1-N-ethyl gentamicin C1a sulfate intermediate and preparation method thereof

Abstract

The invention discloses a 1-N-ethyl gentamicin C1a sulfate intermediate 3, 2 ', 6'-tri-N-t-butyloxycarboryl gentamicin C1a and a preparation method thereof.The preparation method comprises the steps that gentamicin C1a and zinc acetate are subjected to a coordination reaction in methyl alcohol, and a gentamicin C1a-Zn complex is generated; reacting the gentamicin C1a-Zn complex with di-tert-butyl dicarbonate ester under the catalysis of potassium carbonate; dissolving the obtained concentrated solution in an organic solvent, adding water, shaking, standing for layering, taking an organic phase, adding an inert solvent, separating out a product solid, performing suction filtration, and drying to obtain the 3, 2 ', 6'-tri-N-t-butyloxycarboryl gentamicin C1a. The 3, 2 ', 6'-tri-N-t-butyloxycarboryl gentamicin C1a is mild in preparation condition, reaction is performed at room temperature, reaction selectivity is high, purification operation is convenient, the product yield is 98% or above, and the purity is 99% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular to a 1-N-ethyl gentamicin C1a sulfate intermediate 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a and a preparation method thereof. BACKGROUND

[0002] 1-N-ethyl gentamicin C1a sulfate is a semi-synthetic aminoglycoside antibiotic, which has good antibacterial effect on most gram-positive bacteria and gram-negative bacteria, especially on Escherichia coli, Klebsiella pneumoniae, Serratia, Salmonella and Portacoccus, and has good antibacterial activity on some gentamicin, small nystatin and cefazolin-resistant Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and has certain antibacterial activity on some penicillinase-producing Staphylococcus and some low-level methicillin-resistant Staphylococcus.

[0003] As for the preparation method of 1-N-ethyl gentamicin C1a sulfate, the common preparation method is to use gentamicin Cla as a starting material, adopt transition metal Zn complex to protect part of the amino group in gentamicin Cla, react with acetic anhydride, and then obtain the intermediate 3,2',6'-tri-N-acetyl gentamicin Cla after post-treatment, then protect the hydroxyl group by silanization, condense with acetaldehyde, reduce with potassium borohydride, hydrolyze with sodium hydroxide, and then purify to obtain 1-N-ethyl gentamicin Cla, and then obtain 1-N-ethyl gentamicin C1a sulfate by salifying with sulfuric acid. For example, Chinese patent document CN101928312B (application number 201010132963.9).

[0004] In addition to the above route, there are some routes that directly use 3,2',6'-tri-N-acetyl gentamicin Cla as a starting material, and then obtain 1-N-ethyl gentamicin Cla by protecting the hydroxyl group by silanization, condensing with acetaldehyde, reducing with potassium borohydride, hydrolyzing with sodium hydroxide, and then purifying, and then obtain 1-N-ethyl gentamicin C1a sulfate by salifying with sulfuric acid. For example, Chinese patent document CN101928311B (application number 201010132962.4).

[0005] The prior art such as CN 101928312 B (application number 201010132963.9) is to obtain 3,2',6'-tri-N-acetyl gentamicin C1a by post-treatment of a concentrated solution of 3,2',6'-tri-N-acetyl gentamicin C1a-Zn complex in the preparation of 3,2',6'-tri-N-acetyl gentamicin C1a: the concentrated solution is fed into a chromatographic separation column, then the column is washed with purified water, and then the effective component (i.e. the optically active part) at the outlet of the chromatographic separation column is collected and placed in a rotary evaporator for vacuum concentration, and finally the concentrated solution is placed in a freeze dryer for freeze drying to obtain powdered 3,2',6'-tri-N-acetyl gentamicin C1a. That is, the preparation of 3,2',6'-tri-N-acetyl gentamicin C1a requires column chromatographic separation, concentration and freeze drying, which is complicated, produces a large amount of organic solvent waste liquid, is time-consuming, has low product yield and low purity, and affects the yield and purity of the final 1-N-ethyl gentamicin C1a sulfate synthesis. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a 1-N-ethyl gentamicin C1a sulfate intermediate 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a and a preparation method of 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a with mild reaction conditions, simple operation, high yield and high purity.

[0007] The technical solution for achieving the first object of the present application is a 1-N-ethyl gentamicin C1a sulfate intermediate, which is 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a, and the structural formula is as follows: .

