A method for preparing cefprozil by enzymatic method

Abstract

The present invention discloses a method for preparing cefprozil by an enzymatic method, comprising the following steps: (1) enzyme conversion; (2) separation and preparation of a DMF complex; (3) refining; and (4) recovery. The present invention shortens the reaction time and improves the conversion rate by increasing the substrate concentration of the enzyme conversion and adding seed crystals and sodium pyrosulfite during the conversion process. After the conversion is completed and separated, a DMF complex and refined crystals are prepared and the crystallization mother liquor is recovered, thereby improving the total yield and ensuring the product quality. The enzymatic process is simple and easy to operate, with a high product yield, good purity, low production cost, and is convenient for large-scale green chemical production. Compared with the chemical method, it has significant yield, quality and environmental protection advantages.

Description

Technical Field

[0001] The present invention relates to a method for preparing a medicine, in particular to a method for preparing cefprozil by an enzymatic method. Background Art

[0002] Cefprozil is a second-generation cephalosporin drug first developed by Bristol-Myers Squibb. It is the first oral cephalosporin antibiotic approved by the U.S. FDA for the treatment of otitis media and sinusitis in children. It is highly effective, low toxic, enzyme-resistant, and has a broad antibacterial spectrum, and is widely used in the clinical treatment of infants and young children.

[0003] At present, the synthesis method of cefprozil mainly includes chemical method and biological enzyme method. The reaction process of chemical synthesis of cefprozil is cumbersome, the conditions are harsh, the cost is high, the environmental pollution is serious, and the total yield is only about 130%. The enzymatic synthesis is simple to operate, reacts in pure water, the conditions are mild, the cost is low, and it is green and environmentally friendly, so it is increasingly concerned by pharmaceutical companies and drug researchers. However, there are still a series of problems in the existing enzymatic synthesis of cefprozil, such as the substrate parent nucleus 7-APRA concentration is low in the enzymatic synthesis of cefprozil disclosed in patent CN104928340A, and the conversion efficiency is not high. After the reaction is completed, the immobilized enzyme is separated by a sieve, the reaction solution is dissolved with acid, and then directly adjusted to alkaline crystallization and washed and dried to obtain the finished product. The finished product has more residual impurities and lower purity, and the residual amount of cefprozil in the crystallization mother liquor is high, resulting in a low yield. In the process of enzymatic synthesis of cefprozil disclosed in patent CN1045368910A, the mass volume ratio of the nucleus to water is 1:9-15, and the substrate concentration is also low. During the separation and purification process, 2.5-4 times of N, N-dimethylformamide (DMF) is added to prepare the complex, and cefprozil is obtained by recrystallization. The DMF consumption is large and the residual propylene in the crystallization mother liquor is large, resulting in a loss of yield. In short, the existing enzymatic synthesis of cefprozil technology fails to solve the problems of low yield and high cost. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for preparing cefprozil by enzymatic method, comprising a complete set of preparation processes such as enzyme reaction conversion synthesis of cefprozil, separation and preparation of DMF complex, refining and recovery, etc. The preparation process is safe and environmentally friendly, and the obtained finished product has high yield, low cost, good purity and light appearance color.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A method for preparing cefprozil by an enzymatic method, wherein cefprozil seed crystals and sodium pyrosulfite are added during the enzymatic conversion process, the reaction pH is controlled to be 6.0-7.0 (for example, 6.1, 6.3, 6.5, 6.7, 6.9), and the reaction temperature is 12-22° C. (for example, 14° C., 16° C., 18° C., 20° C.);

[0007] Preferably, the reaction pH is 6.0-6.8, and the reaction temperature is 14-20°C;

[0008] More preferably, the reaction pH is 6.3-6.6 and the reaction temperature is 16-18°C.

[0009] Preferably, the amount of sodium metabisulfite added is 0.1-0.2% (w / v) (for example, 0.12%, 0.14%, 0.16%, 0.18%);

[0010] More preferably, the addition amount is 0.15-0.2% (w / v);

[0011] More preferably, the added amount is 0.18-0.2% (w / v).

[0012] Preferably, the amount of cefprozil seed crystals added is 0.05-0.1% (w / v) (for example, 0.06%, 0.07%, 0.08%, 0.09%);

[0013] More preferably, 0.06-0.08% (w / v).

