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

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

 

The invention relates to the field of biochemistry and radiochemistry.

Folic acid belongs to the class of vitamins of group of Century

Folic acid and its derivatives, such as aminopterin and methotrexate, effectively absorbed by certain types of cancer. This makes possible the use of folic acid and its derivatives, labeled with radioactive isotopes for medical diagnostics and radiotherapy. Because folic acid in the body is contained in very small quantities (concentration in blood plasma is a fraction of micromole per liter), cooked preparations must possess high specific radioactivity, and the method of synthesis must be acceptable to work with short-lived isotopes such as iodine-121 or bromine-75 with a half-life of about 2 hours. These isotopes recently found increasingly wide application in positron emission tomography, as are positron emitters.

The literature describes methods of obtaining labeled with iodine different derivatives on the basis of solutions of the connection, which should be marked with radioactive iodine in the form of iodide ions and chloramine.

There is a method of tagging biological compounds, described in [1]is: F..Greenwood & W.M.Hunter, Biochem. J. (1963) 89, 114. The reaction is carried out at a pH of 7.5

However, this too is in the case of applications for tagging iodine folic acid gives a very small outputs radioactivity (less than 1%).

To work around its impotency of folic acid in this reaction, was patented ways in which a glutamine residue folic acid joined easily audiruemaya tyrosine or histidine.

A method of obtaining derivatives of folic acid, labeled with iodine, as described in [2]: US Patent No. 3989812,; [3] US Patent No. 4276280). When this happened compounds corresponding to the formula (1), iodine label held the position of R.

The method described in US Patente No. 3989812 is the following. To a solution consisting of 100 μl of 0.1 M sodium hydroxide and containing 1 MCI NaI125, 20 μl of 0.5 M NaH2PO4and 10 μl of 0.5 M sodium phosphate pH 7.4, add 10 μg of pteroyl-γ-glutamyl-tyrosine in phosphate buffer (0.05 M, pH 7.4) and 20 µl of freshly prepared solution of chloramine T in 0.05 M phosphate buffer pH 7.4 (5 mg/ml). After 15 seconds added 20 μl of a solution of sodium metabisulfite in 0.05 M phosphate buffer pH 7.4. The reaction mixture was separated on a column of BioGel-P2, and then DEAE ion-exchange column.

The disadvantage is that it is not formed folic acid labeled with iodine, and a derivative of folic acid, labeled with iodine, which significantly differs from folic acid on the structure, charge distribution, the presence of additional hydrophobic groups. This may change the conditions of his transport the cell compared to natural folic acid.

It is known, for example, what if instead of histidine or tyrosine is another residue glutamic acid, is a derivative of folic acid in the cell does not penetrate despite the fact that the molecule of glutamic acid takes up less space than a molecule of histidine or tyrosine.

In this connection, it seems to be a promising method to mark the isotopes of iodine and bromine itself folic acid, and not the products of its accession to other easily odirueyr molecules, i.e. method

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

Moreover, this method should be suitable to work with quantities of iodine or bromine, lower micrograms.

The literature describes a method of labelling isotope I131compounds similar in structure to folic acid, aminopterin [4]: D.G.Johus et al, J. Nuclear Med., v.9, No. 10, 1968, 530-536). In this way we obtain a 3'-etaminophen, i.e. iodine label is attached to the compound (formula (3)), and not to the structure, facilitating iodination.

To a solution of 10 MCi NaI131in 0.4 ml of sodium sulfate were added when mixing the following reagents in the order listed: powder aminopterin (20 mg); iodic acid 4.5 mg; iodine - 7.5 mg; dimethylformamide - 0.5 ml; and tetrachloride angle of the ar - 0.1 ml Vessel was closed and stirred for 18 hours. Then the reaction mixture was separated on a column with DEAE-cellulose.

However, the method gives a low output (4÷10%), low specific activity (in the above publication in several experiments was obtained a dispersion of 50-300 microcure to micromoles), the formation of large amounts of contaminants, including radioactive waste, and the duration of the procedure of synthesis (18 hours).

This method is close to the claimed the fact that mark directly the connection that close structural formula, and not its derivative.

Declare the object relates to a method for labeling isotopes of iodine or bromine directly folic acid.

In the patent and scientific literature, we found no way of tagging folic acid, so the prototype is missing.

We the inventive method for the preparation of 3'-itpolicy and 3'-bromelias acids labeled with radioactive isotopes of iodine and bromine, which consists in the fact that prepared the original solution containing folic acid, radioactive iodine or radioactive bromine in the form of iodide ion or bromide ion at pH 7, is added to the original solution was acidified solution of the oxidizing agent from the group of chlorinated amides of the acid and bring the pH of the solution to 3.5÷4.9, optimally up to 4.7, converting folic acid in the gel. The selection of the target product is carried out using reverse-phase chromatography. As the oxidant used chloramine-T or chloramine-Century

Our research has shown that the cause of failure when you try tagging the folic acid in the methods of [1] is the slowness of the reaction of folic acid with ignominity acid HIO, which means [1] is an active agent and is formed by the reaction of iodide ion with bleach. Because of the slowness of the reaction HIO with folic acid, the predominant system reactions become disproportionation HIO and its oxidation by excess chloramine, lead to a radical reduction of the yield of the target product and the growth of output products.

Conducting the reaction under conditions conducive to the formation of cations of the I+and Br+that has allowed us to develop efficient synthesis of itpolicy and bromelias acids labeled with isotopes of iodine and bromine.