[0008] The technical solution for achieving the second object of the present application is a preparation method of the 1-N-ethyl gentamicin C1a sulfate intermediate according to claim 1, comprising the following steps: ① gentamicin C1a and zinc acetate are subjected to a coordination reaction in methanol to generate gentamicin C1a-Zn complex; ② the gentamicin C1a-Zn complex is subjected to a reaction with di-tert-butyl dicarbonate under the catalysis of potassium carbonate to generate 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a-Zn complex, and the solvent methanol is removed after the reaction to obtain a concentrated solution; ③ The concentrated solution obtained in step 2 is dissolved in an organic solvent, water is added, and after shaking and standing, the organic phase is separated, an inert solvent is added, and the product solid is precipitated, and the solid is obtained by filtration and drying.

[0009] In the above step 1, the weight ratio of zinc acetate to gentamicin C1a is 1:1 to 2:1, and the weight ratio of methanol to gentamicin C1a is 7:1 to 15:1.

[0010] In the above step 2, the weight ratio of di-tert-butyl dicarbonate to gentamicin C1a is 1.5:1 to 2:1, and the weight ratio of potassium carbonate to gentamicin C1a is 0.1:1 to 0.5:1.

[0011] In the above step 2, the reaction temperature is 15°C to 35°C.

[0012] In the above step 2, the reaction time is 10 to 20 hours.

[0013] As preferred, in the above step 3, the organic solvent is dichloromethane.

[0014] In the above step 3, the inert solvent is one or a combination of one or more of diethyl ether, n-hexane, n-heptane, and methyl tert-butyl ether.

[0015] In the above step 3, the volume ratio of water to dichloromethane added is 1 to 2:1.

[0016] The technical solution for achieving the third object of the present application is the use of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a as an intermediate for 1-N-ethyl gentamicin C1a sulfate.

[0017] The present application has the following positive effects: (1) The 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a of the present application can be used as an intermediate for 1-N-ethyl gentamicin C1a sulfate, and the 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a is soluble in most organic solvents, so after dissolving it, water can be added to extract the water-soluble impurities, and column chromatography is not required. Then, the organic phase is added with an inert solvent to precipitate the solid product, and the solid product is obtained by filtration and drying. The operation is simple, and the column chromatography, concentration, and freeze-drying operations are avoided, and the solid product obtained has a purity of 99% or more.

[0018] (2) When 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a is used as an intermediate to prepare 1-N-ethyl gentamicin C1a sulfate, the silanized hydroxyl group is protected, acetaldehyde is condensed, and potassium borohydride is reduced, and then the hydrolysis in sulfuric acid and the salt formation reaction are completed at the same time, without the need for long-time (more than 20 hours) hydrolysis under the condition of sodium hydroxide and high-temperature reflux, the reaction can be hydrolyzed under acidic conditions at room temperature, the generated gas is discharged, there is no residue in the reaction system, the side reactions are greatly reduced, and the hydrolysis solution obtained has high purity, without the need for column chromatography of macroporous resin or ion exchange resin, and high-purity 1-N-ethyl gentamicin C1a sulfate product can be obtained only by crystallization.

[0019] (3) The preparation condition of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a is mild, the reaction is carried out at room temperature, the selectivity is strong, the operation is convenient, the yield of the product is more than 98%, and the purity is more than 99%. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The HPLC spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0021] Figure 2 The mass spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0022] Figure 3 The infrared spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0023] Figure 4 The H spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0024] Figure 5 The C spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0025] Figure 6 The DEPT45 spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0026] Figure 7 The DEPT90 spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0027] Figure 8 The DEPT135 spectrum of 3, 2', 6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared for Example 1.

[0028] Figure 9 HSQC spectrum of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a prepared in Example 1.

[0029] Figure 10 HSQC spectrum of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a prepared in Example 1.

[0030] Figure 11 HMBC spectrum of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a prepared in Example 1.

[0031] Figure 12 NMR H spectrum of 1-N-ethyl gentamicin C1a sulfate prepared in the application example. DETAILED DESCRIPTION

[0033] The following describes some of the multiple possible embodiments of the present application, which are intended to provide a basic understanding of the present application and are not intended to identify key or decisive elements or limit the scope of protection. It is easily understood that, according to the technical solutions of the present application, other implementation manners can be proposed by those skilled in the art without changing the essential spirit of the present application. Therefore, the following detailed description is only an exemplary description of the technical solutions of the present application, and should not be regarded as the whole or as a limitation or restriction of the technical solutions of the present application.

