RU2725666C1 - Method of producing 5-, 6-amino-fluoresceins - Google Patents

Abstract

FIELD: medicine; chemistry.SUBSTANCE: invention relates to organic chemistry and medicine, namely to a method of producing 5- and 6-aminofluoresceins by reacting 4-nitrophthalic acid with resorcinol taken in molar ratio 1:2, flowing in medium of orthophosphoric acid, taken in ratio to weight of 4-nitrophthalic acid as 1:2.5 (weight ratio of orthophosphoric acid – 1.75 g/ml) at temperature of 135 °C to form mixture of 5- and 6-nitrofluoresceine isomers and its separation and separation through formation of 5- and 6-nitrofluorescein dipropionates, followed by reduction of each of them to form corresponding amine derivatives, characterized by that in order to separate a mixture of isomers of nitrofluorescein (5- and 6-), propionic anhydride is used in amount of 1:8, which is mixed with a mixture of isomers, the obtained mixture is boiled for 2.5 hours, cooled, filtered and deposited 5-nitrofluorescein dipropionate is washed with ethyl alcohol, dried and subjected to boiling in aqueous solution of sodium sulphide, taken in molar ratio 1:3.3 to 5-nitrofluorescein dipropionate, and 5-aminofluorescein is recovered, and separated after filtration mother solution containing 6-nitrofluorescein dipropionate, treated with distilled water to obtain oily residue, it is crystallized by grinding, crystalline precipitate is separated by filtration and washed with ethanol, dried and recrystallised from benzene, after which obtained 6-nitrofluorescein dipropionate is also subjected to boiling in aqueous solution of sodium sulphide, taken in molar ratio 1:3.3 to 6-nitrofluoresceine dipropionate and isolating 6-aminofluorescein.EFFECT: described is a method of producing which eliminates use of the opioid series as a precursor in production of drugs, which makes the method more readily available for mass production of fluorescein nitrated derivative; these compounds can be used in synthesis of various fluorescent compounds, such as 5-isothiocyanate of fluorescein (FITC), fluorescein lysicol (CFL), 5-[4,6-dichlorotriazinyl] aminofluorescein (5-DATF) used in medicine for diagnosis and treatment diseases.1 cl, 1 ex

Description

The invention relates to the field of organic chemistry and medicine, namely to fluorescein nitro derivatives that can be used in the synthesis of various fluorescent compounds, such as fluorescein 5-isothiocyanate (FITC), fluorescein lysicol (CFL), 5- [4,6-dichlorotriazinyl] aminofluorescein (5-DATF) used in medicine for the diagnosis and treatment of diseases,

It is known that fluorescein derivatives are actively used in medicine for the diagnosis and treatment of diseases. For example, fluorescein lysicol is used in pharmacokinetics studies and in the diagnosis of liver cirrhosis, hepatitis, viral diseases, non-alcoholic steatohepatitis in humans and non-alcoholic fatty liver disease [Mills S.O. et al. Cholyl-lysylfluorescein: synthesis, biliary excretion in vivo and during single-pass perfusion of isolated perfused rat liver // Biochimica et Biophysica Acta (BBA) -General Subjects. - 1991. - V. 1115. - No. 2. - P. 151-156.], For the treatment of atherosclerosis [US 2007116754, A61K 49/00,, 2007]. Fluorescein-5-isothiocyanate has a wide range of biological applications, including use as a fluorescent marking agent for proteins, as a fluorescent reagent for protein tracking, and as a reagent in the technique of fluorescent antibodies for the rapid identification of pathogens [US 0220787, C07D 493/10,. 2012]. For example, the use of FITC for the diagnosis of in vivo prostate cancer is known [RU 2450832, A61K 49/14, 2012]. 5- [4,6-Dichlorotriazinyl] aminofluorescein (DATF) has a high protein reactivity. Unlike other reactive fluoresceins, 5-DTAF not only reacts with amino groups, but also reacts with thiol groups and even directly reacts with hydroxy groups, such as polysaccharides and other alcohols in an aqueous solution at pH above 9. Due to its high reactivity , 5-DTAF is also used to label carbohydrates as well as biologically significant molecules in vivo [GB 2081257, C07D 405/04, 1982].