The essence of our proposed method consists in the reaction of the salt with the use of oxidants type of bleach that all patents is carried out at a pH higher than 7, go to conduct the reaction at a pH much lower. However, if you perform the reaction under conditions in which synthesis is relatively large amounts of the unlabeled itpolicy acid, described in patents [5] - US Patent 2570391 (1951); [6] US Patent 2570392 (1951), where folic acid is in a solution of 5M HCl at low pH, as shown by our experiments, the majority (oxidant) chloramine expended on the formation of 3'-chlorpryifos and 3',5'-dichlorophenol acids. The latter cannot be separated from the desired product by reversed-phase chromatography, which is used to separate in our proposed method. We have found the pH value in the range of 3.5 and 4.9, in which both conditions are satisfied small reaction time (which is important for working with short-lived isotopes), large output and negligible output 3',5'-dichlorophenoxy acid. The difficulty lies in the fact that under such pH folic acid very slightly. Therefore, in the present method preparation (initial) solutions have a pH of about 7, and simultaneously with the beginning of the reaction the pH is reduced to 4.7. While folic acid turns into gel when all reagents have been added. In the gel persist for some time the reactivity of folic acid, sufficient for the passage of the target reaction to the end.

If the pH is below 3, 5, dramatically increases the proportion of 3',5'-dichlorophenoxy acid, which contaminates the target product and virtually inseparable by the method of reversed-phase chromatography; if the pH is higher 4,9, significantly reduces the yield of the target product.

Example 1.

A solution of 0.1 M sodium salt of folic acid is prepared by restorani is 480 mg of folic acid, pre-purified by recrystallization in 8 ml of a 0.28 M solution of sodium hydroxide. Then the pH of the solution was adjusted to 7 by adding about 1.2 ml of 0.1 M sulfuric acid solution in portions with vigorous stirring so that the formed in place of the addition of acid, the gel was immediately dissolved. Then the volume was adjusted to 10 ml of water and this solution is used for preparing a solution of the desired concentration.

Poured into a test tube and 30 μl of 5×10-2M sodium salt of folic acid, 10 μl of 5×10-4M KI and 50 μl of a solution of NaI containing 2 mCi of iodine-125 without media in 10-3M NaOH (solution A)is the initial solution.

After that, separately prepare a solution B. To do this consistently add 0.3 ml of water and 10 μl of 0.1 M solution of chloramine T (pre-cleaned from impurities dichloramine by extraction with carbon tetrachloride) +0.4 ml of acetate buffer (0.8 M acetic acid and 0.4 M sodium acetate). 30 μl of solution B is added to the solution And quickly mix. Within seconds, a gel is formed. Stand the mixture for 30 seconds, add 100 μl solution of 1 M potassium phosphate and 7×10-3M sodium sulfite dissolving gel and remove excess oxidant, mix. Next, add 1 ml of water. The resulting solution was injected into HPLC column 150×8 mm with a sorbent To-18 with a grain size of 5 microns, prefix is Ino balanced buffer solution: 0.05 M NaH 2PO4and 0.05 M Na2HPO4. After making the solution in the column it was washed with 15 ml of equilibrating buffer, then served eluent consisting of the same buffer, to which was added 4.5% (by volume) of acetonitrile. The flow rate of eluent 2.4 ml/min to Measure the optical density of the solution passed through the column at a wavelength of 260 nm. The output time of the peak 3'-itpolicy acid pre-determined using unlabeled 3'-itpolicy acid. In the experiment it was 25 minutes. Target product, released this peak, collected in a volume of 5 ml, then evaporated to a volume of 3 ml, with all of the acetonitrile is evaporated. The resulting solution contained 70% of all introduced into the reaction radioactivity. The specific activity of the preparation was 300 miles/μm. In the control experiment, instead of KI solution and a solution of NaI125added water. Received on the chromatograph peak 3',5'-dichlorophenoxy acid area was two hundred times less than the peak from 3'-itpolicy acid obtained in example 1.

Example 2.

Poured into a test tube and 30 ál of 0.05 M solution of sodium salt of folic acid (prepared as in example 1) and 60 μl of 0.0012 M solution of potassium bromide containing 0.04 MCI Br-82 (solution AI- source solution). After that, separately preparing a solution of BI. Why consistently add 0.3 ml of water and 30 μl of 0.1 M is astora chloramine T (pre-cleaned from impurities dichloramine by extraction with carbon tetrachloride) + 0.4 ml of acetate buffer (0.8 M acetic acid and 0.4 M sodium acetate). Then add 30 ál of solution BIto a solution of AI. Subsequent processing solutions and the selection of the target product produced as in example 1. Time vyhozhdeniya 3'-bromelias acid pre-determined by the unlabeled drug. The peak labeled 3'-bromelias acid contained 50% of the initial activity. The specific activity of the preparation was 350 Micros/ám.

The proposed method can be applied to isotope dilution, and without it. In the latter case, in the reaction mixture instead of the solution of non-radioactive isotope of iodine or bromine should be added the same volume of water. If you want to add more non-radioactive isotope than is indicated in the examples, it should be increased accordingly and the concentration of bleach and sodium sulfite. However, the concentration of chloramine cannot be increased more than twice compared to solution BIas will begin the precipitation. If you still need more bleach, you should take a larger volume of solution BIusing progressively more diluted buffer.

The inventive method can find application in diagnostics and therapy of oncological diseases in biological research.

1. Method for the preparation of 3'-itpolicy and 3'-bromelias acids labeled with radioactive isotopes of iodine or bromine, the bookmark is different in that, what to aqueous solution of alkali salt of folic acid added source of radioactive iodine or radioactive bromine so that the pH of this solution was about 7, to obtain a solution containing folic acid and iodide ion or bromide ion, and then lower the pH of the solution to 3.5 and 4.9, adding to the above solution of the oxidizing agent from the group of N-chlorosulfonic acid in the buffer solution.

2. The method according to claim 1, characterized in that as the oxidant used chloramine-T or chloramine-Century



 

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