Method and intermediate for preparing tulathromycin

Abstract

A method and an intermediate for preparing a tulathromycin. The method includes the following step: in an organic solvent, subjecting a compound represented by formula (II) and an n-propylamine to a ring-opening addition shown below to obtain a tulathromycin represented by formula (I), wherein the organic solvent is a 1,2-propandiol. Tulathromycin obtained using the method has a high purity, with an HPLC purity being 95% and above, and up to 99% and above, satisfying a required purity for preparing a tulathromycin as a pharmaceutical formulation. The method has a high yield, is simple to operate, and is more suitable for industrial production. ##STR00001##

Claims

1. A method for preparing tulathromycin represented by formula I, comprising the following steps: subjecting a compound represented by formula II and n-propylamine to ring-opening addition reaction shown below in an organic solvent to produce tulathromycin represented by formula I; wherein the organic solvent is 1,2-propandiol; ##STR00015##

2. The method according to claim 1, wherein the mole ratio of the compound represented by formula II to n-propylamine is from 1:5 to 1:30; the temperature of the ring-opening addition reaction is between 30 and 90 C.; the duration of the ring-opening addition reaction is from 15 to 40 hours; the method for preparing tulathromycin represented by formula I comprises the following steps: mixing a mixed solution of the compound represented by formula II and the organic solvent with n-propylamine, and performing the ring-opening addition reaction; the ring-opening addition reaction is performed in the condition of gas protection; the gas for the gas protection is nitrogen.

3. The method according to claim 1, which further comprises a post-processing treatment after the completion of the ring-opening addition reaction; the post-processing treatment comprises the following steps: removing n-propylamine and the organic solvent after the completion of the ring-opening addition reaction to obtain crude tulathromycin represented by formula I; and performing recrystallization to obtain tulathromycin represented by formula I.

4. The method according to claim 3, wherein method of the removing n-propylamine and the organic solvent in the post-processing treatment is concentration under reduced pressure; and/or, the recrystallization comprises the following steps: mixing the crude tulathromycin represented by formula I with a solvent A, then adding an anti-solvent, crystallizing to obtain a product of tulathromycin represented by formula I; wherein, the solvent A is a mixed solvent of acetone and C.sub.1-3 alcohol; and the anti-solvent is water or a mixed solvent of water and acetone.

5. The method according to claim 4, wherein the C.sub.1-3 alcohol in recrystallization is 1,2-propandiol; and/or, the volume to mass ratio of the solvent A to the crude tulathromycin represented by formula I is from 2 mL/g to 50 mL/g; and/or, the adding of the anti-solvent is dropwise adding; and/or, the temperature of adding the anti-solvent is between 35 and 45 C.; and/or, the volume to mass ratio of the anti-solvent to the crude tulathromycin represented by formula I is from 2 mL/g to 60 mL/g; and/or, when the anti-solvent is the mixed solvent of water and acetone, the volume ratio of the water to acetone is from 0.5:1 to 3:1.

6. The method according to claim 4, wherein during adding the anti-solvent in the recrystallization, the adding is finished until the solution A containing tulathromycin represented by formula I turns turbid, the resulting mixture is stirred and crystalline grain culturing is carried out for 10 to 30 minutes, and the remaining anti-solvent is added; the temperature of the crystallization is between 0 and 45 C.; and the duration of the crystallization is from 1 to 6 hours.

7. The method according to claim 4, wherein during the recrystallization, the crystallization comprises an early stage of crystallization and a late stage of crystallization, wherein, the early stage of crystallization is carried out at a temperature between 35 and 45 C. for 0.5 to 3 hours, and the late stage of crystallization is carried out at a temperature between 0 and 35 C. for 0.5 to 3 hours.

