Title of Invention

"A METHOD OF PURIFICATION OF A PHTHALEIN"

Abstract

The invention converns high purity phthalein derivatives enabling their use for medical applications or in the field of biotechnology, as well as their preparation method whereby a phathalic anhydride derivative is condensed with a naphthoi or phenol derivative in an organic acid ester and the crystals of the resulting condensate are converted by action of a strong acid or one of its precursors in anhydrous medium.

Full Text

FILED OF THE INVENTION The present invention relates to high purity phthaleins and to the method for preparing them. It relates more particularly to high purity fluorescein. In the present description, the term "high purity phthalein" is intended to mean a phthalein containing at most 1% by weight, preferably at most 0.5% by weight, of impurities. BACKGROUND OF THE INVENTION Phthaleins are molecules having the following xanthcnc unit: (Figure Removed) These products are useful as dyes in various industries, in particular the textile industry, the paper industry, printing, .reprography, the food industry, the cosmetics industry and the pharmaceutical industry. Phthaleins are, currently, the subject of a considerable resurgence of interest in the health field, for their diagnostic use, in particular in the context of medical imaging and in the field of the biotechnology for labeling biological molecules (nucleic acids, proteins, lipoproteins, membrane lipids) and following the intracellular or extracellular biochemical activity of biological molecules. For example, fluorescein is a phthalein commonly used in ophthalmology for performing retinal angiography by fluorescence. The diagnostic advantage of fluorescein angiography is currently increased by the appearance of new medicinal products for treating vascular patholo- STATEMENT OF THE INVENTION The present invention relates to a phthalein of general formula (I) : (Formula Removed) in which Rl, R2, R3, R4 and R5, which are identical to or different from one another, are chosen from the group comprising the following radicals or groups: hydrogen, hydroxyl, halogen, acetyl, amino, phosphate, nitrn, sulfonate, carboxyl, alkylcarboxyl having from 2 to 30 carbon atoms, alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkyl having from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester having from 2 to 40 carbon atoms, nitroalkyl having from 1 to 30 carbon atoms, carboxyalkyl having from 2 to 30 carbon atoms, aminoalkyl having from 1 to 30 carbon atoms, sulfoalkyl having from 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, haloaryl, aryl ester, succinimidyl ester, isothiocyanate, maleimide, iodoacetamide, haloacet-amide, chiorosulfonic, purine or pyrimidine bases, monosaccharides, preferably hexoses or pentoses, oligosides and polyosides, polypeptides, proteins and phospholipids, R3 and R5 are not hydrogen when Rl. is a group -CH2-CH2-COOH, R2 is a hydroxyl group and R4 is a group -COOH, these phthaleins containing no more than 1% by weight, preferably no more than 0.5% by weight, and even more preferably not more than 0.2% by weight, of residual impurities - DETAILED DESCRIPTION OF THE INVENTION The high purity phthaleins according to the invention have the general formula (I): (Formula Removed) in which Rl, R2, R3, R4 and R5, which are identical to or . di I f eren form one another, are oho sen from the group comprising the following radicals or groups: hydrogen, hydroxyl, halogen, acetyl, amino, phosphate, nitro, sulfonate, carboxyl, alkylcarboxyl having from 2 to 30 carbon atoms, alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkyl having from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester having from 2 to 40 carbon atoms, nitroalkyl having from 1 to 30 carbon atoms, carboxyalkyl having from 2 to 30 carbon atoms, aminoalkyl having from 1 to 30 carbon atoms, sulfoalkyl having from 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, haloaryl, aryl ester, succinimidyl ester, isothiocyanate, maleimide, ioduaceLcuuide, haloacet-amide, chlorosulfpnic, purine or pyrimidine bases, monosaccharides, preferably hexoses or pentoses, oligosides and polyosides, polypeptides, proteins and phospholipids, R3 and R5 not each representing hydrogen when Rl represents a group -CH2-CH2-COOH, R2 represents a hydroxyl group and R4 represents a group -COOH, these phthaleins containing no more than 1% by weight, preferably no more than 0.5% by weight, and even more preferably not more than 0.2% by weight, of residual impurities. A phthalein that is particularly advantageous, in particular for ophthalmic applications, is fluorescein having such a purity. It is known practice