitated. 1.0 L of n-heptane was added to the rotary evaporator bottle, the vacuum was turned off, and the mixture was slurried at 25-30 ° C for 20 minutes under normal pressure. The mixed solution was filtered, the filter cake was rinsed once with 0.5 L of n-heptane, the filter cake was transferred to a tray, and dried in a vacuum drying oven (45 ° C, pressure <-0.08 MPa) for 4 h. Compound 1-6 was obtained. 1 H NMR (400MHz, CDCl3) δppm 7.14 (d, J = 9.05Hz, 1H), 6.55 (d, J = 9.05Hz, 1H), 5.13 (s, 1H), 4.13 (q, J = 6.97Hz, 2H), 1.76 (s, 6H), 1.50 (t, J = 6.97Hz, 3H). LCMS(ESI)m / z:239.0(M+1).

[0071] Step 6: Preparation of Compound 1-7

[0072] To a 50L high-low temperature reactor, add 11.5L of acetonitrile, start stirring at 150 rpm, start nitrogen flow, and maintain the internal temperature at 20-25°C. Add compound 1-6 (2300.04g, 9.65mol, 1.0eq), potassium carbonate (1338.50g, 9.68mol, 1.0eq), and iodomethyl naphthalene borate (3267.42g, 11.59mol, 1.2eq) separately. Heat in a circulating oil bath to an internal temperature of 50-55°C and stir under a nitrogen flow for 10 hours. Close the circulating oil bath, maintain the internal temperature at 20-25°C, and continue stirring under a nitrogen flow for 11 hours. The reaction solution was filtered, the filter cake was rinsed once with 2.0 L of ethyl acetate, the filtrate was collected, and concentrated under reduced pressure (45°C, vacuum <-0.08 MPa) until 4-5 L of solvent remained. 15.0 L of ethyl acetate, 5.0 L of water, and 5.0 L of saturated sodium chloride were added to a rotary evaporator. When the solution was dissolved and clarified, it was transferred to a 50 L separator and allowed to stand for separation. 5.0 L of ethyl acetate was added to the aqueous phase and stirred. The pH was adjusted to 3-4 with 1N dilute hydrochloric acid. After stirring, the solution was allowed to stand for separation and the organic phases were combined. The separated organic phase was washed once with 5.0 L of saturated sodium chloride, dried over 500.02 g of anhydrous sodium sulfate for 20 min, filtered under reduced pressure, the filter cake was rinsed once with 0.5 L of ethyl acetate, and the filtrate was transferred to a 50 L rotary evaporator and concentrated under reduced pressure (45°C, vacuum <-0.08 MPa) to obtain a white solid. Add 8.0 L of n-heptane to the rotary evaporator, turn off the vacuum, and rotate at atmospheric pressure at 20-25°C for 10 minutes to form a uniform turbid solution. Filter the suspension, rinse the filter cake once with 1.0 L of n-heptane, transfer the filter cake to a tray, and dry it in a vacuum drying oven (30°C, vacuum <-0.08 MPa) for 7 hours to obtain compound 1-7. 1 H NMR (400MHz, CDCl3) δppm 7.11(d,J=9.05Hz,1H),6.51(d,J=9.17Hz,1H),4.12(q,J=6.97Hz,2H),3.91(s,2H),1.76(s,6H),1.50(t,J=6.97Hz,3H),1.30(s,12H). LCMS(ESI)m / z:297.0(M-82+1).

