RU2398774C2 - Method for synthesis of 3'-iodofolic acid and 3'-bromofolic acid labelled with radioactive isotopes of iodine and bromine - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: source of radioactive iodine or radioactive bromine is added to an aqueous solution of an alkaline salt of folic acid so that pH of this solution is approximately 7 in order to obtain a solution which contains folic acid or an iodide or bromide ion. PH of this solution is then lowered to 3.5-4.9 by adding an oxidising agent selected from N-chlorosulfamides of acids in a buffer solution to the said solution. The oxidising agent is mainly chloramines T or chloramines B.

EFFECT: possibility of adding isotopic tags to folic acid through radicals.

2 cl, 2 ex

Description

The invention relates to the field of biochemistry and radiochemistry.

Folic acid belongs to the class of vitamins of group B.

Folic acid and its derivatives, such as aminopterin and methotrexate, are effectively absorbed by certain types of cancerous tumors. This makes it possible to use folic acid and its derivatives labeled with radioactive isotopes for medical diagnosis and radiotherapy. Since folic acid in the human body is contained in very small quantities (the concentration in the blood plasma is hundredths of a micromole per liter), the prepared preparations must have a high specific radioactivity, and the synthesis method should be acceptable for working with short-lived isotopes such as iodine-121 or bromine-75 with a half-life of about 2 hours. Recently, these isotopes are finding wider application in positron emission tomography, since they are positron emitters.

The literature describes methods for producing iodine-labeled various derivatives based on solutions of the compound to be labeled using radioactive iodine in the form of iodide ions and chloramine.

A known method for the labeling of biological compounds described in [1.]: F. C. Greenwood & W. M. Hunter, Biochem. J. (1963) 89, 114. The reaction is carried out at pH 7.5

However, this method, when folic acid is used for iodine labeling, gives very low radioactivity yields (less than 1%).

To circumvent the inertness of folic acid in this reaction, methods were patented in which easily iodinated tyrosine or histidine were added to the glutamine residue of folic acid.

A known method of producing derivatives of folic acid labeled with iodine, described in [2]: US Patent No. 3989812 ,; [3] US Patent No. 4276280). In this case, the compounds corresponding to formula (1) were obtained, the iodine label occupied the position R.

The method described in US Patente No. 3989812, is as follows. To a solution consisting of 100 μl of 0.1 M sodium hydroxide and containing 1 mCi NaI ¹²⁵ , 20 μl of 0.5 M NaH ₂ PO ₄ and 10 μl of 0.5 M sodium phosphate pH 7.4, add 10 μg pteroyl-γ-glutamyl-tyrosine in phosphate buffer ( 0.05 M, pH 7.4) and 20 μl of a freshly prepared solution of chloramine T in 0.05 M phosphate buffer pH 7.4 (5 mg / ml). After 15 seconds, 20 μl of a solution of sodium metabisulfite in 0.05 M phosphate buffer pH 7.4 was added. The reaction mixture was separated on a BioGel-P2 column, and then on a DEAE ion-exchange column.

The disadvantage is that this does not produce iodine-labeled folic acid, but iodine-labeled folic acid derivative, which differs significantly from folic acid in structure, charge distribution, and the presence of additional hydrophobic groups. This can change the conditions of its transport in the cell compared to natural folic acid.

It is known, for example, that if instead of histidine or tyrosine there is another glutamic acid residue, then such a folic acid derivative does not penetrate into the cell despite the fact that the glutamic acid molecule occupies a smaller volume than the histidine or tyrosine molecule.

In this connection, it seems to be a promising method that allows the folic acid itself to be labeled with iodine and bromine isotopes, and not the products of its addition to other easily iodized molecules, i.e. method allowing

to obtain compounds corresponding to the formula (2), where bromine or iodine occupies the position R.

Moreover, this method should be suitable for working with amounts of iodine or bromine less than a microgram.

The literature describes a method for labeling isotope I ^{131 of a} compound close in structure to folic acid, α-aminopterin [4]: DGJohus et al J. Nuclear Med., V.9, No. 10, 1968, 530-536). In this method, 3'-iodaminopterin, i.e. the iodine label is attached to the compound itself (formula (3)), and not to the structure that facilitates iodization.

