WO2008059516A2 - Procédé de purification de macrolides - Google Patents

Procédé de purification de macrolides Download PDF

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Publication number
WO2008059516A2
WO2008059516A2 PCT/IN2007/000352 IN2007000352W WO2008059516A2 WO 2008059516 A2 WO2008059516 A2 WO 2008059516A2 IN 2007000352 W IN2007000352 W IN 2007000352W WO 2008059516 A2 WO2008059516 A2 WO 2008059516A2
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WO
WIPO (PCT)
Prior art keywords
tacrolimus
aqueous
solvent
purification
water
Prior art date
Application number
PCT/IN2007/000352
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English (en)
Other versions
WO2008059516A3 (fr
Inventor
Pardeep Narula
Jayesh Patel
Sudhir Vaid
Pragnesh Patel
Original Assignee
Concord Biotech Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Concord Biotech Limited filed Critical Concord Biotech Limited
Publication of WO2008059516A2 publication Critical patent/WO2008059516A2/fr
Publication of WO2008059516A3 publication Critical patent/WO2008059516A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/08—Bridged systems

Definitions

  • the present invention relates to a novel process for purification of macrolides such as tacrolimus, rapamycin, ascomycin and immunomycin using a polymer resin and copper salts.
  • the present invention also includes the use of a novel polymer resin and cupric ions in column chromatography for the purification of macrolides. More particularly, it relates to process for preparing highly pure Tacrolimus with separation of four impurities. Separation is obtained by adsorbing crude Tacrolimus over nonionic adsorption resin pretreated with cupric ions. '
  • Macrolides such as tacrolimus, rapamycin, ascomycin and immunomycin have difficulties associated with removal of impurities from the final compound.
  • Tacrolimus FK 506 is macrolide compound, known for its immunosuppressant activity. This compound is used for the treatment of autoimmune disorder, infectious diseases and prevention of resistance occurs during organ transplantation.
  • Tacrolimus was first described in US 4,894,366 and EP 184,162 patent.
  • the compound was produced from Streptomyces tsukubaensis, Streptomyces hygroscopicus subsp. yakushimaenis naturally contain several impurities; which were purified with column chromatography using reverse phase silica gel column, non-ionic silica gel column, non- ionic HP-20 resin with silver ions, cation exchange resin with silver ions and using Preparative HPLC with different solvents for elution. Removal of impurities from the active pharmaceutical compound is very important for the prevention of health hazards to the patients. Many techniques are used to remove the impurities from the active compound.
  • J. Chromatography 149(1978)417 describes the use of silver ions for separating cis-trans isomers of unsaturated aliphatic acid having same number of carbon atoms.
  • Higaki et al in WOO 1/18007 disclosed a method for separating of lactone containing high molecular weight compound Tacrolimus.
  • Compound having at least one of a lower alkenyl group and lower alkoxy group as its side-chain were separated from analogues of Tacrolimus in one or two steps; first step of adsorption was through a nonionic adsorption resin, eluting with silver ion containing aqueous solvent, and in the second step adsorbing to a basic active alumina eluting with organic solvent or in reverse order.
  • the method involved silver ions which are difficult to use at production scale due to its light sensitive nature and it being highly corrosive.
  • WO/2005/010015 Al describes method of purifying Tacrolimus includes loading macrolide to a bed of sorption resin and eluting with water and organic solvent tetrahydrofuran and acetonitrile.
  • WO05/019226 claims a process for the recovery of substantially pure form of rapamycin, tacrolimus or immunomycin. The process comprised of: macrolide treated with water immiscible solvent followed by concentration; mixed with water, water immiscible solvent or mixture thereof then passed through hydrophobic interaction chromatography, fractions collected thereof were concentrated, further water miscible solvent was added, thereafter passed through silica gel chromatography and from the collected fractions macrolide was isolated in pure form. It is known to practitioners that silica gel works better with solvents rather than aqueous solvents.
  • WO05/054253 describes a process for the purification of macrolides.
  • the process comprised of: crude macrolide was treated with water immiscible solvent followed by optional concentration; treated with ammonia gas, separated the impurities and concentrate it, thereafter loaded on silica gel chromatography optionally reverse phase or pretreated with silver and pure form of macrolide were collected, further optional repetition of silica gel and reverse phase chromatography to obtain pure macrolide.
  • US2006/0142565 Al describes pure Tacrolimus and method for purifying by loading charge of tacrolimus in juxtaposition on bed of resin, eluting with tetrahydrofuran (THF), acetonitrile or combination thereof, water and optionally one additional organic solvent.
