This invention relates to an improved process for the preparation of porphyrin C which allows isolation of very pure material without the need for chromatography.

BACKGROUND TO THE INVENTION

It has been known for many years that certain porphyrins are preferentially 5i, accumulated by tumour tissue. It is also well established that porphyrins have a photodynamic effect on tissues, that is, their presence confers on the tissue a sensitivity to light so that considerable damage can be caused to exposed tissue.

It follows that a tumour which is enriched in porphyrin can be destroyed by ro. applying light, whereas the surrounding tissue, which contains little porphyrin, survives. The possibilities of a cancer therapy based on this selective tumour destruction, via the intravenous injection of porphyrins and exposure to light have been recognised for some years (I Diamond et al Lancet, 1972, ii 1175).

The relatively high concentrations of certain porphyrins in malignant tumours is. can also be used for cancer detection. The excitation of the porphyrins with violet light leads to fluorescence in the red. Thus, exposure of both the tumour and the surrounding tissue to violet light will give a strong red fluorescence from the normal tissue. Very small early stage tumours can be detected in this way.

To try to improve the degree of porphyrin localisation by tumours, a number of ^20. different porphyrin derivatives have been studied. Lipson and co-workers (R.L.

Lipson et al, Cancer 1967, 23., 2255) developed a haematoporphyrin derivative (Hpd) with enhanced localization properties and this particular derivative Hpd is now the most widely used photosensitiser for photoirradiation therapy.

Hpd is a complex mixture obtained by treating haematoporphyrin (HP) with acetic and sulfuric acids and dissolving the acetylated product (HPA) in alkali:

CH3CO2H ^' Alkali

HP H PA Hpd

H2SO4

HPLC analysis reveals the presence of at least 15 major components in a typical sample of Hpd and the exact composition has been found to vary 5. according to small changes in the preparative procedure or in the method of storage.

A number of problems in photodynamic therapy stem from the complex and variable nature of Hpd. Thus, it is difficult to determine the optimum doze of Hpd, the method of administration and the tuning of photoirradiation therapy, to 10. obtain the best possible tumour selectivity.

One of the disadvantages in the use of Hpd for cancer therapy and diagnosis is the risk of phototoxic reactions induced by daylight in the human skin after the injection of the high doses of the haematoporphyrin derivative used for tumour identification.

15. For example, after patients have been treated with Hpd they are subject to serious photosensitivity which forces them to remain indoors for four weeks or - even longer.

To overcome the various problems associated with the use of Hpd, new photosensitisers are required which are

20. (a) pure, stable and discrete compounds which combine in a highly selective manner with tumour tissue; and

(b) are less prone to induce prolonged photosensitivity in the patient.

One compound which has recently been shown to localise in tumours and to sensitise them to photoirradiation, both in vivo and in vitro is porphyrin C (see for example, B. Liang et al Dalian Gongxuevuan Xuebao. 1984, 2 , 128; Chemical Abstracts 1984, 1_Q_2. 181627c).

5. Previous workers have prepared porphyrin C in a two-step procedure in which haematoporphyrin dichloride II is first converted into the dibromide adduct III of protoporphyrin. Treatment of this 2,4-di-(1 -bromoethyl) deuteroporphyrin in III with L-cysteine by fusion at 160°C gave crude porphyrin C which required chromatographic purification before being isolated in about 60% yield (H. Goft lu - et al, Inorganic chemistr ^y . 15 2062 (1976).

Another proςess for the preparation of porphyrin C has been described but this latter process is not suitable for commercial scale production of the compound (Dalian Gongxuevuan Xuebao 1984, 21 29-32; Chemical Abstracts 1984, UH 16.0268s) .

15. It has now been found that an alternative process is possible in which porphyrin

C may be prepared in high yield and with high purity.

The invention is therefore said to reside in a process for the preparation of porphyrin C which comprises condensing haematoporphyrin dihydrohalide with L-cysteine hydrohalide in the presence of a hyrdrogen halide in acetic acid or 20.. formic acid under mild conditions and in a single step.

