FORM 2 THE PATENTS ACT, 1970
(Act 39 of 1970)
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
Title: “An improved process for the preparation of
Tigecycline intermediate and process for the preparation of Tigecycline therefrom”.

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of Tigecycline intermediate i.e. 9-amino minocycline of formula-1(C). More particularly, the present invention relates to a process for the preparation of 9-amino minocycline of formula 1(C) and a process for the preparation of Tigecycline of formula 1 from 9-nitro minocycline of formula 1(B).

BACKGROUND OF THE INVENTION
Tigecycline chemically known as (4S,4aS,5aR,12aS)-9-[2-(tert-butylamino)acetamido]-4,7- bis(dimethylamino)- 1 ,4,4a,5,5a,6, 11 , 12a-octahydro-3,l0,12,12a-tetrahydroxy-l,11-dioxo-2-naphthacene carboxamide represented by Formula-1.

Tigecycline is an antibiotic used for a number of bacterial infections. It is a glycylcycline administered intravenously. It was developed in response to the growing rate of antibiotic resistant bacteria such as Staphylococcus aureus, Acinetobacter baumannii, and E. coli. As a tetracycline derivative antibiotic, its structural modification has expanded its therapeutic activity to include Gram-positive and Gram-negative organisms, including those of multi-drug resistance.
Tigecycline is marketed by Pfizer under the brand name Tygacil. It was given a U.S. Food and Drug Administration (FDA) fast-track approval and was approved on 17 June 2005.
Formula-1 Tigecycline is reported to be active against methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycm-resistant enterococci and against organisms carrying either of the two major forms of tetracycline resistance: efflux and πbosomal protection (U.S. Patent Application No. 2007/0049562).
Tigecycline and its use are disclosed in U.S. Patent Nos. 5,494,903 and 5,529,990, respectively. U S. Patent Nos. 5,281,628; 5,401,863; 5,284,963; 5,675,030, U.S. 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.
There are several methods available for preparation of 9-amino minocycline of formula 1(C). Also the references are available for preparation of Tigecycline of formula (1) from 9-nitro minocycline of formula 1(B).

The methods disclosed in the cited literature teach three important steps i.e. nitration, reduction and acylation for the preparation of Tigecycline of formula 1 starting from minocycline of formula KSM-01.
One of the known methods involves nitration of minocycline to obtain 9-mtrominocycline of formula 1(B) which is subsequently reduced to 9-aminominocycline of formula 1(C). Finally acylation of 9-aminominocycline with N-t-butylglycyl chloride provides Tigecycline of formula 1 (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 1 (U.S. Patent No. 5,284,963; Drugs of the future 2001, 26(9), 851-858).
US20040214801A1 of Paratek Pharmaceuitcals Inc. has disclosed 9-substituted minocycline compounds. The said US Patent pertains, at least in part, to novel 9- substituted minocycline compounds. These minocycline compounds can be used to treat numerous tetracycline compound-responsive states, such as bacterial infections and neoplasms, as well as other known applications for minocycline and tetracycline compounds in general, such as blocking tetracycline efflux and modulation of gene expression.
According to scheme-1 of the said US Patent, it has disclosed several processes for producing novel 9-substituted minocycline compounds. In the patent it has been provided a general process for 9-amino minocycline of formula 1(C).
Generally, 9-substituted minocycline compounds can be synthesized as shown in Scheme-1 by treating minocycline (1 A), with sulfuric acid and sodium nitrate. The resulting product is 9-nitro minocycline (1B). The nitro minocycline compound is then treated with hydrogen gas and a platinum catalyst to yield the 9-amino minocycline compound, (1 C).


Almost all references have used a process for preparing 9-amino minocycline of formula 1(C) from –NO2 using Hydrogenation reaction. Hydrogenation reaction uses costly catalyst either H2/Pt or H2/Pd.
The said reaction is very hazardous and tedious process and separation of product out from Hydrogenation catalyst involves several stages which need to follow very carefully as it may cause fire and thus there is a huge risk involved in conducting the said reaction.
Further, Hydrogenation reaction has certain limitation like scale up is limited to few kilos of production and hence multiple commercial batches to be taken regularly. Thus time, men power, money and risk is very much associated with this hydrogenation reaction.
So it is necessary to avoid such tedious, risky, complex and hazardous reaction in order to achieve aimed commercial production without facing any risk or complexity.

