Method for preparation of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or bromide

Abstract

The present invention relates to: a process for preparing 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide or chloride; a method of converting 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide to 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride; and the purification of 7-bis-(dimethylamino)-phenothiazin-5-ium chloride by crystallization from aqueous solution of hydrochloric acid, leading to a pharmaceutically acceptable 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (methylthioninium chloride, methylene blue, MTC) of formula I below reported. ##STR00001##

Claims

1. A 3,7-dichlorophenothiazin-5-ium chloride.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) One objective of the present invention is to provide an improved method for producing 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide or chloride within the standards set by the US and European Health Agencies, suitable for pharmaceutical use.

(2) The present invention provides a process for producing 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide or chloride (Scheme 6 and Scheme 7) comprising the steps of: (a) reacting chlorine or bromine with phenothiazine, optionally in the presence of a suitable metal catalyst, to produce 3,7-dichloro-phenothiazin-5-ium chloride or 3,7-dibromo-phenothiazin-5-ium bromide respectively; (b) adding dimethylamine to the reaction mixture of step (a) to produce 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide respectively; (c) optionally, purification of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide obtained in step (b), preferably by slurring these compounds in a suitable solvent or solvent mixture; (d) optionally, eluting the bromide material of step (b) or (c) into an ion exchange column, to exchange bromide with chloride; (e) optionally, dissolving 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide of step (b) or (c) in aqueous solution of hydrochloric acid and purifying the products; and (f) optionally, crystallizing 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or 3,7-dibromo-phenothiazin-5-ium bromide of step b), c) or d).
wherein the said first (a) and second (b) steps are carried out without isolation and/or purification of the intermediates formed in step (a) (3,7-dichloro-phenothiazin-5-ium chloride or 3,7-dibromo-phenothiazin-5-ium bromide).

(3) According to the present invention, the steps (a) and (b) of the process are a one pot reaction, which is carried out in the same reaction vessel and using one solvent system common for both reaction steps. Step (d), which is the conversion from the bromide form to the chloride form, is optional and is carried out when the material obtained in step (c) is 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide.

(4) To obtain the 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC), the step (d) of eluting the material of step (c) into an ion exchange column to exchange bromide with chloride is necessary when the material obtained in step (c) is 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide.

(5) ##STR00008##

(6) ##STR00009##

(7) Advantageously, the process of this invention produces MTC in two reaction steps, (a) and (b), which are carried out in the same reaction vessel without for the necessity of separation and purification of the intermediates between these steps, allows for the elimination of the time needed for isolation and purification of intermediates and allows for an increase in the space-time yields of reactors. In addition, since both reaction steps (a) and (b) utilize the same solvent system, the process of the invention does not require a process of solvent change after the step (a) to change it with a different solvent to carry out step (b). Moreover, the process of the invention allows a reduction of the molar excess of chlorine or bromine in reaction step (a) with respect to phenothiazine compared to what is reported in the prior art. The reduction of the molar equivalents of bromide or chlorine in step (a) and the unique solvent system for steps (a) and (b) allows a significant reduction of the solvent wastes, which represents, working on industrial scale, a huge cost reduction. A further advantage of the process of the invention is that the elimination of the need to isolate and purify the 3,7-dibromophenothiazin-5-ium bromide or 3,7-dichlorophenothiazin-5-ium chloride after step (a) allows for reducing the risk that those substances, which are known to be quite unstable, especially in solution, could degrade during the isolation and/or purification step. A further advantage of the process of the invention is that it allows the use of solvents with low toxicity, for example, ethylacetate. This last aspect of the present invention is particularly favorable when the product of the chemical process is an active pharmaceutical ingredient for which the use of solvents with low toxicity is strongly recommended by the guidelines of the main health agencies like EMEA and FDA (ref. ICH guideline Q3(R5)). The process uses cheap purification procedures of the final product affording MTC with a very low content of by-products and metals, therefore the product is suitable for pharmaceutical use.

(8) In one embodiment, the present invention is the “not isolated” intermediate obtained from the step (a) of the Scheme 6, namely 3,7-dichlorophenotiazin-5-ium chloride. This compound has been isolated and characterized by the present inventors for analytical purpose only.

(9) One embodiment of the present invention is 3,7-dichlorophenotiazin-5-ium chloride.

(10) One embodiment of the present invention is 3,7-dichlorophenotiazin-5-ium chloride for use as an intermediate.

