Preparation process of prazomicin

Abstract

The invention provides a preparation process of prazomicin, which comprises the following steps: directly using sisomicin alkali as a starting material, firstly carrying out PNZ protection on a 6 '-amino group, and then selectively protecting other amino groups by using t-butyloxycarbonyl (Boc). Iodoethanol and Boc-(S)-HABA are selected as side chains for condensation. And finally, carrying out deprotection under an acidic condition, and crystallizing by using a spray drying method to obtain the Plazomicin sulfate. Compared with freeze-drying crystallization, the crystallization method has the advantages of high drying speed, high production efficiency and high purity of the final product.

Description

Technical Field

[0001] The present invention relates to the field of drug synthesis, and particularly to a preparation process of plazomicin. Background Art

[0002] Plazomicin is a newly launched semi-synthetic aminoglycoside antibiotic in recent years. In June 2018, the plazomicin injection (Zemdri) was approved by the US Food and Drug Administration for marketing. It is applicable to the treatment of patients over 18 years old with complicated urinary tract infections, including pyelonephritis. Compared with previous generations of aminoglycoside drugs, its risk of renal toxicity is relatively low.

[0003] The mechanism of action of plazomicin is to bind to the 30S ribosomal subunit of bacteria, interfere with bacterial protein synthesis, and thus play a bactericidal role. It can avoid being inactivated by the main aminoglycoside antibiotic inactivating enzymes. Plazomicin has good activity against many Gram-negative bacteria and Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus isolates, etc.

[0004]

[0005] Plazomicin is derived from the fermentation product sisomicin through chemical structure modification. Its preparation process mainly involves introducing a hydroxy-aminobutyric acid substituent at the 6'-position amino group of sisomicin and a hydroxyethyl substituent at the 7''-position amino group, thereby obtaining a new aminoglycoside antibiotic.

[0006] As the active ingredient of a new aminoglycoside antibacterial drug, the chemical synthesis method of plazomicin has also attracted extensive attention and reports, mainly in the process development and optimization of the selection of the types of protecting groups and the reaction sequence in the synthesis process. Patents such as CN110878108B and CN112079882B introduce that the synthesis route of plazomicin is to use sisomicin sulfate as the starting material, free it through an anion exchange resin, and then sequentially perform selective protection of amino groups, including protection with hexamethyldisilazane, p-nitrobenzyloxycarbonyl (PNZ) or trifluoroacetyl (TFA), selective tert-butoxycarbonyl (Boc) protection, fluorenylmethoxycarbonyl (Fmoc) protection, and re-selective tert-butoxycarbonyl (Boc) protection, etc.; then remove the protecting groups at the corresponding positions, condense with N-tert-butoxycarbonyl-4-amino-2(S)-hydroxybutyric acid (Boc-(S)-HABA), remove the PNZ protecting group and other reaction steps, and finally remove the protecting groups under acidic conditions. Then, the final product plazomicin or its salt is prepared by freeze-drying or solvent pulping. However, the reported routes are all difficult to avoid a long synthesis route, many side reactions, and complicated post-treatment. Summary of the Invention

[0007] The present invention aims to overcome the above-mentioned defects. Using sisomicin base as the starting material directly, first protect the amino group at the 6'-position with PNZ, and then selectively protect other amino groups with tert-butoxycarbonyl (Boc). Select iodoethanol and Boc-(S)-HABA as the side chains for condensation. Finally, deprotect under acidic conditions and use spray drying to crystallize to obtain Plazomicin sulfate. The advantages of this crystallization method compared with freeze-drying crystallization are faster drying speed, higher production efficiency, and higher purity of the final product.

[0008] A preparation process of Plazomicin provided by the present invention is characterized in that: using sisomicin base as the starting material, and obtaining Plazomicin sulfate through the process shown in the following reaction equation;

[0009]

[0010] Furthermore, a preparation process of Plazomicin provided by the present invention is further characterized in that:

[0011] Plazomicin sulfate is obtained by crystallization through spray drying method.

