HU193381B - Process for determining the adenosin-3',5'-cyclophosphate content by radioimmunology and for the preparation of radioligand applied thereto - Google Patents

Abstract

Method is based on an antigen-autitest reaction used for the detm. of adenosine 3'-5'cyclo-phosphate (C-AMP) concn. employs a radio ligand of a novel structure. The ligand is a 2'-O-(2'',3''-3H2-) succinyl C-AMP deriv. (I) exhibiting excellent moler activity acid extended half life. - R is a hydroxy or L-(3,5-3H)Me-tyrosine group. (I) is prepd. by reaction of tritium-contg. succinic acid anhydride (II) and adenosine 3,-5' cyclophosphate or its salts. A product (1A) being similar to (I) can if required react with a tyrosine deriv.

Description

The present invention relates to a method for the radioimmunoassay of adenosine 3 ', 5'-cyclo-phosphate (hereinafter c-AMP) content of biological tissue samples (tissue, blood, urine, etc.) in known amounts of c-AMP derivatives of various concentrations (preferably acetylated). to be determined in the presence (preferably acetylated) biological media sample (s) by measuring the binding of the highly specific activity radioactive tracer (hereinafter radioligand) to a specific antibody and the change in binding caused by a known amount of (preferably acetylated) c-AMP dilution series. preferably, based on the change in binding caused by the acetylated medium sample, the active substance content of each medium sample is calculated.

According to the invention, the radioligand used in this immunochemical reaction is a novel compound obtained by trituration of a known c-AMP derivative according to the invention.

The present invention further relates to a process for the preparation of this novel structure radioligand.

C-AMP is a biologically active compound in living organisms: the so-called hormone and neurotransmitter. secondary messenger [discussed in detail in Recent Prog. Hormones Res., 21, 623-646 (1965). Sensitive, accurate quantification of c-AMP is extremely important for endocrinological, neurochemical and pharmacological research. Several methods for the determination of c-AMP are described in the literature.

AJ. Biol. Chem., 1977, 247, 1106-1133 and Biochem. J., 121, pp. 561-562 (1971) disclose two variants of the so-called protein binding assay method, which isolates protein fractions from skeletal muscle or adrenal gland and monitors tritium-labeled ³ Hc-AMP on the one hand and unlabeled c -AMP competition at the binding sites of the isolated protein fraction.

The disadvantage of the method is that it is not sensitive enough and has a measuring range of 1 to 16 picomoles; the advantage is the durability of the active ingredient: the half-life is about 12 years.

Another source [Adv. in Cyclic Nucleotide Res., 10 (1979) 1-33; Raven Press, New York, discloses a method in which the formation of a specific antibody used as a binding protein in radioimmunassay is induced in animals by conjugates of 2'-O-succinyl-c-AMP with foreign proteins. The radioligand used is the 2'-O-succinyl tyrosine methyl ester of c-AMP, 125 weighted by iodine. The assay is based on competition between this radioligand and unlabeled (optionally acetylated) c-AMP for antibody binding sites. The advantage of this method is that it has a better sensitivity and measuring range of approx. 0.01-0.2 picomoles, and the antibody used can be produced at low cost, in large quantities, and can be stored for long periods, thus allowing continuous use; The disadvantage of this method is that the half-life of iodine is short, only 60 days.

The present invention is based on the discovery that the advantages of these two methods can be combined without the disadvantages of reacting the 2'-O- [2,3 '- of the formula (I) as a radioligand by novel labeling of the known derivatives of c-AMP in the immunochemical reaction. ^{One of 3} H ₂ ] succinyl-c-AMP derivatives wherein R is hydroxy or L- [3,5- ³ H] -methyl-tyrosyl; these radioligands have a long half-life (12.3 years), high molar activity [70-130 Ci / mmole], and can also be prepared under favorable conditions by the synthesis process of the present invention.

Experience shows that the c-AMP parent molecule binds poorly to the specific antibody; tritiation of the parent molecule is problematic and the tritiated form of the parent molecule would not be useful as a radioligand in this immunochemical reaction even if its specific activity could be increased.