(11) In one embodiment, the invention is a process for producing 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or bromide comprising the steps of: a) reacting chlorine or bromine with phenothiazine, to produce 3,7-dichloro-phenothiazin-5-ium chloride or 3,7-dibromo-phenothiazin-5-ium bromide respectively; b) adding dimethylamine to the reaction mixture of step a), to produce 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide respectively;
wherein steps a) and b) are carried out without isolation and/or purification of the intermediate formed in the step a). In one embodiment, in step (a) of the process of the invention, phenothiazine is dissolved in a solvent system which is contacted with chlorine or bromine to yield a 3,7-dichlorophenothiazine chloride or 3,7-dibromophenothiazine bromide intermediate. All molar ratios of chlorine or bromine:phenothiazine higher than 1:1 are suitable for the step (a) of the process of this invention, provided that 3,7-dichlorophenothiazine chloride or 3,7-dibromophenothiazine bromide are obtained as intermediates. Suitable molar ratios of chlorine or bromine to phenothiazine in step (a) are typically in the range from about 1:1 to about 4:1 bromine or chlorine:phenothiazine, preferably from about 2.2:1 to about 2.8:1 bromine or chlorine:phenothiazine. In some embodiments, the molar ratio of chlorine or bromine to phenothiazine in step (a) is about 2:1 bromine or chlorine:phenothiazine. In some embodiments, the molar ratio of chlorine or bromine to phenothiazine in step (a) is about 2.5:1 bromine or chlorine:phenothiazine. In some embodiments, the molar ratio of chlorine or bromine to phenothiazine in step (a) is about 3:1 bromine or chlorine:phenothiazine. In a preferred embodiment, the molar ratio of chlorine or bromine to phenothiazine in step (a) is about 2.5:1 bromine or chlorine:phenothiazine. In a further preferred embodiment, the molar ratio of chlorine or bromine to phenothiazine in step (a) is about 3:1 bromine or chlorine:phenothiazine.

(12) The step (a) of the process of the invention can, in some embodiments, be carried out in the presence of an inert solvent.

(13) The term “inert” means that the solvent is non-reactive towards the reagents and products and does not interfere with the process of this invention. Any inert solvent is suitable so long as it provides the intended solubilization, and so long as the 3,7-dichlorophenothiazine chloride or 3,7-dibromophenothiazine bromide are obtained as intermediates.

(14) Suitable inert solvents for step (a) include polar protic and aprotic organic solvents, such as dichloromethane, acetic acid, methylacetate, ethylacetate, butylacetate, chloroform or mixtures thereof. In one embodiment, the solvent is dichloromethane. In one embodiment, the solvent is methyl acetate. In one embodiment, the solvent is butyl acetate. In a preferred embodiment, the solvent is ethyl acetate. In some embodiments, bromine or chlorine are added to the reaction mixture of step (a) in form of solutions in said inert solvent. In other embodiments, bromine or chlorine are added into the reaction mixture of step (a) directly as pure substances, i.e. in liquid or in gaseous form, respectively. As a general rule, the volume/mass ratio between solvent and phenothiazine is between about 1 and about 40, and is preferably between about 2.5 and about 25. In one embodiment, the reaction of phenothiazine with bromine or chlorine in step (a) is carried out in the absence of catalyst. In one embodiment, the reaction of phenothiazine with bromine or chlorine in step (a) takes place in the presence of a metal catalyst. The metal catalyst can be used in its anhydrous form or in all its states of hydration. The metal catalyst which is employed in step (a) of the reaction can be any which produces desired intermediates. In some embodiments, said metal catalyst is selected from the Group VIII metals of the Periodic Table, for example, iron, cobalt, nickel. In other embodiments, said metal catalyst is selected in the group IB metals of the Periodic Table, for example, copper and/or silver. In other embodiments, said metal catalyst is selected in the group IIIB metals of the Periodic Table, such as, by way of example, aluminum. In some embodiments, the metal catalyst is added to the reaction mixture in step (a) in elemental form. In other embodiments, the metal catalyst is added to the reaction mixture in step (a) in form of salt. According to the latter embodiments, suitable salts of the above metals may be selected from the group comprising, but not limited to: chlorides, bromides, iodides, fluorides, carbonates, nitrates, sulfates, phosphates, citrates, acetates, maleates. In other embodiments, the metal catalyst is in the form of an oxide. In one embodiment, the metal catalyst of step (a) is iron. In a preferred embodiment, the metal catalyst of step (a) is an iron salt. According to the latter embodiment, said iron salt can be an iron (II) salt or an iron (III) salt, preferably an iron (III) salt. The iron (III) salt can be used in its anhydrous form or in all its states of hydration forms. In a more preferred embodiment, the Iron(III)chloride is used in its hexahydrate form. In some embodiments, said iron (II) salt can be selected from the group comprising, but not limited to: iron (II) chloride, iron (II) bromide, iron (II) iodide, iron (II) fluoride, iron (II) carbonate, iron (II) nitrate, iron (II) sulfate, iron (II) phosphate, iron (II) acetate, iron (II) maleate. In some embodiments, said iron (III) salt can be selected from the group comprising, but not limited to: iron (III) chloride, iron (III) bromide, iron (III) iodide, iron (III) fluoride, iron (III) carbonate, iron (III) nitrate, iron (III) sulfate, iron (III) phosphate, iron (III) acetate, iron (III) maleate. In a preferred embodiment, said iron (II) salt is iron (II) sulfate. In a preferred embodiment, said iron (III) salt is iron (III) chloride.