8. The method according to claim 1, wherein the method for preparing the compound represented by formula I further comprises the following method A or method B: the method A comprises the following steps: with the action of a catalyst and a hydrogen source, subjecting a compound represented by formula Ill to de-protection reaction shown below in the organic solvent to produce the compound represented by formula II; ##STR00016## the method B comprises the following steps: in a mixed solvent of water and a non-polar organic solvent, subjecting a salt IIa of the compound represented by formula II and a base to acid-base neutralization reaction to produce the compound represented by formula II; ##STR00017## and wherein in the compound IIa, X is an organic acid or an inorganic acid, and n is 1, 2 or 3.

9. The method according to claim 8, wherein in the method A, the organic solvent is a C.sub.1-3 alcohol and/or a ketone; the C.sub.1-3 alcohol is selected from the group consisting of methanol, ethanol and isopropanol, or a mixture thereof; the ketone is acetone; the catalyst is palladium-carbon; the mass percentage of palladium in the palladium-carbon is from 3 to 20%; the percentage refers to the percentage of the mass of palladium in the total mass of the palladium-carbon; the hydrogen source is ammonium formate; the amount of the catalyst is 5% to 15% of the mass of the compound represented by formula III; the amount of the hydrogen source is more than 1 fold of the mole quantity of the compound represented by formula II; the temperature of the de-protection reaction is between 20 and 25 C.; the duration of the de-protection reaction is from 1 to 6 hours; the method for preparing the compound represented by formula II comprises the following steps: mixing the mixed solution of the compound represented by formula III and the organic solvent with the hydrogen source and the catalyst, performing the de-protection reaction; the de-protection reaction is performed in the condition of gas protection; the gas for the gas protection is nitrogen; and/or, in the method B, the organic acid is trifluoroacetic acid; the non-polar organic solvent is a halocarbon solvent; the halocarbon solvent is dichloromethane and/or trichloromethane; the base is an inorganic base; and the inorganic base is selected from the group consisting of potassium carbonate, sodium carbonate, sodium hydroxide and potassium carbonate, or a mixture thereof.

10. The method according to claim 8, wherein in the method A, the method for preparing the compound represented by formula II further comprises the following steps: in the organic solution, with the action of the base, subjecting trimethylsulfonium halide and a compound represented by formula IV to epoxidation reaction shown below to produce the compound represented by formula III; ##STR00018##

11. The method according to claim 10, wherein in the method for preparing the compound represented by formula III, the trimethylsulfonium halide is trimethylsulfonium bromide; the organic solvent is an ether; the ether is tetrahydrofuran; the base is potassium tert-butoxide; the mole ratio of the trimethylsulfonium halide to the compound represented by formula IV is from 2:1 to 10:1; the mole ratio of the trimethylsulfonium halide to the base is from 1:1 to 1.2:1; the temperature for mixing the trimethylsulfonium halide with the base is between 15 and 5 C.; after mixing the trimethylsulfonium halide with the base, stirring the mixed solution at a temperature between 15 and 5 C. for 0.5 to 3 hours; after mixing the mixed solution of trimethylsulfonium halide and the organic solvent with the base, controlling the temperature of the reaction system between 75 and 65 C., and then adding an organic solution of the compound represented by formula IV; the organic solvent in the organic solution of the compound represented by formula IV is a halohydrocarbon solvent; the halohydrocarbon solvent is dichloromethane; the temperature of the epoxidation reaction is between 75 and 65 C.; the method for preparing the compound represented by formula III comprises the following steps: mixing the mixed solution of trimethylsulfonium halide and the organic solvent with the base, adding the organic solvent of the compound represented by formula IV, and performing the epoxidation reaction; and the epoxidation reaction is performed in the condition of gas protection, wherein the gas for the gas protection is nitrogen.

12. The method according to claim 8, wherein in the method B, the method for preparing the compound represented by formula II further comprises the following steps: in the organic solvent, with the action of the catalyst and the hydrogen source, subjecting a salt IIIa of the compound represented by formula III to the de-protection reaction to produce the salt IIa of the compound represented by formula II; ##STR00019## wherein definitions of X and n are as defined in claim 8; the conditions of the de-protection reaction are the same as that of the method A of the method for preparing the compound represented by formula II.