To a solution of 10 MCi NaI ¹³¹ in 0.4 ml of sodium sulfate, the following reagents were added with stirring in the indicated order: aminopterin powder (20 mg); iodic acid - 4.5 mg; iodine - 7.5 mg; dimethylformamide - 0.5 ml; and carbon tetrachloride - 0.1 ml. The vessel was closed and stirred for 18 hours. Then, the reaction mixture was separated on a DEAE cellulose column.

However, the method gives a small yield - (4 ÷ 10%), low specific activity (in this publication, in several experiments, a spread of 50-300 microcuries per micromole was obtained), the formation of a large number of contaminants, including radioactive ones, and the duration of the synthesis procedure ( 18 hours).

This method is close to that claimed by the fact that they directly label a compound that is close in structural formula, and not its derivative.

The inventive object relates to a method for labeling isotopes of iodine or bromine directly with folic acid.

In the patent and scientific and technical literature, we did not find methods for labeling folic acid, so there is no prototype.

We declare a method for producing 3'-iodopholic and 3'-bromofolic acids labeled with radioactive isotopes of iodine and bromine, which consists in preparing an initial solution containing folic acid, radioactive iodine or radioactive bromine in the form of an iodide ion or bromide ion with pH 7, add an acidified solution of an oxidizing agent from the group of chlorinated acid amides to the initial solution, adjust the pH of the solution to 3.5–4.9, optimally to 4.7, converting folic acid into a gel. The selection of the target product is carried out by reverse phase chromatography. Chloramine T or chloramine B is used as an oxidizing agent.

Our studies showed that the cause of failure when trying to label folic acid itself according to the methods [1] is the slowness of the reaction of folic acid with iodic acid HIO, which in methods [1] is the active agent and is formed during the reaction of the iodide ion with chloramine. Due to the slow reaction of HIO with folic acid, the dominant reactions in the system are the disproportionation of HIO and its oxidation with an excess of chloramine, leading to a radical decrease in the yield of the target product and an increase in the yield of by-products.

Carrying out the reaction under conditions conducive to the formation of I ⁺ and Br ⁺ cations allowed us to develop an effective synthesis of iodopholic and bromofolic acids labeled with isotopes of iodine and bromine.

The essence of our proposed method is that in the iodination reaction using oxidizing agents such as chloramine, which in all patents is carried out at a pH above 7, go to the reaction at a pH significantly lower. However, if the reaction is carried out under conditions in which the synthesis of relatively large amounts of unlabeled iodopholic acid is carried out, described in the patents [5] - US Patent 2570391 (1951); [6] US Patent 2570392 (1951), where folic acid is in a solution of 5M HCl at low pH, then, as our experiments have shown, most of the (oxidizing agent) of chloramine is spent on the formation of 3'-chlorofolium and 3 ', 5'-dichlorofolic acids. Moreover, the latter cannot be separated from the target product by reverse phase chromatography, which is used for separation in our proposed method. We found a pH value in the range of 3.5–4.9, at which the conditions of short reaction time (which is important for working with short-lived isotopes), high yields and negligible yields of 3 ', 5'-dichlorofolate acid are simultaneously satisfied. The difficulty is that folic acid is very slightly soluble at such pH. Therefore, in the claimed method, the preparatory (initial) solutions have a pH of about 7, and only at the same time as the start of the reaction, the pH is lowered to 4.7. In this case, folic acid passes into the gel when all reagents have already been added. The gel retains for some time the folic acid reactivity sufficient to undergo the desired reaction to the end.

If the pH is lower than 3, 5, then the proportion of 3 ', 5'-dichlorofolate acid, which contaminates the target product and is practically inseparable by reverse phase chromatography, sharply increases; if the pH is above 4.9, the yield of the target product is significantly reduced.

Example 1

A solution of 0.1 M folic acid sodium salt is prepared by dissolving 480 mg of folic acid, previously purified by recrystallization, in 8 ml of a 0.28 M sodium hydroxide solution. Then, the pH of the solution was adjusted to 7 by adding about 1.2 ml of a 0.1 M sulfuric acid solution in portions with vigorous stirring so that the gel formed at the site of acid addition immediately dissolved. Next, the volume is adjusted to 10 ml with water and this solution is used to prepare a solution of the required concentration.

