Claims:We claim:
1. A process for preparation of Gamithromycin comprising the steps of:
a. treating a solution of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) in polar protic solvent or mixture thereof; with aqueous hydrazine hydrate (50-80%) or anhydrous hydrazine hydrate in the presence of oxygen gas in polar protic solvent;
b. stirring the solution of step (a) for 24-36 hrs;
c. work up and adjust pH 3.0-9.5 range with hydrochloric acid or acetic acid or formic acid;
d. filtering and washing the solid with polar protic solvent or mixture thereof;
wherein the step (a) involves treatment of Allylated-Gamithromycin (i.e. Structure- 5 in Scheme-I) with hydrazine hydrate; and wherein the reaction steps (a) and (b) are carried out at temperature range about 25-80 oC.
2. The process as claimed in claim 1, wherein the solvent in step (a) and step (d) is a single solvent or a mixture of the solvents selected from the group comprising of lower alkyl alcohols such as ethanol, methanol, isopropyl alcohol, n-propanol and n-butanol. Also, polar aprotic solvents such acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide.
3. The process as claimed in claim 1, wherein the said dimide source used is hydrazine hydrate with oxygen gas. Number of equivalents of sodium salt of alkyl carboxylic acid used ranges from 3 to 15 equivalents and one particular reaction the quantity used is from about 10 to 15 equivalents.
4. A process for the preparation of Gamithromycin comprising the steps of:
a. treating a solution of Allylated-Gamithromycin (i.e. Structure-5 in Fig:1) in polar protic solvent or mixture thereof with hydrazine hydrate solution in the presence of oxygen gas in polar protic solvent or mixture thereof at 50-55 oC;
b. heating the solution for 10-12 hrs at 50-55 oC;
c. work up and adjust pH 3-9.5 range with hydrochloric acid or acetic acid or formic acid;
d. filtering and washing the solid with polar protic solvent or mixture thereof.
5. The process as claimed in claim 4, wherein the quantity of hydrazine hydrate in step (a) is in an amount ranging from 3 to 15 equivalents.
6. The process as claimed in claim 5, wherein the quantity of hydrazine hydrate in step (a) is in an amount ranging from 10 to 15 equivalents.
7. The process as claimed in claim 4, wherein the dimide source used is hydrazine hydrate with oxygen gas. Number of equivalents of hydrazine hydrate used ranges from 3 to 15 equivalents and one particular reaction the quantity used is from about 10 to 15 equivalents.
8. The process as claimed in claim 4, wherein the solvent in step (a) and step (d) is a single solvent or a mixture of the solvents selected from the group comprising of lower alkyl alcohols such as ethanol, methanol, isopropyl alcohol, n-propanol and n-butanol. Also, polar aprotic solvents such acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide.
9. The process as claimed in claims 1 or 4, wherein the gamithromycin obtained by the process has greater than 99% purity and comprises less than 0.15% w/w of known impurities of formula II, formula III, formula IV, formula V, formula VI and formula VII.
10. A process for preparing highly pure crystalline form of gamithromycin comprising the steps of:
a. treating a solution of gamithromycin in polar aprotic or protic solvents at 25-30 oC;
b. heating the solution for 2-3 hrs at 70-80 oC;
c. cooling the suspension and filtering the solid at 25-30 oC;
d. washing and slurring the solid in polar aprotic or protic solvents or mixtures thereof;
e. isolating Gamithromycin of Formula-I and drying at below 60-65 oC.
11. The process as claimed in claim 10, wherein the crystalline form of gamithromycin is characterized by X-ray diffraction pattern having peaks at 7.0, 9.7, 10.3, 11.6, 15.1 and 20.7 degrees two theta.
12. The crystalline form of claim 10, which has a differential scanning calorimetry spectrum an event with an onset at about 177.2 oC.
13. The process as claimed in claim 10, wherein the dimide source used is hydrazine hydrate with oxygen gas. Number of equivalents of hydrazine hydrate used ranges from 3 to 15 equivalents and one particular reaction the quantity used is from about 10 to 15 equivalents.
14. The process as claimed in claim 10, wherein the solvent in step (a) and step (d) is a single solvent or mixture of any two solvents selected from the group comprising lower alkyl alcohols such as ethanol, methanol, isopropyl alcohol, n-propanol and n-butanol. Also, polar aprotic solvents such acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide.
15. The process as claimed in claim 10, wherein the quantity of hydrazine hydrate in step (a) is in an amount ranging from 3to 15 equivalents.
16. The process as claimed in claim 10, wherein the step (d) is performed at a temperature range of -5 to 30 oC for a period of 1-2 hrs.
17. The process as claimed in claim 10, wherein the crystalline form of gamithromycin obtained by the process has greater than 99% purity and comprises less than 0.15% w/w of known impurities of formula II, formula III, formula IV, formula V, formula VI and Formula VII.
18. The highly pure crystalline form of gamithromycin characterized by X-ray diffraction pattern having peaks at 7.0, 9.7, 10.3, 11.6, 15.1 and 20.7 degrees two theta obtained by the process as claimed in claim 10, wherein said crystalline form has greater than 99% purity and comprises less than 0.15% w/w of known impurities of formula II, formula III, formula IV, formula V, formula VI and Formula VII.
, Description:TECHNICAL FIELD OF THE INVENTION
The invention relates to a novel and improved process for the preparation of gamithromycin of Formula-I, which involves conversion of Allylated-Gamithromycin (i.e. Structure-5) to gamithromycin (i.e. Formula-I) with hydrazine hydrate in the presence of oxygen in organic polar solvents. More particularly, the invention relates to a novel process for the preparation of gamithromycin of Formula I in a substantially pure form with a purity level greater than 99% wherein known impurities illustrated below as Formula II, Formula III, Formula IV, Formula V, Formula VI and Formula VII are either absent in the final product or are very well controlled and under the limit of as low as below 0.15% w/w as depicted in Scheme-2. The invention also relates to highly pure crystalline form of gamithromycin and a process for the preparation thereof.