(15) Despite the knowledge in the field, it has in fact surprisingly been discovered by the present inventors that the metal catalysis above described can be usefully obtained also using an iron (II) salt, preferably iron (II) sulfate.

(16) Any hydrate form of the iron salts above can be used according to the invention.

(17) Any operable process conditions can be employed in the process of this invention provided that the desired intermediate products are formed. According to the process of the invention, the process temperature ranges for step (a) are from about −35° C. to about 45° C., preferably from about −30° C. to about 35° C., more preferably from about −20° C. to about 15° C., much more preferably from about −20° C. to about −10° C. In one preferred embodiment, the temperature for step (a) is of about −15° C. In one embodiment, the temperature of step (a) is maintained in the range between about −25° C. to about 0° C. In another embodiment, the temperature of step (a) is maintained in the range between about −20° C. to about −5° C. In a preferred embodiment, the temperature of step (a) is maintained in the range between about −20° C. to about −10° C. In one preferred embodiment, the temperature of step (a) is maintained at about −15° C. In one embodiment, step (a) is conducted at a temperature of less than about 45° C., 35° C., 25° C., 15° C., 10° C., 5° C., 0° C., −5° C., −10° C., −15° C., −20° C. or −25° C. In one embodiment, step (a) is conducted at a temperature of less than about 25° C. In one embodiment, step (a) is conducted at a temperature of less than about 0° C. In one embodiment, step (a) is conducted at a temperature of less than about −10° C. In one preferred embodiment, step (a) is conducted at a temperature of between about −20° C. and about −10° C. In one more preferred embodiment, step (a) is conducted at a temperature of about −15° C.

(18) The reaction times of step (a) depend on the reaction temperature, the rate of mixing of reactants and the concentration of reactants in the reaction zone. In one embodiment, optionally in a batch reactor, the reaction time of step (a) is equal to or less than about 6 hours. In one embodiment, the reaction time of step (a) is about 5 hours. In one embodiment, the reaction time of step (a) is about 4 hours. In one embodiment, the reaction time of step (a) is about 3 hours. In one embodiment, the reaction time of step (a) is about 2 hours. In some embodiments, the reaction time of step (a) is about 3 hours, preferably about 2 hours, more preferably about 1 hour. In a preferred embodiment, the reaction time of step (a) is about 1 hour. The reaction times may be properly adapted based on the reaction vessels and the process scale (for example, passing from pilot to industrial scale)

(19) In one embodiment of the present invention, the step (b) of the process of the present method can be carried out by addition of dimethylamine into the reaction mixture rich in 3,7-dichlorophenothiazine chloride or 3,7-dibromophenothiazine bromide formed in step (a), without any intermediate purification or isolation step. In one embodiment, said dimethylamine is added to the reaction mixture of step (b) as pure dimethylamine. According to such embodiment, said pure dimethylamine can be in gaseous or liquid form. According to another embodiment, said dimethylamine is added to the reaction mixture of step (b) in form of a solution in a suitable solvent. According to such embodiment, said suitable solvent should be inert and can be selected from the group comprising, but not limited to: methanol, water, butyl acetate and ethyl acetate, and mixtures thereof. According to a preferred embodiment, said dimethylamine is added to the reaction mixture of step (b) in form of solution in ethyl acetate. According to a preferred embodiment, said dimethylamine is added to the reaction mixture of step (b) in form of solution in methanol. Suitable molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is in the range of about 1:1 to about 1:5, preferably in the range of about 1:1.5 to about 1:4.5, more preferably in the range of about 1:2.0 to about 1:4.0 bromine or chlorine:dimethylamine. In one embodiment, the molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is about 1:2. In another embodiment, the molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is about 1:2.6. In another embodiment, the molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is about 1:3.0. In another embodiment, the molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is about 1:3.4. In another embodiment, the molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is about 1:4.0. In a preferred embodiment, the molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is about 1:3.0. In a preferred embodiment, the molar ratio of chlorine or bromine used in step (a) to dimethylamine used in step (b) is about 1:3.1.