to prepare the phthaleins of formula (I) by condensation of a phthalic anhydride derivative and of a phenol derivative having a free ortho-position with respect to a hydroxyl group. This condensation is carried out by heating at the melting temperature of a mixture of the phthalic anhydride and of the phenol derivative, in the desired proportions. This condensation can optionally be carried out in a dilution solvent. It can also be carried out in the presence of a catalyst. In the absence of solvent, during the heating, the reaction medium rapidly thickens and has a tendency to harden. Within the reaction medium, areas where the temperature is too high and areas where the temperature is not high enough then form. In the areas where the temperature is too high, the reactants and/or the reaction product are degraded and, in the areas where the temperature is not high enough, the reaction is not complete. The product obtained is of mediocre quality since it contains by-products that are very difficult to remove. In order to improve this known method, the addition of an inert solvent or the use of a catalyst has been considered. Thus, US patent 1,931,04 9 describes the addition to the reaction medium of an inert solvent consisting of a benzene-based or aliphatic hydrocarbon, more particularly ortho-dichlorobenzene. However, the condensation reaction is then incomplete, and generate intermediate products that are subsequently difficult to remove. Therefore the method described in US patent 1,931,04 9 does not make it possible to obtain a high purity phthalein according to the definition of the present application. Moreover, higher aliphatic hydrocarbons are not miscible with the reaction medium; consequently, they introduce no improvement in terms of thermal transfer and impair the elimination of the water formed, thereby slowing down the reaction, which takes place exclusively in a hydrophobic medium. Regarding the catalysts that are used to improve the reaction yield, these are concentrated sulfuric acid, anhydrous zinc chloride and tin chloride. The German patent DE 360691, describes the use of an aromatic sulfonic acid as a catalyst, in particular benzenesulfonic acid, alone or combined with one of the abovementioned three catalysts. It so happens, however, that the addition of these catalysts leads to a reaction product that sets and hardens with entrapment of impurities, which can no longer be eliminated from the desired product. In order to eliminate both the generated by-products and the impurities, isolation and purification methods have been developed, but none have made it possible to substantially improve the quality of phthalcins. A conventional method consists in basifying the phthalein in an aqueous medium so as to dissolve it, and then in acidifying it so as to allow it to precipitate. These two steps are repeated successively in an attempt to eliminate the impurities. However, this method does not bring about a noticeable improvement in the purity of the product since, while the impurities dissolve with the phthalein during the basifying step, they precipitate again with the phthalein during the acidification step. In addition, this purification method has the drawback of producing a considerable amount of salts which are difficult and expensive to subsequently eliminate. US patent 1,965,842 describes the purification of phthaleins, derived from hydroxybenzene, by direct extraction of the by-products with dichlorobenzene alone or in a mixture with other solvents. This direct extraction of the crude product with the solvent in question does not, however, make it possible to obtain extensive elimination of impurities, which remain partly trapped in the phthalein crystals. None of the purification methods described in r.he prior art makes it possible to reach a level of purity that is sufficient to allow the pharmaceutical use of phthaleins. Given the advantage of these molecules for medical diagnosis, the production of high purity phthaleins is a real medical need and would satisfy a high demand, in particular for pharmaceutical applications in ophthalmology, for diagnosis, in particular in medical imaging, or in the field of biotechnological applications (for example, as dye to label molecules). The inventors have, to their credit, at the end of extensive research, found that it is possible to obtain high purity products by the condensation of a phthalic anhydride with a phenol or naphthol derivative in a specific solvent which consists of an organic acid ester. They have also found that the use of organic acid esters as solvents makes it possible to carry out this condensation with an excellent yield, greater