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WO2002012267A1 - Procede de production de derives d'acide biliaire a fluoresceine - Google Patents

Procede de production de derives d'acide biliaire a fluoresceine Download PDF

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Publication number
WO2002012267A1
WO2002012267A1 PCT/GB2001/003559 GB0103559W WO0212267A1 WO 2002012267 A1 WO2002012267 A1 WO 2002012267A1 GB 0103559 W GB0103559 W GB 0103559W WO 0212267 A1 WO0212267 A1 WO 0212267A1
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WIPO (PCT)
Prior art keywords
compound
general formula
formula
salt
alkali metal
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/GB2001/003559
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English (en)
Inventor
Charles Oswald Mills
Ian David Cox
David John Hartley
Ian Burley
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Norgine Europe BV
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Norgine Europe BV
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 Norgine Europe BV filed Critical Norgine Europe BV
Priority to AU2001276541A priority Critical patent/AU2001276541A1/en
Publication of WO2002012267A1 publication Critical patent/WO2002012267A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J41/00—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring

Definitions

  • This invention relates to the production of bile acid derivatives and is particularly concerned with the production of bile acid derivatives containing a fluorescein moiety.
  • Bile acid derivatives containing a fluorescein moiety are absorbed by the liver after injection into the blood and have a variety of uses.
  • WO 99/07325 discloses the use of a cholyl lysyl fluorescein (CLF) derivative for determining liver function in a patient
  • WO97/06829 disclose the use of a CLF derivative for visualising the bile duct and biliary leakage during an operation as an aid to the surgeon.
  • the cholyl-lysine formate is then treated with NaOH, incubated and acidified with HCI, before the resultant solution is passed through a Sep-Pak C18 column, eluted with methanol and then evaporated to dryness to form the free cholyl-lysine.
  • the cholyl-lysine is then dissolved in 0.1 M methanolic NaOH followed by addition of ether and trituration with ethyl acetate to form a sodium salt.
  • the sodium salt is then dissolved in bicarbonate buffer and FITC (fluorescein isothiocyanate) in bicarbonate buffer followed by stirring for 18 hours at room temperature in the dark.
  • Unreacted free FITC is removed by percolating the reaction mixture through Sep-Pak-C18 cartridges, the desired product is recovered by elution with methanol and further purification by TLC (thin layer chromatography) on silica gel plates using a solvent mixture of 3:1 v/v chloroform/methanol.
  • TLC thin layer chromatography
  • A is ⁇ -OH or ⁇ -OH
  • B is ⁇ -H or ⁇ -H
  • C is -H, ⁇ -OH or ⁇ -OH
  • B and C together form a double bond
  • D is -H, ⁇ -OH or ⁇ -OH
  • E is -H, ⁇ - OH or ⁇ -OH
  • Q is S or O
  • X is an alkali metal (preferably Na or K) or ammonium ion
  • n is 0 or 1
  • m is 1 to 8, preferably 3 to 5 and most preferably 4; said method comprising the step of reacting (a) a bile acid derivative of the general formula (II):-
  • A, B, C, D, E, Q, n and m are as defined above and each of X' and X" is -H, alkali metal or ammonium ion, provided that at least one of X' and X" is an alkali metal or an ammonium ion; with (b) an alkali of the formula X-OH, where X is as defined above, so as to produce said salt of the formula (I), and purifying the resultant mixture using reverse phase chromatography so as to isolate said compound of the formula (I).
  • the reaction between compound (II) and the alkali of formula X-OH is effected in an aqueous-based reaction system to enable accurate control of pH. It is preferred for the pH of the aqueous-based reaction system to be at least 8 and more preferably in the range of 8 - 8.2.
  • the bile acid derivative of the general formula (II) is produced by reacting a compound of the general formula (III)
  • the compound of the general formula (III) may be produced by mixing a compound of the general formula (V):-
  • A, B, C, D, E and n are as defined above with a non-polar solvent (such as acetone) and a tertiary amine (such as triethylamine), mixing ethyl chloroformate with the resultant mixture, and then adding an aqueous alkaline solution containing a compound of the general formula (VI):- H
  • a non-polar solvent such as acetone
  • a tertiary amine such as triethylamine
  • BL is a blocking group (such as N-e-CBZ), followed by acidification, extraction and removal of the blocking group BL.
  • a blocking group such as N-e-CBZ
  • the purity of said salt is preferably at least 96%.
  • the steroid moiety in the compound of the formula (I) may be based on cholic acid, chenodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid, hyocholic acid, ⁇ -, ⁇ -, or ⁇ -muricholic acid, a nor-bile acid, lithocholic acid, 3 ⁇ -hydroxycholenoic acid, ursodeoxycholic acid, allocholic acid (5 ⁇ -cholan-24-oic acid), or the like
  • the salt has the formula (VII):-