[0014] Preferably, in the enzymatic conversion process, cefprozil nucleus 7-APRA (7-amino-3-propenyl-4-cephalosporanic acid) and D-p-hydroxyphenylglycine methyl ester are added, mixed and dissolved in water, immobilized penicillin acylase is added for catalytic reaction, and then cefprozil seed crystals and sodium pyrosulfite are added. When the conversion rate of cefprozil nucleus 7-APRA is greater than 98%, the reaction is stopped, and the reaction time is 2-5 hours.

[0015] The mother nucleus 7-APRA conversion rate = (the mass of the mother nucleus 7-APRA input before the reaction - the mass of the mother nucleus 7-APRA remaining in the solution after the reaction / the mass of the mother nucleus 7-APRA input before the reaction);

[0016] Preferably, the molar ratio of the sporophyll nucleus 7-APRA to D-p-hydroxyphenylglycine methyl ester is 1:(1.1-1.2).

[0017] Preferably, the amount of water added is 4-6 mL of water per 1 g of cefprozil core 7-APRA.

[0018] Preferably, the method for preparing cefprozil further comprises the steps of separating and preparing DMF (N,N-dimethylformamide) complex, refining and recovering.

[0019] Preferably, the separation and preparation of the DMF complex comprises the following steps: separating the immobilized enzyme from the coarse powder and the clear liquid with a sieve, washing the immobilized enzyme with deionized water and then reusing it, adding the coarse powder to the clear liquid and the enzyme washing water, adjusting it with hydrochloric acid until it is dissolved, adding DMF, decolorizing with activated carbon and then filtering, adding ammonia water to crystallize and prepare the DMF complex, and vacuum drying;

[0020] The immobilized enzyme is washed with deionized water having a mass of 1.5 to 2.5 times that of the enzyme;

[0021] After the clear liquid, coarse powder and enzyme washing water are combined and dissolved, DMF is added in an amount of 1-2 times the volume of the combined liquid.

[0022] Preferably, the refining comprises the following steps: adding deionized water to the DMF complex, adjusting the pH to 0.5-1.0 with hydrochloric acid until it is completely dissolved, adding activated carbon, stirring to decolorize, and then filtering, collecting the clear liquid, rinsing the filter cake once with a small amount of deionized water, combining it with the clear liquid, rinsing it once with a small amount of deionized water, collecting and recycling it for later use; adjusting the pH of the combined clear liquid to 5.0-6.5 with 10% ammonia water, cooling it to below 5°C, growing crystals for 1 hour, filtering it with suction, washing it with deionized water, collecting the mother liquor and the washing liquid for later use; then rinsing it with acetone, draining it, and vacuum drying the filter cake at 40-45°C;

[0023] The amount of water added during the refining process is 1.0-2.0 times (for example, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times) of the DMF complex;

[0024] The recovery comprises the following steps: collecting activated carbon washing water from the refining process and mother liquor and water washing liquid after refined crystallization, adding DMF, controlling the pH to 5.0-6.0, cooling to below 10° C. and growing crystals for 30-90 minutes (for example, 35 minutes, 40 minutes, 50 minutes, 55 minutes, 65 minutes, 70 minutes, 75 minutes, 85 minutes), suction filtering and washing with a small amount of DMF and acetone, vacuum drying the filter cake at 40-45° C. to obtain a DMF complex, and recycling it to the next batch for refined crystallization;

[0025] The amount of DMF added during the recovery process is 1.4-2.0 times (for example, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times) the volume of the recovered combined liquid.

[0026] The point values ​​in the data range described in the present invention can all be selected and will not be described in detail here.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The substrate concentration during the enzyme reaction conversion process is more than doubled, and the parent core and side chains do not need to be dissolved. Water and enzyme are directly added to carry out the conversion reaction. At the same time, a small amount of sodium pyrosulfite is added to reduce losses and ensure the color grade of the product. Seed crystals are added during the conversion process to shorten the reaction time and improve the conversion rate.

[0029] (2) During the separation and purification process, the types and amounts of solvents are reduced, and the amount of DMF used is reduced by half compared with the existing technology, which reduces the preparation cost and safety and environmental protection pressure.