BACKGROUND ART
Gamithromycin, the active pharmaceutical ingredient of ZACTRAN is a novel 7a-azalide that has been developed for treatment and prevention of bovine respiratory disease (BRD). Gamithromycin is indicated for the treatment of bovine respiratory disease (BRD) associated with Mannheimia haemolytica, Pasteurella multocida and Histophilus somni in beef and non-lactating dairy cattle. Gamithromycin is also indicated for the control of respiratory disease in beef and non-lactating dairy cattle at high risk of developing BRD associated with Mannheimia haemolytica and Pasteurella multocida. The compound belongs to the 15-membered semi-synthetic macrolide antibiotics of the azalide sub-class with a uniquely positioned alkylated nitrogen at the 7a-position of the lactone ring. structurally as represented in Formula I above and chemically known as 1-Oxa-7-azacyclopentadecan-15-one,13-[(2,6-dideoxy-3-C-methyl-3-Omethyl-.alpha.-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy3,5,8,10,12,14-hexamethyl-7propyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-.beta.-D-xylo-hexopyranosyl]oxy]-,(2R,3S,4R,5S,8R,10R,11R,12S,13S,14R).
Gamithromycin drug substance is a white to beige fine powder with antimicrobial properties. It is soluble in most of the organic solvents such as alcohols but very slightly soluble in water. Gamithromycin is insoluble in water, reversibly hygroscopic and not sensitive to light.
Scheme-III: Method for Synthesis of Gamithromycin in US US5985844

US5985844 discloses a method for preparing gamithromycin from erythromycin A as illustrated in Scheme-III. The key transformation of this approach is reduction of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) with Pd/C in ethanol as solvent under high pressure of hydrogen gas, which leads mixture of compounds. During the reduction, observed major by-product as Structure-4 in Scheme-I along with Gamithromycin. Hence, this process is difficult for scale-up production. Furthermore, this patent does not disclose the formation or preparation of any process impurities or degradation impurities.

Scheme-IV: Method for Synthesis of Gamithromycin in US8314218 B2

US8314218 B2, discloses alternative and scalable process for gamithromycin from erythromycin A as illustrated in Scheme-IV. They have used expensive platinum catalyst for synthesis of Structure-4 in Scheme-I. Then, treated Structure-4 with Pd/C in the presence of propanal with high pressure of hydrogen gas to give gamithromycin. The maximum purity achieved by this process is more than 98%. All the known methods for the synthesis of gamithromycin used highly expensive metal catalysts such as Platinum/Carbon, Palladium/Carbon. In addition, they used, pressure reactions with hydrogen gas in two steps. Furthermore, this patent does not disclose the formation or preparation of any process impurities or degradation impurities as well as process for the preparation of crystalline form of gamithromycin.
Therefore, there is a need to develop an efficient and cost effective method for the commercial scale production of substantially pure stable form of Gamithromycin, which is free from the above mentioned problems and known impurities as disclosed in Scheme-II.

