CS212652B1 - C 2 substituted derivatives of barbiturates - Google Patents

Description

The invention relates to C? substituted derivatives of barbiturates intended for the preparation of specific immunogenic agents against barbiturets and labeled haptenes.

The specificity of the determination of barbiturates by immunospecific methods mainly depends on the specificity of the antibodies used. All of the methods described so far have used to immunize semi-synthetic antigens where a determinant group of barbiturates has been attached to a support using a substituent on C 1. The determinant groups thus attached stimulate the production of antibodies which do not distinguish well the barbiturates with the various C 1-6 substituents and also do not distinguish the barbiturates and their physiologically inactive metabolites resulting from the C 2 substitution. The hitherto known barbiturate derivatives could not be attached via a substituent at the Cg position to a protein carrier or other carrier used in immunology to stimulate the production of antibodies against low molecular weight substances. Such binding of barbiturates to a carrier is necessary for the preparation of antigens that stimulate the production of specific antibodies against C 1 substituted barbiturets. The hitherto known barbiturate derivatives did not allow the preparation of Cg-labeled haptenes which are required for the preparation of isotopically-labeled haptenes.

These drawbacks are overcome by the invention which is based on Cg substituted barbiturate derivatives for the preparation of specific immunogenic agents against barbiturates and labeled haptenes. These C 6 substituted barbiturate derivatives are represented by the formula ^R 1

z ^R 3

C = N (CH 3) _n A ^R 2 ^Z CN //

O

R, where η ^R 1 ^{r r} 2 ^r 3, ^r 4 A

More particularly, the invention is 1-6 denotes alkyl of 1-6 carbon atoms or phenyl denotes hydrogen or methyl denotes a group capable of reacting with the side chains of amino acids in proteins, including carboxyl, hydroxyl, amino, mercapto, imidazolyl, p-hydroxyphenyl, and can be chemically prepare, for example, the methods of the examples.

the following specific examples illustrate.

Example 1

2- (carboxymethylimino) -5-ethyl-5- (1-methylbutyl) -dihydro-4,6- (1H, 5H) -pyrimidinedione of formula I,

HO \ / \ ^ ₂ Η ₅ c

(CH _Z JN = Ch ho ⁷ \ -ch 'ch ₂ .ch ₂ -ch ₃ / \ i (I)

CH 2 was prepared by heating 2.4 g (10 mmol) of 5-ethyl-5- (1-methylbutyl) -2-thiobarbituric acid with 1 g of glycerin and 4 ml of triethylemine for 16 hours at 85 ° C. The contents of the flask were then evaporated in vacuo. The syrupy residue was dissolved in 10 ml of water, the reaction was adjusted to pH 7.6 with sodium bicarbonate and the solution was shaken twice with 20 ml of ether. The pH was adjusted to 5.0 with hydrochloric acid and the solution was extracted twice with 30 mL of ether. The ether extracts were combined, shaken with 2 ml of water, dried over anhydrous sodium sulfate, filtered with a small amount of activated carbon and evaporated. The colorless syrupy residue which was dried over θ2θ5 · solidified in 1.2 g of brittle product.

This product was applied in thin-layer chromatography in an amount of 50 µg onto silufol in a butanol: ammonia solvent system 8: 2 under UV irradiation at a wavelength of 254 the only strong spot containing 0.15.

Glycine was not detected by detection with ninhydrin, barbiturates (pentobarbital, thiopental) were not detected by detection with mercury sulphate and diphenylcarbazone. The titration curve indicates the presence of one carboxyl group in the molecule. Heating the substance with 1 ml of hydrochloric acid in a sealed tube at 120 ° C for two hours decomposes the substance into glycine and pentobarbital.

Compounds of formula I were used in the preparation of the immunogen. The compound of formula I was bound to bovine albumin via the hydroxysuccinimide active ester. A 25-fold molar amount of active ester relative to albumin was used, a molar ratio of 5: 1 bound groups to albumin was determined from the differential spectrum of albumin and the albumin conjugate of formula I. This ratio was not altered by truncating the immunogen with ethanol. Eight rabbits were immunized by injecting paws in an amount of 00 µg of rabbit immunogen with complete Freund's adjuvant. After 4 weeks the animals were re-immunized with 50 µg of immunogen and after 14 days the first collection was performed. Subsequent harvesting was performed on the four larvae who had the highest antibody titers at the first harvest, 4 to 6 weeks each 10 to 14 days after the administration of 10 µg of immunogen.