[0012] Furthermore, a preparation process of Plazomicin provided by the present invention is further characterized in that:

[0013] The preparation process of Plazomicin sulfate is as follows: Add intermediate A000007 and dichloromethane to the reaction kettle, control the temperature at 0-5°C during the dropping process and drop trifluoroacetic acid. After dropping, raise the temperature of the reaction solution to 20-30°C, stir for 1-2 h, then concentrate and extract to obtain the aqueous phase. Adjust the pH to 6.8-7.0, remove ammonia under the condition of controlling the temperature not higher than 0°C, then adjust the pH to 6.3-6.5 with sulfuric acid solution, raise the temperature to 20-25°C, and carry out spray drying.

[0014] Furthermore, a preparation process of Plazomicin provided by the present invention is further characterized in that:

[0015] The parameters of the spray drying are as follows:

[0016] Feed flow rate: 8-12 kg / h, liquid temperature: 2-8°C, drying gas flow rate: 320-480 kg / h, outlet temperature: 85-95°C, condenser temperature: 4-6°C, nitrogen sweep flow rate: 9-11 kg / h, nitrogen sweep temperature: 30-50°C, inlet air temperature: 160-180°C, pressure: 20-40 bar.

[0017] Furthermore, a preparation process of Plazomicin provided by the present invention is further characterized in that:

[0018] The preparation process of intermediate A000002 is as follows: Add PNZ-Bt and dichloromethane into a reaction kettle, stir to dissolve, dropwise add sisomicin solution, and then keep the temperature for reaction for 2 - 3 hours to obtain A000002.

[0019] Furthermore, a preparation process of plazomicin provided by the present invention is further characterized in that:

[0020] The process of preparing intermediate A000003 from intermediate A000002 is as follows: Add methanol and triethylamine to A000002, keep the temperature at 25 - 35 °C, add zinc acetate dihydrate and stir for 1 - 2 hours. Dropwise add di-tert-butyl dicarbonate solution and methanol to the reaction solution. After the addition is completed, keep the temperature for reaction for 2 - 3 hours. Add ammonia water to the reaction solution, keep the temperature at 20 - 30 °C, stir for more than 1 h, and then obtain A000003 through extraction and concentration.

[0021] Furthermore, a preparation process of plazomicin provided by the present invention is further characterized in that:

[0022] The process of preparing intermediate A000004 from intermediate A000003 is as follows: Under the condition of 25 - 35 °C, add purified water, Boc-(S)-HABA, and HOBt monohydrate to A000003, adjust the pH to 4.5 - 5.5, add EDAC, and keep the temperature for reaction for 3 - 4 hours. After the reaction is completed, adjust the reaction to pH 8 - 10.0, compress the organic phase, add acetonitrile to the residue to control the reaction temperature not exceeding -5 °C, keep the temperature and stir for 10 - 12 h to obtain intermediate A000004.

[0023] Furthermore, a preparation process of plazomicin provided by the present invention is further characterized in that:

[0024] The process of preparing intermediate A000005 from intermediate A000004 is as follows: Add intermediate A000004 and alcohol to a reaction kettle, heat to 40 - 50 °C, dropwise add di-tert-butyl dicarbonate solution, and then keep the temperature for reaction for 4 - 5 hours to obtain intermediate A000005.

[0025] Furthermore, a preparation process of plazomicin provided by the present invention is further characterized in that:

[0026] The process for preparing Intermediate A000006 from Intermediate A000005 is as follows: Cool the aqueous sodium hydroxide solution to no higher than 0 °C, add sodium dithionite, then keep stirring at a constant temperature for 10 - 30 min. After dropping in A000005, heat the mixture to no lower than 30 °C and keep the reaction at a constant temperature for 5 hours. After the reaction is completed, concentrate, extract, and then concentrate again. At 30 - 40 °C, add purified water and dichloromethane to the residue, and during this process, keep the temperature of the reaction solution no lower than 25 °C. After the addition is complete, cool the solution to no higher than -5 °C and continue stirring at a constant temperature for 12 - 18 hours to obtain Intermediate A000006.