[0073] Step 7: Preparation of crude compound 1

[0074] Add 17.3 L of isopropanol to a 50 L high- and low-temperature reactor. Start stirring at 200 rpm and nitrogen flow, maintaining the internal temperature at 20-25°C. Add compound 1-7 (1725.02 g, 4.56 mol, 1.0 eq) all at once and stir to obtain a pale yellow suspension. Cool the mixture in a circulating oil bath to an internal temperature of 0-5°C. Add sodium hydroxide (437.84 g, 10.95 mol, 2.4 eq) to 5.48 L of water to create a 2.0 M aqueous sodium hydroxide solution. Stir and dissolve until dissolved, then cool the mixture to 20-25°C. Once the reaction mixture cools to an internal temperature of 0-5°C, slowly add the 2.0 M aqueous sodium hydroxide solution dropwise. The reaction mixture becomes clear after halfway through the addition, and solids precipitate after the addition is complete. After the addition is complete, close the circulating oil bath and slowly raise the reaction temperature to 15-20°C. Maintain the internal temperature at 15-20°C and stir for 1.5 hours. Control the circulating oil bath to heat up, the internal temperature is 35-40°C, and continue stirring under nitrogen flow for 16 hours. Turn off the circulating oil bath and cool to an internal temperature of 20-25°C. The reaction solution is filtered under reduced pressure, and the filter cake is rinsed with 2.0L acetone twice to obtain a filter cake as an off-white solid. Add 12.5L of acetone to a 50L high and low temperature reactor, turn on stirring, speed (220 rpm) and nitrogen flow, control the internal temperature to 20-25°C, add the above filter cake at one time, stir and beat for 1h, filter under reduced pressure, rinse the filter cake twice with acetone (1.0L×2), transfer the filter cake to a tray, and dry it in a vacuum drying oven (30°C, vacuum degree <-0.08Mpa) for 37h to obtain crude compound 1.

[0075] Step 8: Preparation of compound 1

[0076] To a 50L high-low temperature reactor, add 7.0L of water. Start stirring at 180 rpm and nitrogen flow. Add crude compound 1 (1400.10g, 3.95mol, 1.0eq) all at once. Stir for 10 minutes to dissolve, maintaining the internal temperature at 20-25°C. Filter under reduced pressure to obtain a clear filtrate. Transfer the filtrate to a reactor, start stirring at 180 rpm, maintaining the internal temperature at 20-25°C. Slowly add 21.0L of acetone. During addition, a large amount of solid will precipitate. Maintain the internal temperature at 20-25°C. Continue stirring for 0.5h after addition. Filter the mixture under reduced pressure, rinse the filter cake twice with 2.0L of acetone, and drain to obtain the filter cake. Add 10.0 L of acetone to a 50 L high-low temperature reactor, start stirring at 220 rpm and nitrogen flow, control the internal temperature at 20-25°C, add the filter cake all at once, and stir and slurry for 1 hour. The mixture is filtered under reduced pressure, and the filter cake is rinsed twice with acetone (1.0 L x 2). The filter cake is transferred to a tray and dried in a vacuum drying oven (30°C, vacuum <-0.08 MPa) for 62 hours to obtain the compound of formula (I). 1H NMR (400MHz, D2O) δppm 6.52 (d, J = 8.68Hz, 1H), 6.25 (d, J = 8.68Hz, 1H), 3.90 (q, J = 7.09Hz, 2H), 3.20 (s, 2H), 1.19 (t, J = 7.03Hz, 3H). LCMS(ESI)m / z:239.0(M-54-46-16+1).

[0077] Example 2: Preparation of Crystalline Form A of Compound (I)

[0078] Approximately 15 mg of each compound of Formula (I) was weighed and added to a 20 mL vial. The solid was completely dissolved with 0.2-3.0 mL of each solvent (Table 3). Any undissolved sample was filtered through a 0.45 μm PTFE filter to obtain a clear solution. The antisolvent listed in Table 3 was added dropwise to the clear solution with stirring (1000 rpm) until solid precipitated. Alternatively, when the total antisolvent volume reached 10 mL, the sample without solid precipitation was suspended and stirred at 5°C. The clear sample was transferred to -20°C and suspended and stirred. If still clear, the sample was transferred to room temperature for evaporation. The precipitated solid was isolated and analyzed by XRPD. The results are shown in Table 3, yielding Form A of the compound of Formula (I).