OBJECTS OF THE INVENTION
An object of the invention to provide a novel process for the preparation of highly pure Gamithromycin of formula I.
Another object of the invention is to provide a novel process for preparing highly pure Gamithromycin of formula-I, with purity level greater than 99%.
Another object of the invention is to provide a highly pure crystalline form of Gamithromycin of formula-I.
A further object of the invention is to provide novel processes for preparing highly pure crystalline Gamithromycin of formula-I.
Yet another object of the invention is to provide substantially pure crystalline form gamithromycin of formula-I with known impurities represented by formula II, III, IV, V, VI and formula VII less than 0.15% w/w.

SUMMARY OF THE INVENTION
Herein disclosed is an improved process for the preparation of highly pure gamithromycin of formula I as shown in below reaction Scheme-I:

Scheme-I: Method for Synthesis of Gamithromycin by Using Novel Process

Scheme-2: Chemical Structures of Impurities formula-II, III, IV, V, VI and VII

The improved process for the preparation of highly pure gamithromycin of formula I comprises the steps of:
a. treating a solution of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) in polar protic solvent or mixture thereof; with aqueous hydrazine hydrate (50-80%) or anhydrous hydrazine hydrate in the presence of oxygen gas in polar protic solvent;
b. stirring the solution for 24-36 hrs;
c. work up and adjust pH 3.0-9.5 range with hydrochloric acid or acetic acid or formic acid;
d. filtering and washing the solid with polar protic solvent or mixture thereof;
wherein the step (a) involves treatment of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) with hydrazine hydrate; and wherein the reaction steps (a) and (b) are carried out at temperature range about 25-80 oC.
In another aspect, the invention provides an improved process for the preparation of highly pure Gamithromycin of formula I comprising the steps of:
a. treating a solution of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) in polar protic solvent or mixture thereof with hydrazine hydrate solution in the presence of oxygen gas in polar protic solvent or mixture thereof at 50-55 oC;
b. heating the solution for 10-12 hrs at 50-55 oC;
c. work up and adjust pH 3-9.5 range with hydrochloric acid or acetic acid or formic acid; and
d. filtering and washing the solid with polar protic solvent or mixture thereof.
In another aspect the invention provides a process for preparing highly pure crystalline form of Gamithromycin of formula I as shown in below reaction Scheme-I, comprising the steps of:
a. treating a solution of gamithromycin in polar aprotic or protic solvents at 25-30 oC;
b. heating the solution for 2-3 hrs at 70-80 oC;
c. cooling the suspension and filtering the solid at 25-30 oC;
d. washing and slurring the solid in polar aprotic or protic solvents or mixtures thereof; and
e. isolating Gamithromycin of Formula-I and drying at below 60-65 oC.
The crystalline form obtained above is characterized by X-ray diffraction pattern having characteristic peaks expressed in 2??at about 7.0, 9.7, 10.3, 11.6, 15.1 and 20.7.
In any of the above said processes the quantity of hydrazine hydrate (50-80%) in step (a) is in an amount ranging from about 3 to 15 equivalents; preferably in an amount ranging from about 10 to 15 equivalents.
In any of the above said processes the in-situ generation of dimide from aqueous hydrazine hydrate or anhydrous hydrazine hydrate in protic or aprotic solvents with oxidizing agents such as H2O2, NaIO4, K3[(FeCN)6] and oxygen gas.
In another aspect, above process(es) the in-situ generation of dimide from benzenesulfonylhydrazide, O-nitro benzenesulfonylhydrazide in protic or aprotic solvents with trimethylamine as oxidizing agent. The dimide source for reduction of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) to Gamithromycin of formula I: preferably from hydrazine hydrate with oxygen gas.
In any of the above said processes the solvent(s) is/are a single solvent or mixture of any two solvents selected from the group comprising lower alkyl alcohols such as ethanol, methanol, isopropyl alcohol, n-propanol and n-butanol; preferably when mixture is used the solvent is mixture of isopropyl alcohol and methanol. Also, polar aprotic solvents such acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide.
The final product Gamithromycin of formula I as obtained by any of above said processes is greater than 99% pure and comprises less than 0.15% w/w of known impurities of formula II, formula III, formula IV, formula V, formula VI and formula VII.
The above said isolation step (e) comprises the steps of filtration, washing and drying or the combinations thereof. The above said step (c) is performed at a temperature range of -5 to 30 oC or preferably at 10 to 15 oC for a period of 1-2 hrs.
In another aspect the present invention further provides a crystalline form of gamithromycin of formula I, wherein said crystalline form is characterized by X-ray diffraction pattern having peaks at 7.0, 9.7, 10.3, 11.6, 15.1 and 20.7 degrees two theta and X-ray diffraction as depicted in figure-1 and differential scanning chromatogram (DSC) as depicted in Figure-2.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
Figure-1: Illustrates a powder X-ray diffraction pattern of crystalline form of Gamithromycin of formula I
Figure-2: Illustrates a differential scanning chromatogram of crystalline form of Gamithromycin of formula I.