[0027] Furthermore, a preparation process of plazomicin provided by the present invention is further characterized in that:

[0028] The process for preparing Intermediate A000007 from Intermediate A000006 is as follows: Add Intermediate A000006 and acetonitrile to the reaction kettle, heat the reaction solution to 30 - 40 °C, add acetone, sodium bicarbonate, and 2-iodoethanol, then keep the reaction at a constant temperature for 4 - 6 hours. After the reaction is completed, cool the solution to no higher than 20 °C, add DABCO to the reaction solution and continue stirring at a constant temperature for 16 - 20 hours. After complete quenching, add isopropyl acetate to the concentrated solution obtained by extraction and concentration, continue heating to 80 - 100 °C until completely dissolved, and then cool the solution to no higher than 0 °C and keep stirring at a constant temperature for 8 - 10 hours to obtain Intermediate A000007.

[0029] The functions and effects of the present invention:

[0030] The process flow of the solution of the present invention is simple, the equipment cost is low, the drying speed is fast, the obtained product has high purity and better quality, and it is more suitable for industrial production. Description of the Drawings

[0031] Figure 1 、The chromatogram of the product prepared in this example;

[0032] Figure 2 、The mass spectrum of the product prepared in this example;

[0033] Figure 3 、The hydrogen spectrum of the product prepared in this example; Specific Embodiment

[0034] This embodiment is carried out through the conditions and steps shown in the following equations:

[0035]

[0036] Example 1: Preparation of Intermediate A000002: At room temperature, add PNZ-Bt (0.696 kg, 1.175 mol) and dichloromethane (18.59 kg) to a reaction kettle and stir to dissolve. Then, dropwise add sisomicin solution (sisomicin free base (1 kg, 1.125 mol), methanol (3.96 kg), dichloromethane (6.64 kg)). After the dropping is completed, rinse the pipeline with 1.33 kg of dichloromethane, keep the temperature for reaction for 3 hours. After the reaction is completed, concentrate under reduced pressure at T < 40 °C until the volume (L) is 5 × batch ± 5% to obtain A000002. Purity: 99.22%.

[0037] Example 2: Preparation of Intermediate A000003: Add methanol (5.54 kg) and triethylamine (1.08 kg) to A000002 under stirring. When T = 35 °C, add zinc acetate dihydrate (1.63 kg) and stir for more than 1 h. Dropwise add di-tert-butyl dicarbonate solution (Boc2O (2.55 kg, 11.5 mol), methanol (1.58 kg)) to the reaction solution. After the dropping is completed, keep the temperature for reaction for 3 hours. Under stirring, add 25% ammonia water (5.46 kg) to the reaction solution, keep T = 20 °C, and stir for more than 1 h. Add dichloromethane (13.28 kg) for extraction. The organic layer is washed twice with purified water (5 kg) and methanol (0.79 kg), and the organic layer is separated. Concentrate under reduced pressure at T < 40 °C until the volume (L) is 9 × batch ± 5% to obtain A000003. Purity: 97.03%.

[0038] Example 3: Preparation of Intermediate A000004: At T = 25 °C, add purified water (0.5 kg) and Boc-(S)-HABA (0.512 kg, 2.35 mol) to A000003, and add HOBt monohydrate (0.057 kg). Adjust the pH to 5.0 by dropwise adding dilute hydrochloric acid, then add EDAC (0.447 kg), and keep the temperature for reaction for 3 hours. After the reaction is completed, add methanol (1.58 kg), dichloromethane (15.94 kg), and purified water (5.0 kg) to the reaction solution, and adjust the pH to 10.0 by dropwise adding sodium hydroxide solution. Separate the organic phase and concentrate under reduced pressure at T < 40 °C. Add acetonitrile to the residue until the final volume (L) is 20 × batch. Raise the temperature of the reaction, stir to dissolve clearly, cool the reactant to -5 °C, keep the temperature and stir for 12 h. Filter. The filter cake is rinsed twice with acetonitrile. Dry under vacuum to obtain Intermediate A000004 (1.56 kg, yield 69%). Purity: 98.57%.