30 μl of 5 × 10 ^-2 M folic acid sodium salt, 10 μl of 5 × 10 ^-4 M KI and 50 μl of a NaI solution containing 2 mCi iodine-125 without a carrier in 10 ^-3 M NaOH (solution A) stock solution.

After this, solution B is separately prepared. For this, 0.3 ml of water + 10 μl of a 0.1 M solution of chloramine T (previously purified from dichloramine impurities by extraction with carbon tetrachloride) + 0.4 ml of acetate buffer (0.8 M in acetic acid and 0.4 M in sodium acetate) are successively added. ) 30 μl of solution B is added to solution A, mix rapidly. Within a few seconds, a gel forms. After keeping the mixture for 30 seconds, add 100 μl of a solution of 1 M potassium hydrogen phosphate and 7 × 10 ^-3 M sodium sulfite to dissolve the gel and remove excess oxidizing agent, mix. Then add 1 ml of water. The resulting solution was introduced into a 150 × 8 mm HPLC column with an S-18 sorbent with a grain size of 5 microns, previously balanced by a buffer solution: 0.05 M NaH ₂ PO ₄ and 0.05 M Na ₂ HPO ₄ . After the solution was added to the column, it was washed with 15 ml of equilibration buffer, and then an eluent consisting of the same buffer was added to which 4.5% (volume) acetonitrile was added. The consumption of eluent 2.4 ml / min. The optical density of the solution passed through the column at a wavelength of 260 nm is measured. The peak exit time of 3'-iodopholic acid is preliminarily determined using unlabeled 3'-iodopholic acid. In the experiment, it amounted to 25 minutes. The target product, released at this peak, is collected in a volume of 5 ml, then evaporated to a volume of 3 ml, while all acetonitrile evaporates. The resulting solution contained 70% of all radioactivity introduced into the reaction. The specific activity of the drug was 300 milliCi / μM. In the control experiment, water was added instead of KI solution and NaI ¹²⁵ solution. The peak of 3 ', 5'-dichlorofolate obtained by chromatography was two hundred times smaller in area than the peak from 3'-iodopholic acid obtained in Example 1.

Example 2

30 μl of a 0.05 M solution of folic acid sodium salt (prepared as in Example 1) and 60 μl of a 0.0012 M solution of potassium bromide containing 0.04 mCi Br-82 (solution A ^I — initial solution) are added to a test tube. After this, solution B ^{I is} separately prepared. For this purpose, 0.3 ml of water +30 μl of a 0.1 M solution of chloramine T (previously purified from dichloramine impurities by extraction with carbon tetrachloride) + 0.4 ml of acetate buffer (0.8 M in acetic acid and 0.4 M in sodium acetate) are successively added. Then add 30 μl of solution B ^I to solution A ^I. Subsequent processing of the solutions and the isolation of the target product are carried out in the same manner as in Example 1. The exit time of 3'-bromofolic acid is preliminarily determined by the unlabeled preparation. The peak of labeled 3'-bromofolic acid contained 50% of all initial activity. The specific activity of the drug was 350 microCi / μM.

The proposed method can be used both with isotopic dilution and without it. In the latter case, the same amount of water should be added to the reaction mixture instead of a solution of the non-radioactive isotope of iodine or bromine. If it is necessary to add more non-radioactive isotope than indicated in the examples, then the concentration of chloramine and sodium sulfite should be increased accordingly. However, the concentration of chloramine can not be more than doubled compared with solution B ^I , since it will begin to precipitate. If you still need more chloramine, then you should take a larger volume of solution B ^I , using a correspondingly more diluted buffer.

The inventive method can find application in the diagnosis and treatment of cancer, in biological research.

Claims (2)

1. The method of obtaining 3'-iodopholic and 3'-bromofolic acids labeled with radioactive isotopes of iodine or bromine, which consists in adding a source of radioactive iodine or radioactive bromine to an aqueous solution of alkaline folic acid so that the pH of this solution is about 7 , to obtain a solution containing folic acid and iodide ion or bromide ion, and then lower the pH of this solution to 3.5-4.9, adding to the above solution an oxidizing agent from the group of N-chlorosulfamide acids in a buffer solution. 2. The method according to claim 1, characterized in that chloramine T or chloramine B is used as an oxidizing agent.