(20) According to the process of the invention, the reaction of step (b) can be carried out at a temperature of from about −35° C. to about 45° C., preferably at a temperature from about −30° C. to about 35° C., more preferably at a temperature from about −25° C. to about 20° C., much more preferably at a temperature from about −20° C. to about 10° C. In one preferred embodiment, step (b) is carried out at a temperature of about −15° C.

(21) In one embodiment, the temperature of step (b) is maintained in the range between about −25° C. to about 25° C. In another embodiment, the temperature of step (b) is maintained in the range between about −20° C. to about 20° C. In another embodiment, the temperature of step (b) is maintained in the range between about −20° C. to about 15° C. In a preferred embodiment, the temperature of step (b) is maintained in the range between about −20° C. to about 10° C. In one preferred embodiment the temperature of step (b) is maintained at about −15° C.

(22) In one embodiment step (b) is conducted at a temperature of less than about 45° C., 40° C., 35 C, 30° C., 25° C., 20° C., 15° C. or 10° C. In one embodiment step (b) is conducted at a temperature of less than about 25° C. In one embodiment step (b) is conducted at a temperature of less than about 20° C. In one embodiment step (b) is conducted at a temperature of less than about 10° C. In one preferred embodiment, step (b) is conducted at a temperature of between about −15° C. and about 10° C.

(23) In one embodiment, step (a) is conducted at a temperature of less than about 0° C., preferably less than about −10° C., more preferably between about −20° C. and about −10° C. and step (b) is conducted at a temperature of less than about 20° C., preferably less than about 10° C., more preferably between about −20° C. and about 10° C. In one preferred embodiment, step (a) is conducted at a temperature of about −15° C. and step (b) is conducted at a temperature of about −15° C.

(24) Reaction times of step (b) depends on the reaction temperature, the rate of mixing of reactants and the concentration of reactants in the reaction zone. In one embodiment, optionally in a batch reactor, the reaction time of step (b) is equal to or less than about 6 hours. In one embodiment, the reaction time of step (b) is about 5 hours. In one embodiment, the reaction time of step (b) is about 4 hours. In one embodiment, the reaction time of step (b) is about 3 hours. In one embodiment, the reaction time of step (b) is about 2 hours. In some embodiments, the reaction time of step (b) is about 3 hours, preferably about 2 hours, more preferably about 1 hour. In a preferred embodiment, the reaction time of step (b) is about 2 hours.

(25) The reaction times may be properly adapted based on the reaction vessels and the process scale (for example, passing from pilot to industrial scale).

(26) In one embodiment of the present invention, steps (a) and (b) are carried out in the same reaction vessel and in the same solvent system. Since the regioselectivity of reaction in the step (a) of the process is high, the separation and purification of the main intermediates formed in the step (a) is not needed. Not wishing to be bound by theory it is believed that the high regioselectivity of the step (a) is due to at least in part by the selected temperature ranges or by the combination of the temperature ranges and the selected solvent. The reaction mixture from the step (a) is directly used for reaction with dimethylamine.

(27) In the process of the invention, the final reaction products of step (b), 3,7-bis-(dimethylamine)-phenothiazin-5-ium chloride or bromide, are solids which are only slightly soluble in the inert solvent used for both reactions of steps (a) and (b). Said reaction products can be separated from the reaction mixture by using common solid-liquid separation techniques well known in the art, such as, by way of example, filtration, or sedimentation, or centrifugation.

(28) In one embodiment, after separation of the raw product obtained after step (a) and (b) from the reaction mixture by using a common solid-liquid separation technique, the solid raw product is washed with the used inert solvent system and subsequently with a diluted aqueous solution of hydrochloric acid. The concentration of hydrochloric acid is in the range from about 0.05M to about 2.0M, preferably from about 0.1M to about 1.0M, more preferably from about 0.1M to about 0.4M. After washing, dimethylamine hydrobromide or hydrochloride, generated in process, are removed from the desired products.