[0039] Example 4: Preparation of Intermediate A000005: Add Intermediate A000004 (1 kg, 0.942 mol) and methanol (7.92 kg) to a reaction kettle, heat to T = 50 °C, and dropwise add a solution of di-tert-butyl dicarbonate (Boc2O (0.255 kg, 1.17 mol), methanol (0.2 kg)). After the dropwise addition is completed, keep the reaction at a constant temperature for 5 hours. Obtain Intermediate A000005. Purity, 92.91%.

[0040] Example 5: Preparation of Intermediate A000006: Add sodium hydroxide (0.39 kg) to purified water (7.78 kg), stir to dissolve and then cool to 0 °C, add sodium dithionite (1.196 kg), and keep stirring at a constant temperature for 30 min after the addition is complete. Dropwise add the A000005 reaction solution into the above solution, and after the addition is complete, raise the temperature of the reaction solution to 30 °C and keep the reaction at a constant temperature for 5 hours. After the reaction is completed, T < 40 °C, and concentrate under reduced pressure to a volume (L) of 13 × batch ± 5%. At T = 40 °C, add isopropyl acetate (3.49 kg) and purified water (7.0 kg) to the residue, and perform extraction and liquid separation. At T < 50 °C, concentrate under reduced pressure to a volume (L) of 3 × batch ± 5%. At T = 40 °C, add purified water (0.135 kg) to the residue, add dichloromethane (5.98 kg), keep the temperature of the reaction solution at 25 °C, and then cool to T = -5 °C and keep stirring at a constant temperature for 12 h. Filter, wash the filter cake with dichloromethane (1.33 kg), and dry under vacuum to obtain Intermediate A000006 (1.28 kg, yield 90.1%). Purity, 97.33%.

[0041] Example 6: Preparation of Intermediate A000007: Add Intermediate A000006 (1 kg, 1.054 mol) and acetonitrile (3.94 kg) to a reaction kettle, heat the reaction solution to T = 30 °C, add acetone (2.36 kg), add sodium bicarbonate (0.177 kg), and add 2-iodoethanol (0.226 kg, 1.31 mol). Keep the reaction at a constant temperature for 6 hours. After the reaction is completed, cool to T = 20 °C, add DABCO (0.24 kg) to the reaction solution, keep the reaction at a constant temperature and stir for 20 h. After complete quenching, add purified water (5 kg) and isopropyl acetate (4.36 kg) to the reaction solution, and perform extraction and liquid separation. Wash the organic phase with 3 L of saturated brine twice and separate the organic layer. Concentrate under reduced pressure at T < 50 °C to 7.2 × batch. Add isopropyl acetate (0.7 kg) to the residue, heat to T = 80 °C until completely dissolved. Then cool to 0 °C and keep stirring at a constant temperature for 10 h. Filter, wash the filter cake with acetonitrile (2.83 kg), and dry under vacuum to obtain Intermediate A000007 (1.08 kg, yield 87.5%). Purity, 98.1%.

