PROCESS FOR THE PREPARATION OF TIGECYCLINE
Field of the Invention
The present invention relates to improved process for the preparation of Tigecycline.
Background of the Invention
Tigecycline chemically is (4S,4aS,5aR,12aS)-9-[2-(tert-butylamino)acetamido]-4,7-bis(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,10,12,12a-tetrahydroxy-1,11 -dioxo-2-naphthacenecarboxamide represented by Formula I.
(Formula Removed)
Tigecycline is a glycylcycline antibiotic developed and marketed by Wyeth under the brand name Tygacil. It has expanded broad-spectrum antibacterial activity both in vitro and in vivo. Glycylcycline antibiotics, like tetracycline antibiotics, act by inhibiting protein translation in bacteria.
Tigecycline may be used as a treatment against drug-resistant bacteria, and it has been shown to work where other antibiotics have failed. For example, it is active against methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant enterococci and against organisms carrying either of the two major forms of tetracycline resistance: efflux and ribosomal protection (US 2007/0049562).
Tigecycline may be used in the treatment of many bacterial infections, such as complicated intra-abdominal infections, complicated skin and skin structure infections, Community Acquired Pneumonia, and Hospital Acquired Pneumonia indications, which may be caused by gram-negative and gram-positive pathogens, anaerobes, and both methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus (MSSA and MRSA). Additionally, tigecycline may be used to treat or control bacterial infections in warm-blooded animals caused by bacteria having the TetM and TetK resistant determinants. Also, tigecycline may be used to treat bone and joint infections, catheter-related Neutropenia, obstetrics and gynecological infections, or to treat other resistant pathogens, such as VRE, ESBL, enterics, rapid growing mycobacteria, and the like (US 2007/0049562).
Tigecycline and its use for treating bacterial infections are disclosed in US patents 5.494,903 and 5,529,990, respectively. US patents 5,281.628; 5,401,863; 5,284,963; 5,675,030; US patent application 2007/0049562 and PCT patent application WO 05/056538 disclose either process for preparation of tigecycline or intermediate compounds thereof. Two synthetic methods for preparing tigecycline are disclosed by Drugs of the future 2001, 26(9), 851-858. The preparation of intermediate compounds is also disclosed by J. Med. Chem. 1994, 37(1), 184-188.
The methods disclosed in these patents/applications or journal articles mention three important steps i.e. nitration, reduction and acylation for the preparation of tigecycline starting from minocycline.
One of the known methods involves nitration of minocycline to obtain 9-nitrominocycline which is subsequently reduced to 9-aminominocycline. Finally acylation of 9-aminominocycline with N-t-butylglycyl chloride provides tigecycline of formula I {Drugs of the future 2001, 26(9), 851-858).
Another method describes reaction of 9-aminominocycline with chloroacetyl chloride to produce an acylated intermediate which on reacting with t-butylamine provides tigecycline of formula I (US patent 5,284,963; Drugs of the future 2001, 26(9), 851-858).
The acylation step as mentioned in patents/applications or journal articles for the preparation of tigecycline of formula I is either performed in presence of water alone or in a mixture of l,3-dimethyl-3,4,5,6-tetrahydro-2(lH)-pyrimidinone (hereinafter called DMPU) and acetonitrile.
The prior processes give tigecycline along with several impurities which are difficult to separate. The overall reaction time is lengthy and yield obtained at various steps is also less. During acylation step, the reagents used such as DMPU and acetonitrile are expensive and further work-up for isolation of tigecycline is tedious. In case where water alone is used during acylation, lot of frothing is observed during pH adjustment while extracting out tigecycline.
Summary of the Invention
The process of present invention for preparing tigecycline is advantageous in many
aspects over prior art processes as there is less formation of degradation products or
impurities. The pH of the reaction mixture at the acylation stage is maintained below 3 for the
effective production of tigecycline in good yields. Use of a mixture of organic solvent and
water during the acylation stage is advantageous as there is no frothing observed during pH adjustment while extracting out tigecycline.
The present invention provides improved process for the preparation of tigecycline comprising reacting 9-aminominocycline of formula II with N-t-butylglycyl chloride of formula III in presence of water and organic solvent at pH below 3. The acylation reaction is performed at temperature level of 0-10°C.