13. The method according to claim 12, wherein the method for preparing the salt of the compound represented by formula II further comprises the following steps: in a halohydrocarbon solvent, subjecting the compound represented by formula III and an acid X to salt formation reaction to produce the salt Illa of the compound represented by formula III; ##STR00020## wherein X is an organic acid or an inorganic acid, and n is 1, 2 or 3, and the salt IIIa of the compound represented by formula III is a trifluoroacetate IIIa1 of the compound represented by formula III: ##STR00021##

14. The method according to claim 13, wherein the method for preparing the trifluoroacetate IIIa1 of the compound represented by formula III further comprises the following steps: mixing a mixed solution of the compound represented by formula III and a halohydrocarbon solvent with trifluoroacetic acid, and performing the salt formation reaction; ##STR00022##

15. The method according to claim 14, wherein in the method for preparing the trifluoroacetate IIIa1 of the compound represented by formula III, the halohydrocarbon solvent is dichloromethane; and/or, the mole ratio of the compound represented by formula Ill to trifluoroacetic acid is from 1:2 to 1:3; and/or, the temperature of the mixing is between 0 and 40 C.

16. The method according to claim 14, wherein the method for preparing the trifluoroacetate IIIa1 of the compound represented by formula III further comprises a post-processing treatment; and the post-processing treatment comprises the following steps: adding an anti-solvent to the reaction solution after the salt formation reaction, mixing, and crystallizing; the anti-solvent is isopropyl ether; the volume ratio of the halohydrocarbon solvent to the anti-solvent is from 1:0.8 to 1:2; the adding of the anti-solvent is dropwise adding; the temperature of the dropwise adding of the anti-solvent is between 0 and 40 C.; the temperature of the crystallization is between 5 and 40 C.

17. A compound represented by formula IIIa1: ##STR00023##

18. A method for purifying tulathromycin represented by formula I, comprising the following steps: mixing a crude tulathromycin represented by formula I with a solvent A, adding an anti-solvent, and crystallizing to obtain a product of tulathromycin represented by formula I; wherein the purity of the crude tulathromycin represented by formula I measured by HPLC is more than 65%; the solvent A is a mixed solvent of acetone and a C.sub.1-3 alcohol; and the anti-solvent is water or a mixed solvent of water and acetone; ##STR00024##

19. The method according to claim 18, wherein the purity of the crude tulathromycin represented by formula I measured by HPLC is from 65 to 99.5%; and/or, the C.sub.1-3 alcohol is 1,2-propandiol; and/or, the volume to mass ratio of the solvent A to the crude tulathromycin represented by formula I is from 2 mL/g to 50 mL/g; and/or, the adding of anti-solvent is dropwise adding; the temperature of the dropwise adding of the anti-solvent is between 35 and 45 C.; and/or, the volume to mass ratio of the anti-solvent to the crude tulathromycin represented by formula I is from 2 mL/g to 60 m L/g.

20. The method according to claim 18, wherein when the anti-solvent is a mixed solvent of water and acetone, the volume ratio of water to acetone is from 0.5:1 to 3:1; when adding the anti-solvent, the adding is performed until the solution A containing tulathromycin represented by formula I turns turbid, the resulting mixture is stirred and crystalline grain culturing is carried out for 10 to 30 minutes, and the remaining anti-solvent is added; the temperature of the crystallization is between 0 and 45 C.; the duration of the crystallization is from 1 to 6 hours; the crystallization comprises an early stage of crystallization and a late stage of crystallization, wherein, the early stage of crystallization is carried out at a temperature between 35 and 45 C. for 0.5 to 3 hours, and the late stage of crystallization is carried out at a temperature between 0 and 35 C. for 0.5 to 3 hours.

Description

DETAILED DESCRIPTION

(1) In the following examples, the HLPC content refers to the purity measured by HPLC.