[0030] (3) A method for recovering cefuroxime has been added, and the preparation method is simple and easy. Compared with the chemical method and the existing enzymatic method, the product quality is ensured, while the total yield of the product is increased by nearly 10 percentage points, significantly reducing the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The HPLC spectrum of Example 1 after the conversion is completed and the national standard HPLC spectrum of the finished product.

[0032] Figure 2 The HPLC spectrum of the finished product of Example 1 is as shown in the national standard method.

[0033] Figure 3 The HPLC spectrum of Example 2 after the conversion is completed and the national standard HPLC spectrum of the finished product.

[0034] Figure 4 The HPLC spectrum of the finished product of Example 2 is as shown in the national standard method.

[0035] Figure 5 The HPLC spectrum of Example 3 after the conversion is completed and the national standard HPLC spectrum of the finished product.

[0036] Figure 6 The HPLC spectrum of the finished product of Example 3 is as shown in the national standard method.

[0037] Figure 7 The HPLC spectrum of Example 4 after the conversion is completed and the national standard HPLC spectrum of the finished product.

[0038] Figure 8 The HPLC spectrum of the finished product of Example 4 is as shown in the figure. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the examples, which are only typical descriptions of the present invention, but the present invention is not limited thereto. The test methods used in the following examples are conventional methods unless otherwise specified, and the raw materials, reagents, etc. used are raw materials and reagents that can be obtained from conventional commercial sources such as commercial purchases unless otherwise specified.

[0040] The conversion rate and total weight yield calculation formulas involved in the implementation method are as follows:

[0041]

[0042] Total weight yield = 100% * finished product mass / mother core mass

[0043] Example 1

[0044] (1) Enzymatic reaction conversion: In a 500 mL three-necked flask, add 40 g 7-APRA and 33.2 g D-p-hydroxyphenylglycine methyl ester, then add 170 mL deionized water, stir and mix, add 52 g immobilized penicillin acylase, then add 0.15 g seed crystals and 0.3 g sodium pyrosulfite, use 6 mol / L hydrochloric acid and 3 mol / L ammonia water to control the reaction pH to 6.0-6.3, the reaction temperature to 12-15°C, and HPLC tracking to detect the residual 7-APRA. After reacting for 4 hours, the 7-APRA conversion rate was 98.1%. Stop the reaction, and the HPLC spectrum is as follows: Figure 1 shown.

[0045] (2) Separation and purification: After the reaction, the immobilized enzyme and the reaction mixture were separated with a 100-mesh screen, and the crude powder was collected by suction filtration. The clear liquid after suction filtration was used to repeatedly wash the immobilized enzyme four times until the clear liquid was clear, and then the immobilized enzyme was washed with 100 mL of deionized water, combined with the clear liquid, and added to a 1000 mL three-necked flask, and then the crude powder was added, and 6 mol / L hydrochloric acid was used to dissolve it. The total volume was 310 mL, and 500 mL of DMF was slowly added. 2 g of activated carbon was added and stirred for decolorization for 30 minutes. After suction filtration, the clear liquid was collected, and the filter cake was rinsed once with 30 mL of deionized water and combined with the clear liquid. The pH was adjusted to 5.8-6.0 with ammonia water for crystallization, and the temperature was lowered to below 10°C. The crystals were grown for 1 hour, and suction filtration was performed, and the immobilized enzyme was washed with 100 mL of DMF and 100 mL of acetone respectively. After being drained, the filter cake was vacuum dried at 40-45°C for 2 hours to obtain 87 g of DMF complex.

[0046] (3) Refining: Add the DMF complex to a 500mL three-necked flask, add 110mL of deionized water, and adjust the pH to 0.5-1.0 with 6mol / L hydrochloric acid until it is completely dissolved. Add 1.2g of activated carbon and stir to decolorize for 30 minutes, then filter and collect the clear liquid. Rinse the filter cake once with 50mL of deionized water, combine it with the clear liquid, and then rinse it once with 50mL of deionized water, collect and recycle it for later use. Adjust the pH of the combined clear liquid to 5.8-6.0 with 10% ammonia water, cool it to below 5°C, grow the crystals for 1 hour, filter it, wash it with 50mL of deionized water, collect the mother liquor and 225mL of the washing liquid by suction and recycle it for later use. Rinse it with 80mL of acetone and drain it. The filter cake was vacuum dried at 40-45°C for 2 hours to obtain 54.4g of ceftriaxone fine product. The HPLC spectrum is as follows: Figure 2As shown, the purity is 99.74% and the total weight yield is 136.0%.