The succinyl derivatives of the c-AMP molecule, on the other hand, provide orders of magnitude better binding [Anal. Biochem., 56, 383-393 (1973)]; We have discovered that the succinyl derivatives of c-AMP can be tritiated well in a novel synthesis and the efficiency of the immunochemical reaction can be improved by using one of the compounds of formula (I) thus obtained as a radioligand. It has now been found that tritiated c-AMP derivatives of formula (I) can be prepared under favorable conditions by first treating a compound that is convertible to a succinyl group of a compound of formula (I) with tritium, followed by tritiated succinic anhydride of formula (II) reacting the inactive c-AMP parent molecule or a salt thereof, preferably its sodium salt. This yields the target product of formula Ia, which can be used directly as a radioligand, and optionally converted to an even higher molar activity target product of formula Ib by reaction with tritiated tyrosine methyl ester of formula IV. Thus, the two types of radioligand of Formula (I) can be prepared by using c-AMP as a starting material in its inactive structure: avoiding the radiochemical synthesis of the c-AMP molecule and trituration of the parent molecule. The steps for the synthesis of the target product of formula (Ia) are illustrated in Scheme A and the steps of conversion (Ia) to (Ib) are illustrated in Scheme B.

The present invention therefore relates to a method for determining an antibody-antigen reaction, which differs from the known radioimmunoassay method outlined above by the use of a novel tritium-labeled c-AMP derivative of formula (I) as the radioligand.

This involves measuring the binding of the radioligand to a specific antibody in the presence of a known amount of c-AMP at various concentrations (preferably acetylated), and then calculating the c-AMP concentrations for binding changes caused by unknown (preferably acetylated) media samples.

To do this, incubate the radioligand and buffer containing different amounts of inactive c-AMP and specific antibody according to the dilution series, and separate the bound and non-bound fractions of the antibody and measure the radioactivity of the bound fractions.

Depending on the particular composition of the solution, the incubation temperature is between 0 and 40 ° C and the incubation time is between 1 and 50 hours, preferably between 3 and 20 hours.

Generally, a radioligand having a cpm count of between three thousand and thirty thousand, preferably between six thousand and twelve thousand, is preferably prepared by filling into serially numbered test tubes containing a buffer solution, e.g. ten thousand cpm (500 Bq radioactivity) radioligand formula (I) and pipetted into the appropriate tube serial number corresponding to the respective dilution ratios 0-25-50-100-250-500-1000 fmol (10 ', ¹⁵ mol) of a (preferably acetylated) c -AMP and the biological sample to be determined (preferably acetylated); preferably 3 to 3 samples are tested in parallel with the same dilution.

The buffer solution is preferably 0.01-0.2 M, preferably 0.05 M sodium acetate, containing 0.1 to 1% (v / v) serum albumin, preferably bovine serum albumin (BSA). %, preferably 0.5% by volume. Generally, this antibody-antigen reaction is conveniently carried out in an acidic medium and may be carried out in a slightly alkaline medium so that the pH of the buffer solution can be adjusted to between 4 and 8, preferably about 4.75.

The specific antibody is preferably a high titre (e.g., 10 ^{3 to} 10 ⁴ fold dilution) of anti-c-AMP antiserum used in the amount of the radioligand 40-60, preferably ca. It can bind 50%.

Samples and inactive c-AMP are preferably acetylated with acetic anhydride reagent in the presence of triethylamine, e.g. so that they are app. 10% acetic anhydride in 0.005 M sodium acetate buffer (pH 4.75) containing 20% v / v triethylamine; the reaction temperature is preferably 20 to 25 ° C, the reaction time is preferably about 30 minutes.

For the preparation of anti-c-AMP immune serum and for the acetylation of samples and inactive c-AMP, it is preferred that Brooker et al., Adv. in Cyclic Nucleotide Res., 10, 1979 (1).

Preferably, 0.1 M potassium phosphate buffer solution (pH 6.8) containing 0.25% BSA and 0.2% charcoal is used to separate the bound and unbound radioligand fractions. Charcoal at a final concentration of 0.15% binds the unbound fraction of the radioligand and the bound fraction obtained after centrifugation as a supernatant is measured by liquid distillation.

The present invention also relates to a process for the preparation of 2'-O- [2 ', 3' - ³ H ₂ ] -succinyl-c-AMP derivatives of formula (I) for use as a radioligand.