EXAMPLE 1

Preparation of the Compound Represented by Formula V

(2) 25 g of dihydroerythromycin and 350 mL of dichloromethane were added to a 1L three-necked flask, stirred, and dissolved. Nitrogen was filled in the flask for protection, and the temperature was cooled to 0 to 10 C. by an ice-salt water bath. A mixed solution of 12.2 mL of carbobenzoxy chloride and 50 mL of dichloromethane was added dropwise over 20 minutes, during which the internal temperature was kept between 0 and 10 C. The reaction was traced by TLC+HPLC until the reaction was completed, and the duration of the reaction was 3 hours. 125 mL of saturated NaHCO.sub.3 aqueous solution was added to quench the reaction. The organic layer was separated from the water layer, and 50 mL of dichloromethane was added to the water layer for one more extraction. The organic layers were combined, washed with 100 mL of saturated sodium bicarbonate solution once, and dried with anhydrous sodium sulfate. The mixture was filtered, concentrated under reduced pressure at a temperature between 35 and 40 C., and drained with an oil pump to give 29.1 g of a bubble compound represented by formula V, MS(ESI)868.53.

EXAMPLE 2

Preparation of the Compound Represented by Formula IV

(3) The bubble compound represented by formula V produced in the Example 1 was dissolved with 150 mL of dichloromethane, and 50 mL of DMSO was added under stirring. Nitrogen was filled in for protection and the mixture was cooled to 75 C. with liquid nitrogen. A mixed solution of 11.5 mL of trifluoroacetic anhydride and 25 mL of CH.sub.2Cl.sub.2 was added dropwise, reaction was carried out for 45 minutes, and the internal temperature was kept between 65 and 75 C. Then a mixed solution of 25 mL of triethylamine and 25 mL of CH.sub.2Cl.sub.2 was added dropwise, reaction was carried out for 30 minutes, and the temperature was kept between 65 and 75 C. Water was added to quench the reaction. The mixture was stratified, and the water layer was separated out. The organic layer was washed with 100 mL of saturated salt water once. After stratification, anhydrous sodium sulfate was added to the organic layer for drying. The mixture was filtered, concentrated under reduced pressure, and drained with an oil pump to give 24.3 g of a bubble compound represented by formula IV, MS(ESI)866.51.

EXAMPLE 3

Preparation of the Compound Represented by Formula III

(4) 17.6 g (0.11 mol) of trimethylsulfonium bromide was added to a 1000 mL three-necked flask. Under nitrogen protection and mechanical agitation, 150 mL of THF was added and the temperature was cooled to 15 C. with liquid nitrogen. 12.5 g (0.11 mol) of potassium tert-butoxide was added, the internal temperature was kept between 15 and 5 C., and the reaction was carried out for 1 hour under stirring. The temperature was then cooled to 75 C., a solution of 24.3 g (0.028 ml) of the compound represented by formula IV obtained in Example 2 and 250 mL of CH.sub.2Cl.sub.2 was added dropwise for 1 hour, and the temperature was kept between 75 and 65 C. After the adding, the reaction was carried out for 2 hours while the temperature was kept between 75 and 65 C. The reaction was traced by HPLC until the completion of the reaction. Ammonium chloride aqueous solution was added to quench the reaction. The temperature of the reaction solution was raised to room temperature, and the water layer and the organic layer were separated. The water layer was extracted with CH.sub.2Cl.sub.2 once. The organic layers were combined and washed with saturated salt water once. Anhydrous sodium sulfate was added to the organic layer for drying. The mixture was filtered, concentrated at a temperature between 35 and 40 C. to almost dry, and drained with an oil pump for 1 h to give 24.7 g of a bubble compound represented by formula III, MS(ESI)880.53.