  • a 10 litre stirred reaction vessel equipped with a lid, mechanical stirrer, anchor stirrer rod, dropping funnel, condenser, thermometer and funnel is charged sequentially with 858g acetone, 146g cholic acid and 50g triethylamine at ambient temperature with stirring. Stirring is continued for 30 minutes and 39g ethyl chloroformate is added dropwise over 20 minutes to the vessel while maintaining an internal temperature of 15- 25°C using ice/water bath cooling as necessary. Stirring is continued for 90 minutes. Following this the reaction vessel is charged with an aqueous solution containing 100g N-e-CBZ-L-lysine and 15g sodium hydroxide in 800g water. The resultant mixture is stirred vigorously for a further three hours, following which 550g 1M hydrochloric acid are added so as to attain a pH of 2.0-2.5.
  • the contents of the reaction vessel are then transferred to a separating funnel and extracted twice with 900g ethyl acetate, combining the organic layers after each extraction.
  • the combined organic extracts are then vacuum filtered.
  • the filtered solution is then mixed with 5000g water and acidified with 1 M HCI to a pH of 2.0-2.5 before separation of the organic layer from the aqueous layer.
  • the upper organic layer is then transferred to a container and charged with 150g magnesium sulphate, followed by filtration.
  • the filtered organic layer is then dried in an evaporating flask on a rotary evaporator at a temperature of 45-50°C and then allowed to cool for twenty minutes.
  • the yield of the Compound (VIII) is 200-21 Og, having a purity of > 90% (HPLC area%), typically 91-93%.
  • a two litre reaction vessel equipped with mechanical stirrer, thermometer, dropping funnel and water-cooled condenser is charged with 1520g methanol at ambient temperature, and 190g N-e-CBZ-cholyl-L-lysine and 19g of 10% Pd/C (on a dry weight basis) at 15-20°C are added with stirring. Then, 342g formic acid is added, and the resultant reaction mixture is stirred for 24 hours at an ambient temperature. The reaction mixture is then vacuum filtered through 285g Celite into a flask. The reaction vessel is then washed with 475g methanol and the resultant washing combined with the filtered reaction mixture. The combined mixture is then poured into 13.3kg water to give a solution having a pH of 2.0-2.5.
  • the resultant solution is then extracted twice with 3420g ethyl acetate in a separating funnel following which the resultant aqueous solution is adjusted to pH 4.5-5.0 by addition of 1283g 2.5M sodium hydroxide solution, followed by cooling of the solution to 0-4°C for four hours and then removing the resultant precipitate by vacuum filtration.
  • the filtered precipitate is then dried in a vacuum oven at 45-50°C for four hours to afford cholyl-L-lysine (IX) as a white solid.
  • the yield of cholyl-L- lysine (IX) is about 1 10-120g having a purity > 90%> (HPLC area %), typically 92-98%.
  • a 2 litre glass reaction vessel equipped with a mechanical stirrer and a thermometer is charged with 750 ml 0.5 M sodium methoxide in methanol. After commencement of stirring, 10Og cholyl-L-lysine is added to the reaction vessel and the contents are stirred for 15 minutes. Then, 72g of fluorescein isothiocyanate isomer of 90 + % purity is added at ambient temperature. After stirring for one hour, a sufficient quantity of a 0.5M solution of sodium methoxide in methanol is added to ensure complete solution of the reaction mixture. Then, the contents of the reaction vessel are stirred at ambient temperature in the absence of light for 24 hours.
  • a 20 litre glass flask equipped with a mechanical stirrer and a thermometer is charged with 9000g ethyl acetate.
  • the contents of the first-mentioned reaction vessel are then transferred to the 20 litre vessel dropwise with stirring.
  • the reaction mixture is stirred for a further 30 minutes and the resultant orange precipitate is vacuum filtered off.
  • the resulting solid is then mixed with 500g methanol in a 2 litre reaction vessel with stirring, and stirring is continued until all the solid residue has dissolved.
  • the resultant solution is transferred dropwise to a 20 litre vessel charged with 4500g acetone with stirring.
  • the mixture is then stirred for a further 30 minutes, after which the precipitate is vacuum filtered and the methanol-acetone treatment is repeated once more followed by vacuum filtration.
  • the resultant precipitate is dried under vacuum 45-50°C to constant weight to product compound (X).
  • the yield of cholyl-L-lysine fluorescein di-sodium salt (X) is about 150- 170g having a purity > 80 HPLC area %, typically 83-89 HPLC area %.
  • a column having a 4 inch (100 mm) bore is charged with 1.80kg Envi 18 reverse phase silica and pre-conditioned with 9 litres of 5% v/v solution of acetonitrile in water using nitrogen pressure and a bottom run-off valve as required to maintain a flow of approximately 200ml/min through the silica bed. This mobile phase is run through the bed until the top of the bed is just wetted.
  • the collected fractions can then be combined according to purity so that one set of combined fractions contains compound (VII) at a purity of at least 96% with no single impurity being present in an amount greater than 0.5%, another set of combined fractions contains compound (VII) at a purity of at least 96% with at least one impurity being present in an amount greater than 0.5%, and further fractions of lesser purity.
  • the pooled fractions are then freeze-dried or rotary evaporated to afford homogeneous compound (VII).
  • the chromatography yields about 18g (40g loading) of cholyl-L-lysine fluorescein tri-sodium salt (compound VII) having a purity > 96 HPLC area %, typically 98-99.5 HPLC area %.
  • the UV spectrum, the FT-IR spectrum, the DSC data and the NMR data for compound (VII) are respectively shown in accompanying Figs. 1 to 4.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Steroid Compounds (AREA)