None of the prior art reference has disclosed use of water and inorganic catalytic reagent Sodium dithionite for preparation of 9-amino minocycline of formula 1(C).
The process uses water and sodium dithionite during the reaction which is less cumbersome and easy to carry on commercial scale.
There is a demand of providing environment friendly process which is a simple, easy to operate, economically viable process for the preparation of 9-amino minocycline of formula 1(C) and process for the preparation of Tigecycline of formula 1 from 9-amino minocycline of formula 1(C).
OBJECTS OF THE INVENTION
Accordingly, the main object of the present invention is to overcome the problems faced by the prior art processes in the preparation of Tigecycline of formula 1.
There is an object of the present invention is to provide a simple, environment friendly, robust, easy to operate, economically viable process for the preparation of 9-amino minocycline of formula 1(C).
There is an object of the present invention is to provide a simple, environment friendly, robust, easy to operate, economically viable process for the preparation of Tigecycline of formula 1 from 9-nitro minocycline of formula 1(B).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process for the preparation of Tigecycline intermediate i.e. 9-amino minocycline of formula 1(C). More particularly, the present invention relates to a process for the preparation of 9-amino minocycline of formula 1(C) and a process for the preparation of Tigecycline of formula 1 from 9-Nitro minocycline of formula 1(B).


According to the embodiments (Scheme-2) of the present invention, there is provided a process for the preparation of preparation of 9-amino minocycline of formula 1(C) and preparation of Tigecycline of formula 1 from 9-nitro minocycline of formula 1(B).
In one of the embodiments, a process for the preparation of 9-amino minocycline of formula 1(C) is described, a process for the preparation of 9-amino minocycline of formula 1(C) comprises the stages of:
(1) Conducting nitration reaction of Minocycline HCl using HNO3:H2SO4 to give 9-nitro minocycline of formula 1(B);
(2) Dissolving 9-nitro minocycline of formula 1(B) in water and adjusted pH to 6.5 to 7.5 to get neutral reaction mixture;

(03) Reacting above mixture with sodium dithionite (Na2S2O4) in solvent for 2-5 hours at a temperature between 15-400C to give 9-amino minocycline of formula 1(C).
Scheme-2:

In stage-i, synthesis of 9-nitro minocycline of formula 1(B) is prepared by nitration reaction. In the said reaction, Minocycline hydrochloride of formula KSM-01 is dissolved in concentrated sulfuric acid (H2SO4)

at temperature between 0-50C. After dissolving minocycline HCl (KSM-01) in sulfuric acid (H2SO4), mixture of nitric acid (HNO3) and sulfuric acid (H2SO4) is added slowly and cautiously. The addition of HNO3 into the reaction is exothermic so extra care to be taken while addition of mixture of nitric acid (HNO3) and sulfuric acid (H2SO4). Reaction mass is stirred for 2-4 hrs. After completion of reaction, organic solvent is added and again stirred for 1-2 hrs and pour the same onto a cold Isopropyl alcohol (IPA) to get solid mass of stage-01. The reaction mass is then filtered and washed with IPA to get 9-nitro minocycline of formula 1(B).
Organic solvent is selected from alcohols such as methanol, ethanol or IPA.
The whole reaction is carried out at a temperature between 0-200C. 9-nitro minocycline of formula 1(B) is hygroscopic in nature so it is suggested to proceed further for stage-02 immediately after obtaining stage-01.
Table-1 provides comparative study of process known in the prior art references and the use of HNO3, H2SO4, mole ratio of KSM-01, solvent & HPLC purity of stage-01 according to the present invention.
In stage-02, Stage-01 is used as a wet cake in the reaction. 2 to 10 volume of water is added into the stage-01 and pH is adjusted between 6.5 to 7.7 using inorganic bases selected from sodium hydroxide (NaOH), sodium carbonate (Na2CO3), ammonia and sodium hydrogen carbonate (NaHCO3) to make the neutral reaction mixture. Adjusting pH is an important criteria for the next stage for reaction completion. In case if pH is more or less than 6.5 to 7.5, the impurities will be higher and thus it affects the overall yield and purity of 9-amino minocycline of formula 1(C) of stage-02 and final product Tigecycline of formula 1.