EXAMPLE 4

Preparation of Trifluoroacetate of the Compound Represented by Formula III

(5) 24.7 g of the bubble compound produced by Example 3 was dissolved in 80 mL of CH.sub.2Cl.sub.2, and a mixed solution of 7.2 g trifluoroacetic acid and 20 mL of CH.sub.2Cl.sub.2 was added dropwise at temperature between 25 and 35 C. Then 150 mL of isopropyl ether was added dropwise at 30 C.; the solution turned turbid when about 100 mL isopropyl ether was added, and the adding was stopped. The mixture was slowly stirred for 0.5 hour, and the remaining isopropyl ether was added dropwise. The mixture was stirred at 30 C. to crystallization for 1 hour, followed by cooling crystallization in an ice-water bath (between 5 and 0 C.) for 1 hour. After filtration, the filter cake was washed with a mixed solvent of 10 mL of CH.sub.2Cl.sub.2 and 15 mL of isopropyl ether, and the filter cake was subjected to vacuum drying at 45 C. to give 26.2 g of a loose white solid, i.e., trifluoroacetate of the compound represented by formula III with a purity of 88.2% measured by HPLC, .sup.1H-NMR(400 MHz, DMSO); =10.85(s, 1H); 8.70(s, 1H); 8.38(t, J=9.7 Hz, 1H); 7.38(s, 5H); 6.08(s, 1H); 5.18(d, J=12.3 Hz, 1H); 5.06(d, J=12.2 Hz, 1H); 5.01(d, J=3.9 Hz, 1H); 4.90(dd, J=10.1 Hz, 2.0 Hz, 1H); 4.70(q, J=6.5 Hz, 1H); 4.60-4.67(m, 2H); 4.00(s, 1H); 3.67-3.72(m, 1H); 3.55-3.63(m, 1H); 3.47(d, J=6.0, 1H); 3.41(s, 1H); 3.23-3.30(m, 4H); 3.00-3.06(m, 1H); 2.67-2.76(m, 10H); 2.59-2.64(m, 1H); 2.39(d, J=14.8 Hz, 1H); 2.13-2.15(m, 1H); 1.73-1.89(m, 4H); 1.58-1.67(m, 1H); 1.35-1.44(m, 1H); 1.24-1.31(m, 8H); 1.15(d, J=5.8 Hz, 3H); 1.10(d, J=6.0, 3H); 1.07(s, 3H); 1.01-1.03(m, 3H); 0.93-0.94(m, 6H); 0.82(t, J=7.4 Hz, 3H); 0.74(d, J=7.1 Hz, 3H). MS(ESI)880.53.

(6) Results of experiments under other conditions:

(7) TABLE-US-00001 Number 1 2 3 Amount of compound 2 g 5 g 5 g of formula III Compound of formula 1:2 1:2.5 1:3 III: trifluoroacetic acid (mole ratio) Amount of CH.sub.2Cl.sub.2 20 ml 50 ml 50 ml Amount of isopropyl 16 ml 100 ml 60 ml ether Reaction temperature 30-35 C. 0-10 C. 35-40 C. Crystallization Crystallizing Crystallizing Crystallizing temperature at 30-35 C. at 10-20 C. at 30-40 C. for 1 hour; for 1 hour; for 1 hour; then then then crystalizing at crystalizing crystalizing 5~0 C. at 5~0 C. at 0~10 C. for 1 hour for 1 hour for 1 hour Amount of compound 2.0 g 5.4 g 5.3 g of formula IIIa1 HPLC purity of 90.1% 86.1% 88.5% compound of formula IIIa1

EXAMPLE 5

Preparation of the Compound Represented by Formula II

(8) 15 g of trifluoroacetate of the compound represented by formula III produced in Example 4 was dissolved in 150 mL of methanol under stirring. Under the protection of nitrogen, 7.5 g of ammonium formate was added. After ammonium formate was absolutely dissolved, 1.1 g of 10% Pd/C was added, and the reaction was carried out at temperature between 20 and 25 C. The reaction was traced by HPLC until the reaction was completed, which lasted for about 2 to 3 hours. The mixture was filtered, the filter cake was washed with methanol and the filtrate was concentrated at 40 C. under reduced pressure. 40 mL of water and 60 mL of dichloromethane were added to dissolve the mixture under stirring. 2N sodium hydroxide aqueous solution was used to adjust the pH to between 9 and 10 under stirring for 30 minutes. After stratification, the upper water layer was extracted with 40 mL of dichloromethane, the organic layers were combined and washed with saturated salt water. After stratification, anhydrous sodium sulfate was added to the organic layer for drying. The mixture was filtered, concentrated under reduced pressure at a temperature between 35 and 40 C., and drained with an oil pump to give 11.7 g of a bubble compound represented by formula II with a purity of 86.1% measured by HPLC, MS(ESI)746.49.