than 75%. Specifically, these solvents have the particularity firstly, of leading to specific crystallization of the phthalein that derives from the condensation and of excluding, from the generated crystals, all the impurities that remain dissolved in the reaction medium, and secondly, of allowing a complete condensation that is predominant, to detrimental and unwanted side reactions, which results in the total consumption of the reactants and minimizes the: formation of by-products. The method in accordance with the invention thus makes it possible to obtain high purity phthaleins with a yield that is satisfactory from an inductrial point of view. More particularly, the invention is related to a method for preparing phthaleins, from which the residual impurities have been removed, having general formula (I) : (Formula Removed) in which Rl, R2, R3, R4 and R5, which are identical to or different from one another, are chosen from the group comprising the following radicals or groups: hydrogen, hydroxyl, halogen, acetyl, amino, phosphate, nitro, sulfonate, carboxyl, alkylcarboxyl having from 2 to 30 carbon atoms, alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkyl having from 1 to 3 0 carbon atoms, hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester having from 2 to 4 0 carbon atoms, nitroalkyl having from 1 to 3 0 carbon atoms, carboxyalkyl having from 2 to 3 0 carbon atoms, aminoalkyl having from 1 to 30 carbon atoms, sulfoalkyl having from 1 to 30 carbon atoms, aryl, aryloxy arylalkyi, haloaryl, aryl ester,. succinimidyl ester, isothiocyanate. maleimide, iodoacetamide, haloacet-amide, chlorosulfonic, purine or pyrimidine bases, monosaccharides, preferably hexoses or pentoses, oligosides and polyosides, polypeptides, proteins and nospholipids, alkylamino, arylamino; two of Rl, R2 or R3 form together an optionally substituted aryl group; R4 and R5 form together an optionally substituted aryl group; R3 and R5 are not hydrogen when Rl is a group -CH2-CH2-COOH, R2 is a hydroxyl group and R4 is a group -COOH, By condensation of a phthalic anhydride derivative of formula (II) with a compound of formula (III) (Formula Removed) in which Rl, R2, R3, R4 and R5 have the same meanings as above, in a solvent consisting of an organic acid ester. Particulary advantageously, the phthalic anhydride (II), is condensed with a compound chosen from the group comprising in particular resorcinol, orcinol, naphthol, pyrogallol, alkylamino-phenol and arylaminophenol. When resorcinol is used as starting product, the method in accordance with the invention makes it possible to prepare fluorescein. Advantageously, the solvent used in the method in accordance with the invention is an organic acid ester of formula (IV) (Formula Removed) in which R6 is chosen from the group comprising the following radicals or groups: hydrogen, alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, haloalkyl having from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 3 0 carbon atoms, nitroalkyl having from 1 to 30 carbon atoms, aryl, aryloxy, alkylaryl, arylalkyl, substituted arylalkyl, haloaryl, aryl ester, alkyl ester having from 2 to 4 0 carbon atoms, and alkyloxy having from 1 to 3 0 carbon atoms, R7 representing one of the following groups: alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, haloalkyl having from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 3 0 carbon atoms, nitroalkyl having from 1 to 3 0 carbon atoms, aryl, aryloxy, alkylaryl, arylalkyl, substituted arylalkyl, haloaryl, aryl ester, alkyl ester having from 2 to 4 0 carbon atoms, or alkyloxy having from 1 to 3 0 carbon atoms. Particularly advantageously, the abovementioned solvent is chosen from the group comprising methyl, ethyl, propyl or butyl benzoates, methyl, ethyl, propyl or butyl heptanoates, methyl, ethyl, propyl or butyl octanoates, methyl, ethyl, propyl or butyl laurates, methyl, ethyl, propyl or butyl myristates and methyl, ethyl, propyl or butyl palmitates, and mixtures thereof. The solvent is chosen according to its boiling point, so as to make it possible to carry out the condensation reaction at a temperature of between 150°C and 250°C. The condensation reaction can be carried out at atmospheric pressure or under a pressure that is adjusted according to the difference that exists between the temperature corresponding to the boiling point of the solvent and the temperature necessary to carry out the reaction, in particular under a pressure greater than atmospheric pressure when the boiling point of the compound (IV) is lower than the reaction temperature. The condensation reaction can be carried out in the presence of a catalyst chosen from the