Detailed Description of the Invention
Following terminology(s) can be used to understand the processes of the present invention. These terminology(s) are not intended in any way to limit the scope of the present invention. Several variants of these terminology(s) would be evident to persons ordinarily skilled in the art.
The terms 'minocycline', '9-nitrominocycline' or '9-aminominocycline' as used herein is meant to present as free base or include pharmaceutically acceptable salt form thereof. For example, the term minocycline wherever mentioned includes minocycline as free base or in pharmaceutically acceptable salt thereof and if the term minocycline hydrochloride is mentioned, it specifically mean hydrochloride salt form of minocycline.
The term 'pharmaceutically acceptable salt' as used herein refers to acid addition salt. Exemplary salt include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, phosphoric, nitric, sulfuric, acetic, benzoic, citric, fumaric, glycolic, maleic, succinic, tartaric, sulfate, alkyl (C1-C6) or aryl (C6-C10) sulfonates and chlorobenzenesulfonate salt. Preferred pharmaceutically acceptable salt is hydrochloride or sulfate salt.
A first aspect of present invention provides process for the preparation of tigecycline comprising reacting 9-aminominocycline of formula II
(Formula Removed)
with N-t-butylglycyl chloride of formula III

(Formula Removed)
in presence of water and organic solvent.
In an embodiment of this aspect the reaction can be performed at pH below 3.
In another embodiment of this aspect the reaction can be performed at temperature level of 0-10°C.
In another embodiment of this aspect the organic solvent is selected from the group comprising of dichloromethane (DCM), ethylene dichloride, chloroform, ethyl acetate, DMPU, acetonitrile and mixtures thereof. Preferably, the organic solvent selected is dichloromethane (DCM).
In another embodiment of this aspect, the organic solvent is dichloromethane (DCM).
Accordingly, 9-aminominocycline is dissolved in deionized water purged with nitrogen gas followed by addition of organic solvent (for example, dichloromethane). The pH of the resultant solution is adjusted to 2 to 3 by addition of sodium carbonate solution and then N-t-butylglycyl chloride is added to the solution while controlling pH to 2-3 with sodium carbonate solution at temperature of 0-10°C. The resultant reaction mixture is stirred and tigecycline is isolated from the mixture.
In another embodiment of this aspect, the tigecycline is isolated by adjusting the pH of the reaction mixture to 6.7 ±0.2, separating the organic layer and recovering organic solvent from the organic layer.
The crude tigecycline obtained by aforementioned manner can be then crystallized or purified using methanol.
In another embodiment of this aspect, the 9-aminominocycline is prepared by nitrating minocycline at -1° to -10°C followed by subsequent reduction of formed 9-nitrominocycline with hydrogen in presence of palladium on carbon.
A second aspect of the present invention provides process for isolation of tigecycline from reaction mixture thereof comprising
a) adjusting pH of the reaction mixture to 6.7 ±0.2 followed by separation of the organic layer
b) treating the organic layer obtained in step a) with activated carbon followed by filtration
c) recovering solvent from the filtrate of step b) to obtain crude tigecycline
d) dissolving crude tigecycline into methanol followed by filtration to obtain clear solution
e) crystallizing tigecycline from the clear solution of step d).
In an embodiment of this aspect, the reaction mixture comprises of water, organic solvent and tigecycline. More specifically, the reaction mixture is the resultant of reaction of 9-aminominocycline with N-t-butylglycyl chloride in presence of water and organic solvent.
In another embodiment of this aspect, the pH of the reaction mixture is adjusted to 6.7 ±0.2 by adding sodium carbonate solution into it.
In another embodiment of this aspect, the organic layer comprises tigecycline and organic solvent.
In another embodiment of this aspect the organic solvent is selected from the group comprising of dichloromethane (DCM), ethylene dichloride, chloroform, ethyl acetate, DMPU, acetonitrile and mixtures thereof. Preferably, the organic solvent selected is dichloromethane (DCM).
In another embodiment of this aspect, the organic solvent is dichloromethane (DCM).
In another embodiment of this aspect, the solvent is recovered from the filtrate of step b) under vacuum.
In another embodiment of this aspect, the crude tigecycline is dissolved in methanol by heating at 40 to 60°C.
The term clear solution in this aspect refers to the solution of tigecycline in methanol wherein undissolved tigecycline or other solid particle(s) is not present.
In another embodiment of this aspect, the crystallization of tigecycline from the clear solution thereof is performed either through cooling of the solution or adding chilled methanol into the solution.
