Bile acid derivatives containing a fluorescein moiety are absorbed by the liver after injection into the blood and have a variety of uses. For example, WO 99/07325 discloses the use of a cholyl lysyl fluorescein (CLF) derivative for determining liver function in a patient, whilst W097/06829 disclose the use of a CLF derivative for visualising the bile duct and biliary leakage during an operation as an aid to the surgeon.

C. O. Mills et al in Biochemica et Biophysica Acta., 1115 (1991), 151- 156 observe that a CLF derivative has a fluorescent property similar to that of fluorescein and that upon injection into rats, it is similar in biliary output and hepatic extraction to the natural bile acid, cholylglycine. They describe a method of producing the derivative which involves first forming cholyl-lysine by reaction of excess N-E-CBZ-L-lysine with cholic acid in the presence of EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline) to form the protected cholyl-N-e-CBZ-L-lysine. Following purification, the protecting group is removed via production of the formate using formic acid, methanol and palladium black under nitrogen. 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. The process described produces only about 50mg of a product which is not a high purity single compound salt but a mixture consisting predominantly of the disodium salt (as determined by HPTLC). Also, the process cannot be scaled up to produce production scale batches of the product.

It is an object of the present invention to provide an improved method of producing a high purity product containing a bile acid linked to fluorescein in substantially higher quantities than is possible by the above- described known method.

According to one aspect of the present invention, there is provided a method of producing a salt of the general formula (I) :- wherein A is a-OH or R-OH, B is a-H or ß-H, C is-H, a-OH or ß-OH, or B and C together form a double bond, D is-H, a-OH or P-OH, E is-H, oc- OH or R-OH, Q is S or O, X is an alkali metal (preferably Na or K) or ammonium ion, n is 0 or 1 and 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 (11) :- wherein 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).

Most preferably, 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 above process can be used to produce the compound of the formula (I) at a purity level in excess of 96% even when starting from compound (II) in relatively impure form (>75% pure).

Preferably, the bile acid derivative of the general formula (II) is produced by reacting a compound of the general formula (III) with a compound of the general formula (IV) :- in the presence of a solution of an X-alkoxide (where X is an alkali metal or an ammonium group) in an alcohol, eg sodium methoxide in methanol.

The above reaction scheme will generally produce mostly the di-salt but with some of the mono-salt and, in addition, some of the tri-satt (i. e. compound (I)). However, if an excess of the X-alkoxide is used in an endeavour to maximise synthesis of the tri-salt at this stage, poor yields and purity are observed.

It is possible to use compound (IV) of regular grade purity, with the result that no special purification techniques are required before this compound is used in the reaction with compound (III).

The compound of the general formula (III) may be produced by mixing a compound of the general formula (V):- where 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 (Vl) :- where BL is a blocking group (such as N-E-CBZ), followed by acidification, extraction and removal of the blocking group BL.

According to another aspect of the present invention, there is provided a substantially pure salt of the general formula (I) as defined above.

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, α-, ß-. or-muricholic acid, a nor-bile acid, lithocholic acid, 3p-hydroxycholenoic acid, ursodeoxycholic acid, allocholic acid (5a-cholan-24-oic acid), or the like Preferably, the salt has the formula (Vll) :- The present invention will now be described in further detail in the following Example.

Example Preparation of N-e-CBZ-cholyl-L-lysine (Compound Vlil) 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 1 00g 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 HCf 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 (VlII) is 200-21 Og, having a purity of > 90% (HPLC area%), typically 91-93%.

Preparation of Cholyl-L-lysine (Compound IX) 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 110-120g having a purity > 90% (HPLC area %), typically 92-98%.

Preparation of cholyl-L-lysine fluorescein disodium salt (Compound X) 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, 1 OOg 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. Then, 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 %.

Preparation of Cholyl-Lysyl-Fluorescein-trisodium salt (compound (Vi I) 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 200m !/min through the silica bed. This mobile phase is run through the bed until the top of the bed is just wetted.

To a 250ml beaker furnished with a magnetic stirrer bar, there are charged 1 oog 5% v/v solution of acetonitrile in water, and 40g of the cholyl-lysyl- fluorescein disodium salt (compound (X)). The mixture is stirred until a complete solution is formed and then the pH is adjusted to 8.0-8.2 using 1 M NaOH solution. The solution containing compound (X) is then loaded on to the column by applying nitrogen pressure. The column is then charged with 300moi 5% v/v solution of acetonitrile in water solution and nitrogen pressure is applied until the mobile phase has been run to the top of the silica bed. Then 9000ml 5% v/v solution of acetonitrile in water is run through the column using nitrogen pressure and the bottom run-off valve as required to maintain a flow of approximately 200ml/min through the bed until the top of the silica bed is just wetted. The eluent is collected as a single"fronts"fraction. Then, 6000ml of 20v/v solution of acetonitrile in water is run through the column using nitrogen pressure and the bottom run-off valve as required to maintain a flow of approximately 200ml/min through the bed until the top of the silica bed is just wetted.

The first litre of eluent is collected and combined with the previously collected"fronts"fraction and the remainder of the eluent is collected in 250moi fractions and submitted for HPLC purity analysis.

18 litres of methanol for flushing the column are charged to the top of the column, taking care not to disturb the silica bed. Using the bottom run- off valve and nitrogen pressure as required to maintain a flow of approximately 200ml/min through the silica bed, the mobile phase is run through until the top of the silica bed is just wetted. The eluent is collected as 2 x 2.5 litre fractions and then the head of the mobile phase is run to within one inch (25 mm) of the silica bed and the eluent collected as a single fraction and discarded. The 2 x 2.5 litre fractions are also submitted for HPLC purity analysis.

The collected fractions can then be combined according to purity so that one set of combined fractions contains compound (Vil) 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 (Vil).

The chromatography yields about 18g (40g loading) of cholyl-L-lysine fluorescein tri-sodium salt (compound Vil) 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.

It will be appreciated from the above that very pure product can be obtained even when using regular grade FITC having a purity of about 90% and without taking any special measures to purify compound (X) before it is used to produce compound (Vil).