Further, if pH is adjusted with ammonia or sodium hydroxide (NaOH), it creates impurities during the reaction so preferred inorganic base is 1 to 1.5 mole of sodium carbonate (Na2CO3) or sodium hydrogen carbonate (NaHCO3) to adjust pH between 6.5 to 7.5.
After adjusting pH between 6.5 to 7.5, reaction is directly taken for stage-03, reduction stage. In stage-03, reaction is carried out in 2 to 15 volumes of solvent, the said solvent is selected from water, ethyl acetate, 1:4-dioxane or mixture thereof. The reaction is carried out at an ambient temperature; preferred temperature is between 15-400C.
In order to complete the reaction, 3-4 moles of reduction catalyst i.e. sodium dithionite (Na2S2O4) is used, sodium dithionite (Na2S2O4) is added lot wise slowly to maintain the temperature of the reaction. Inventors have surprisingly found that usage of water as solvent along with inorganic bases gives similar or better yield and purity compared to the prior art processes. Further use of water as solvent avoids the use of organic solvents and thus contributes towards greener chemistry and process for the preparation of stage-03.
After completion of reaction, reaction mass it taken for worked up to get 9-amino minocycline of formula 1(C). Table-2 provides combined stage-02 and stage-03 comparative study on processes known in the prior art references and the change of catalyst, base, solvent and selection of water as solvent in the reaction according to the present invention.
In a second embodiment, a process for the preparation of Tigecycline of formula 1 from 9-nitro minocycline of formula 1(B) is provided wherein a process for the preparation of Tigecycline of formula 1 from 9-nitro minocycline of formula 1(B) comprises the stages of:
(01) Conducting nitration reaction of Minocycline HCl using HNO3:H2SO4 to give 9-nitro minocycline of formula 1(B);

(2) Dissolving 9-nitro minocycline of formula 1(B) in water and adjusted pH to 6.5 to 7.5 to get neutral reaction mixture;
(3) Reacting above mixture with sodium dithionite (Na2S2O4) in solvent for 2-5 hours at a temperature between 15-400C to give 9-amino minocycline of formula 1(C).
(4) Reacting compound of formula 1(d) with 9-amino minocycline of formula 1(C) in water as solvent in presence of hydrochloric acid to give Tigecycline of formula 1.
In stage-i, synthesis of 9-nitro minocycline of formula 1(B) is prepared by nitration reaction. In the said reaction, Minocycline hydrochloride of formula KSM-01 is dissolved in concentrated sulfuric acid (H2SO4) at temperature between 0-50C. After dissolving minocycline HCl (KSM-01) in sulfuric acid (H2SO4), mixture of nitric acid (HNO3) and sulfuric acid (H2SO4) is added slowly and cautiously. The addition of HNO3 into the reaction is exothermic so extra care to be taken while addition of mixture of nitric acid (HNO3) and sulfuric acid (H2SO4). Reaction mass is stirred for 2-4 hrs. After completion of reaction, organic solvent is added and again stirred for 1-2 hrs and pour the same onto a cold Isopropyl alcohol (IPA) to get solid mass of stage-01. The reaction mass is then filtered and washed with IPA to get 9-nitro minocycline of formula 1(B).
Organic solvent is selected from alcohols such as methanol, ethanol or IPA.
The whole reaction is carried out at a temperature between 0-200C. 9-nitro minocycline of formula 1(B) is hygroscopic in nature so it is suggested to proceed further for stage-02 immediately after obtaining stage-01.
Table-1 provides comparative study of process known in the prior art references and the use of HNO3, H2SO4, mole ratio of KSM-01, solvent & HPLC purity of stage-01 according to the present invention.