[0047] (4) Recovery: 50 mL of activated carbon washing water from the refining process and 225 mL of the mother liquor and water washing liquid after refining, totaling 275 mL, were collected and added to a 1000 mL three-necked flask, and then 470 mL of DMF was added. The pH was controlled to 5.8-6.0, and the temperature was lowered to below 10° C. to grow crystals for 30 minutes. Then, the mixture was filtered and washed with a small amount of DMF and acetone. The filter cake was vacuum dried at 40-45° C. for 2 hours to obtain 7.5 g of cefprozil DMF complex, which was recovered and used in Example 2 for refining and crystallization.

[0048] Example 2

[0049] Enzyme reaction conversion: In a 500mL three-necked flask, add 40g 7-APRA and 34.7g D-p-hydroxyphenylglycine methyl ester, then add 200mL deionized water, stir and mix, add 56g immobilized penicillin acylase, then add 0.23g seed crystals, 0.45g sodium pyrosulfite, use 6mol / L hydrochloric acid and 3mol / L ammonia water to control the reaction pH to 6.3-6.6, the reaction temperature is 16-18°C, the enzyme reaction conversion is 4 hours, and the 7-APRA conversion rate is 98.9%. After separation and purification, 86.6g of DMF complex is obtained, and 8g of DMF complex recovered in Example 1 is added, 120mL deionized water is added for refining, and 235mL of mother liquor and water washing liquid are recovered for standby use, and 58.2g of cefprozil fine product is obtained by vacuum drying, and the HPLC spectrum is as follows: Figure 4 As shown, the purity is 99.76% and the total weight yield is 145.5%.

[0050] Collect and combine 50 mL of activated carbon washing water from the refining process and 235 mL of the mother liquor and water washing liquid after refining, totaling 285 mL, add 480 mL of DMF, control the pH to 5.8-6.0, cool to below 10°C to grow crystals for 60 minutes, then filter and wash with a small amount of DMF and acetone, and vacuum dry the filter cake at 40-45°C for 2 hours to obtain 8.0 g of cefprozil DMF complex after crystallization and drying.

[0051] Figure 3 The HPLC spectrum of Example 2 after the conversion is completed and the national standard HPLC spectrum of the finished product.

[0052] Example 3

[0053] (1) Enzymatic reaction conversion: In a 500 mL three-necked flask, add 40 g 7-APRA and 36.2 g D-p-hydroxyphenylglycine methyl ester, then add 220 mL deionized water, stir and mix, add 60 g immobilized penicillin acylase, then add 0.3 g seed crystals and 0.6 g sodium pyrosulfite, use 6 mol / L hydrochloric acid and 3 mol / L ammonia water to control the reaction pH to 6.7-7.0, the reaction temperature to 20-22°C, HPLC tracking detection of 7-APRA residue, react for 4 hours, the 7-APRA conversion rate is 98.5%, stop the reaction, the HPLC spectrum is as follows: Figure 5 shown.

[0054] (2) Separation and purification: After the reaction, the immobilized enzyme and the reaction mixture were separated with a 100-mesh screen, and the crude powder was collected by suction filtration. The immobilized enzyme was repeatedly washed with the filtered clear liquid four times until the clear liquid was clear, and then the immobilized enzyme was washed with 70 mL of deionized water, combined with the clear liquid, added to a 1000 mL three-necked flask, and the crude powder was added, and 6 mol / L hydrochloric acid was used to dissolve it. The total volume was 340 mL, and 500 mL of DMF was slowly added. 2 g of activated carbon was added and stirred for decolorization for 30 minutes. After suction filtration, the clear liquid was collected, and the filter cake was rinsed with 30 mL of deionized water and combined with the clear liquid. The pH was adjusted to 5.8-6.0 with ammonia water for crystallization, and the temperature was lowered to below 10°C. The crystals were grown for 1 hour, and then filtered, and washed with 100 mL of DMF and 100 mL of acetone respectively. After drying, the filter cake was vacuum dried at 40-45°C for 2 hours to obtain 86.8 g of DMF complex.