(9) Results of experiments under other conditions:

(10) TABLE-US-00002 Number 1 2 3 Amount of trifluoroacetate 5 g 5 g 5 g of formula III Amount of Pd/C 0.5 g of 0.5 g of 0.5 g of 5% Pd/C 10% Pd/C 15% Pd/C Solvent and the amount Ethanol, Isopropanol, Methanol, thereof used in reaction 50 mL 50 mL 50 mL Temperature and duration 20-25 C., 3 h 20-25 C., 3 h 20-25 C., 3 h of the reaction Mass of compound of 3.7 g 3.4 g 3.8 g formula II HPLC purity of the 84.6% 82.2% 86.7% compound of formula II

EXAMPLE 6

Preparation of the Compound Represented by Formula II

(11) 5 g of the compound represented by formula III produced in Example 3 was dissolved in 50 mL of methanol under stirring. Under the protection of nitrogen, 2.5 g of ammonium formate was added. After ammonium formate was absolutely dissolved, 0.4 g of 10% Pd/C was added, and the reaction was carried out at temperature between 20 and 25 C. The reaction was traced by HPLC until the reaction was completed, which lasted for about 2 to 3 hours. The mixture was filtered, the filter cake was washed with methanol and the filtrate was concentrated at 40 C. under reduced pressure. 20 mL of water and 50 mL of dichloromethane were added to dissolve the mixture under stirring. 2N sodium hydroxide aqueous solution was used to adjust the pH to between 9 and 10 under stirring for 30 minutes. After stratification, the upper water layer was extracted with 20 mL of dichloromethane, the organic layers were combined and washed with saturated salt water. After stratification, anhydrous sodium sulfate was added to the organic layer for drying. The mixture was filtered, concentrated under reduced pressure at a temperature between 35 and 40 C., and drained with an oil pump to give 4.1 g of a bubble compound represented by formula II with a purity of 78.9% measured by HPLC, MS(ESI)746.49.

EXAMPLE 7

Preparation of the Compound Represented by Formula I

(12) 11.7 g of the bubble compound represented by formula II produced in Example 5 was dissolved in 6.0 mL of 1,2-propandiol and 12 mL of n-propylamine. Under the protection of nitrogen, the reaction was carried out at temperature between 45 and 55 C. and traced by HPLC until the reaction was completed, which lasted for 20 hours. The mixture was concentrated at 40 C. under reduced pressure until no liquid drop to give an oil-like product containing crude 1,2-propaniol tulathromycin with a tulathromycin content of 78.3% measured by HPLC.

(13) Results of experiments under other conditions:

(14) TABLE-US-00003 Compound of formula II: n-propyl- Temperature Duration HPLC amine of the of the purity of Number (mole ratio) reaction ( C.) reaction tulathromycin 1 1:5 55-65 C. 16 h 77.3% 2 1:20 45-50 C. 26 h 75.8% 3 1:30 45-50 C. 35 h 73.9%

EXAMPLE 8

(15) 65 mL of acetone was added to the oil-like crude 1,2-propandiol tulathromycin produced in Example 7. The mixture was disposed in water bath at about 40 C. and 85 mL of water was slowly added dropwise. When the crystallization liquid turned turbid, the adding was stopped, and crystalline grain culturing was carried out for 10 minutes under stirring, and then the remaining water was added. After the adding of water, the mixture was hold in a water bath at temperature between 35 and 40 C. for 1 hour with stirring. The mixture was cooled to 0 to 10 C. in an ice-water bath, and quickly stirred for 1 hour. After filtration, the resulting solid was washed with an appropriate amount of 40% acetone aqueous solution to give a loose white solid, which was the wet product of tulathromycin after first crystallization, with a content of 95.8% measured by HPLC, MS(ESI)805.57.