In stage-02, Stage-01 is used as a wet cake in the reaction. 2 to 10 volume of water is added into the stage-01 and pH is adjusted between 6.5 to 7.5 using inorganic bases selected from sodium hydroxide (NaOH), sodium carbonate (Na2CO3), ammonia and sodium hydrogen carbonate (NaHCO3) to make the neutral reaction mixture. Adjusting pH is an important criteria for the next stage for reaction completion. In case if pH is more or less than 6.5 to 7.5, the impurities will be higher and thus it affects the overall yield and purity of 9-amino minocycline of formula 1(C) of stage-02 and final product Tigecycline of formula 1.
Further, if pH is adjusted with ammonia or sodium hydroxide (NaOH), it creates impurities during the reaction so preferred inorganic base is 1 to 1.5 mole of sodium carbonate (Na2CO3) or sodium hydrogen carbonate (NaHCO3) to adjust pH between 6.5 to 7.5.
After adjusting pH between 6.5 to 7.5, reaction is directly taken for stage-03, reduction stage. In stage-03, reaction is carried out in 2 to 15 volumes of solvent, the said solvent is selected from water, ethyl acetate, 1:4-dioxane or mixture thereof. The reaction is carried out at an ambient temperature; preferred temperature is between 15-400C.
In order to complete the reaction, 3-4 moles of reduction catalyst i.e. sodium dithionite (Na2S2O4) is used, sodium dithionite (Na2S2O4) is added lot wise slowly to maintain the temperature of the reaction. Inventors have surprisingly found that usage of water as solvent along with inorganic bases gives similar or better yield and purity compared to the prior art processes. Further use of water as solvent avoids the use of organic solvents and thus contributes towards greener chemistry and process for the preparation of stage-03.
After completion of reaction, reaction mass it taken for worked up to get 9-amino minocycline of formula 1(C).

Table-2 provides combined stage-02 and stage-03 comparative study on processes known in the prior art references and the change of catalyst, base, solvent and selection of water as solvent in the reaction according to the present invention.
Compound of formula 1(d) is prepared by reacting t-butyl amine of formula KSM-02 with t-butyl bromo acetate of formula KSM-03 as per the process reported in the prior art references.
In Stage-04, reaction of 9-amino minocycline of formula 1(C) with compound of formula 1(d) in water as solvent in presence of hydrochloric acid to give Tigecycline of formula 1. Inventors have surprisingly found that usage of water as solvent along with hydrochloric acid gives similar or better yield and purity compared to the prior art processes. Further use of water as solvent avoids the use of organic solvents and thus contributes towards greener chemistry and process for the preparation of stage-04.
After completion of the reaction, crude Tigecycline of formula 1 is purified using dichoromethane (MDC) and Methanol solvent to give pure Tigecycline of formula 1.
It is also possible to prepare crystalline form of Tigecycline of formula 1 using the methods known in the prior art technologies.
The reaction of all stages was monitored by HPLC. The present invention demonstrated examples cited below, which are provided as illustration only and therefore should not be construed as limitation of the possible and future invention.
EXAMPLES: Preparation of Tigecycline of formula 1: Stage-01: Preparation of 9-nitro minocycline of formula 1(B):
1 mole of Minocycline hydrochloride (KSM-01) is dissolved slowly in 5V of concentrated Sulfuric acid at 0-5 °C. Prepared a solution of 1V of 10% Nitric acid with concentrated sulfuric acid and added the said