According to the invention, the tritiated c-AMMP derivatives of formula I are prepared by reacting the tritiated succinic anhydride of formula II with adenosine 3 ', 5'-cyclophosphate or a salt thereof, preferably the sodium salt thereof, and optionally the reaction product (IV). ) wherein [3,5- ³ H] tyrosine methyl ester in a polar solvent, a tertiary amine, e. triethylamine and ethyl chloroformate. (Scheme A illustrates an embodiment of reacting the starting material of formula II with the c-AMP sodium salt of formula III).

The starting material of formula (II) can also be prepared by the two process variants described below, one having a molar activity of 30-50 Ci / mmol and a product having another molar activity of 70-80 Ci / mmol. Thus, the molar activity of the target product (1a) synthesized in the first step may also vary within a range of 30 to 80 Ci / mmol. The starting material of formula (IV) can be prepared by the synthesis described below having a molar activity of 40 to 50 Ci / mmol, so that the target product of formula (Ib) obtained in the second step has a molar activity of 70 to 130 Ci / mmol.

A particular advantage of the process according to the invention is that there is no need for the radiochemical synthesis (c) of c-AMP in the preparation of the starting materials and that the total synthesis of the target product of formula Ib can be carried out using the two tritiated reagents.

Suitably the solvent for the synthesis of the compound of formula (Ia) is triethylamine / acetone, preferably in a ratio of 1: 4 by volume, from 8 to 15, preferably 10 mol, per mole of starting material (II). The c-AMP (salt) is used in an amount of 0.1 to 0.3, preferably 0.15 mol, per mole of starting material of formula (II). The reaction mixture was acidified and the precipitate was filtered off. Preferably 2M hydrochloric acid is used for acidification. The precipitate is washed and dried, and the resulting compound of formula (Ia) can be used as a radioligand in the immunochemical reaction or as a starting material for the synthesis of compound (Ib).

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In the synthesis of the compound of formula (1b), the reaction time is from four to ten hours, preferably six hours, depending on other reaction conditions. Preferably, the starting material of formula (Ia) is dissolved in dimethylformamide (DMF) to give a 0.3% w / v solution in% by weight of a solution of tertiary amine in 10% v / v DMF. 3% v / v and 3 to 4 molar, preferably 3.5 molar, of a 2 to 2.5% v / v DMF solution of ethyl chloroformate; the mixture is then thoroughly mixed, preferably ca. 0 ° C for about 15 minutes. Then, the starting material (IV), also dissolved in DMF, is added in an amount of 2 to 2.5 moles. During the reaction, which is preferably carried out for a period of six hours, the reaction mixture (anhydrous) is maintained, starting from the premix of the mixture, e.g. under argon and the mixture was stirred for the complete reaction.

After completion of the reaction, the solvent was distilled off. This operation is conveniently carried out in a vacuum bridge, which is also used for some of the steps in the preparation of the intermediates, at a temperature of about 40 ° C and a pressure of 0.00133 bar (1 mm Hg) in a liquid nitrogen cooled flask.

The residue was dissolved and chromatographed. Preferably, the solvent is ethyl alcohol, preferably a plastic film cellulose layer for chromatography and a 12: 3: 5 by volume mixture of ternary n-butanoacetic acid / water, which is chromatographed in the upper phase of the reaction product. The product is identified by radiographic chromatography (e.g., Berthold LB 2001 Dünnschicht Scanner), the product is leached from the layer (eg, the ternary mixture already indicated) and the solvent is distilled off, preferably at about 40 ° C, 0.02 bar (15 mm Hg). pressure. The resulting product, which has a molar activity in excess of 70 and 110 Ci / mmole, respectively, depending on the method used for trituration of the intermediate, provides radioimmunoassay efficiency.

The tyrosine derivative of formula (IV) may be prepared by trituration of a 3,5-dibromo-L-tyrosine methyl ester of formula (V) in a polar solvent in the presence of a reduced, e.g. At a pressure of 0.933 bar (700 mmHg). Preferably, the palladium catalyst is supported on an alumina support and the solvent is a mixture of DMF and dioxane (1: 5 to 1:20, preferably 1: 8). The reagent is carrier-free tritium gas. The catalyst is separated by filtration, and the solvent, e.g. using a vacuum bridge as mentioned above, distilling off the residue at 0.00133 bar (1 mm Hg) and drying under reduced pressure over phosphorus pentoxide.

The solid residue is chromatographed and identified by radiographic chromatography, similar to the post-treatment of the target product of formula (Ib).