(29) According to the invention, further purification of the products obtained from step (a) and (b) can be carried out to obtain a very low concentration of by-products and metals (within the specifications of the US and European pharmacopoeia monographs). Such purification (step (c)) may comprise different steps of slurring 3,7-bis-(dimethylamine)-phenothiazin-5-ium chloride or bromide under stirring at different temperatures and with suitable solvent. In one embodiment, said purification comprises the following steps: 1) slurring said products from steps (a) and (b) with a solvent or solvent mixtures at a temperature within the range from about 2° C. to about 35° C., preferably from about 5° C. to about 30° C., more preferably from about 20° C. to about 25° C.; slurring said products from steps (a) and (b) with a solvent or solvent mixtures, at a temperature within the range from about 25° C. to about 75° C., preferably from about 30° C. to about 65° C., more preferably from about 40° C. to about 60° C.; and, optionally, 2) slurring again said products from steps (a) and (b) with a solvent or solvent mixtures at a temperature within the range from about −5° C. to about 20° C., preferably from about 0° C. to about 15° C., more preferably from about 5° C. to about 10° C.

(30) Any of the above steps may be repeated several times in order to obtain a product of the desired quality, having very low concentration of by-products and metals (within US and European pharmacopoeia monographs). After each step, the solid material can be separated from the washing solvent through a common solid-liquid separation technique well known in the art, such as, by way of example, filtration, or sedimentation, or centrifugation. Advantageously, before any step of separation of solids from the liquid phase, the mixture to be filtered off must be at a temperature equal to or lower than about 30° C., in order to maximize the yield of the solid. The separation step is carried out at a temperature equal to or below about 30° C., preferably within the range from about −5° C. to about 25° C., more preferably from about 0° C. to about 15° C., much more preferably from about 5° C. to about 10° C.

(31) Suitable solvents or solvent mixtures for purification include polar aprotic and/or protic solvents. Said polar protic and aprotic solvents comprise, but are not limited to: dichloromethane, methylacetate, ethylacetate, butylacetate, methanol, ethanol, 2-propanol, hydrochloric acid, water or mixtures thereof. Preferably, pure alcohols or aqueous alcohols mixtures with a relative v/v ratio ranging from 1/4 to 5/1 are used as solvents. In some embodiments, the solvent for the washing is an alcohol, preferably methanol, ethanol or 2-propanol. In some embodiments, said alcohol is pure. In other embodiments, said alcohol contains a percentage of water, preferably in the range from about 5% to about 80, more preferably from about 10% to about 40% much more preferably from about 10% to about 30%. In some embodiments, the solvent is a solution of hydrochloric acid in water. According to such embodiments, the concentration of hydrochloric acid in water is preferably in the range from about 0.05M to about 2.0M, more preferably from about 0.1M to about 0.4M. In some embodiments, the purification is carried out using a mixture of the aforementioned solvents.

(32) The purifications stages can be repeated several times and can use a single solvent or solvent mixture systems.

(33) In one embodiment purification of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or 3,7-dibromo-phenothiazin-5-ium bromide obtained from step (b), (c) or (d), can be carried out by crystallization.

(34) Suitable solvents or solvent mixtures for crystallization include methanol, ethanol, 2-propanol, hydrochloric acid, water or mixtures thereof.

(35) The crystallization can be repeated several times and can use a single solvents or solvent mixture systems.

(36) In one embodiment, when the product obtained after steps (a), (b) and optionally (c) is 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide, in order to obtain 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) said product must be eluted into an ion exchange column to exchange the bromide ion with the chloride ion. According to such embodiment, after the optional purification step (c), the product obtained from steps (a) to (b) is dissolved in a suitable solvent and is eluted through a column containing a macroporous anion exchange resin in an activated chloride form, to perform an anion exchange on the product (step (d)).

(37) Suitable solvents to dissolve the product can be methanol, ethanol, 2-propanol, water or mixtures thereof. In some embodiments, a mixture of ethanol and water is used, the mixture having a v/v percentage of water ranging from about 1% to about 40%, more preferably from about 5% to about 35%, more preferably from about 8% to about 25%, much more preferably from about 9% to about 20%. In some embodiments, a mixture of methanol and water is used, the mixture having a v/v percentage of water ranging from about 1% to about 40%, more preferably from about 5% to about 35%, more preferably from about 8% to about 25%, much more preferably from about 9% to about 20%. In some embodiments, a mixture of 2-propanol and water is used, the mixture having a v/v percentage of water ranging from about 1% to about 40%, more preferably from about 5% to about 35%, more preferably from about 8% to about 25%, much more preferably from about 9% to about 20%. This solution is then eluted on a column filled with anion exchange resin in activated chloride form.