group comprising in particular Lewis acids, such as ZnCl2 or Alcl3, Bronsted acids such as H2S04, polyphosphoric acid, or salts thereof. Advantageously, the catalyst used is an alkali metal, salt of hydrogen sulfate. Particularly advantageously, the catalyst used is potassium hydrogen sulfate (KHSOJ or sodium hydrogen sulfate. The use of hydrogen sulfate as catalyst makes it possible to obtain an excellent yield for the condensation reaction and has the advantage, unlike other catalysts, of making it possible to obtain complete condensation of the reactants, of being able to be completely eliminated from the phthalein obtained, and of not inducing the formation of tars in the reaction medium. At the end of the condensation reaction, a crude product is obtained, the organic purity of which is already much higher than that of the products obtained with the methods of the prior art, since it is equal to or greater than 95%. However, this purity is not yet sufficient to allow pharmaceutical use, in particular by injection. Furthermore, as shown above, the purification methods described in the prior art do not result in a process that makes it possible to notably improve this purity. At the end of extensive research, surprisingly and unexpectedly, the inventors have found a method that considerably increases the purity of crude phthaleins resulting from the condensation reaction, by treating them with a strong acid or one of its precursors in an anhydrous organic medium. According to an advantageous embodiment, the method in accordance with the invention consequently comprises, after the condensation reaction, a step consisting in acidifying the product resulting from the condensation, in an anhydrous organic medium, by addition of a strong acid or one of its precursors, chosen from the group comprising in particular sulfuric acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydriodic acid, polyphosphoric acid, pyrophosphate (P205) , and mixtures thereof. This acidification is carried out until the phthalein crystals resulting from the condensation are converted to phthalein crystals having a different structure. The effect of this acidification step is to convert the crude phthalein that is insoluble and dispersed in the organic medium into a form that has a different crystalline structure and is slightly soluble in this same medium. This conversion is very rapid, and takes place via an intermediate solubilization phase during which the impurities are released and completely eliminated from the phthalein crystals. This new purification method is very advantageous since it requires very little solvent and yields to a very high purity in a very short time. According to the method of the present invention, this purification step is carried out by dispersing the crude phthalein resulting from the condensation, in an anhydrous solvent, preferably in an alcohol, a ketone, an ether or a halogenated solvent, that is either used alone or as a mixture, even more preferably in absolute ethanol or acetone, alone or as a mixture. The dispersion of crude phthalein thus obtained is acidified by the addition of a strong acid or one of its precursors, chosen from the group comprising in particular sulfuric acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydriodic acid, polyphosphoric acid, pyrophosphate (P205) , and mixtures thereof. According to a particularly advantageous embodiment:, the acidification is carried out either by sparging gaseous hydrochloric acid into the phthalein dispersion, or by adding hydrochloric acid dissolved beforehand in an organic solvent. According to another advantageous embodiment of the method in accordance with the invention, the product obtained after acidification is washed with a washing solution chosen from the group comprising water, polar solvents such as alcohols or ketones, or slightly polar solvents such as ethers and halogenated solvents, used, pure or as a mixture. At the end of the purification step, the phthaleins thus treated have a purity of greater than 98%, preferably greater than 99%, more preferably greater than 99.5%, and even more preferably of 99.8%. The phthaleins thus obtained have the suitable quality to prepare other phthaleins of formula (I) by chemical modification of the groups Rl, R2, R3, R4 and R5 according to the conventional methods of the art, in particular to obtain phthaleins that can be used for labeling biological molecules (nucleic acids, proteins, lipoproteins, membrane lipids) and in the field of biotechnological applications (for example, for the labeling of molecules and of their intracellular or extracellular biochemical activity). The invention is particularly