[0079] Table 3 Antisolvent addition test *: Solid obtained by stirring at 5°C. #: Solid obtained by evaporation at room temperature.

[0080] Approximately 15 mg of each compound of formula (I) was weighed into a glass vial, and 0.5 mL of each solvent listed in Table 4 was added. The resulting turbid solution was magnetically stirred (1000 rpm) at room temperature for approximately 3 days. The solid was then collected by centrifugation and subjected to XRPD analysis. The results are shown in Table 4, yielding Form A of the compound of formula (I).

[0081] Table 4 Room temperature suspension stirring test

[0082] Approximately 15 mg of each compound of formula (I) was weighed into a glass vial. 0.5 mL of each solvent listed in Table 5 was added. The resulting suspension was magnetically stirred (1000 rpm) at 50°C for approximately 3-7 days. The solid was then collected by centrifugation and analyzed by XRPD. The test results are shown in Table 5, yielding Form A of the compound of formula (I).

[0083] Table 5 50℃ suspension stirring test

[0084] Approximately 15 mg of the compound of formula (I) was weighed into a 3 mL vial. 0.2-3.0 mL of the solvent listed in Table 6 was added for dissolution (unclear residue was filtered through a 0.45 μm PTFE filter). The vial was sealed with parafilm and pierced with four pinholes. The vial was allowed to evaporate slowly at room temperature. The resulting solid was collected and analyzed by XRPD. The results are shown in Table 6, yielding Form A of the compound of formula (I).

[0085] Table 6 Slow volatilization test

[0086] Approximately 15 mg of each portion of the compound of formula (I) was weighed into a 3 mL vial and dissolved with 0.2-1.0 mL of the solvent listed in Table 7 (the undissolved solution was filtered using a 0.45 μm PTFE filter). Approximately 4 mL of antisolvent was added to another 20 mL vial. The 3 mL vial containing the clear solution was placed open inside the 20 mL vial. The 20 mL vial was then sealed and allowed to stand at room temperature. The resulting solid was collected and analyzed by XRPD. The test results are shown in Table 7, yielding Form A of the compound of formula (I).

[0087] Table 7 Gas-liquid diffusion test

[0088] Example 3: Solid Preliminary Stability Test of Crystalline Form A of Compound of Formula (I)

[0089] According to the "Guidelines for Stability Testing of APIs and Preparations" (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 9001), the stability of the crystalline form of compound A of formula (I) was investigated under high temperature (60°C, open), high humidity (25°C / relative humidity 75%, open), high humidity (25°C / relative humidity 92.5%, open), strong light (5000lx and ultraviolet intensity 90μw / cm 2 , open) and stability under high temperature and high humidity conditions (40℃ / relative humidity 75% and 60℃ / relative humidity 75%, open).

[0090] Weigh about 250 mg of Form A of the compound of formula (I), place it in a dry and clean glass bottle, spread it into a thin layer, and use it as a test sample. Place it under influencing factors and accelerated conditions, and the sample is completely exposed. Samples are taken for analysis after 5 days and 10 days under high temperature and high humidity, and after 1 month, 2 months, and 3 months under accelerated conditions. The sample placed under light conditions is completely exposed at room temperature. XRPD is performed on the samples placed under different conditions, and the test results are compared with the initial test results on day 0. The crystal form results are shown in Table 8 below, which show that Form A of the compound of formula (I) is a stable crystal form.

[0091] Table 8 Pre-stability crystal form results

[0092] Example 4: Single crystal X-ray diffraction analysis of the compound of formula (I)

[0093] 1. Instrument Parameters and Data Collection

[0094] Rigaku Oxford Diffraction XtaLAB Synergy four-circle diffractometer equipped with a HyPix-6000HE area detector.

[0095] Cryogenic system: Oxford Cryostream 800

[0096] Cu: 50W, microfocus source with multilayer mirrors (μ-CMF).