[0034] (Example 1) The 1-N-ethyl gentamicin C1a sulfate intermediate of this embodiment is 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a, and the structural formula is as follows: .

[0035] The preparation reaction formula of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a of this embodiment is as follows: .

[0036] The preparation method of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a of this embodiment includes the following steps: ① In a 3L reaction bottle, 700g of methanol, 100g of gentamicin C1a and 150g of zinc acetate are added, and the reaction is stirred at 15°C to 35°C (20°C in this embodiment) for 2 hours to form a gentamicin C1a-Zn complex.

[0037] (2) To the system after the reaction of step (1), 10 g of potassium carbonate and 170 g of di-tert-butyl dicarbonate were added, and the stirring reaction was continued at 15-35 °C for 10-20 h (15 h in this example) to generate 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a-Zn complex. After the reaction was completed, methanol was removed by distillation to obtain a concentrated solution.

[0038] (3) The concentrated solution obtained in step (2) was dissolved in 300 mL of dichloromethane, and 300 mL of water was added. After shaking, the layers were separated by standing.

[0039] The dichloromethane phase was taken, 600 mL of ether was added, and the product solid was precipitated. After suction filtration and drying, 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a was obtained in a yield of 164.4 g, 98.6% and a purity of 99.89%.

[0040] The HPLC spectrum of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a prepared in this example is shown in Figure 1 , the mass spectrum is shown in Figure 2 , the infrared spectrum is shown in Figure 3 , the nuclear magnetic resonance H spectrum is shown in Figure 4 , the nuclear magnetic resonance C spectrum is shown in Figure 5 , the DEPT45 spectrum is shown in Figure 6 , the DEPT90 spectrum is shown in Figure 7 , the DEPT135 spectrum is shown in Figure 8 , the COSY spectrum is shown in Figure 9 , the HSQC spectrum is shown in Figure 10 , and the HMBC spectrum is shown in Figure 11 .

[0041] (Example 2) The preparation method of 3,2',6'-tris-N-tert-butyloxycarbonyl gentamicin C1a in this example includes the following steps: (1) In a 3 L reaction bottle, 1000 g of methanol, 100 g of gentamicin C1a and 100 g of zinc acetate were added, and the stirring reaction was continued at 15 °C for 2 hours to generate gentamicin C1a-Zn complex.

[0042] (2) To the system after the reaction of step (1), 30 g of potassium carbonate and 150 g of di-tert-butyl dicarbonate were added, and the stirring reaction was continued at 15-35 °C for 18 °C. After the reaction was completed, methanol was removed by distillation under reduced pressure.

[0043] (3) The concentrated solution obtained in step (2) was dispersed in 300 mL of dichloromethane, and 500 mL of water was added. After shaking, the layers were separated by standing.

[0044] The dichloromethane phase is taken, 500 mL of n-hexane is added, the product solid is precipitated, and is extracted by filtration and dried to obtain 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a product 163.7 g, with a yield of 98.2% and a purity of 99.6%.

[0045] (Example 3) The preparation method of 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a of this example comprises the following steps: ①In a 3L reaction bottle, 1500 g of methanol, 100 g of gentamicin C1a and 200 g of zinc acetate are added, and the mixture is stirred at 30°C for 2 hours to form a gentamicin C1a-Zn complex.

[0046] ②50 g of potassium carbonate and 200 g of di-tert-butyl dicarbonate are added to the reaction system after step ①, and the mixture is continuously stirred at 15°C to 35°C for 20 hours. After the reaction is completed, the methanol is removed by distillation under reduced pressure to obtain a concentrated solution.

[0047] ③The concentrated solution obtained in step ② is dispersed in 300 mL of dichloromethane, 600 mL of water is added, and the mixture is shaken and then allowed to stand to separate into layers.

[0048] The dichloromethane phase is taken, a mixture of methyl tert-butyl ether (300 ml) and n-heptane (300 ml) is added, the product solid is precipitated, and is extracted by filtration and dried to obtain 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a product 163.9 g, with a yield of 98.3% and a purity of 99.4%.

[0049] (Example 3) The 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared by the method of the present application is used to prepare 1-N-ethyl gentamicin C1a sulfate, which comprises the following steps: S1, 650 g of trichloromethane is added to a dry 2L flask, 130 g of 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a prepared in Example 1 is added, and the mixture is stirred to dissolve, 200 g of hexamethyldisilazane, 2.6 g of concentrated sulfuric acid are added, and the mixture is heated to reflux for 6 hours, and then the trichloromethane is concentrated and evaporated by rotary evaporation.