In another embodiment of this aspect, the cooling of the solution can be performed till the temperature of the solution is reached at or in between -10° to about 5°C.
The crystals obtained are isolated by removing methanol either through filtration, drying, flushing with nitrogen or evaporation.
Accordingly, the reaction mixture comprising tigecycline which is formed by the acylation reaction of 9-aminominocycline with N-t-butylglycyl chloride in presence of mixture of organic solvent and water is subjected to pH adjustment using sodium carbonate solution. When the pH of the reaction mixture attains 6.7 ±0.2 than organic layer is separated and it is subjected to carbon treatment followed by filtration through hyflo having sodium sulphate bed. The organic solvent is then completely recovered from the filtrate leaving a residue. The residue is dissolved in methanol by heating at 40-60°C. The solution formed is cooled to obtain crystals of tigecycline which are separated and dried.
The N-t-butylglycyl chloride used hereinabove for the preparation of tigecycline is prepared by reacting chloroacetyl chloride with benzyl alcohol to obtain benzyl chloroacetate which upon treatment with t-butylamine produces benzyl N-t-butyl glycinate. Benzyl N-t-butyl glycinate is hydrolyzed to obtain corresponding acid. The acid is then chlorinated to obtain N-t-butylglycyl chloride hydrochloride.
While the present invention has been described in terms of its specific aspects, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.
In the following section aspects are described by way of examples to illustrate the processes of the invention. However, these are not intended in any way to limit the scope of the present invention. Several variants of the examples would be evident to persons ordinarily skilled in the art.
EXAMPLE-1
Preparation of Tigecycline
To a solution of 9-aminominocycline (75 g) in de-ionized water (225 ml) purged with nitrogen gas, dichloromethane (375 ml) was added. Sodium carbonate solution was drop-wise added to it at 0-5°C to adjust pH of the solution to 2.5-3. To this solution, N-t-butyl glycyl chloride (95 g) was added while controlling pH at 2-3 with sodium carbonate solution at 0-5°C. The reaction mixture obtained was stirred for 15 minutes and added to dichloromethane (5 L) at 5-10°C under nitrogen. The pH of the resultant mixture was adjusted to 6.8 with sodium carbonate solution and it was stirred for 20 minutes. Organic layer was separated and subjected to carbon treatment with activated carbon (15 g). To the aqueous layer, dichloromethane (1 L) was added followed by pH adjustment at 6.8. It was stirred and allowed to settle. The organic layer was separated and mixed with the previous organic layer. The combined organic layer after carbon treatment was filtered through hyflo having sodium sulphate bed. The bed was washed with dichloromethane (250 ml). Dichloromethane was completely recovered from the filtrate at 39-40°C under vacuum and the obtained residue was degassed for 15 minutes [wet weight of the residue (crude tigecycline) after complete recovery of dichloromethane was 52.5 g].
To the obtained residue, methanol (225 ml) was added and it was stirred. The slurry was cooled to 0-5°C and stirred for 30-45 minutes. The slurry was then filtered through
sintered funnel under nitrogen. The cake obtained was washed with chilled methanol (300 ml) at -5 to 0°C and flushed with nitrogen till the removal of maximum solvent. The product so obtained was kept for drying under vacuum at ambient temperature (20-30°C) for 1 hour. It was then dried at 62-65°C for 10-12 hours under vacuum.
Wet weight: 60 g
Dry weight: 33 g
Chromatographic purity: 99.49% (by HPLC)
EXAMPLE-2 Preparation of Tigecycline
To a solution of 9-aminominocycline (25 g) in de-ionized water (75 ml) purged with nitrogen gas, dichloromethane (150 ml) was added under nitrogen atmosphere. Sodium carbonate solution was drop-wise added to it at 0-5°C to adjust pH of the solution to 2.5-3. To this solution, N-t-butyl glycyl chloride hydrochloride (32 g) was added while controlling pH at 2-3 with sodium carbonate solution at 0-5°C. The reaction mixture obtained was stirred for 15 minutes and transferred to dichloromethane (1.75 L) at 5-10°C under nitrogen. The pH of the resultant mixture was adjusted to 6.8 with sodium carbonate solution and it was stirred for 20 minutes. Organic layer was separated and subjected to carbon treatment with activated carbon (5 g). To the aqueous layer, dichloromethane (750 ml) was added followed by pH adjustment at 6.8. It was stirred and allowed to settle. The organic layer was separated and mixed with the organic layer which was under carbon treatment. The combined organic layer was filtered through hyflo having sodium sulphate bed. The bed was washed with dichloromethane (250 ml). Dichloromethane was completely recovered from the filtrate at 39-40°C under vacuum and the obtained residue was degassed for 20 minutes [chromatographic purity of the obtained residue (crude tigecycline) was 95.54%].