mixture to the reaction mass and allowed to stir for 2-3hrs at 0-50C.
After completion of the reaction methanol is added to the reaction
mass and stirred for 1.0 hr & pour on cold IPA solution to get solid
material, filtered the reaction mass and suck dried well to get 9-nitro
minocycline of formula 1(B).
Yield: 95%
HPLC Purity: NLT 88%
Wet cake is used in next stage without drying.
Example 1 to 4 of stage-01 of formula 1 (B):
In a similar manner, Table-1 provides comparative study of process known in the prior art references and the use of HNO3, H2SO4, mole ratio of KSM-01, solvent & HPLC purity of stage-01 according to the present invention.
Table-1
Ex. No. KSM-01
(in
moles) Nitration using Solvent(s) (in volume) Remarks HPLC purity
1 1 HNO3+H2SO4 IPA:Haptane (10:1 V) As per prior art process 92
2 1 HNO3+H2SO4 IPA:Haptane (15:2 V) Reaction completed 93
3 1 HNO3+H2SO4 IPA 1(5V) Reaction completed 95
4 1 HNO3+H2SO4 IPA (15V) Reaction completed 96
Ex. KSM-01 Nitration Solvent(s) Remarks HPLC
No. (in using (in volume) purity
moles)
1 1 HNO3+H2SO4 IPA:Haptane As per prior 92
(10:1 V) art process
2 1 HNO3+H2SO4 IPA:Haptane Reaction 93
(15:2 V) completed
3 1 HNO3+H2SO4 IPA 1(5V) Reaction 95
completed
4 1 HNO3+H2SO4 IPA (15V) Reaction 96
completed
Stage-02 and Stage-03: Preparation of 9-amino minocycline of formula 1(C): Combined process of Stage-02 & Stage-03:
Tigecycline stage-01 500gm wet cake (100gm minocycline on dry basis) is dissolved in purified water 10V and sodium carbonate 1.5mole is added to get PH between 6.5 to 7.5 of the reaction mass. Sodium dithionite 3.5mole is added lot wise to the reaction mass and allowed to stir for 4hrs at a temperature between 15-400C. . After completion of the reaction, 1,4-dioxane and ethyl acetate 12V is added to the reaction to extract the product and distilled to get 9-amino minocycline of formula 1(C).

9-amino minocycline of formula 1(C) shall be taken for purification
using methanol, IPA and n-heptane to give pure 9-amino minocycline
of formula 1(C).
Yield: 70%
HPLC Purity: 94%
Example 1 to 7 of 9-amino minocycline of formula 1(C):
In a similar manner, Table-2 provides combined stage-02 and stage-03 comparative study on processes known in the prior art references and the change of catalyst, base, solvent and selection of water as solvent in the reaction according to the present invention.
Table-2

Ex. No. Stage-01 (in moles) catalyst Base Solvent(s) and
remarks
(in volume) HPLC
purity
(%)
1 1 Pd/C Not used IPA (Prior art process); reaction complied 90
2 1 Sodium dithionite (Na2S2O4) Not used Water; reaction not completed. NA
3 1 Sodium
dithionite
(Na2S2O4) Ammonia Water; Impurity is higher 75
4 1 Sodium
dithionite
(Na2S2O4) NaOH Water; Impurity is higher 70
5 1 Sodium
dithionite
(Na2S2O4) Na2CO3 Water; reaction
complied with less
impurity 85
6 1 Sodium
dithionite
(Na2S2O4) Na2CO3 Water; reaction
complied with less
impurity 90
7. 1 Sodium
dithionite
(Na2S2O4) Na2CO3 Water; reaction
complied with less
impurity 92
9-amino minocycline of formula 1(C) is used directly in next stage-04 of Tigecycline of formula 1 synthesis.