[0042] Example 7: Preparation of Plazomicin Sulfate: Add intermediate A000007 (1 kg) and dichloromethane (6.6 kg) into a reaction kettle, stir and cool down to T = 0 °C. Slowly add trifluoroacetic acid (4.5 kg) dropwise, control the temperature at T = 0 - 5 °C during the dropping process. After dropping, raise the temperature of the reaction solution to T = 30 °C and stir for 2 h. Concentrate under reduced pressure at T < 30 °C to 3 * batch volume. Dropwise add purified water (2 kg) to the residue, add isopropyl acetate (2.6 kg), extract, and combine the aqueous phases. Add purified water to make the total volume 9 × batch volume. Adjust the pH to 6.8 with an aqueous ammonia solution at T = 0 °C, remove ammonia from the aqueous phase through a reverse osmosis membrane. After ammonia removal, measure the content of the free base to ensure the content is 2 - 4%. Cool down to T = 0 °C, add sulfuric acid solution to adjust the pH to 6.3, raise the temperature of the solution to T = 25 °C, pass the solution through an activated carbon filter element, and perform spray drying. Feed flow rate: 8 - 12 kg / h, feed liquid temperature: 2 - 8 °C, drying gas flow rate: 320 - 480 kg / h, outlet temperature: 85 - 95 °C, condenser temperature: 4 - 6 °C, nitrogen purge flow rate: 9 - 11 kg / h, nitrogen purge temperature: 30 - 50 °C, inlet air temperature: 160 - 180 °C, pressure: 20 - 40 bar. Obtain the finished product of plazomicin. (0.73 kg, yield 81.1%). Purity, 98.72%.

[0043] Example 8: The process is the same as that in Example 7, only changing the spray drying parameters. When the feed flow rate: 8 - 12 kg / h, feed liquid temperature: 2 - 8 °C, drying gas flow rate: 320 - 480 kg / h, outlet temperature: 85 - 95 °C, condenser temperature: 4 - 6 °C, nitrogen purge flow rate: 9 - 11 kg / h, nitrogen purge temperature: 30 - 50 °C, inlet air temperature: 180 - 200 °C, pressure: 20 - 40 bar. Obtain the finished product of plazomicin. (0.76 kg, yield 84.4%). Purity, 91.33%.

[0044] Example 9: The process is the same as that in Example 7, only changing the spray drying parameters. When the feed flow rate: 6 - 8 kg / h, feed liquid temperature: 2 - 8 °C, drying gas flow rate: 320 - 480 kg / h, outlet temperature: 85 - 95 °C, condenser temperature: 4 - 6 °C, nitrogen purge flow rate: 9 - 11 kg / h, nitrogen purge temperature: 30 - 50 °C, inlet air temperature: 140 - 160 °C, pressure: 20 - 40 bar. Obtain the finished product of plazomicin. (0.67 kg, yield 74.4%). Purity, 96.75%.

[0045] Comparative Example 1: Plazomicin adopts the freeze-drying process of sulfate

[0046] Preparation of Plazomicin Sulfate: Add intermediate A000007 (1 kg) and dichloromethane (6.6 kg) into a reaction kettle, stir and cool down to T = 0 °C. Slowly add trifluoroacetic acid (4.5 kg) dropwise, control T = 0 - 5 °C during the dropping process. After dropping, heat the reaction solution to T = 30 °C and stir for 2 h. Concentrate under reduced pressure at T < 30 °C to 3 * batch volume. Drop purified water (2 kg) into the residue, add isopropyl acetate (2.6 kg), extract, and combine the aqueous phases. Add purified water to make the total volume 9 × batch volume. Adjust the pH to 6.8 with aqueous ammonia solution at T = 0 °C, pass the aqueous phase through a reverse osmosis membrane to remove ammonia. After the ammonia removal is completed, measure the content of free base to ensure it is 2 - 4%. Cool down to T = 0 °C, add sulfuric acid solution to adjust the pH to 6.3, raise the solution temperature to T = 25 °C, pass the solution through an activated carbon filter element, and perform freeze-drying. Obtain the finished product of Plazomicin. (0.79 kg, yield 87.7%). Purity, 96.68%.

Claims

1. A process for preparing prazomicin, characterized in that: Using sisomicin base as a starting material, Plazomicin sulfate is obtained through the process shown in the following reaction equation; A000007 was crystallized by spray drying to obtain Plazomicin sulfate.

2. A process for preparing prazomicin according to claim 1, characterized in that: The preparation process of plazomicin sulfate is as follows: add intermediate A000007 and dichloromethane into a reaction kettle, add trifluoroacetic acid dropwise while controlling the temperature to be 0-5°C during the dropping process, heat the reaction solution to 20-30°C, stir for 1-2h, concentrate and extract to obtain an aqueous phase, adjust the pH to 6.8-7.0, remove ammonia while controlling the temperature not higher than 0°C, adjust the pH to 6.3-6.5 with a sulfuric acid solution, heat to 20-25°C, and spray dry.