To the obtained residue, methanol (750 ml) was added and it was stirred. The slurry was cooled to 0°C and stirred for 35-45 minutes. The slurry was then filtered through sintered funnel under nitrogen. The cake obtained was washed with chilled methanol (100 ml) at -2 to 0°C and flushed with nitrogen till the removal of maximum solvent. The product so obtained was kept for drying under vacuum at ambient temperature (20-30°C) for 1 hour. It was then dried at 62-65°C for 12-14 hours under vacuum.
Wet weight: 16 g (chromatographic purity 99.6%)
Dry weight: 12 g
EXAMPLE-3 Purification of Tigecycline
To de-ionized water (80 ml) purged with nitrogen gas, crude tigecycline (10 g) was added at 10°C under nitrogen atmosphere. It was stirred to dissolve and then cooled to 0°C under nitrogen. Methanol (20 ml) was added to the resultant solution and stirred for 5 minutes (pH of the solution was 8.1) followed by addition of dichloromethane (700 ml) into it. To adjust the pH of the solution to 6.8, a mixture of concentrated hydrochloric acid and deionized water (1:10) (2 ml) was added to the solution. The pH of the solution was 6.5. The pH was adjusted to 6.8 by addition of ammonia solution (1:10) (1 ml). The solution was stirred for 10 minutes at 5°C under nitrogen atmosphere and then allowed to settle for 5 minutes. The organic layer was separated. To the aqueous layer, dichloromethane (200 ml) was added at 8-10°C under nitrogen and again organic layer was separated and combined with previously obtained organic layer. The combined organic layer was filtered through sodium sulphate bed followed by washing of the bed with dichloromethane (100 ml) at 20-30°C. The solvent was completely recovered from the filtrate at 30-5°C leaving behind wet product which was degassed under high vacuum for 10 minutes. Hot methanol of 45-50°C (700 ml) was added to the degassed product and the resultant solution was filtered through hyflo bed followed by washing of the bed with methanol (300 ml) under nitrogen pressure. The clear solution (filtrate) obtained was stirred at 20-25°C for 3 hours under nitrogen atmosphere. The crystals so formed were obtained through filtration followed by washing with methanol (40 ml) at 20-30°C. These were flushed with nitrogen for the removal of solvent and kept for drying at 20-30°C for 30 minutes and then for 12 hours at 60-65°C.
Weight of dried product: 6.9 g
Chromatographic purity: NLT 99.6%

WE CLAIM:
1. A process for the preparation of tigecycline comprising reacting 9-aminominocycline
of formula II
(Formula Removed)
with N-t-butylglycyl chloride of formula III
(Formula Removed)
in presence of water and organic solvent.
2. The process of claim 1 wherein the reaction is performed at pH below 3.
3. The process of claim 1 wherein the reaction is performed at temperature range of 0-10°C.
4. The process of claim 1 wherein organic solvent is selected from the group comprising of dichloromethane, ethylene dichloride, chloroform, ethyl acetate, DMPU, acetonitrile and mixtures thereof.
5. A process for isolation of tigecycline from reaction mixture thereof comprising

a) adjusting pH of the reaction mixture to 6.7 ±0.2 followed by separation of the organic layer
b) treating the organic layer obtained in step a) with activated carbon followed by filtration
c) recovering solvent from the filtrate of step b) to obtain crude tigecycline
d) dissolving crude tigecycline into methanol followed by filtration to obtain clear solution
e) crystallizing tigecycline from the clear solution of step d).
6. The process of claim 5 wherein the reaction mixture comprises of water, organic
solvent and tigecycline.
7. The process of claim 6 wherein the organic solvent is selected from the group
comprising of dichloromethane, ethylene dichloride, chloroform, ethyl acetate,
DMPU, acetonitrile and mixtures thereof.
8. A process for the preparation, isolation and purification of tigecycline substantially as
described and exemplified herein.