Further the invention provides a one-step process for the preparation of porphyrin C (formula I)

by condensing haematoporphyrin dihydrochloride and L-cysteine hydrochlorrde in the presence of hydrogen bromide in acetic or formic acid.

5. In a preferred form of the invention L-cysteine hydrochloride and haematoporphyrin dihydrochloride in a mole ratio of from 2:1 to 10:1 are dissolved in a solution of hydrogen bromide (5 to 50%) in acetic or formic acid. The concentration of haematoporphyrin in the acid solution may vary from 0.1 M to 0.4 M, i.e. from 0.06g/ml to 0.25g/ml.

ιo. Preferably a solution is prepared of L-cysteine hydrochloride and haematoporphyrin dihydrochloride in a mole ratio of from 2.5:1 to 4:1 in a mixture of hydrogen bromide (10 to 50%) in acetic acid. The preferred concentration of haematoporphyrin in the solution is between 0.1 and 0.15g/ml.

The solution thus formed is stirred and maintained at room temperature for a 15. short period (0.25 to 2 hours) with careful exclusion of oxygen and light.

The solvent and excess hydrogen bromide may then be removed under high vacuum at room temperature (10-30°C). This evaporation may take several hours depending on the pressure used, the ratio of solution to flask size, the efficiency- of stirring and the exact temperature. The length of time taken is not 20. critical provided precautions are made against photodecomposition of the material.

The reaction may then be driven to completion by heating the mixture at a temperature of 80° to 120°C, preferably 100°C, for a period of from 0.25 to 2 hours, preferably 0.5 to 1 hour, all the time keeping a reduced pressure on the mixture and stirring to allow evaporation of volatile material.

⁵ - The crude porphyrin C thus prepared may be isolated in crystalline form by dissolving the residue in ice-cold aqueous hydroxide solution of from 1 M to 5 M strength and then precipitating the product by the careful addition of ice-cold dilute hydrochloric acid (1 M to 5 M) to give a final pH of between 3 and 5.

Alternatively, the residue may be dissolved in the dilute hydrochloric acid and 10. reprecipitated with the dilute sodium hydroxide solution. It is preferable to use oxygen-free solutions of the hydroxide and hydrochloric acid.

The precipitated porphyrin C is collected by filtration and dried under reduced pressure with exclusion of light.

The advantages of the present process are that porphyrin C is obtained from 15. haematoporphyrin in a single step and in higher purity than previous methods.

No chromatographic purification is required to obtain material of up to 95% purity. The preferred conditions of the process also allow preparation of porphyrin C with little excess of L-cysteine and the use of less hydrogen bromide than with earlier methods.

20. Example

Preparation Of pQrphyrjη C

A one-necked round-bottom flask (100 ml) was flushed with nitrogen and then haematoporphyrin dihydrochloride (1g, 1.5 m mole) (Roussel UCLAF), L- cysteine hydrochloride (2g, 12.7 m mole) (BDH) and hydrogen bromide in acetic $5. acid (8 ml of 45% solution, 62.5 m mole) were added to the flask in turn. The flask was stoppered and the mixture was stirred vigorously for 0.5 hours at 21 °C. The solvent was then evaporated under reduced pressure (10-30 Pa) as the mixture was stirred and maintained at room temperature until a viscous glass was formed. The flask was then heated at 100°C for 0.5 hours and kept

under reduced pressure. The residue was allowed to cool to room temperature, nitrogen was admitted to the flask and ice-cold oxygen-free sodium hydroxide solution (2 M; 25 ml) was added to dissolve the crude porphyrin C. Ice-cold oxygen-free dilute hydrochloric acid (2 M) was then added slowly with stirring to 5. pH 3 to precipitate the porphyrin C which was collected by filtration and washed with cold water (3 x 20 ml). The solid was dried under vacuum to give porphyrin C (1.3g, 92%) as a dark purple solid which on analysis by HPLC was 95% pure. The identity of the product was confirmed by HPLC comparison with an authentic sample, by elemental analysis and by proton and ^{1 3} C nuclear 10. resonance spectroscopy.