(38) In one embodiment, the macroporous anion exchange resin is a macroporous polystyrene crosslinked with divinylbenzene containing quaternary ammonium groups.

(39) In one embodiment the macroporous anion exchange resin is Purolite A500® in a chloride form.

(40) In one embodiment, a final crystallization step of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride can be carried out in an aqueous solution of hydrochloric acid, preferably in the concentration range from about 0.05M to about 2.0M, more preferably from about 0.1 M to about 0.4M (steps (e) and (0)

(41) In one embodiment, the purity of obtained products (3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide and 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) falls within the current, draft version and following updates for standards set out in the US and European pharmacopoeia monographs of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC).

(42) In one embodiment, the product of the present invention has very low metal content, for example, less than about 1 ppm arsenic, less than about 2 ppm copper and/or less than about 2 ppm zinc.

(43) In one embodiment, the present invention therefore provides also for a 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide having a purity higher than about 97% and less than about 1 ppm of arsenic.

(44) In another embodiment, the present invention also provides for a 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide having a purity higher than about 97% and less than about 2 ppm copper and less than about 2 ppm zinc.

(45) In another embodiment, the present invention also provides for a 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide having a purity higher than about 97%, less than 1 about ppm arsenic, less than about 2 ppm copper and less than about 2 ppm zinc.

(46) In one further embodiment, the present invention also provides for a 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide obtained or obtainable from the process for producing 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or bromide as above disclosed.

(47) In one further embodiment, the present invention provides the compound obtained from the step (a) of the Scheme 6, namely 3,7-dichlorophenotiazin-5-ium chloride as such.

(48) In one further embodiment, the present invention provides the compound obtained from the step (a) of the Scheme 6, namely 3,7-dichlorophenotiazin-5-ium chloride as intermediate of a process for the production of 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride.

(49) In one further embodiment, the present invention provides for a pharmaceutical composition containing a 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or the 3,7-bis-(dimethylamino)-phenothiazin-5-ium bromide having the characteristics as above disclosed in term of purity/metals and/or obtained or obtainable from the process for producing 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride or bromide as above disclosed.

(50) In one further embodiment, the present invention provides for a 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride (MTC) or bromide as above disclosed or a pharmaceutical composition as above disclosed for use in diagnostic field, preferably in the gastro-intestinal endoscopy evaluation of inflammatory, ulcerative, pre-neoplastic, neoplastic, dysplastic pathologies and/or lesion of the gastro-intestinal tract. According to such an embodiment, the invention is able to ensure enhancing the detection of intestinal mucosal lesions as, for example, cancerous forms, pre-cancerous forms, interval cancers, adenomas, carcinomas, serrated lesions, intraepithelial neoplasia, dysplasia, polyps, pseudo-polyps, pre-polyps or different inflammatory pathologies, lesions sessile, flat or pedunculated shaped.

EXAMPLES

Example 1. Crude 3,7-bis-(dimethylamino)-phenothiazin-5-ium-bromide

(51) Into a cooled reaction vessel (under 10° C.) phenothiazine (10.86 kg), ethyl acetate (216 liters) and catalyst, iron (III) chloride (30 g), were added. The reaction mixture was cooled (under 10° C.) and bromine (21.84 kg, 7.0 liters) in ethyl acetate (21.0 liters) was then added. The reaction temperature was kept within the range −10° C. to −15° C. After addition of all the bromine, the reaction mixture was stirred for an additional one hour at a temperature of −10° C. to −15° C.

(52) Then, dimethylamine (19.0 kg) in ethyl acetate (77 liters) was added and the reaction temperature was kept within the range from −15° C. to +10° C. After addition of all of the dimethylamine, the reaction mixture was stirred for an additional two hours at a temperature of −15° C. to +10° C.

(53) The raw product was filtered off, washed with cooled ethyl acetate and with 0.1 M hydrochloric acid. The isolated product was then purified using the below described methods.

Example 1 BIS. Crude 3,7-bis-(dimethylamino)-phenothiazin-5-ium-bromide

(54) Into a cooled reaction vessel (under 10° C.) phenothiazine (10.86 kg), ethyl acetate (216 liters) and catalyst, iron (Ill) chloride (30 g), were added. The reaction mixture was cooled (under 10° C.) and bromine (21.84 kg, 7.0 liters) in ethyl acetate (21.0 liters) was then added. The reaction temperature was kept within the range −10° C. to −15° C. After addition of all the bromine, the reaction mixture was stirred for an additional one hour at a temperature of −10° C. to −15° C.