It was crystallized from the methanol solution as a cyclohexylamine salt, m.p. 194-195 ° C with decomposition. Elemental analysis showed good agreement with the proposed structure:

C Η N

59,66% 8,96% 14,65%

59,23% 9,03% 14,4% theory: found:

The cyclohexylamine salt in methanol has (d »250 nm) = 1.518.10 ⁴ , the dicyclohexylamine salt has f (d = 250 nm) - 1.523.10 ⁴ .

Example 2

2- (2-Imidazolylethylimino) -5-ethyl-5- (1-methylbutyl) -dihydro-4,6- (1H, 5H) -pyrimidinedione of Formula II

H ch ₂ .

_CH2 H W _{C H} / \ / ^2H5 cc \ | _z CH. CH ₂ . CH ₂ . CH3

⁷ H \ CH3 (II) was prepared by heating a solution of one mmol (0.24 g) thiopental, 1 mmol (0.18 g) of histamine dihydrochloride and 0.6 ml of triethylamine in 5 ml of ethylene glycol for 24 hours at 85 ° C . The solvent was evaporated in vacuo, the residue was dissolved in 3 ml of water and at pH 3 the solution was shaken twice with 10 ml of ether. The pH of the solution was then adjusted to 7.6 with sodium bicarbonate, and the solution was extracted twice with 10 mL of ethyl acetate. The combined ethyl acetate extracts were extracted with 2 mL of water, dried over anhydrous sodium sulfate. The dry extract was filtered after addition of a small amount of activated carbon and the filtrate was evaporated.

Evaporation gave 85 mg of a white solid. In the system of butanol: ammonia 8: 2 to giving the substance Silufol diazopozitivní single spot, absorbs at 254 nm with Rp ³ 0.45. Heating with 1 mol / L hydrochloric acid in a sealed tube at 120 ° C for two hours decomposes to histamine and pentobarbital. Mass spectrometry is in accordance with the assumed structure (M ⁺ 319).

Example 3

2- (2-Aminoethylimino) -5-ethyl-5- (methylbutyl) -dihydro-4,6- (1H, 5H) -pyrimidinedione

It was prepared by heating 2.4 g of thiopental with 13 ml of 1,2-diaminoethane (200 mml) at 100 ° C for 4 h. The diaminoethane was then distilled off under reduced pressure. The syrupy residue was rubbed with a small amount of anhydrous ethanol. A white precipitate was formed. The yellowish alcoholic solution was separated from the precipitate, decolourised by the addition of activated carbon, filtered and evaporated in vacuo. The syrup-like consistency residue was dried in a vacuum desiccator above 1? 2 ^Ο 5 · Thin layer chromatography showed acetone: chloroform:: ammonia 27: 3: 2 on Silufol as the only UV absorbing spot at R _F 0.2.

The amorphous, slightly yellowish mass, after drying over PgO ^, was easily crushed to a sieve, which in contact with air humidity turned into a sticky mass. In aqueous solution, the picric acid substance gave a yellow precipitate. It dissolved in alcohol and precipitated with water, was dried over P ₂ ° 5 ° ^te Pl ^herein pools 105 ° C and elemental composition corresponding dipikrátu prepared compounds.

C Η N theory: 41.3% 4.2% 19.2% found: 41.15% 4.7% 18.94%

Claims (1)

SUBJECT OF THE INVENTION C ₂ substituted derivatives of the barlturates of the general formula C = N (CH ₂ ) _n A wherein n is t to 6 H 1, R ₂ represents alkyl of 1 to 6 carbon atoms or phenyl R 6, R 6 denotes hydrogen or methyl A denotes a group capable of reacting with amino acid side chains in proteins, including carboxyl, hydroxyl, amino, mercapto, imidazolyl, p-hydroxyphenyl