[0097] Distance from crystal to CCD detector: d = 35 mm

[0098] Tube voltage: 50kV

[0099] Tube current: 1mA

[0100] The diffraction experiment collected 25762 diffraction points, of which 2603 were independent diffraction points (Rint=0.0636). The diffraction collection range was 2θ=5.9to133.168°, and the diffraction index range was -8≤h≤9, -35≤k≤35, -7≤l≤7. The structure was solved using SHELXT (Sheldrick, GM2015. ActaCryst.A71,3-8), and the structure was refined using SHELXL (against F2) (Sheldrick, GM2015. ActaCryst.C71,3-8). Among the 2603 independent diffraction points, 259 parameters were used for structure refinement. After refinement, S=1.094, R1=0.0513, wR2=0.1404. The residual electron density values ​​were 0.50 and

[0101] 2. Crystal Cultivation

[0102] A sample of the compound of formula (I) (20 mg) was weighed and dissolved in a 300 μL acetone / water mixture (1:2, V / V) at room temperature. The sample solution was placed in a 4 mL sealed sample vial and allowed to stand at 10°C. Colorless prismatic crystals were obtained the next day.

[0103] 3. Crystal data list

[0104] Table 9 Single crystal structure data of compound of formula (I)

[0105] 4. Conclusion

[0106] The detected crystal is colorless prism (0.30×0.10×0.04mm 3 ), belongs to the monoclinic P21 / c space group. Unit cell parameters α=90°, β=104.934(2)°, γ=90°, Z = 4. Calculated density Dc = 1.598 g / cm 3 , the number of electrons in the unit cell F(000) = 736.0, the linear absorption coefficient of the unit cell μ(Cu Kα) = 1.705 mm –1 , diffraction experiment temperature T = 293 (2) K. Single crystal data show that the single crystal is the compound of formula (I). The single crystal SXRD stereoscopic structure ellipsoid diagram of the compound of formula (I) is shown in Figure 4. The single crystal structure data and parameters of the compound of formula (I) are shown in Table 9.

[0107] Biological test data:

[0108] Experimental Example 1: In vitro activity test of the crystal form A of compound of formula (I)

[0109] 1. Culture medium

[0110] CAMHB, Cation-Adjusted Mueller-Hinton Broth

[0111] 2. Tested strains

[0112] The test bacteria included 20 strains of Escherichia coli (10 each of KPC-type and NDM-type carbapenem-resistant strains), 30 strains of Klebsiella pneumoniae (10 each of KPC-type, NDM-type, and OXA-type carbapenem-resistant strains), and 10 strains of carbapenem-resistant Acinetobacter baumannii (CRAB) isolated from clinical sites in China over the past two to three years, for a total of 60 strains. Details are shown in Table 10 below.

[0113] Table 10 Strain information list

[0114] 3. Experimental Methods

[0115] According to the recommendations of the relevant documents of the Clinical and Laboratory Standards Institute (CLSI), the MIC of the combination of the compound of formula (I) and the test drug against clinical isolates was determined by the broth microdilution method.

[0116] (1) Preparation of antimicrobial drugs:

[0117] The compound of formula (I) and meropenem were dissolved and diluted with sterile water; cefepime was dissolved and diluted with phosphate buffer (pH 6.0); ceftazidime was dissolved with phosphate buffer (pH 6.0) and diluted with sterile water; aztreonam was dissolved with saturated sodium bicarbonate and diluted with sterile water; ertapenem was dissolved and diluted with phosphate buffer (pH 7.2); the antibacterial drug concentration range was 128 mg / L-0.06 mg / L.

[0118] (2) Inoculation amount:

[0119] The test bacteria cultured overnight were prepared into 0.5 McFarland turbidimeter using direct colony suspension method, and diluted 100 times. The final inoculum volume was 10 5 CFU / mL.

[0120] (3) Culture conditions:

[0121] Enterobacteriales: Culture in air at (35±2)℃ for 16-20 hours.