EXAMPLE 9

(16) 25 mL of acetone and 3.0 mL of 1,2-propandiol were added and mixed with 5.0 g of the first crystallization product of tulathromycin obtained in Example 8. The mixture was hold in water bath at 40 C. with stirring for dissolution. An anti-solvent of 25 mL of acetone and 60 mL of water was slowly added dropwise. When the crystallization liquid turned turbid, the adding was stopped, and crystalline grain culturing was carried out for 10 minutes under stirring, and then the remaining anti-solvent was added. After the adding of anti-solvent, the mixture was hold in a water bath at 40 C. for 1 hour with stirring. The mixture was cooled to 0 to 10 C. in an ice-water bath, and quickly stirred for 1 hour. After filtration, the resulting solid was washed with an appropriate amount of 40% acetone aqueous solution to give a wet solid of tulathromycin crystal, with a purity of 99.2% measured by HPLC, MS(ESI)805.57.

(17) Results of experiments under other conditions.

(18) Therein, (1) tulathromycin product produced in Example 7 was dried to obtain a dried tulathromycin product by drying; (2) mixed solvent and amount; (3) anti-solvent and amount; (4) crystallization process; (5) yield of recrystallization; (6) purity of tulathromycin product obtained after the recrystallization measured by HPLC.

(19) TABLE-US-00004 Number 1 2 3 4 (1) 1 g 1 g 1 g 1 g (2) 2.5 mL 11 mL 15 mL 30 mL (2 mL of acetone (10 mL of acetone (10 mL of acetone (25 mL of acetone and 0.5 mL of and 1 mL of and 5 mL of and 5 mL of 1,2-propandiol) 1,2-propandiol) 1,2-propandiol) 1,2-propandiol) (3) 2 mL of water 50 mL 47 mL 45 mL (40 mL of water and (27 mL water and (25 mL water and 20 mL 10 mL of acetone) 20 mL of acetone) of acetone) (4) Slowly add the Slowly add the Slowly add the Slowly add the anti-solvent anti-solvent dropwise; anti-solvent dropwise; anti-solvent dropwise; dropwise; stop stop adding when the stop adding when the stop adding when the adding when the crystallization liquid crystallization liquid crystallization liquid turn crystallization liquid turn turbid; stir and turn turbid; stir and turbid; stir and culture turn turbid; stir and culture the crystalline culture the crystalline the crystalline grain for culture the crystalline grain for 30 minutes; grain for 20 minutes; 20 minutes; add the grain for 10 minutes; add the remaining add the remaining remaining mixed add the remaining mixed solution; stir and mixed solution; stir and solution; stir and water; stir and incubate in a water bath incubate in a water bath incubate in a water bath incubate in a water at 45 C. for 3 hours; at 35 C. for 1 hour; cool at 35 C. for 1 hour; cool bath at 40 C. for 1 cool to 0-10 C. in to 0-5 C. in ice-water to 10 C. in ice-water bath hour. ice-water bath and stir bath and stir for 1 hour. and stir for 1 hour. for 3 hours. (5) 98.2% 96.4% 95.3% 93.7% (6) 97.6% 99.0% 99.3% 99.5%

EXAMPLE 10

(20) 2.5 mL of acetone and 0.3 mL of 1,2-propandiol were added to 0.5 g of tulathromycin product represented by formula I (obtained in Example 9 with a purity of 99.2% measured by HPLC). The mixture was hold in water bath at 40 C. with stirring for dissolution. A mixed solution of 2.5 mL of acetone and 6 mL of water was slowly added, dropwise. When the crystallization liquid turned turbid, the adding was stopped, and crystalline grain culturing was carried out for 10 minutes under stirring, and then the remaining mixed solution was added. After the adding of mixed solution, the mixture was hold in a water bath at 40 C. for 1 hour with stirring. The mixture was cooled to 0 to 10 C. in an ice-water bath and stirred for 1 hour. After filtration, the resulting solid was washed with an appropriate amount of 40% acetone aqueous solution to give a wet solid of tulathromycin crystal, with a purity of 99.5% measured by HPLC.