Abstract

L'invention concerne un procédé de production d'un sel représenté par la formule générale (I) dans laquelle A représente α-OH ou β-OH, B représente α-H ou β-H, C représente H, α-OH ou β-OH, ou B et C ensemble forment une double liaison, D représente H, α-OH ou β-OH, E représente H, α-OH ou β-OH, Q représente S ou O, X représente un métal alcalin (par ex. sodium ou potassium) ou un ion ammonium, n représente 0 ou 1 et m vaut de 1 à 8. Ce procédé consiste à faire réagir (a) un dérivé d'acide biliaire représenté par la formule générale (II), dans laquelle A, B, C, D, E, Q, n et m sont tels que définis ci-dessus et chaque X' et X'' représente H, un métal alcalin ou un ion ammonium, à condition qu'au moins un parmi X' et X'' soit un métal alcalin ou un ion ammonium; avec (b) un alcali de formule X-OH, dans laquelle X est tel que défini ci-dessus, de manière à produire ledit sel représenté par la formule (I), et à purifier le mélange résultant au moyen de la chromatographie à phase inversée afin d'isoler ledit composé de formule (I).
PCT/GB2001/003559 2000-08-10 2001-08-08 Procede de production de derives d'acide biliaire a fluoresceine Ceased WO2002012267A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001276541A AU2001276541A1 (en) 2000-08-10 2001-08-08 Method for the production of fluorescein bile acid derivatives

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0019593A GB0019593D0 (en) 2000-08-10 2000-08-10 Production of bile acid derivatives
GB0019593.3 2000-08-10

Publications (1)

Publication Number Publication Date
WO2002012267A1 true WO2002012267A1 (fr) 2002-02-14

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PCT/GB2001/003559 Ceased WO2002012267A1 (fr) 2000-08-10 2001-08-08 Procede de production de derives d'acide biliaire a fluoresceine

Country Status (3)