[0122] Acinetobacter: Culture in air at (35±2)℃ for 20-24 hours.

[0123] (IV) Reading and judging the results:

[0124] Refer to CLSI 2021 version M100 31st Edition standard.

[0125] (V) Quality control strains:

[0126] Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC700603, Acinetobacter baumannii ATCC19606.

[0127] (6) Statistics:

[0128] WHONET version 5.6 software was used to perform statistical analysis on the results of drug susceptibility testing.

[0129] 4. Experimental Results

[0130] The specific MIC results are shown in Table 11.

[0131] Table 11 MIC (μg / mL) results of the combination of the compound of formula (I) and β-lactam compounds

[0132] 5. Conclusion

[0133] The combined susceptibility test results of the compound of formula (I) with meropenem, ertapenem, cefepime, and ceftazidime showed significant synergistic effects against all three carbapenem-resistant test bacteria. The combined susceptibility test results of the compound of formula (I) with aztreonam showed synergistic effects against carbapenem-resistant Escherichia coli and Klebsiella pneumoniae, but had essentially no effect on carbapenem-resistant Acinetobacter baumannii.

[0134] Experimental Example 2: In vivo efficacy test of the crystal form A of compound of formula (I)

[0135] 1) Thigh muscle model

[0136] Thirty-six CD-1 female mice were divided into 12 cages, with 3 mice in each cage; the day of infection was designated as day 0.

[0137] On day -4, 150 mg / kg of the immunosuppressant cyclophosphamide was intraperitoneally injected, and on day -1, 100 mg / kg of the immunosuppressant cyclophosphamide was intraperitoneally injected again to produce immunodeficient mice.

[0138] On day -1, Klebsiella pneumoniae ATCC BAA-1705 was revived on MHA plates. Revived colonies were selected and dissolved in sterile saline to prepare a bacterial solution with a concentration of 5.6E+07 CFU / mL for infection of the thigh muscles of mice. With infection starting at 0 h, 100 μL of the bacterial solution was injected into the thigh muscles of each mouse at 0 h, resulting in an inoculum of 5.6E+06 CFU / mouse. Two hours after infection, dosing was performed according to the experimental protocol. See Table 12 for the specific experimental protocol.

[0139] (1) 2 h after infection: At the end of the first cage of mice, thigh muscle tissue was collected and placed in 10 mL of sterile saline. The homogenate was homogenized using a homogenizer and the homogenate was graded and plated, with two replicates for each mouse.

[0140] (2) 2 hours after infection: Cages 2 to 12 were treated at a volume of 10 mL / kg of mouse body weight. Cage 2 mice were given the vehicle intravenously every 2 hours, with a 12-hour endpoint; Cage 3 mice were given 100 mg / kg of meropenem intravenously every 2 hours, with a 12-hour endpoint; Cage 4 mice were given 100 mg / kg of meropenem and 20 mg / kg of QPX7728 intravenously every 2 hours, with a 12-hour endpoint; Cage 5 mice were given 100 mg / kg of meropenem and 100 mg / kg of the compound of formula (I) intravenously every 2 hours, with a 12-hour endpoint; Cage 6 mice were given 100 mg / kg of meropenem and 20 mg / kg of the compound of formula (I) intravenously every 2 hours, with a 12-hour endpoint; Cage 7 mice were given 100 mg / kg of meropenem and 4 mg / kg of the compound of formula (I) intravenously every 2 hours. The mice in the eighth cage were given 100 mg / kg of cefepime by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the ninth cage were given 100 mg / kg of cefepime and 20 mg / kg of QPX7728 by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the tenth cage were given 100 mg / kg of cefepime and 100 mg / kg of the compound of formula (I) by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the eleventh cage were given 100 mg / kg of cefepime and 20 mg / kg of the compound of formula (I) by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the twelfth cage were given 100 mg / kg of cefepime and 4 mg / kg of the compound of formula (I) by intravenous injection every 2 hours, with a 12-hour endpoint. At the 12-hour endpoint, thigh muscle tissue was placed in 10 mL of sterile saline and homogenized using a homogenizer. The homogenate was then serially diluted and plated, with two replicates per mouse. The bacterial load in the thigh muscle tissue of the mice was counted. The results are summarized in Table 13.