EXAMPLE 11

(21) 5.0 mL of acetone and 0.6 mL of 1,2-propandiol were added to 1.0 g of crude tulathromycin with a purity of 68.9% measured by HPLC (prepared by the method in Example 1-6 of the U.S. Pat. No. 6,825,327B2). The mixture was hold in water bath at 41 C. with stirring for dissolution. A mixed solution of 5 mL of acetone and 12 mL of water was slowly added, dropwise. When the crystallization liquid turned turbid, the adding was stopped, and crystalline grain culturing was carried out for 10 minutes under stirring, and then the remaining mixed solution was added. After the adding of mixed solution, the mixture was hold in a water bath at 40 C. for 1 hour with stirring. The mixture was cooled to 0 to 10 C. in an ice-water bath and stirred for 1 hour. After filtration, the resulting solid was washed with an appropriate amount of 40% acetone aqueous solution to give a wet solid of tulathromycin crystal with a purity of 95.2% measured by HPLC, MS(ESI)805.57.

EXAMPLE 12

(22) 5 mL of acetone was added to 0.5 g of tulathromycin represented by formula I with a purity of 99.2% measured by HPLC obtained in Example 7. The mixture was hold in water bath at 35 C. with stirring for dissolution. 7.5 mL of water was slowly added, dropwise. When the crystallization liquid turned turbid, the adding was stopped, and crystalline grain culturing was carried out for 10 minutes under stirring, and then the remaining water was added. After the adding of mixed solution, the mixture was cooled for 1 hour with stirring. The mixture was cooled to 0 to 10 C. in an ice-water bath and stirred for 1 hour. After filtration, the resulting solid was washed with an appropriate amount of 40% acetone aqueous solution to give a wet solid of tulathromycin crystal with a purity of 92.4% measured by HPLC.

EXAMPLE 13

(23) 0.5 mL of 1,2-propandiol and 5 mL of methanol were added to 0.5 g of tulathromycin represented by formula I with a purity of 99.2% measured by HPLC obtained in Example 7. The mixture was hold in water bath at 40 C. with stirring for dissolution. 6 mL of water was slowly added, dropwise. When the crystallization liquid turned turbid, the adding was stopped, and crystalline grain culturing was carried out for 10 minutes under stirring, and then the remaining water was added. After the adding of mixed solution, the mixture was hold in a water bath at 40 C. for 1 hour with stirring. The mixture was cooled to 0 to 10 C. in an ice-water bath and stirred for 1 hour. After filtration, the resulting solid was washed with an appropriate amount of 40% acetone aqueous solution to give a wet solid of tulathromycin crystal with a purity of 94.6% measured by HPLC.

COMPARATIVE EXAMPLE 1

(24) 5 g of the bubble compound represented by formula II produced by Example 5 was dissolved in 2.5 mL of isopropanol and 5 mL of n-propylamine. Under the protection of nitrogen, the reaction was carried out at temperature between 45 and 55 C. and traced by HPLC until the reaction was completed, which lasted for 20 hours. The mixture was concentrated at 40 C. under reduced pressure until no liquid drop to give an oil-like crude tulathromycin with a tulathromycin content of 60.3% measured by HPLC.

COMPARATIVE EXAMPLE 2

(25) 3 g of the bubble compound represented by formula II produced by Example 5 was mixed with 15 mL of isopropanol and 6.1 g of n-propylamine. The mixture was reacted at a temperature between 50 and 55 C. for 30 hours, and then was concentrated to about 8 mL in a water bath at 50 C. under vacuum. The sample was taken and measured by HPLC, and the purity of the target compound tulathromycin was 68.9%.

(26) Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, numerous variations or modifications may be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the protection scope of the present invention is defined by the appended claims.