[0055] (3) Refining: Add the DMF complex to a 500mL three-necked flask, add 110mL of deionized water, and adjust the pH to 0.5-1.0 with 6mol / L hydrochloric acid until it is completely dissolved. Add 1.2g of activated carbon and stir to decolorize for 30 minutes, then filter and collect the clear liquid. Rinse the filter cake once with 50mL of deionized water, combine it with the clear liquid, and then rinse it once with 50mL of deionized water, collect and recycle it for later use. Adjust the pH of the combined clear liquid to 5.8-6.0 with 10% ammonia water, cool it to below 5°C, grow the crystals for 1 hour, filter it, wash it with 50mL of deionized water, collect the mother liquor and 225mL of the washing liquid by suction and recycle it for later use. Rinse it with 80mL of acetone and drain it. The filter cake was vacuum dried at 40-45°C for 2 hours to obtain 54.1g of ceftriaxone fine product. The HPLC spectrum is as follows: Figure 6 As shown, the purity is 99.76% and the total weight yield is 135.2%.

[0056] (4) Recovery: 50 mL of activated carbon washing water from the refining process and 230 mL of the mother liquor and water washing liquid after refining, totaling 280 mL, were collected and added to a 1000 mL three-necked flask, and then 500 mL of DMF was added. The pH was controlled at 5.8-6.0, and the temperature was lowered to below 10° C. to grow crystals for 90 minutes. Then, the mixture was filtered and washed with a small amount of DMF and acetone. The filter cake was vacuum dried at 40-45° C. for 2 hours to obtain 7.8 g of cefprozil DMF complex, which was recovered and used in Example 2 for refining and crystallization.

[0057] Figure 5 The HPLC spectrum of Example 3 after the conversion is completed and the national standard HPLC spectrum of the finished product.

[0058] Example 4

[0059] The operation steps of Example 4 are the same as those of Example 3, and the DMF complex recovered in Example 3 is added during the refining process. The enzyme reaction is converted for 4 hours, and the 7-APRA conversion rate is 98.4%. After separation and purification, 86.9 g of DMF complex is obtained, and 7.8 g of DMF complex recovered in Example 3 is added, and 120 mL of deionized water is added for refining, and 240 mL of mother liquor and water washing liquid are recovered for standby use, and 58.1 g of cefprozil fine product is obtained by vacuum drying. The HPLC spectrum is as follows: Figure 8 As shown, the purity is 99.74% and the total weight yield is 145.2%.

[0060] Collect and combine 50 mL of activated carbon washing water from the refining process, 240 mL of the mother liquor after refining and the water washing liquid, a total of 290 mL, add 480 mL of DMF, and after crystallization and drying, obtain 7.7 g of cefprozil DMF complex.

[0061] Figure 7 The HPLC spectrum of Example 4 after the conversion is completed and the national standard HPLC spectrum of the finished product.

[0062] Comparative Example 1

[0063] Except that sodium pyrosulfite was not added during the enzymatic conversion process, the other steps of Comparative Example 1 were the same as those of Example 1.

[0064] Comparative Example 2

[0065] Except that the amount of sodium metabisulfite added during the enzyme conversion process was 0.3%, the other steps of Comparative Example 2 were the same as those of Example 1.

[0066] Comparative Example 3

[0067] Except that no seed crystals were added during the enzymatic conversion process, the other steps of Comparative Example 3 were the same as those of Example 1.

[0068] Comparative Example 4

[0069] Except that the amount of seed crystal added during the enzyme conversion process was 0.15%, the other steps of Comparative Example 4 were the same as those of Example 1.

[0070] Comparative Example 5

[0071] In this comparative example, cefprozil was prepared by the method of Example 1 of patent CN105368910A, 40 g of cefprozil mother nucleus was used for reaction, and other amounts were increased in the same proportion.