[0141] Table 12. Thigh muscle model experimental plan

[0142] Table 13. Experimental results of bacterial load in mouse thigh muscle tissue

[0143] 2) Lung infection model

[0144] Thirty-six CD-1 female mice were divided into 12 cages, with 3 mice in each cage; the day of infection was designated as day 0.

[0145] On day -4, 150 mg / kg of the immunosuppressant cyclophosphamide was intraperitoneally injected, and on day -1, 100 mg / kg of the immunosuppressant cyclophosphamide was intraperitoneally injected again to produce immunodeficient mice.

[0146] On day -1, Klebsiella pneumoniae ATCC BAA-1705 was revived on MHA plates. Revived colonies were selected and dissolved in sterile saline to prepare a bacterial solution at a concentration of 2.75E+08 CFU / mL. Lung infection was initiated via nasal instillation. The infection start time was calculated as 0 h. At 0 h, 50 μL of the bacterial solution was injected into each mouse via nasal instillation, resulting in an inoculum of 1.38E+07 CFU / mouse. Two hours after infection, dosing was performed according to the experimental protocol. The specific experimental protocol is shown in Table 14.

[0147] (1) 2 h after infection: At the end of the first cage of mice, lung tissue was collected and placed in 10 mL of sterile saline. The tissue was homogenized using a homogenizer and the homogenate was graded and plated, with two replicates for each mouse.

[0148] (2) 2 hours after infection: Cages 2 to 12 were treated at a volume of 10 mL / kg of mouse body weight. Cage 2 mice were given the vehicle intravenously every 2 hours, with a 12-hour endpoint; Cage 3 mice were given 100 mg / kg of meropenem intravenously every 2 hours, with a 12-hour endpoint; Cage 4 mice were given 100 mg / kg of meropenem and 20 mg / kg of QPX7728 intravenously every 2 hours, with a 12-hour endpoint; Cage 5 mice were given 100 mg / kg of meropenem and 100 mg / kg of the compound of formula (I) intravenously every 2 hours, with a 12-hour endpoint; Cage 6 mice were given 100 mg / kg of meropenem and 20 mg / kg of the compound of formula (I) intravenously every 2 hours, with a 12-hour endpoint; Cage 7 mice were given 100 mg / kg of meropenem and 4 mg / kg of the compound of formula (I) intravenously every 2 hours. The mice in the eighth cage were given 100 mg / kg of cefepime by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the ninth cage were given 100 mg / kg of cefepime and 20 mg / kg of QPX7728 by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the tenth cage were given 100 mg / kg of cefepime and 100 mg / kg of the compound of formula (I) by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the eleventh cage were given 100 mg / kg of cefepime and 20 mg / kg of the compound of formula (I) by intravenous injection every 2 hours, with a 12-hour endpoint; the mice in the twelfth cage were given 100 mg / kg of cefepime and 4 mg / kg of the compound of formula (I) by intravenous injection every 2 hours, with a 12-hour endpoint. At the endpoint, lung tissue was placed in 10 mL of sterile saline and homogenized using a homogenizer. The homogenate was then serially diluted and plated, with two replicates per mouse. The bacterial load in the mouse lung tissue was counted. The results are summarized in Table 15.

[0149] Table 14. Experimental plan for lung infection model

[0150] Table 15. Experimental results of bacterial load in mouse lung infection model tissue

[0151] Conclusion: The mouse thigh muscle infection model and the mouse lung infection model show that the compound of formula (I) of the present invention has a significant inhibitory effect on infection in vivo when combined with meropenem or cefepime.