Derivatives of fluorescein, in particular 5-isothiocyanate fluorescein (FITC), fluorescein lysicol, can be obtained from 5-aminofluorescein synthesized by a known method from 5-nitrofluorescein. The latter, in turn, is obtained according to the scheme described below by the reaction of 4-nitrophthalic acid with resorcinol catalyzed by phosphoric acid [Coons AN, Kaplan MN Localization of antigen in tissue cells: II. Improvements in a method for the detection of antigen by means of fluorescent antibody // Journal of Experimental Medicine. - 1950. - T. 91. - No. 1. - C. 1-13 .; Glushko V.N., Blokhina L.I., Sadovskaya N.Yu. Studies on the production of fluorescein-5-isothiocyanate-fluorescent nanomarker for the creation of biosensors // Russian Chemical Journal. - 2014. - T. 58. - No. 1. - S. 40-45.], Or methanesulfonic acid [US 0220787 A1, C07D 493/10, 2012].

The result is a mixture of isomers of 5- and 6-nitrofluorescein, which is separated using acetic anhydride,

The preparation of 5-aminofluorescein is carried out either by first saponification of the obtained 5-nitrofluorescein acetates with alcohol alkali, followed by reduction of the obtained 5-nitrofluorescein in a solution of alcohol alkali with hydrazine hydrate on a nickel catalyst to obtain 5-aminofluorescein [Coons A.N., Kaplan M.N. Localization of antigen in tissue cells: II. Improvements in a method for the detection of antigen by means of fluorescent antibody // Journal of Experimental Medicine. - 1950. - T. 91. - No. 1. - C. 1-13.], Or by boiling in an aqueous solution of sodium sulfide [Glushko VN, Blokhina LI, Sadovskaya N.Yu. Studies on the production of fluorescein-5-isothiocyanate-fluorescent nanomarker for the creation of biosensors // Russian Chemical Journal. - 2014. - T. 58. - No. 1. - S. 40-45.]. The restoration of the nitro group and the saponification of acetates in the second case occurs in one stage. Below is a reaction scheme characterizing the course of this process of synthesis of 5-aminofluorescein:

The main disadvantage of the above methods for the synthesis of 5-aminofluorescein is the use of acetic anhydride, which is a precursor in the production of heroin and other drugs of the opioid group, at the stage of separation of the mixture of isomers of 5 and 6-nitrofluoresceins. In this regard, acetic anhydride is included in the lists of narcotic drugs, psychotropic substances and their precursors subject to control that have been approved in a number of states, including the Russian Federation [In Russia, it was included in Decree of the Government of the Russian Federation of June 30, 1998 N 681 “On approval of the list of narcotic drugs, psychotropic substances and their precursors subject to control in the Russian Federation” (with amendments and additions)].

So, for example, in the method chosen by us as a prototype [Glushko V.N., Blokhina L.I., Sadovskaya N.Yu. Studies on the production of fluorescein-5-isothiocyanate-fluorescent nanomarker for the creation of biosensors // Russian Chemical Journal. - 2014. - T. 58. - No. 1. - P. 40-45.], 5-aminofluorescein by the reaction of 4-nitrophthalic acid with resorcinol taken in a molar ratio of 1: 2, proceeding in phosphoric acid medium, taken in the ratio to the mass of 4-nitrophthalic acid as 1: 2.5 (the concentration of phosphoric acid is 70%, which corresponds to a specific gravity of 1.52) at a temperature of 135 ° C until a mixture of 5- and 6-nitrofluoresceins isomers is formed and is isolated. Acetic anhydride is used to separate the mixture of 5- and 6-nitrofluorescein diacetates. 5-nitrofluorescein diacetate is reduced by boiling in an aqueous solution of sodium sulfide taken in a molar ratio of 1: 3.3 to 5-nitrofluorescein diacetate and 5-aminofluorescein is isolated.