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AU (1) AU2001276541A1 (fr)
GB (1) GB0019593D0 (fr)
WO (1) WO2002012267A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2467902A (en) * 2009-02-12 2010-08-18 Norgine Bv Preparation of Cholyl-L-lysine from N-E-CBZ-cholyl-L-lysine
EP2773653A4 (fr) * 2011-11-02 2015-08-05 Broad Inst Inc Substrats fluorescents destinés à la détermination de l'activité enzymatique modifiée par la lysine
US9745613B2 (en) 2014-07-22 2017-08-29 The Broad Institute, Inc. Compounds, substrates and methods related to histone deacetylases
WO2018221479A1 (fr) * 2017-05-29 2018-12-06 五稜化薬株式会社 Nouvelle substance fluorescente pour analyse de fonction de transport

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997006829A1 (fr) * 1995-08-19 1997-02-27 The University Of Birmingham Utilisation de derives de l'acide biliaire
US5952187A (en) * 1995-12-01 1999-09-14 Oxis International, Inc. Topiramate immunoassay
US6013802A (en) * 1995-02-06 2000-01-11 Molecular Probes, Inc. Fluorescent conjugates of metal-chelating nitrogen heterocycles

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013802A (en) * 1995-02-06 2000-01-11 Molecular Probes, Inc. Fluorescent conjugates of metal-chelating nitrogen heterocycles
WO1997006829A1 (fr) * 1995-08-19 1997-02-27 The University Of Birmingham Utilisation de derives de l'acide biliaire
US5952187A (en) * 1995-12-01 1999-09-14 Oxis International, Inc. Topiramate immunoassay

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
M. K. HERRLEIN ET AL: "57. 3 -Amino-Modified Nucleotides Useful as Potent Chain Terminators for Current DNA Sequencing Methods", HELVETICA CHIMICA ACTA, vol. 77, no. 2, 23 March 1994 (1994-03-23), BASEL CH, pages 586 - 596, XP002183276 *
MILKIEWICZ, P. ET AL: "Canalicular secretion of lysyl-fluorescein conjugated bile acids in 17.beta.-estradiol glucuronide (17.beta.EG) induced cholestasis: protective effect of s-adenosyl-L-methionine (SAMe)", BILE ACIDS HEPATOBILIARY DIS., PROC. FALK WORKSHOP (2000), MEETING DATE 1999, 72-84. EDITOR(S): NORTHFIELD, TIM C. PUBLISHER: KLUWER ACADEMIC PUBLISHERS, DORDRECHT, NETH., XP001039804 *
MILLS C O ET AL: "CHOLYL-LYSYLFLUORESCEIN: SYNSTHESIS, BILIARY EXCRETION IN VIVO AND DURING SINGLE-PASS PERFUSION OF ISOLATED PERFUSED RAT LIVER", BBA - GENERAL SUBJECTS, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 1115, no. 2, 1991, pages 151 - 156, XP000615215, ISSN: 0304-4165 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2467902A (en) * 2009-02-12 2010-08-18 Norgine Bv Preparation of Cholyl-L-lysine from N-E-CBZ-cholyl-L-lysine
WO2010092381A1 (fr) 2009-02-12 2010-08-19 Norgine Bv Procédé de préparation de cholyl-l-lysine
CN102317304A (zh) * 2009-02-12 2012-01-11 诺金公司 胆酰-l-赖氨酸的制备方法
JP2012517463A (ja) * 2009-02-12 2012-08-02 ノージーン ビーブイ コリル−ｌ−リシンの製造方法
EP2773653A4 (fr) * 2011-11-02 2015-08-05 Broad Inst Inc Substrats fluorescents destinés à la détermination de l'activité enzymatique modifiée par la lysine
US9856509B2 (en) 2011-11-02 2018-01-02 The Broad Institute, Inc. Fluorescent substrates for determining lysine modifying enzyme activity
US9745613B2 (en) 2014-07-22 2017-08-29 The Broad Institute, Inc. Compounds, substrates and methods related to histone deacetylases
WO2018221479A1 (fr) * 2017-05-29 2018-12-06 五稜化薬株式会社 Nouvelle substance fluorescente pour analyse de fonction de transport

Also Published As

Publication number Publication date
AU2001276541A1 (en) 2002-02-18
GB0019593D0 (en) 2000-09-27

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