3. A process for preparing prazomicin according to claim 1, characterized in that: The parameters of the spray drying are: Feed flow rate: 8-12kg / h, feed liquid temperature: 2-8℃, drying gas flow rate: 320-480kg / h, outlet temperature: 85-95℃, condenser temperature: 4-6℃, nitrogen sweep flow rate: 9-11kg / h, nitrogen sweep temperature: 30-50℃, inlet temperature: 160-180℃, pressure: 20-40bar.

4. A process for preparing prazomicin according to claim 1, characterized in that: The preparation process of the intermediate A000002 is as follows: PNZ-Bt and dichloromethane are added into a reaction kettle, stirred and dissolved, sisomicin solution is added dropwise, and the mixture is kept warm for reaction for 2-3 hours to obtain A000002.

5. A process for preparing prazomicin according to claim 1, characterized in that: The process of preparing intermediate A000003 from intermediate A000002 is as follows: methanol and triethylamine are added to A000002, the temperature is maintained at 25-35°C, zinc acetate dihydrate is added and stirred for 1-2 hours, di-tert-butyl dicarbonate solution and methanol are added dropwise to the reaction solution, after the addition is complete, the temperature is kept for reaction for 2-3 hours, ammonia water is added to the reaction solution, the temperature is maintained at 20-30°C, stirring is performed for more than 1 hour, and A000003 is obtained by extraction and concentration.

6. A process for preparing prazomicin according to claim 1, characterized in that: The process of preparing intermediate A000003 from intermediate A000004 is as follows: at 25-35°C, purified water, Boc-(S)-HABA and HOBt monohydrate are added to A000003, the pH is adjusted to 4.5-5.5, EDAC is added, and the reaction is kept warm for 3-4 hours. After the reaction is completed, the pH is adjusted to 8-10.0, the organic phase is compressed, acetonitrile is added to the residue to control the reaction temperature not to exceed -5°C, and the reaction is kept warm and stirred for 10-12 hours to obtain intermediate A000004.

7. A process for preparing prazomicin according to claim 1, characterized in that: The process of preparing intermediate A000004 from intermediate A000005 is as follows: add intermediate A000004 and alcohol into a reaction kettle, heat to 40-50°C, dropwise add di-tert-butyl dicarbonate solution, and react at this temperature for 4-5 hours to obtain intermediate A000005.

8. A process for preparing prazomicin according to claim 1, characterized in that: The process of preparing intermediate A000005 from intermediate A000006 is as follows: cooling the sodium hydroxide aqueous solution to no higher than 0°C, adding sodium dithionite and stirring for 10-30 minutes, dropping A000005 and heating to no lower than 30°C and reacting for 5 hours, concentrating, extracting and re-concentrating after the reaction, adding purified water and dichloromethane to the residue at 30-40°C, during which the temperature of the reaction liquid must be kept no lower than 25°C, and after the addition is completed, cooling to no higher than -5°C, and continuing to stir for 12-18 hours to obtain intermediate A000006.

9. A process for preparing prazomicin according to claim 1, characterized in that: The process of preparing intermediate A000007 from intermediate A000006 is as follows: add intermediate A000006 and acetonitrile into a reactor, heat the reaction solution to 30-40°C, add acetone, sodium bicarbonate and 2-iodoethanol, and then keep the temperature for reaction for 4-6 hours. After the reaction is completed, cool the temperature to no higher than 20°C, add DABCO to the reaction solution and continue to keep the temperature for reaction for 16-20 hours. After complete quenching, add isopropyl acetate to the concentrated solution obtained by extraction and concentration, continue to heat to 80-100°C for complete dissolution, cool again to no higher than 0°C, keep the temperature for stirring for 8-10 hours to obtain intermediate A000007.

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