Preparation of Compound of formula 1(d): Preparation of compound of formula 1(d) is as per the process disclosed in the prior art references, the same is provided for reference purpose.
In to a round bottom flask, t-Butyl amine (KSM-02) and toluene are mixed at 250C to 350C and stirred for 10-15mins.The reaction mass is heated to 450C to 500C, t-Butyl bromoacetate (KSM-03) is added though addition funnel slowly to portion wise at 450C -500C and stirred the reaction mass for 2 hrs at 700C to 750C. cooled it to 250C -350C and filtered the product and wash it with toluene, filtrate contained the product. Tale the filtrate for distillation under vacuum at 450C-500C to obtain crude oil. In the said crude oil add toluene into it and hydrochloric acid is added slowly at 200C to 250C and stirred the reaction mass for 1 hrs. at 200C to 250C . Allow the content to settle for 20 minute and separate layers to collect toluene layer. Toluene is removed from the mass through distillation under vacuum, cooled the mass to 250C -300C, acetone is added and stirred it for 10-15 minutes at 00C to 50C to get white solid. Into the said product, chloroform and dimethyl formamide (DMF) is added and stirred at room temperature. Into the said reaction mass, thionyl chloride (SOCl2) is added slowly and then heated to reflux 600C -650C for 3-4 hrs under stirring. Cool the mass, filter the mass under nitrogen atmosphere and wash it with dichloromethane (MDC) to give compound of formula 1(d). Preserve it under nitrogen and use as such in the next step.
Stage-04: Tigecycline of formula 1:
1 mole of 9-amino minocycline of formula 1(C) of stage-03 is dissolved in 5 volume purified water, hydrochloric acid (HCl) is added to adjust pH between 4 to 5. 1.5mole of compound of formula 1(d) is added portion wise to the reaction mass and allow to stir for 3-4hrs at room temperature. Reaction mass is extracted with 10Volume dichloromethane, distilled it to get crude Tigecycline of formula 1.

Crude Tigecycline is purified using mixture of solvent MDC:Methanol
(2volume:3volume) and dried in oven to get pure Tigecycline of
formula 1.
Yield: 85%
HPLC purity: 99.7%.
ADVANTAGES OF THE INVENTION
1) Tigecycline of formula 1 obtained by this process is of high purity.
2) The process for the preparation of Tigecycline of formula 1 is cost-effective, environment friendly and commercially viable.
3) Stage-02 of the process is carried out in water as solvent by avoiding the use of organic or hazardous solvents and chemicals.
4) The present process does not require any chromatographic
purification.

We claim:
1. A process for the preparation of 9-amino minocycline of formula
1(C) comprises the stages of:
(1) Conducting nitration reaction of Minocycline HCl using HNO3:H2SO4 to give 9-nitro minocycline of formula 1(B);
(2) Dissolving 9-nitro minocycline of formula 1(B) in water and adjusted pH to 6.5 to 7.5 to get neutral reaction mixture;
(3) Reacting above mixture with sodium dithionite (Na2S2O4) in solvent for 2-5 hours at a temperature between 15-400C to give 9-amino minocycline of formula 1(C).

2. The process for the preparation of Tigecycline of formula 1 as claimed in claim-1 wherein stage-01 is carried at a temperature between 0-200C.
3. The process for the preparation of Tigecycline of formula 1 as claimed in claim-1 wherein in stage-02, pH is adjusted using inorganic base selected from sodium hydroxide (NaOH), sodium carbonate (Na2CO3), ammonia and sodium hydrogen carbonate (NaHCO3).
4. A process for the preparation of Tigecycline of formula 1 from 9-nitro minocycline of formula 1(B) comprises the stages of:

(1) Conducting nitration reaction of Minocycline HCl using HNO3:H2SO4 to give 9-nitro minocycline of formula 1(B);
(2) Dissolving 9-nitro minocycline of formula 1(B) in water and adjusted pH to 6.5 to 7.5 to get neutral reaction mixture;
(3) Reacting above mixture with sodium dithionite (Na2S2O4) in solvent for 2-5 hours at a temperature between 15-400C to give 9-amino minocycline of formula 1(C);
(4) Reacting compound of formula 1(d) with 9-amino minocycline of formula 1(C) in water as solvent in presence of hydrochloric acid to give Tigecycline of formula 1.

5. The process for the preparation of Tigecycline of formula 1 as claimed in claim-4 wherein stage-01 is carried at a temperature between 0-200C.
6. The process for the preparation of Tigecycline of formula 1 as claimed in claim-4 wherein in stage-02, pH is adjusted using inorganic base selected from sodium hydroxide (NaOH), sodium carbonate (Na2CO3), ammonia and sodium hydrogen carbonate (NaHCO3).
7. The process for the preparation of Tigecycline of formula 1 as claimed in claim-4 wherein in stage-03, solvent is selected from water, ethyl acetate, 1:4-dioxane or mixture thereof.