(55) Then, dimethylamine (19.0 kg) in methanol (77 liters) was added and the reaction temperature was kept within the range from −15° C. to +10° C. After addition of all of the dimethylamine, the reaction mixture was stirred for an additional two hours at a temperature of −15° C. to +10° C.

(56) The raw product was filtered off, washed with cooled ethyl acetate and with 0.1 M hydrochloric acid. The isolated product was then purified using the below described methods.

Example 2. Crude 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride

(57) To a cooled reaction vessel (under 10° C.) phenothiazine (10.86 g), ethyl acetate (250 ml) and catalyst, iron (Ill) chloride (30 mg) were added. The reaction mixture was cooled (under 10° C.) and gaseous chlorine (9.66 g, condensed by −30° C.) was added. The reaction temperature was kept within the range −10° C. to −15° C. After addition of all of the chlorine the reaction mixture was stirred for an additional hour at temperature within the range −10° C. to −15° C.

(58) Then, dimethylamine (19.0 g) in ethylacetate (77 ml) was added and the reaction temperature was kept within the range −15° C. to +10° C. After addition of all of the dimethylamine, the reaction mixture was stirred for additional two hours at a temperature within the range −15° C. to +10° C.

(59) The raw product was filtered off, washed with cooled ethyl acetate and with 0.1 M hydrochloric acid. The isolated product was then purified using the below described methods with aliquot volumes of solvents.

Example 3. Purified 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride from crude 3,7-bis(dimethylamino)-phenothiazin-5-ium bromide

(60) The isolated product from Example 1 (3,7-bis(dimethylamino)-phenothiazin-5-ium bromide) was washed under stirring in 2-propanol (60 liters) at room temperature for one hour, and filtered off. 2-propanol was added and washing under stirring was repeated at temperature of about 50° C., the mixture was cooled to room temperature and filtered off.

(61) At the end a mixture of 2-propanol (12 liters) and 0.5 M hydrochloric acid (48 liters) was added and the slurry procedure was repeated at temperature of about 50° C. The mixture was cooled to room temperature and filtered off.

(62) The product was dissolved in mixture of 2-propanol (100 liters) and water (20 liters) at 60° C. and eluted through an anion exchange resin column (45 liters of Purolite A500, chloride form); then the eluates were concentrated by evaporation. The dry product was dissolved in 0.4 M hydrochloric acid (100 liters) at 60° C. and then cooled at 10° C., the solution was stirred at this temperature for two hours and the product was filtered off and dried. Yield 9.6 kg.

(63) The .sup.13C and .sup.1H-NMR spectra ((CD.sub.3).sub.2SO; 600 MHz) of this product confirm the structure of the compound. .sup.1H-NMR spectrum (values in ppm): 3.34 (s, 12H, 2×N(CH.sub.3).sub.2, 7.43 (m, 4H, aromatics; 2, 4, 6 and 8 CH), 7.83 (d, 2H, aromatics; 1 and 9 CH). .sup.13C NMR spectrum (values in ppm): 41.1 (N(CH.sub.3).sub.2), 106.8 (4 and 6 CH), 119.0 (2 and 8 CH), 133.5 (12 and 13 CS.sup.+), 134.9 (11 and 14 CN), 137.8 (1 and 9 CH) and 153.8 (3 and 7 C—N(CH.sub.3).sub.2).

Example 4. Purified 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride from crude 3,7-bis(dimethylamino)-phenothiazin-5-ium bromide

(64) The isolated product from Example 1 (3,7-bis(dimethylamino)-phenothiazin-5-ium bromide) was slurried in ethanol (60 liters) at room temperature for one hour and filtered off. Ethanol was added and slurry procedure was repeated at temperature of about 50° C. The mixture was cooled to room temperature and filtered off.

(65) At the end a mixture of ethanol (12 liters) was added and 0.5 M hydrochloric acid (48 liters) and the slurry procedure was repeated at temperature of about 50° C. The mixture was cooled to room temperature and filtered off.

(66) The product was dissolved in a mixture of ethanol (100 liters) and water (20 liters) at 60° C. and eluted through a column packed with a anion exchange resin (45 liters of Purolite A500, chloride form); then the eluted fractions were evaporated under vacuum to dryness. The dry product was dissolved in 0.4 M hydrochloric acid (100 liters) at 60° C. and cooled under 10° C., the solution was stirred at this temperature for two hours and the product was filtered off and dried. Yield 10.8 kg.