A very important advantage of the present invention is that the target product of formula (Ia) - and of course the target product of formula (1b) obtained by further conversion thereof - is obtained by reacting inactive (unlabeled) c-AMP with a tritiated starting material which already contains a tricyclic moiety incorporated into the c-AMP side chain. It is clearly shown in Flow Diagram A that the compound containing the side elements to be triturated is first triturated and the resulting tritiated group is further transported from the resulting intermediate.

The tritiated succinic anhydride starting material (II) used in the synthesis of the target product (Ia) can also be prepared from tritiated [2,3-H] succinic acid (VI). The reaction temperature of the acetyl chloride using 3-5 molar, preferably 4 molar ratios, is 80-150, preferably 110 ° C, reaction time is 1-10, preferably 3 minutes. The solvent was distilled off from the product.

(VI) tritiated succinic acid of formula may be prepared in one step (VII) acetylene dicarboxylic triciálásával (the variant) and two stages (IX) acetylene dicarboxylate and the resulting triciálásával (VIII): Formula III [2,3- ³ H ₂ ] -Succinic acid dimethyl ester (variant b).

Intermediate a of formula (VI) obtained in variant a) has a molar activity of 30 to 50 Ci / mmole, and intermediate b of intermediate (VI) obtained in two-stage variant b has a molar activity of between 70 and 80 Ci / mmole. On the other hand, the more efficient b-intermediate can be obtained in a lower total yield than the one-step intermediate.

The α-variant acetylene dicarboxylic acid (VII) is reacted with a carrier-free tritium gas in the presence of palladium catalyst under reduced pressure of about 0.866 bar (650 mm Hg). The solvent used is 2.0% w / v dioxane and the catalyst 5% palladium on charcoal. After completion of the reaction, the catalyst is filtered off, the filtrate is concentrated under reduced pressure, preferably 0.00133 bar (1 mm Hg), and the residue is dried over phosphorus pentoxide, also under reduced pressure.

b-version

The acetylene dicarboxylic acid dimethyl ester starting material (IX) is also reacted with carrier-free tritium gas. The reaction conditions are the same as those of variant a, except that the solvent is preferably 0.67% w / v dioxane. After filtration of the catalyst, the filtrate is diluted with water to about 10 times and stirred with ion exchange resin at room temperature, preferably about 40 ° C for at least 2 hours, preferably 20 hours. The resin is then filtered off and the filtrate is treated as described in variant a.

The invention will now be explained in more detail by the following examples, which are, of course, not limited to the present invention.

Example 1

Preparation of succinic anhydride of formula II from intermediate b

Freshly distilled dimethyl ester of acetylene dicarboxylic acid (16.7 mg, 118 pmol) was dissolved in freshly distilled dioxane (0.25 mL) and then 5% palladium on carbon (1.5 mg) was added. The mixture was reacted with carrier tritium gas at 0.866 bar (650 mmHg) in an evacuation chain until gas uptake ceased; the reaction time is about one hour. The amount of tritium taken up was 4.18 normal ml (volume measured at 273 K and atmospheric pressure), corresponding to 10.86 Ci radioactivity. The catalyst is filtered off with filter paper, the filter is washed with 0.5 ml of water, and 2 ml of water are added to the filtrate and 300 mg of Dowex 50 resin (30-70 µm freshly activated with hydrochloric acid) and stirred at 40 ° C for 20 hours. The resin is then filtered through a glass filter, the filter is washed four times with 0.5 ml of ethanol, and the solvent is distilled off under vacuum at 0.00133 bar (1 mm Hg) in a liquid nitrogen-cooled flask and the residue is treated with phosphorus pentoxide in the same apparatus. hours. There was thus obtained 12.4 mg (yield: 89%) of intermediate (VI) having a radioactivity of 6.36 Ci and a molar activity of 74.9 Ci / mmol. To intermediate b-intermediate (VI) was added 50 µl of freshly distilled acetyl chloride and the mixture was heated at 110 ° C in an oil bath for 3 minutes. The tritiated succinic anhydride of formula (II) thus obtained weighed 10.0 mg (total yield: 84.7%, radioactivity).