suitable for preparing very high purity fluorescein, i.e. such that its content of each of the by-products of the reaction is less than or equal to 0.2%, and preferably less than or equal to 0.1%, the sum of the contents of each of these by-products being less than or equal to 0.5%. The method for preparing a fluorescein having the abovementioned purity comprises the following successive steps: condensing phthalic anhydride with resorcinol, in a solvent which is an ester of an aliphatic or aromatic organic acid, preferably methyl benzoate, in the presence of a catalyst chosen from the group of Bronsted acids, suspending the red-colored crystals obtained in the preceding step in an anhydrous solvent chosen from the group comprising alcohols such as absolute ethanol, ketones such as acetone, ethers, halogenated solvents, or mixtures thereof, acidifying the suspension obtained, by the addition of a strong acid or one of its precursors, chosen from the group comprising sulfuric acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydriodic acid, polyphosphoric acid, pyrophosphate (P205) , or mixtures thereof, until yellow-colored crystals are obtained, washing the crystals thus obtained with a washing solution chosen from the group comprising water, alcohols, ketones, ethers and halogenated solvents, pure or as a mixture, until red-colored crystals are obtained. An X-ray diffraction analysis of the red-colored fluorescein crystals is represented in Figure 1 ,- an X-ray diffraction analysis of the yellow-colored fluorescein crystals is represented in Figure 2 ; these X-ray-diffraction analysis being obtained on the following equipment: Philips 1729 generator, Philips 1050 goniometer, Cu Kα radiation, Gonio acquisition software and Rayon processing software, under the following operating conditions: Voltage : 4 0 mV Intensity : 40 mA Number of points : 4 000 Number of passages : 10 Acquisition period : 250 ms Start angle (°0) : 3.000 End angle (°G) : 23 .000 Standard : silicon According to an advantageous embodiment of the method for preparing fluorescein, the catalyst used for the condensation reaction is the hydrogen sulfate of an alkali metal, preferably potassium hydrogen sulfate or sodium hydrogen sulfate. According to another advantageous embodiment of the method for preparing fluorescein, the acidifiratinn is carried out by sparging gaseous hydrochloric acid into the fluorescein suspension or by the action, on this fluorescein, of hydrochloric acid in solution in an anhydrous organic solvent, preferably chosen from the group comprising alcohols, ketones, ethers and halogenated solvents, used alone or as a mixture, even more preferably isopropanol, absolute ethanol or acetone, pure or as a mixture. Advantageously, the washing step is carried out with a mixture of water, of ethanol and of acetone. By means of this method in accordance with the invention, it is possible to prepare fluorescein having a purity of greater than or equal to 99.7%, which provides unquestionable advantages, in particular for pharmaceutical uses in diagnosis, especially in medical imaging or else in the field of biotechnological applications. The inventors have demonstrated a novel crystallographic form of fluorescein being yellow-colored crystals. This novel crystallographic form is identified by the X-ray diffraction analysis given in figure 2, which was determined under the conditions mentioned above. They have also demonstrated a novel crystallographic form of the following compounds: 4',5'-dihydroxy-fluorescein and 4',5'-dimethylfluorescein. Thus, the invention relates to the yellow-colored fluorescein crystals for which the X-ray diffraction analysis is given in figure 2. The invention also relates to the yellow-colored 4',5'-dihydroxyfluorescein crystals for which the X-ray diffraction analysis is given in figure 4. It also relates to the dark red-colored 4', 5 '-dimethyl fluorescein crystals for which the X-ray diffraction analysis is given in figure 6. All these spectra were determined with the equipment and under the conditions mentioned above. The invention will be described in more detail with the aid of the following examples which are not limiting but relate to advantageous embodiments. EXAMPLES EXAMPLE 1: Preparation of high purity fluorescein Synthesis of fluorescein: A mixture comprising 55 g of resorcinol, 30 g of phthalic anhydride, 2 g of potassium hydrogen sulfate and 500 ml of methyl benzoate is brought to 200°C for 6 hours. After cooling, the red crystals of crude fluorescein are washed with acetone and dried. Mass obtained = 51.8 g (78%). These crystals are analyzed by X-ray crystallography under the following operating