(67) The registered .sup.13C and .sup.1H-NMR spectra ((CD.sub.3).sub.2SO; 600 MHz) confirm the structure of the examined compound: the NMR signals are practically superimposeable to those obtained by the analysis of the sample isolated by the example 3.

Example 5. Purified 3,7-bis-(dimethylamino)-phenothiazin-5-ium chloride from crude 3,7-bis(dimethylamino)-phenothiazin-5-ium bromide

(68) The isolated product from Example 1 (3,7-bis(dimethylamino)-phenothiazin-5-ium bromide) was slurried in methanol (60 liters) at room temperature for one hour and filtered off. Methanol was added and the slurry procedure was repeated at temperature of about 50° C. The mixture was cooled to room temperature and filtered off.

(69) At the end a mixture of methanol (12 liters) and 0.5 M hydrochloric acid (48 liters) was added and the slurry procedure was repeated at temperature of about 50° C. The mixture was cooled to room temperature and filtered off.

(70) The product was dissolved in mixture of methanol (100 liters) and water (20 liters) at 60° C. and eluted through a column filled with an anionic exchange resin (45 liters of Purolite A500, chloride form); then the collected eluates were concentrated by evaporation. The dry product was dissolved in 0.2 M hydrochloric acid (100 liters) at 60° C. and cooled under 10° C. The solution was stirred at this temperature two hours and the product was filtered off and dried. Yield 12.4 kg.

(71) The .sup.13C and .sup.1H-NMR spectra ((CD.sub.3).sub.2SO; 600 MHz) registered on this product confirm the structure of the compound: the NMR signals are practically superimposeable to those obtained by the analysis of the samples isolated by the examples 3 and 4.

Example 6. Comparative Example: Preparation of Purified 3,7-bis-(dimethylamino)-phenothiazin-5-ium-bromide without the Use of FeCl.SUB.3

(72) Into a cooled reaction vessel (under 10° C.) phenothiazine (10.86 kg) and ethyl acetate (216 liters) were added. The reaction mixture was cooled (under 10° C.) and bromine (21.84 kg, 7.0 liters) in ethyl acetate (21.0 liters) was then added. The reaction temperature was kept within the range −10° C. to −15° C. After addition of all the bromine, the reaction mixture was stirred for an additional two hours at a temperature of −10° C. to −15° C.

(73) Then, dimethylamine (19.0 kg) in ethyl acetate (77 liters) was added and the reaction temperature was kept within the range from −15° C. to +10° C. After addition of all of the dimethylamine, the reaction mixture was stirred for an additional two hours at a temperature of −15° C. to +10° C.

(74) The raw product was filtered off, washed with cooled ethyl acetate and with 0.1 M hydrochloric acid.

(75) The isolated raw product (3,7-bis(dimethylamino)-phenothiazin-5-ium bromide) was slurried in methanol (60 liters) at room temperature for one hour and filtered off. Methanol was added and the slurry procedure was repeated at temperature of about 50° C. The mixture was cooled to room temperature and filtered off.

(76) At the end a mixture of methanol (12 liters) and 0.5 M hydrochloric acid (48 liters) was added and the slurry was repeated at temperature of about 50° C. The mixture was cooled to room temperature and filtered off.

(77) The product was dissolved in mixture of methanol (100 liters) and water (20 liters) at 60° C. and eluted through a column filled with an anionic exchange resin (45 liters of Purolite A500, chloride form); then the collected eluates were concentrated by evaporation. The dry product was dissolved in 0.2 M hydrochloric acid (100 liters) at 60° C. and cooled under 10° C. The solution was stirred at this temperature two hours and the product was filtered off and dried. Yield 11.1 kg.

(78) The .sup.13C and .sup.1H-NMR spectra ((CD.sub.3).sub.2SO; 600 MHz) registered on this product confirm the structure of the compound: the NMR signals are practically superimposeable to those obtained by the analysis of the samples isolated by the examples 3, 4 and 5.

(79) TABLE-US-00001 TABLE 1 Relevant Analytical Results of samples obtained according to the above Examples 3-5 Example 3 Example 4 Example 5 Product 97.35 97.65 97.58 % HPLC Impurity A 2.26 2.10 2.06 % HPLC* Total other 0.39 0.25 0.36 impurities % HPLC Arsenic ppm <1 <1 <1 Copper ppm <2 <2 <2 Zinc ppm <2 <2 <2 Iron ppm 35.8 31.5 28.9 Bromides absent absent Absent *HPLC methods and specifications according to PhEur