5.92 Ci, molar activity 73.2 Ci / mmol.

Example 2

Preparation of starting material of formula II from intermediate a

10.0 mg (87.7 pmoles) of acetylene dicarboxylic acid in 0.5 ml of freshly distilled dioxane

Similarly to Example I, we tritiate. The uptake of tritium per hour is 4.0 normal ml, which is adequate

II, 6 Ci radioactivity. The catalyst was filtered off as described in Example 1, the filter was washed with 0.5 mL of dioxane, and the filtrate was de-solvented as in Example 1 and dried. The crude product is then treated without isolation as described in Example 1; the succinic anhydride obtained after drying according to Example 1 weighed 8.5 mg (96.6%), had a radioactivity of 4.21 Ci and a molar activity of 49.5 Ci / mmol.

Example 3

Preparation of the target product of formula (Ia)

The active succinic anhydride obtained in Examples 1 and 2 was dissolved in 0.1 ml of a solvent containing 4: 1 by volume acetone / triethylamine; 25 mg of c-AMP sodium salt (5 mg, 14 pmol) dissolved in distilled water were added and the mixture was stirred at room temperature for 20 minutes and then evaporated to dryness at 0.02 bar (15 mmHg) at 30 ° C. The residue was dissolved in water (0.3 mL), acidified to pH = 3 with 2M hydrochloric acid, and the precipitated white crystalline material was filtered (washed with 0.5 mL ethanol) and pressurized with phosphorus pentoxidone (0.00132 bar (1 mm Hg)). dried. This gives 3.0 mg (47% yield) of the target product of formula (Ia), which has a radioactivity and a molar activity of 0.322 Ci and 48 Ci / mmol, respectively, in intermediate a and 0.476 Ci and 71 Ci in intermediate b, respectively. mmol.

Example 4

Preparation of starting material of formula (IV)

8.9 mg (25 pmoles) of 3,5-dibromo-L-tyrosine methyl ester (V) previously recrystallized from DMF (m.p. 198-205 ° C) are dissolved in 0.05 ml DMF and completely After addition of 0.4 ml of freshly distilled dioxane and 4 mg of 10% Pd / Al ₂ O ₃ catalyst, the reaction is carried out at 0.933 bar (700 mmHg) with anhydrous tritium gas. Gas uptake ceases after about four hours, and the amount of gas taken up is 3.9 normal ml, which corresponds to 11.3 Ci of radioactivity. In the same manner as in Example 1, the catalyst was filtered off and the crude product was dried, which was then dissolved in 0.2 ml of ethanol and chromatographed on DC Alufolien-Kieselgel 60 (Merck, 20x20x0.25 cm), chloroform: methanol: water 90:10: Using a 1 volume mixture. The product, when run in conjunction with unlabeled tyrosine methyl ester, is identified by detecting radioactive peaks (Berthold LB 2001); scrape at appropriate places, add methanol (50 mL) and elute at 4 ° C for 12 hours. The silica gel was filtered off and the filtrate was evaporated to dryness at 0.02 bar (15 mm Hg) at 40 ° C. The starting material of the formula (IV) had a weight of 1.0 mg (21.6%), a radioactivity of 0.225 Ci and a molar activity of 43.8 Ci / mmol.

Example 5

Preparation of the target product of formula Ib

According to Example 1 or 2, b, and applying the obtained intermediate (Ia) 2'-0- formula [2 ', 3' - ³ H _2] -hemiszukcinil c-AMP compound 0.3 mg -of (0.67 pmol) was dissolved in freshly distilled DMF and 3 ml DMF containing 10% v / v triethylamine and 16 µl DMF containing 1:44 ethyl chloroformate; the resulting mixture was cooled to 0 ° C and stirred for 15 minutes. 0.3 mg (1.54 pmol) of the tritiated L-tyrosine methyl ester derivative (IV) prepared in Example 4 and dissolved in 10 µl of triethylamine in 10% by volume are then added and the the resulting mixture was stirred at room temperature for six hours. Formula (Ia) is provided to ensure waterproofing

From the dissolution of starting material -5193381 to the completion of the reaction, an inert atmosphere of argon is maintained in the reaction space. After completion of the reaction, the solvent was distilled off as described in Example 1, and the residue was dissolved in 0.1 mL of ethanol and chromatographed on DC Plastikfolien PEI Cellulose F layer (Merck, 20x20x0.25 cm), ternary - n-butanol: acetic acid: water. 12: 5: 3 mixture in the upper phase separated in a shaking funnel, then scraped off at the radioactive peak of inactive succinyl-c-AMP-tyrosine methyl ester and eluted with 50 ml of a three-component mixture at 4 ° C for 12 hours. The solution is then concentrated at 0.02 bar (15 mm Hg) and the residue is dissolved in 100 ml of ethanol to give a stock solution having radioactivity of 24.88 mCi for intermediate b and 17.45 mCi for intermediate synthesis.