Claims

1. The compound of formula (I) has the following structural formula:

2. Form A of the compound of formula (I), having an X-ray powder diffraction pattern (XRPD) with characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, 23.800±0.200°, and 29.861±0.200°; 3. The crystal form A according to claim 2, wherein its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, 20.660±0.200°, 23.800±0.200°, 29.300±0.200°, 29.861±0.200°, 35.001±0.200° and 36.002±0.200°.

4. The crystal form A according to claim 3, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.959±0.200°, 11.860±0.200°, 18.642±0.200°, 20.660±0.200°, 21.258±0.200°, 22.518±0.200°, 23.800±0.200°, 29.300±0.200°, 29.861±0.200°, 34.061±0.200°, 35.001±0.200° and 36.002±0.200°.

5. The crystal form A according to claim 4, wherein the X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ angles: 5.959°, 11.860°, 14.542°, 16.538°, 17.820°, 18.642°, 20.660°, 21.258°, 22.518°, 23.800°, 25.499°, 27.415°, 27.844°, 29.300°, 29.861°, 30.478°, 31.314°, 32.061°, 33.504°, 34.061°, 35.001°, 36.002° and 38.960°.

6. The crystal form A according to claim 5, whose XRPD pattern is substantially as shown in Figure 1.

7. The crystal form A according to any one of claims 1 to 6, wherein its differential scanning calorimetry (DSC) curve has endothermic peaks at 105.93±3°C and 118.45±3°C.

8. The crystal form A according to claim 7, whose DSC spectrum is shown in Figure 2.

9. The crystal form A according to any one of claims 1 to 6, wherein the thermogravimetric analysis (TGA) curve thereof shows a weight loss of 15.146% at 150.0±3°C.

10. The crystal form A according to claim 9, whose TGA spectrum is shown in Figure 3.

11. A method for preparing the crystalline form A of compound of formula (I) according to claim 1, comprising: 1) adding the compound of formula (I) to an alcohol solvent, acetone, ethyl acetate, tert-butyl methyl ether, tetrahydrofuran, dimethyltetrahydrofuran, 1,4-dioxane, acetonitrile, water, dichloromethane, chloroform, toluene, n-heptane, dimethyl sulfoxide, N,N-dimethylacetamide, N-methylpyrrolidone, or a mixed solvent of any two of the above solvents; 2) stirring at -20 to 50° C. for 1 to 192 hours; 3) filtering, collecting the filter cake, and drying the filter cake at 30-40° C. for 10-12 hours, preferably at 35° C. for 10-12 hours; Wherein, the compound of formula (I) is 12. The method for preparing the crystal form A of compound of formula (I) according to claim 11, wherein: The mixed solvent is selected from an alcohol solvent, a mixed solvent of acetone or acetonitrile and water, and a mixed solvent of an alcohol solvent and methyl tert-butyl ether.

13. The method for preparing the crystal form A of compound of formula (I) according to claim 12, wherein: The volume ratio of the alcohol solvent, acetone, acetonitrile and water is selected from 1:50 to 1:

4.

14. The method for preparing the crystal form A of compound of formula (I) according to claim 12, wherein: The volume ratio of the alcohol solvent to methyl tert-butyl ether is selected from 10:1 to 1:

1.

15. The method for preparing the crystal form A of compound of formula (I) according to claims 11 to 14, wherein: The alcohol solvent is selected from methanol, ethanol and isopropanol.

16. The method for preparing the crystal form A of compound of formula (I) according to claim 11, wherein: The weight-to-volume ratio of the compound of formula (I) to the solvent is selected from 1:1 to 200.

17. Use of the method for preparing the compound according to claim 1, the crystal form A according to any one of claims 2 to 10, or the crystal form A of the compound of formula (I) according to any one of claims 11 to 16 in preparing drugs for treating diseases related to bacterial infections.

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