DETAILED DESCRIPTION OF THE INVENTION
The invention as disclosed herein provides an improved process for the preparation of Gamithromycin of formula I in a substantially pure form with purity level greater than 99%, wherein known impurities such as Formula II, Formula III, Formula IV, Formula V, Formula VI and Formula VIII as shown below are absent in the final product or very well controlled under the limit. The final product comprises less than 0.15% w/w of impurity compounds of structural formula II, III, IV, V, VI and formula VII. Compound of formula I is used as an API in the pharmaceutical preparations/composition used in the treatment and prevention of bovine respiratory disease (BRD).
Thus in one aspect, the invention provides novel processes for the preparation of Gamithromycin formula I in significantly high yield and substantially pure crystalline form as shown in below Scheme-I:
Scheme-I: Novel Route of Synthesis of Gamithromycin

The present process involves treatment of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) with hydrazine hydrate in an organic polar solvent or mixture of organic solvents.
The product formation can be carried out at 25 to 80 oC and the quantity of quantity of hydrazine hydrate (50-80%) be used in amount ranging from 3 to 15 equivalents.
Suitable dimide source for reduction of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) to Gamithromycin of formula I: preferably from hydrazine hydrate with oxygen gas.
Suitable organic solvent or mixture thereof may be selected from polar solvents the group comprising lower alkyl alcohols such as ethanol, methanol, isopropyl alcohol, n-propanol, n-butanol etc and mixtures thereof. Also, polar aprotic solvents such acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide.
In one embodiment, the dimide source used is hydrazine hydrate with oxygen gas. Number of equivalents of hydrazine hydrate used ranges from 3 to 15 equivalents and one particular reaction the quantity used is from about 10 to 15 equivalents.
Temperature of the reaction ranges from about 25 to 80 oC and best results is obtained at temperature range of 25-30 oC.
Finally, the product is isolated from the reaction mixture by cooling it to a temperature range from about ?5 to 30 oC.
In another aspect the obtained Gamithromycin is slurred in polar protic solvents or mixtures thereof. Gamithromycin obtained by the present process is 95-98% pure and is further purified from polar protic or aprotic solvents selected from the group comprising alcohols like Acetone, DMF, acetonitrile, methanol, ethanol, isopropyl alcohol; water; or mixtures thereof.
The major process impurities which are eliminated during the purification of gamithromycin according to the invention herein are impurities of formula II, III, IV, V, VI and VII (as shown above), thus obtaining Gamithromycin in substantially pure form with a purity greater than 99%.
In one aspect the invention provides a novel process for preparing Gamithromycin of formula I comprising the steps of:
a. treating a solution of Allylated-Gamithromycin (i.e. Structure-5 in Scheme-I) in polar protic solvent or mixture thereof; with aqueous hydrazine hydrate (50-80%) or anhydrous hydrazine hydrate in the presence of oxygen gas in polar protic solvent;
b. stirring the solution for 24-36 hrs;
c. work up and adjust pH 3.0-9.5 range with hydrochloric acid or acetic acid or formic acid;
d. filtering and washing the solid with polar protic solvent or mixture thereof;
wherein the step (a) involves treatment of Allylated-gamithromycin (i.e. Structure-5 in Scheme-I) with hydrazine hydrate; and wherein the reaction steps (a) and (b) are carried out at temperature range about 25-80 oC.
The said polar protic solvents or mixture thereof may be selected from polar solvents the group comprising lower alkyl alcohols such as ethanol, methanol, isopropyl alcohol, n-propanol, n-butanol etic and mixtures thereof. Also, polar aprotic solvents such acetonitrile, acetone, dimethyl sulfoxide, N,N-dimethylformamide.
The said dimide source used is hydrazine hydrate with oxygen gas. Number of equivalents of hydrazine hydrate used ranges from 3 to 15 equivalents and one particular reaction the quantity used is from about 10 to 15 equivalents.
In another aspect of the invention provides a crystalline form of Gamithromycin of formula I, wherein the said crystalline form of Gamithromycin of formula I is characterized by powder X-ray diffraction pattern as presented in Figure-1 and differential scanning chromatogram (DSC) as presented in Figure-2.