[0050] S2, the product obtained in S1 is dissolved in 650 g of dichloromethane, cooled to below 10°C, 1.3 g of concentrated sulfuric acid is added, 39 g of acetaldehyde is added, and the mixture is reacted at a temperature of 0°C to 10°C for 0.5 hours, then 13 g of potassium borohydride is added for reduction reaction for 0.5 hours, then 260 g of boric acid buffer is added, and the mixture is reacted for 1 hour. After the reaction is completed, the aqueous phase is removed by separation, and the organic phase is distilled to remove dichloromethane.

[0051] S3, to the system obtained after removing dichloromethane in step 5, 200 mL of 4M sulfuric acid solution was added, and hydrolysis reaction was carried out at room temperature (15-35 °C) for 2 hours. After the reaction was completed, 500 mL of anhydrous ethanol was slowly added dropwise, and white solid was precipitated. After the dropwise addition was completed, stirring was continued for 3 hours. Filtration and drying gave 99.4 g of solid, which was 1-N-ethyl gentamicin C1a.

[0052] The H-NMR spectrum of the product prepared is shown in Figure 12 , which confirms that it is 1-N-ethyl gentamicin C1a.

[0053] As can be seen from the above preparation process, using 3,2',6'-tri-N-tert-butyloxycarbonyl gentamicin C1a as an intermediate to prepare 1-N-ethyl gentamicin C1a sulfate, hydrolysis does not need to be carried out under sodium hydroxide conditions for a long time at high temperature, but can be carried out under acidic conditions at room temperature, greatly reducing the side reactions, the obtained hydrolysis solution has high purity, and does not need to be subjected to column chromatography of macroporous resin or ion exchange resin, but only needs to be crystallized to obtain high-purity 1-N-ethyl gentamicin C1a sulfate product, thus simplifying the preparation process of 1-N-ethyl gentamicin C1a sulfate.

Claims

1. A 1-N-ethylgentamicin C1a sulfate intermediate, characterized in that... The intermediate is 3,2',6'-tris-N-tert-butoxycarbonylgentamicin C1a, with the following structural formula: 。 2. A method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate as described in claim 1, characterized in that... Includes the following steps: ① Gentamicin C1a and zinc acetate undergo a coordination reaction in methanol to form a gentamicin C1a-Zn complex; ② The gentamicin C1a-Zn complex reacts with ditert-butyl dicarbonate under potassium carbonate catalysis to generate 3,2',6'-tris-N-tert-butyloxycarbonylgentamicin C1a-Zn complex. After the reaction is complete, methanol is removed to obtain a concentrated solution. ③ Dissolve the concentrated solution obtained in step ② in an organic solvent, add water, shake and let stand to separate the layers, take the organic phase, add an inert solvent, the product solid precipitates, filter and dry to obtain 3,2',6'-tris-N-tert-butoxycarbonylgentamicin C1a.

3. The method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate according to claim 2, characterized in that: In step ①, the weight ratio of zinc acetate to gentamicin C1a is 1:1 to 2:1; the weight ratio of methanol to gentamicin C1a is 7:1 to 15:

1.

4. The method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate according to claim 2, characterized in that: In step ②, the weight ratio of ditert-butyl dicarbonate to gentamicin C1a is 1.5:1 to 2:1; the weight ratio of potassium carbonate to gentamicin C1a is 0.1:1 to 0.5:

1.

5. The method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate according to claim 2, characterized in that: In step ②, the reaction temperature is 15℃~35℃.

6. The method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate according to claim 5, characterized in that: In step ②, the reaction time is 10–20 h.

7. The method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate according to claim 2, characterized in that: In step ③, the organic solvent is dichloromethane.

8. The method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate according to claim 7, characterized in that: In step ③, the inert solvent is one or a combination of more than one of diethyl ether, n-hexane, n-heptane, and methyl tert-butyl ether.

9. The method for preparing the 1-N-ethylgentamicin C1a sulfate intermediate according to claim 7, characterized in that: The volume ratio of water to dichloromethane added is 1 to 2:

1. Application of 10.3,2',6'-tri-N-tert-butoxycarbonyl gentamicin C1a as an intermediate for 1-N-ethylgentamicin C1a sulfate.

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