  • THF tetrahydrofuran
  • the heart cut of eluent is collected and tacrolimus is collected then crystallized and recrystallized until reduce the impurity level. This method suffers serious drawbacks regarding yield.
  • WO06/031664 Al describes a process for separation of tacrolimus and its analogues, ascomycin and tsucubamycin B; wherein purification of tacrolimus is accomplished in two steps using two different chromatographic sorbents. One is accomplished on normal phase chromatography and another on silica gel column with silver nitrate and organic solvents. This makes it a time and resource consuming process.
  • Reverse phase system and use of silver ion in chromatography are not convenient because their water containing mobile phase facilitates isomerization of Tacrolimus to its tautomers.
  • use of resin HP-20 with silver nitrate to obtain pure form of Tacrolimus is not economical.
  • passing the crude Tacrolimus material multiple times through chromatography is a tedious procedure and commercially not feasible.
  • the procedure for purification of Tacrolimus through polymer resin containing silver ions describes separation of only two analogues such as Ascomycin and Dihydro Tacrolimus, while the procedure described in the present invention using cupric nitrate over the non-ionic adsorption resin segregates other analogues such as Delta lactone, Methyl derivative, Demethomycin and Pyrrole analogue (FK-525) thereby rendering a clean product.
  • cupric nitrate is much easy to handle as compared to all types of silver ion metal salts during use.
  • the resin used contain polyvinyl pyrrolidone, methyl acrylate, divinyl benzene based compounds and metal salt used for purification belongs to transition metals like copper, nickel, lead etc.
  • the same metal salts can also be used along with other neutral non ionic adsorption resins of polyethylene i.e. HP20, HP20SS, Sepabeads SP207 and the like.
  • Tacrolimus obtained after extraction and final crystallization contains any individual impurity is below 0.15% (as per ICH guidelines 0.15% is the qualification limit) and total impurity level is less than 0.50% excluding Tautomers.
  • the invention presents an economical and efficient process to remove impurities from macrolides.
  • the main object of the present invention is to provide a process for separation of analogues of Tacrolimus such as Demethomycin, Pyrrole analogue, Ascomycin, Delta lactone, dihydro and Methyl derivative as shown in following structures
  • Another object of the present invention is process of purification of crude Tacrolimus using a novel polymer resin of Polyvinyl pyrrolidone (K-30) type loaded with aqueous solvent solution of a reagent containing cupric ions.
  • K-30 Polyvinyl pyrrolidone
  • a copper salt contain in the copper ion containing aqueous solvent preferred one is cupric nitrate, cupric chloride or the like.
  • Another object of the present invention is process for purification of crude Tacrolimus using a nonionic adsorbent resin of polyethylene like HP20 loaded with aqueous solvent containing cupric salts.
  • the polymer resin used can be a polyvininyl pyrrolidone resins having partial structure of divinyl benzene and polyvinyl pyrrolidone.
  • the polymer resin used can be a polyethylene resins having partial structure of Divinyl benzene.
  • the concentration of copper ions may be generally 0.20 to 1.5 mol/L, preferably 0.30 to
  • an aqueous medium for the copper ion containing aqueous solvent an aqueous acetone, an aqueous alcohol, an aqueous acetonitrile or the like may be exemplified.
  • the quantity of adsorbent used is about 40 times more than the wt. of the compound.
  • the flow rate of the eluting solvent varies from 0.5 bed volume to 1.2 bed volume per hour, preferably at 0.9 to 1.0 bed volume per hour.
  • the fractions containing pure product were analyzed through HPLC are combined and extracted with water immiscible solvent like ethyl acetate. The solvent was evaporated under reduced pressure and a dried powder obtained was further purified to yield Tacrolimus pure in solvents like Diisopropyl ether and Acetonitrile -water.
  • the present invention encompasses a process of purification of macrolides such as Tacrolimus.
  • polymer resin is prepared and loaded over column and compound is adsorbed over it using aqueous solvents and then a compound is eluted with aqueous salts containing cupric ions for purification of macrolides.
  • a non ionic adsorption polymer resin is prepared and loaded over a column and the macrolide compound is adsorbed over the polymer resin and eluted with solvents such as aqueous solvents containing transition metal ions.
  • solvents such as aqueous solvents containing transition metal ions.
  • the preferred embodiment of the invention covers a process for purification of tacrolimus by adsorbing tacrolimus over non ionic adsorption resin and eluting it with an aqueous solvent containing cupric ions to separate impurities.