conditions: Equipment Philips 1729 generator Philips 1050 goniometer Cu Kα radiation Gonio acquisition software Rayon processing software (Figure Removed) The radiocrystallogram of figure 1 is obtained, for (Figure Removed) Purification of crude fluorescein: 50 g of crude fluorescein obtained in the previous step are stirred into 1000 ml of ethanol/acetone mixture. Concentrated sulfuric acid is added to this mixture, until complete conversion of the red crystals to yellow crystals is obtained. The yellow crystals obtained are analyzed by X-ray crystallography as above. The X-ray diffraction analysis of figure 2 is obtained, for which the peaks are identified below: (Figure Removed) Wavelength: 1.54 051 A These crystals are filtered off and then washed with an ethanol/acetone/water (40/40/20) mixture. The washing turns the yellow fluorescein crystals to red fluorescein crystals, which have a higher purity. Purity by HPLC: 99.8% EXAMPLE 2: Preparation of high purity 4',5'-dimethy1-fluorescein Synthesis of 4',5'-dimethylfluorescein A mixture comprising 62 g of 2-methylresorcinol, 30 g of phthalic anhydride, 2 g of potassium hydrogen sulfate and 500 ml of ethyl laurate is brought to 200°C for 3 hours. After cooling, the crude product is filtered and washed with acetone and then dried. The product obtained is a dark orange powder. Mass obtained = 49.7 g (69%) These crystals are analyzed by X-ray crystallography in the same way as in example 1. The X-ray diffraction analysis of figure 3 is obtained, for which the peaks are identified below. (Figure Removed) Purification of 4',5'-dimethylfluorescem 40 g of crude 4',5'-dimethylfluorescein are added to 800 ml of ethanol/acetone mixture. Concentrated sulfuric acid is added to this mixture, until complete conversion of dark orange-colored crystals to yellow-colored crystals is obtained. The yellow crystals obtained are analyzed by X-ray crystallography as above. The X-ray diffraction analysis of figure 4 is obtained, for which the peaks are identified below. (Figure Removed) Wavelength: 1.54051 A After filtration and recrystallization from an acetone/ water mixture or washing in ethanol/acetone/water, the yellow-colored crystals turn to dark oranqe-colored crystals. EXAMPLE 3; Preparation of 4',5'-dihydroxyfluorescein Synthesis of 4',5'-dihydroxyfluorescein A mixture comprising 63 g of pyrogallol, 30 g of phthalic anhydride, 2 g of potassium hydrogen sulfate and 500 ml of ethyl myristate is brought to 200°C for 3 hours. After cooling, the crude product is filtered and washed with acetone and then dried. The product obtained is a grayish-brown- or anthracite-colored powder. Mass obtained = 43.5 g (59.7%) The crystals obtained are analyzed by X-ray crystallography as above. The X-ray diffraction analysis of figure 5 is obtained, the peaks of which are identified below. (Figure Removed) Purification of 4',5'-dihydroxyfluorescein 40 g of crude 4',5'-dihydroxyfluorescein are added to 800 ml of ethanol/acetone mixture. Concentrated sulfuric acid is added to this mixture, until complete conversion of grayish-brown- or anthracite-colored crystals to reddish-brown- or mahogany-colored crystals is obtained. The crystals obtained are analyzed by X-ray crystallography as above. The X-ray diffraction analysis of figure 6 is obtained, for which the peaks are identified below: (Figure Removed) After filtration and washing in water, the reddish-brown- or mahogany-colored crystals turn to grayish-brown- or anthracite-colored crystals. WE CLAIM: 1. A method for acidifying the product resulting from the condensation of a formula (II) with formula (III), phthalic anhydride derivative of a phenol or naphthol compound of (Formula Removed) in which Rl, R2, R3, R4 and R5, which are identical to or different from one another, are selected from the group comprising the following radicals or groups: hydrogen, hydroxyl, halogen, acetyl, amino, phosphate, nitro, sulfonate, carboxyl, alkylcarboxyl having from 2 to 30 carbon atoms, alkyl having from 1 to 30 carbon atoms, cycloalkyl having from 3 to 12 carbon atoms, alkyloxy having from 1 to 30 carbon atoms, haloalkyl having from 1 to 30 carbon atoms, hydroxyalkyl having from 1 to 30 carbon atoms, alkyl ester having from 2 to 40 carbon atoms, nitroalkyl having from 1 to 30 carbon atoms, carboxyalkyl having from 2 to 30 carbon atoms, aminoaLkyl having from 1 to 30 carbon atoms, sulfoalkyl having from 1 to 30 carbon atoms, aryl, aryloxy, arylalkyl, haloary.l , aryl ester, succinimidyl ester, isothiocyanate, maleimide, iodoacetamide, haloacet-amide, chiorosulfonic, purine or pyrimidine bases, monosaccharides, preferably hexoses or pentoses, oligosides and polyosides, polypeptides, proteins, phospholipids; R3 and R5 are not hydrogen when Rl is a group -CH2-CH?