The radiochemical purity of both products was found to be better than 95% as determined by TLC. If the two kinds of products as markers! efficiency is considered to be the arithmetic mean of the two molar activities, yield 32.2% of intermediate b, 28.6% of intermediate a.

Example 6

Reaction Determination of c-AMP Contents of Brain Tissue with Antibody Antigen Reaction Using a radioligand of formula (Ib), prepare a series of dilutions of a µΜ concentration of c-AMP stock solution in 0.005 M sodium acetate buffer (pH 4.75) to give 1 The concentration of c-AMP is 0.5-1.0-2.0-5.0-10.0-20 nM and 25-50-100-250-2500-1000 fmol / tube. 50 mg of wet tissue was homogenized with 1 ml of 5% trichloroacetic acid, centrifuged and the supernatant was extracted three times with 1 ml of ethyl ether and then at 50 ° C for approx. Shake for 10 minutes (to remove solvent). The biological samples thus obtained are also made up to a volume of 1 to 1 ml in the same buffer. The standard and sample solutions were stirred with 20 µl of triethylamine for 1 minute, then 10 µl of acetic anhydride was added and after one minute of stirring, the mixture was allowed to stand at 20 ° C for half an hour. Then pipette into serially numbered polyethylene or glass test tubes with an automatic pipette for approx. tritiated tyrosine methyl ester c-AMP (Ib) containing tens of thousands of cpm (radioactivity 500 Bq) containing 0.1 ml of buffer solution as above and 0.05 ml of both standard and sample solutions, and 0.2 ml of goat anti-c-AMP bovine serum dissolved in 0.05 M sodium acetate buffer, pH 4.75 containing 0.5% BSA, diluted to a Bo / TC value of 40 to 50% the contents of the test tubes are vortexed (made by KUTESZ 6) for a few seconds. Three to three samples corresponding to each member of the dilution series are tested in parallel. For the determination of nonspecific binding (NSB), three additional test tubes are pipetted with pure buffer solution instead of antiserum and 0.05 ml of pure buffer solution is added to the radioligand instead of standard solutions. An additional three test tubes containing a sample of the same composition as standard 0 are used for total radioactivity (TC) measurement. After mixing, allow the tubes to stand in a refrigerator (at + 4 ° C) overnight and pipette with 1 to 1 ml of 0.25% BSA in ice water, except for TC test tubes. 2% charcoal suspension at 0 ° C [Norit-A, Serva (Germany)! Instead, pipette into the TC measuring tubes an equal volume and composition of pure buffer solution. The test tubes were allowed to stand for 15 minutes in the refrigerator, then centrifuged at 1500 g for 10 minutes at 4 ° C and pipetted into 0.5 ml to 0.5 ml of supernatants. The samples were filled with 10 to 10 ml of a scintillation mixture of 120 g of naphthalene, 4 g of 2,5-diphenyloxazole (PPO) and 0.3 g of p-bis [2- (5-phenyloxazolyl) in 1 liter of dioxane. )] benzene (POPOP) and radioactivity was measured by a known liquid scintillation method. The calculation is also carried out in a manner customary for the radioimmunoassay. Figure 1 shows a plotted diagram of B / Bo as a function of pmo / tube.

Example 7

Reaction Determination of c-AMP Content of a Brain Tissue Sample Using a Radioligand of Formula (Ia)

Example 6 except that the radioligand is not a compound of formula (Ib) but a compound of formula (Ia). As a control, the example is repeated with a radioligand which differs from formula Ib in that the tyrosyl group of the side chain is inactive (prepared by using an inactive tyrosine methyl ester starting material in the synthesis of Scheme B as tritiated tyrosine methyl ester of formula IV). ).

Figure 2 shows a comparison of the B / Bo binding functions obtained with radioligands of formula (Ia), (Ib) and inactive side chain tyrosine methyl ester, while Figure 3 shows the corresponding Bo / TC curves as a function of c-AMP concentration. The improvement in sensitivity is particularly striking.