  • the process for the preparation of a novel polymer resin comprises of the following steps:
  • Polymerized beads from the reactor are transferred into grading vessel by opening the bottom valve.
  • the beads are washed thoroughly with water and then with methanol in the grading vessel.
  • the polymerized beads now filtered through Nutsche Filter (NF) under vacuum.
  • the filtered slurry containing polymerized beads and methanol is use as polymer resin in the column chromatography.
  • polymer resin is charged in column then loaded with crude Tacrolimus which is produced by fermentation process using mutated culture strain of Streptomyces tsukubaensis; under submerged aerobic fermentation conditions culturing in aqueous nutrient medium containing vegetable oil as carbon source and nitrogen, mineral salts and other defoaming agents at temperature between 20 - 40° C, for 48 - 216 hours at pH 7.
  • the fermentation broth obtained is extracted in toluene.
  • the toluene extract then concentrated, column chromatographed over silica gel using ethyl acetate: hexane eluent to obtain crude Tacrolimus.
  • Crude Tacroliums recovered from the broth is further purified over the polymer resin in column chromatography. Purification is done with using aqueous solvents like acetone water, methanol water, THF water and acetonitrile water, preferably with acetone water.
  • Methanol is charged to a column.
  • Polymer resin is added slowly in methanol. Resin is then allowed to stand for 24 hours for activation. After 24 hrs methanol is drained and resin is washed thoroughly with R.O. water till neutral pH is attained.
  • the resin is then equilibrated with a solution of 4BV acetone: water (40:60) at a flow rate of 0.6 - 1.5 BV/hr. After equilibration solution of crude Tacrolimus is prepared at a concentration of 0.30 - 0.60 g/L preferably at 0.30 - 0.50 g/L and most preferably at 0.5 g/L. The solution of compound is then charged over the resin.
  • the column is then washed with 4 By of acetone : water (40:60 ) at a flow rate of 0.6 - 1.5 BV/hr preferably at 0.8 - 1.1 BV/hr and most preferably at 1 BV/hr.
  • the elute is collected and analysed using HPLC. Collection of elute and it's analysis did not show any product.
  • the compound is eluted with a 8 BV solution of acetone: water (50:50) containing cupric ions preferably cupric nitrate, cupric chloride and the like.
  • a solution of copper nitrate at a concentration of 0.25-1.20mole/L in 8 BV acetone : water (50:50) is prepared, preferably at 0.3-0.70mole/L and most preferably at 0.30-0.40 mole/L, The solution is then added over the column and fractions were collected of around half bed volume and analyzed through HPLC to check impurity profile i of Tacrolimus. The fractions containing pure Tacrolimus were pooled and further diluted with water. The compound is then extracted into ethyl acetate and then concentrated to yield Tacrolimus pure in oil form. To the oil added Diisopropyl ether and few mL of water and a mixture was stirred to give Tacrolimus monohydrate as crystalline solid.
  • the compound was filtered and washed with Di isopropyl ether and then dried to give Tacrolimus pure having chromatographic purity more than 98%. Chromatogram purity analysis showed higher content of tautomer and impurity at RRT 1.12 i.e. delta lactone.
  • the compound is then dissolved in acetonitrile (3X) and water (10X). The mixture is stirred for 2 hours at RT and then filtered, dried to yield Tacrolimus having total impurity content less than 1%, excluding Tautomers.
  • Delta lactone impurity in pure Tacrolimus is present at less than about 0.30%, preferably less than 0.10% .
  • Methyl derivative content in pure Tacrolimus is present at less than about 0.3%.
  • the column is washed with 4BV of methanol : water (40:60) at flow rate of lBV/hr.
  • the elute is collected and checked for HPLC.
  • the column is eluted with a solution of 8BV acetone : water (50:50) at flow rate of 1 BV/hrs.
  • Fractions of 0.5 BV are collected and checked through HPLC. Pure fractions from 10-19 are pooled and compound is obtained by extracting it in ethyl acetate and concentrating the solvent to yield oily residue which can be crystallized in
  • the Tacrolimus obtained contains tautomer around 2.77% and delta lactone impurity around 0.25%.
  • the compound is then dissolved in acetonitrile (3X) and crystallized by adding water (10X) at room temperature only.
  • Pure Tacrolimus obtained after filtration and drying contain Tautomer 0.38% and delta lactone impurity below detection level.
  • the product purity of compound is more than 99.0%.
  • one or more additional crystallization steps may be performed to remove the impurity.
  • the elute is collected and checked for HPLC.
  • the column is eluted with a solution of 8BV acetone: water (50:50) at flow rate of 1 BV/hrs. Fractions of 0.5 BV are collected and checked through HPLC. Pure fractions are pooled and compound is obtained in pure form as per the procedure in example-2.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)