The crystalline form of gamithromycin may be formed by recrystallizing the solution of crude gamithromycion in polar aprotic solvents at a temperature in the range of at 70-80 oC for a period of 2-3 hrs. The crystalline form of gamithromycin may be isolated by the steps of filtration, washing and drying or the combinations thereof.
In one embodiment the present invention provides a crystalline form of gamithromycin which may be characterized by X-ray diffraction pattern having peaks at 7.0, 9.7, 10.3, 11.6, 15.1 and 20.7 degrees two theta.
The crystalline form of said Gamithromycin may be characterized by X-ray diffraction as depicted in figure-1.
Thus in one embodiment the invention provides a process for preparing crystalline form of Gamithromycin (Formula I) comprising the steps of:
a. treating a solution of gamithromycin in polar aprotic or protic solvents at 25-30 oC;
b. heating the solution for 2-3 hrs at 70-80 oC;
c. cooling the suspension and filtering the solid at 25-30 oC;
d. washing and slurring the solid in polar aprotic or protic solvents or mixtures thereof; and
e. isolating Gamithromycin of Formula-I and drying at below 60-65 oC.
Another aspect the invention provides substantially pure crystalline form of gamithromycin having impurities less than 0.15% w/w of compounds of structural formula II, III, I, VI, VI and formula VII.
In one embodiment in any of the above said processes, the gamithromycin (formula I) obtained is more than 99% pure and comprises impurities less than 0.15% w/w of compounds of structural formula II, III, I, VI, VI and formula VII.
Moreover, the Gamithromyicn as obtained by the novel process by any of the processes as described above and above Scheme-1 is substantially pure, more specifically greater than 99% pure; and the known impurities such as impurity Formula II, III, IV, V, VI and VII are absent or very well controlled under the limit, more specifically said impurities are less than 0.15% w/w in the final product.
DEFINITIONS
The following terms shall have for the purpose of this application, including the claims appended here to, the respective meanings set forth below.
The term alcoholic solvent when used herein includes lower alkyl alcohols such as ethanol, methanol, isopropyl alcohol, n-propanol, n-butanol or the like.
The term solvent when used herein includes protic solvent, or mixtures thereof.
API: Active Pharmaceutical Ingredient
The following examples further illustrate the present invention, but should not be construed in any way as to limit its scope.
Example-1
Preparation of Gamithromycin of formula I
A solution of Allylated-Gamithromycin (i.e., Structure-5 in Scheme-I) (100 g, 0.126 mol) in isopropyl alcohol (1500 mmL) was added a solution of hydrazine hydrate (60.6, 1.89 mol) at 25-30 oC. The resultant reaction mass was stirred at same temperature for 24-36 hrs. The resulting solution was distilled under vacuum at 50-55 oC. Then, added 1000 mL of water and 1500 mL of ethyl acetate. Then, adjust pH of the reaction mass to 8-9.5 and extracted with ethyl acetate. The combined ethyl acetate layers were distilled under vacuum at 50-55 oC and solid suspension was cooled to room temperature, filtered and washed with isopropyl alcohol (200 mL). The obtained solid was dried for 10-12 hr at below 60-65 oC under reduced pressure to get title compound as white solid.
Yield: (80 g, 80%)
Purity: 99.14 (by HPLC)
Example-2
Preparation of Gamithromycin of formula I
A solution of Allylated-Gamithromycin (i.e., Structure-5 in Scheme-I) (100 g, 0.126 mol) in isopropyl alcohol (1500 mL) was added a solution of hydrazine hydrate (60.6, 1.89 mol) at 25-30 oC. The resultant reaction mass was stirred at 50-55 oC for 10-12 hrs. The resulting solution was distilled under vacuum at 50-55 oC. Then, added 1000 mL of water and 1500 mL of ethyl acetate. The, adjust pH of the reaction mass to 8-9.5 and extracted with ethyl acetate. The combined ethyl acetate layers were distilled under vacuum at 50-55 oC and solid suspension was cooled to room temperature, filtered and washed with isopropyl alcohol (200 mL). The obtained solid was dried for 10-12 hr at below 60-65 oC under reduced pressure to get title compound as white solid.
Yield: (85, 85%)
Purity: 99.19 (by HPLC)
Example-3
Preparation of Crystalline form of Gamithromycin of formula I
A solution of Gamithromycin (100 g, 0.128 mol) in acetonitrile (500 mL) was stirred at 70-80 oC for 2-3 hrs. The solid suspension was cooled to room temperature, filtered and washed with acetonitrile (200 mL). The obtained solid was dried for 10-12 hr at below 60-65 oC under reduced pressure to get title compound as white solid.
Yield: (90, 90%)
Purity: 99.19 (by HPLC)