-COOH, R2 is a hydroxyl group and R4 is a group -COOH, characterized in that it is carried out in an anhydrous organic . medium, by the addition of a strong acid or one of its precursors, chosen from the group comprising sulfuric acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydriodic acid polyphosphoric acid, pyrophosphate (P2O5) , and mixtures thereof, the acidification being carried out until the phthlaein crystals resulting from the condensation are converted to phthalein crystals having a different structure. 2. The method as claimed in claim 2, characterized in that the condensation product is a phthalein of general formula I. 3. The method as claimed in claim 1 or 2, characterized, in that it comprises a step consisting in washing the product obtained after acidification, said washing step being carried out with a washing solution chosen from the group comprising water, alcohols, ketones, ethers and halogenated solvents, pure or as a mixture, until the crystals are reconverted to the structure that they had before the acidification reaction. 4. A method or preparing a fluorescein having a purity such that its content of each of the by-products of the reaction is less than or equal to 0.2%, and preferable less than or equal to 0.1%, the sum of the contents of each of these by-products being less than or equal to 0.5%, said method comprising the following successive steps: - condensing phthalic anhydride with resorcinol, in a solvent consisting of an ester of an aliphatic or aromatic organic acid, preferably ethyl or methyl benzoate or ethyl or methyl palmitate, in the presence of a catalyst chosen from the group comprising in particular Lewis acids or Bronsted acids, and preferably an alkali metal hydrogen sulfate, - suspending the red-colored crystals obtained in the preceding step in an anhydrous solvent chosen from the group comprising alcohols such as absolute ethanol, ketones such as acetone, ethers, halogenated solvents, or mixtures thereof. - acidifying the suspension thus obtained by the addition of a strong acid or one of its precursors, chosen from the group comprising in particular sulfuric acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, hydriodic acid, polyphosphoric acid, phrophosphate (P2O5), and mixtures thereof, until the red-colored crystals are converted to yellow colored crystals exhibiting the X ray diffraction analysis of figure 2. - washing the crystals obtained with a washing solution chosen from the group comprising water, alcohols, ketones, ethers and halogenated solvents, pure or as a mixture, this washing being continued until the yellow-colored crystals are reconverted to red-colored crystals. 5. The method as claimed in one of claims 1 to 4, characterized in that the acidification is carried out by sparging gaseous hydrochloric acid into the phthalein suspension or by the action, on the phthalein, of hydrochloric acid in solution in the anhydrous organic solvent, preferably an alcohols, ketones, ethers and halogenated solvents, pure or as a mixture, this washing being continued until the yellow-colored crystals are reconverted to red-colored crystals. 6. The method as claimed; in one of claims 1 to 5, characterized in that the catalyst used for the condensation reaction consists of the hydrogen sulfate of an alkali metal, prefe'rabiy potassium hydrogen sulfate or sodium hydrogen sulfate. 7. A yellow-colored fluorescein crystal having the x-ray diffraction analysis of figure 2. 8. A yellow-colored 4', 5' -dimethylfluorescein crystal having the X-ray diffraction analysis of figure 4. 9. A reddish-brown- or mahogany-colored 4',5' dihydroxyfluorescein crystal having the X-ray diffract.ion analysis of figure 6. 10. A phthalein obtained by means of the method -as claimed in any one of claims 1 to 6. 11. A fluorescein obtained, by means of a method: as claimed in any one of claims 1 to 6. 12. A 4', 5' -dimethylf luorescein obtained b,y means of a method as claimed in any one of claims 1 to 6. 13. A 4', 5' -dihydroxyfluorescein obtained by means of a method as claimed in any one of claims 1 to ,6. 14. A method for acidifying; the product resulting from the condensation of a phthalic anhydride derivative of formula (II) with a phenol or naphthpl compound of formula (III), a method for preparing a fluorescein, and fluorescein crystal such as herein described with reference to the accompanying drawings and as illustrated in the foregoing examples.

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4792-delnp-2008-Abstract-(01-11-2013).pdf

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