Abstract

L'invention est un nouveau procédé pour la purification de macrolides tel que Tacrolimus à l'aide d'une nouvelle résine polymère et de sels de cuivre contenant des ions cuivrique. La nouvelle résine polymère contient des composés à base de polyvinyl pyrrolidone, de méth acrylate, de divinyl benzène. Dans ce procédé, la fabrication d'une nouvelle résine polymère et l'utilisation d'un sel cuivrique est très rentable. Les mêmes sels métalliques peuvent également être utilisés conjointement avec d'autres résines d'adsorption non ioniques neutres de polyéthylène, à savoir HP2O, HP2OSS, Sepabeads SP207 et similaires. La concentration en ions cuivre peut être d'une manière générale de 0,20 à 1,5 mol/l, de préférence, de 0,30 à 0,40 mol/l. En tant que milieu aqueux pour le solvant aqueux contenant des ions cuivre, une acétone aqueuse, un alcool aqueux, un acétonitrile aqueux ou similaires peuvent être utilisés. L'invention concerne le Tacrolimus ayant une pureté de plus de 99,7%.
PCT/IN2007/000352 2006-08-21 2007-08-16 Procédé de purification de macrolides WO2008059516A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
IN1310MU2006 2006-08-21
IN1310/MUM/2006 2006-08-21
IN838/MUM/2007 2007-04-27
IN838MU2007 2007-04-27

Publications (2)

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WO2008059516A2 true WO2008059516A2 (fr) 2008-05-22
WO2008059516A3 WO2008059516A3 (fr) 2009-04-16

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113087723A (zh) * 2020-01-09 2021-07-09 鲁南制药集团股份有限公司 一种西罗莫司的分离纯化方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4337335A (en) * 1978-05-18 1982-06-29 Schering Corporation Transition metal salt complexes of polyamino organic compounds
US20060142565A1 (en) * 2004-12-22 2006-06-29 Vilmos Keri Method of purifying tacrolimus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4337335A (en) * 1978-05-18 1982-06-29 Schering Corporation Transition metal salt complexes of polyamino organic compounds
US20060142565A1 (en) * 2004-12-22 2006-06-29 Vilmos Keri Method of purifying tacrolimus

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113087723A (zh) * 2020-01-09 2021-07-09 鲁南制药集团股份有限公司 一种西罗莫司的分离纯化方法
CN113087723B (zh) * 2020-01-09 2023-07-28 鲁南制药集团股份有限公司 一种西罗莫司的分离纯化方法

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Publication number Publication date
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