The aim of the invention is to obtain 5-aminofluorescein and 6-aminofluorescein in an affordable manner that would ensure the receipt of a product that in quality would meet all the requirements for reagents for medicine.

The proposed production method consists in the following: 5- and 6-aminofluoresceins are obtained by the reaction of 4-nitrophthalic acid with resorcinol taken in a molar ratio of 1: 2, proceeding in an orthophosphoric acid medium, taken in the ratio to the mass of 4-nitrophthalic acid as 1: 2.5 (specific weight of phosphoric acid - 1.75 g / ml) at a temperature of 135 ° C until a mixture of 5- and 6-nitrofluoresceins isomers is formed and it is isolated and separated through the formation of 5- and 6-nitrofluorescein dipropionates, followed by reduction of each of them to the formation of the corresponding amino derivatives, characterized in that to separate the mixture of nitrofluorescein isomers (5- and 6-) propionic anhydride is used in an amount of 1: 8, which is mixed with the mixture of isomers, the resulting mixture is boiled for 2.5 hours, cooled, filtered and precipitated the precipitate of 5-nitrofluorescein dipropionate is washed with ethyl alcohol, dried and subjected to boiling in an aqueous solution of sodium sulfide, taken in a molar ratio of 1: 3.3 to 5-nitro fluorescein dipropionate and 5-aminofluorescein is isolated, and the mother liquor containing 6-nitrofluorescein dipropionate separated after filtration is treated with distilled water to obtain an oily precipitate, it is crystallized by trituration, the crystalline precipitate is filtered off and washed with ethanol, dried and recrystallized from benzene 6 nitrofluorescein dipropionate is also boiled in an aqueous solution of sodium sulfide taken in a molar ratio of 1: 3.3 to 6-nitrofluorescein dipropionate and 6-aminofluorescein is isolated

The proposed method for producing 5-nitrofluorescein (a precursor of 5-aminofluorescein) by the reaction of 4-nitrophthalic acid with resorcinol is characterized in that more concentrated orthophosphoric acid is used to obtain a mixture of 5-, 6-nitrofluoresceins, and nitrofluorescein isomers (5- and 6- ) use propionic anhydride.

Propionic anhydride - a homologue of acetic anhydride, is not a precursor in the production of heroin and other drugs of the opioid group, is a raw material available for mass production.

The invention is illustrated below by the following example:

Example

1. Obtaining a mixture of 5-, 6-nitrofluoresceins:

220 g (2 mol) of resorcinol, 211 g (1 mol) of 4-nitrophthalic acid and 300 ml of orthophosphoric acid (90%, specific gravity 1, are charged into a 1-liter three-neck round-bottom flask equipped with a thermometer, a mechanical stirrer, and reflux condenser). 75 g / ml). Heated to 135 ° C and maintained at this temperature for 3.5 hours. The reaction mass is cooled to 100 ° C and poured into 1 liter of distilled water heated to 80 ° C. The reaction flask is rinsed with 0.5 l of hot distilled water and attached to the bulk. It is stirred for 1 hour, the precipitate is filtered off, transferred to a 2 L flask, 1 liter of distilled water is added, boiled for 1 hour and filtered off hot. Dried at 110 ° C in a vacuum oven to constant weight. 312 g of a mixture of 5- and 6-nitrofluoresceins are obtained. Yield 90%.

2. Obtaining a mixture of 5-, 6-nitrofluorescein diacetates and its separation into isomers

422 g (1.12 mol) of a mixture of 5-, 6-nitrofluoresceins and 1162 g (1145 ml, 8.94 mol) of propionic anhydride are placed in a 2.0-liter three-necked flask, heated to boiling and boiled for 2.5 hours . Then the reaction mixture was poured into a 2.0 L beaker, the precipitate formed was filtered off with cooling, and washed with 350 ml of ethyl alcohol. After drying, 137 g (0.28 mol) of 5-nitrofluorescein dipropionate are obtained. Yield: 25%.