From the above, one skilled in the art will appreciate the advantages of the invention over the prior art. Among the benefits we highlight:

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The radiochemical syntheses employed can advantageously yield a radioligand, namely the tritiated TME-succinyl-c-AMP derivative of formula (I), whose high molar activity provides an effective radioimmunoassay of the c-AMP concentration in the samples. This is achieved by preliminary labeling of the side chain groups, so there is no need for radiochemical synthesis of the c-AMP molecule.

In the process of the invention, the composition and ratio of solvents, the temperature, the addition of reagents and the catalysts can be optimized in such a way that the parameters of radioactive labeling in radiochemical synthesis are as low as possible.

On the other hand, the parameters of the radiochemical synthesis employed can be selected so that a sufficiently high chemical and radiochemical yield is achieved with as little radioactive activity as possible on the smallest possible scale (up to a few mg doses).

The use of the new tritium-labeled radioligand allows the realization of a c-AMP radioimmunassay that preserves the engineering and manufacturing benefits of the state-of-the-art protein binding assay by using the long-lived tritium isotope, while providing significantly greater sensitivity over the benefits of using specific antibodies. .

Claims (7)

CLAIMS 1. A method for reactively determining the content of adenosine 3 ', 5 ' -cyclophosphate (" c-AMP ") in biological tissue samples (tissues, blood, urine, etc.) in known amounts, preferably acetylated, at various concentrations. In the presence of a -AMP derivative and a biological media sample to be determined (preferably acetylated), which measures binding of a highly specific radioactive tracer (hereinafter radioligand) to a specific antibody and a known amount of (preferably acetylated) c-AMP dilution series. calculating the active substance content of each medium sample, characterized in that the 2'-O- [2 ', 3' - ³ H _{2 '} - succinyl-c-AMP derivative of formula (I) is used as the radioligand, wherein R is hydroxy or L- [ 3,5- ³ H-methyl-tyrosyl residue. The process according to claim 1, wherein the radioligand is cpm of between three thousand and thirty thousand, preferably between six thousand and twelve thousand. 3. The method of claim 1 or 2, wherein the antibody is a goat-produced anti-c-AMP antiserum in an amount sufficient to produce a radioligand 40-4 in a sample containing in5 active (unlabeled) c-AMP. It can bind 60%, preferably 50%. 4. Process according to any one of claims 1 to 3, characterized in that the buffer solution is sodium acetate solution containing a serum albumin, preferably bovine serum albumin, having a pH of between 4 and 8, preferably about 4.75, in an amount of 0.1 to 1 vol. from about 0.5 to about 0.5% by volume. 5. Process according to any one of claims 1 to 3, characterized in that the reaction time is from 1 to 50 hours, preferably from 3 to 20 hours and The reaction temperature is from 0 to 40 ° C. 6. The process according to any one of claims 1 to 3, wherein the acetylation of the media samples and the inactive c-AMP agent with acetic anhydride reagent, tertiary amine, 25 pl. in the presence of triethylamine. 7. A method (I), 2'-0- formula [2 ", 3" ^{-: i} H _2] succinyl-c-AMP derivative, wherein R is hydroxy or L- [3,5- ³ H] methyl-tyrosyl residue, Characterized in that the tritiated succinic anhydride of formula (II) is reacted with adenosine 3 ', 5'-cyclophosphate or a salt thereof, preferably the sodium salt thereof, and if desired, the resulting compound of formula (I) Reaction products of 35 / l) of formula (IV) with the formula [3,5- ³ H] tyrosine methyl ester po'áros solvent, a tertiary amine, e. in the presence of triethylamine and ethyl chloroformate. 8. A process according to claim 7 wherein the starting material of formula II is a mixture of adenosine 3 ', 5'-cyclophosphate (salt thereof) in triethylamine and acetone, preferably in a volume ratio of 1: 4. Preferably, the adenosine 3 ', 5'-cyclophosphate (salt thereof) is used in an amount of 0.1, 3, preferably 0.15 molar to one mole of the starting material (II). 9. The process according to claim 7 or 8, wherein the tritiated reaction product of formula (Ia) is reacted with the tritiated tyrosine derivative of formula (IV) in dimethylformamide. 55 10. A 7-9. Process according to any one of claims 1 to 3, characterized in that the reaction mixture comprising the reaction product of formula (Ia) and the tyrosine derivative of formula (IV) is stirred at room temperature and the reaction time is between four and ten hours, preferably six hours.