[0072] The product color, conversion rate, total weight yield, purity, and DMF dosage of Examples 1-4 and Comparative Examples 1-5 were compared, and the results are shown in Table 1. It can be seen that the present invention reduces losses and ensures product color grade by adding a small amount of sodium pyrosulfite; adding seed crystals during the conversion process shortens the reaction time and improves the conversion rate; the types and dosages of solvents are reduced, and the amount of DMF is reduced by nearly half compared with the prior art, reducing the preparation cost and safety and environmental protection pressure. The total product yield is increased by nearly 10 percentage points, significantly reducing the production cost.

[0073] Table 1 Comparison of the products of the embodiments of the present invention and the comparative examples

[0074]

[0075]

[0076] The cefprozil products obtained in Examples 1-4 were tested according to the national standard test method, and the results are shown in Table 2 below:

[0077] Table 2 Test results of products of Examples 1-4

[0078]

[0079]

Claims

1. A method for preparing cefprozil by enzymatic method, characterized in that: During the enzymatic conversion process, cefprozil seed crystals and sodium metabisulfite were added to control the reaction pH to 6.0-7.0 and the reaction temperature to 12-22°C.

2. The method for preparing cefprozil according to claim 1, characterized in that: The amount of sodium pyrosulfite added is 0.1-0.2% (w / v); Preferably, the addition amount is 0.15-0.2% (w / v); Preferably, the added amount is 0.18-0.2% (w / v).

3. The method for preparing cefprozil according to claim 1, characterized in that: The reaction pH is 5.5-7.2, and the reaction temperature is 20-25°C; Preferably, the reaction pH is 6.8-7.0 and the reaction temperature is 23-25°C.

4. The method for preparing cefprozil according to claim 1, characterized in that: The amount of cefprozil seed crystals added is 0.05-0.1% (w / v), preferably 0.06-0.08% (w / v).

5. The method for preparing cefprozil according to claim 1, characterized in that: In the enzyme conversion process, cefprozil mother nucleus 7-APRA (7-amino-3-propenyl-4-cephalosporanic acid) and D-p-hydroxyphenylglycine methyl ester are added, mixed and dissolved in water, immobilized penicillin acylase is added for catalytic reaction, and then cefprozil seed crystals and sodium pyrosulfite are added. When the conversion rate of cefprozil mother nucleus 7-APRA is greater than 98%, the reaction is stopped.

6. The method for preparing cefprozil according to claim 5, characterized in that: The molar ratio of the sporophyll nucleus 7-APRA to D-p-hydroxyphenylglycine methyl ester is 1:(1.0-1.2).

7. The method for preparing cefprozil according to claim 5, characterized in that: The amount of water added is 4-6 mL of water per 1 g of cefprozil core 7-APRA.

8. The method for preparing cefprozil according to claim 1, characterized in that: The method also includes the steps of separation and preparation of DMF (N, N-dimethylformamide) complex, purification and recovery.

9. The method for preparing cefprozil according to claim 8, characterized in that: The separation and preparation of the DMF complex comprises the following steps: using a sieve to separate the immobilized enzyme from the coarse powder and the clear liquid, and then washing the immobilized enzyme with deionized water and then reusing it; The immobilized enzyme is washed with deionized water having a mass of 1.5 to 2.5 times that of the enzyme; After the clear liquid, coarse powder and enzyme washing water are combined and dissolved, DMF is added in an amount of 1-2 times the volume of the combined liquid.

10. The method for preparing cefprozil according to claim 8, characterized in that: The refining comprises the following steps: adding deionized water to the DMF complex, adjusting the pH to 0.5-1.0 with hydrochloric acid until the complex is completely dissolved, adding activated carbon, stirring to decolorize, and then filtering to collect the clear liquid, washing the filter cake once with a small amount of deionized water, combining it with the clear liquid, and then washing it once with a small amount of deionized water, collecting and recycling it for later use; The recovery comprises the following steps: collecting activated carbon washing water from the refining process and mother liquor and water washing liquid after refined crystallization, adding DMF, controlling the pH to 5.0-6.0, cooling to below 10°C to grow crystals for 30-90 minutes, filtering and washing with a small amount of DMF and acetone, vacuum drying the filter cake at 40-45°C to obtain a DMF complex, and recycling it to the next batch for refined crystallization.

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