T _pl. = 215-218 ° C. TLC (thin layer chromatography) (Toluene / AcOEt 7: 1): Rf 0.64. 1 H NMR (DMSO d6, σ): 8.70 (d 1H); 8.62 (dd, 1H); 7.75 (d, 1H); 7.30 (d, 2H); 7.05-6.52 (m, 4H); 2.62 (q, 4H (CH ₂ )) 1.29 (t, 6H (CH ₃ )).

The mother liquor is poured into 2 l of distilled water. An oily precipitate forms, which crystallizes upon grinding with a glass rod. The precipitate is filtered off, washed with 100 ml of ethyl alcohol, dried and recrystallized from 700 ml of benzene. After drying, 109.5 g of 6-nitrofluorescein dipropionate (0.224 mol) are obtained. Yield: 20%. T _pl. = 191 ° C. TLC (thin layer chromatography) (Toluene / AcOEt; 7: 1) Rf 0.51. 1 H NMR (DMSO, d6): 8.70 (d 1H); 8.40 (dd, 1H); 7.75 (d, 1H); 7.30 (d, 2H); 7.05-6.52 (m, 4H); 2.62 (q, 4H (CH ₂ )) 1.29 (t, 6H (CH ₃ )).

3. The restoration of the nitro group with the simultaneous saponification of diproionates (for example, obtaining 5-aminofluorescein:

In a two-necked flask with a volume of 1000 ml, equipped with a reflux condenser, a dropping funnel, a magnetic stirrer, 50.6 g (0.11 mol) of 5-nitrofluorescein dipropinate and 500 ml of water are placed. To this solution a solution of 87.2 g (0.363 mol) of 9-aqueous sodium sulfide in 220 ml of water from a dropping funnel is added in small portions. The suspension of 5-nitrofluorescein diacetate is completely dissolved and the color of the solution from tan becomes dark red. The solution is boiled for 2 hours, then cooled to room temperature. It is filtered off from the released sulfur and acidified with a small amount of acetic acid (52 g, 50 ml)). A fine red precipitate forms. The precipitate is filtered off, dried on the filter, and dried in a vacuum oven at 90 ° C to constant weight. 26.7 g of a brick-red precipitate of 5-aminofluorescein was obtained. Yield: 70%. ¹ H NMR (DMSO d6, σ) 10.05 (s, 2H); 7.00 - 6.80 (m, 3H); 6.65 - 6.50 (m, 6H); 5.70 (s, 2H).

Claims (1)

The method of producing 5- and 6-aminofluoresceins by the reaction of 4-nitrophthalic acid with resorcinol taken in a molar ratio of 1: 2, proceeding in an orthophosphoric acid medium, taken in the ratio to the mass of 4-nitrophthalic acid as 1: 2.5 (specific weight of orthophosphoric acid - 1.75 g / ml) at a temperature of 135 ° C until a mixture of isomers of 5- and 6-nitrofluoresceins is formed and it is isolated and separated through the formation of 5- and 6-nitrofluorescein dipropionates, followed by reduction of each of them to form the corresponding amino derivatives, characterized in order to separate the mixture of nitrofluorescein isomers (5- and 6-), propionic anhydride is used in an amount of 1: 8, which is mixed with the mixture of isomers, the resulting mixture is boiled for 2.5 hours, cooled, filtered and the precipitate formed is 5-nitrofluorescein dipropionate washed with ethanol, dried and boiled in an aqueous solution of sodium sulfide taken in a molar ratio of 1: 3.3 to 5-nitrofluorescein dipropionate and isolated with 5 nofluorescein, and the mother liquor containing 6-nitrofluorescein dipropionate separated after filtration is treated with distilled water to obtain an oily precipitate, it is crystallized by trituration, the crystalline precipitate is filtered off and washed with ethanol, dried and recrystallized from benzene, after which the obtained 6-nitrofluorescein is also dipropionated boiling in an aqueous solution of sodium sulfide, taken in a molar ratio of 1: 3.3 to 6-nitrofluorescein dipropionate, and 6-aminofluorescein is isolated.