DE2905883A1 - Clonidine derivs. contg. tyrosine fragment - useful for radioimmunoassay of antihypertensive clonidine - Google Patents

Abstract

Clonidine derivs of formula (I) and their acid-addn salts are new. In (I) Ar is p-hydroxyphenyl (Ia) or diiodo-p-hydroxyphenyl (Ib) (Ia) is prepd by reacting 3,5-dichloro-4- (2-imidazoliden)amino -benzoic acid (II) with tyrosine methyl ester (III) in the presence of a carbodiimide, esp N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC). (Ib) is prepd by reacting (Ia) with iodine (espiodine-125), pref. formed in situ from an alkali metal iodide and an oxidising agent, esp Na N-chloro-p-toluenesulphonamide (IV). (Ib) contg radioactive iodine is useful as a tracer for radioimmunoassay of cloridine, giving higher sensitivity than prior art tracers. (Ia) is an intermediate for (Ib).

Description

Dr. Cr / Is Case 1/632 QiHi_BgEHRINGER_SOHN _4ii INGELHEIM _a AM _e RKEIN

Radioimmunoassay for clonidine

080035/0153

The invention relates to a very sensitive and specific one Detection of clonidine, 2- (2,6-dichlorophenylinino) -imidazolidine hydrochloride, a unique, centrally acting antihypertensive, whose special properties e.g. in Schmitt, H., Handbook of Experimental Pharmacology, F. Gross, Ed. Springer Verlag, Berlin, Heidelberg, New York Vol. 39, 299 to 396; Are published in 1977. Because of the high effectiveness of this drug and the resulting very low dosage from 75 to 300 ^ g per day and patient, it needs a lot sensitive detection method for the active ingredient, especially when the pharmacokinetics of galenic substances in body fluids Preparations with a strongly delayed release should be determined. Concentrations of up to 10 pg / ml must be measurable here.

The previously used detection methods such as mass fragmentography or gas chromatography are not sufficiently sensitive for these purposes. In addition, it requires an expensive and large one Workload.

More recently, two radioimmunoassays for clonidine are known-'geworden, the DAR is an improvement of the previous methods ^one place, do not meet, but with regard to sensitivity and specificity the requirements. In DE-OS 27 39 083 a radioimmuno test is proposed which is based on k- ( [6- (2,4-Di-'chlor-3- (4,5-dihydro-1H-imidazol-2-yl) -an ; ino) -hydroxypheny?.] - azoJ, benzoic acid as hapten and C-labeled clonidine as tracer. With this test, a detection limit of up to 500 pg is achieved. «The reason for the relatively low sensitivity of this test is that it is too low Radioactivity of the added C-labeled tracer, which is 43 mCi / mmol.

In the abstracts of the 7th International Congress of Pharmacology, Pergamon Press Ltd., Abstract No. 114 is a radioimmuno test for Clonidine described, which with 3,5-dichloro-4 - [(2-imidazolidinylidene) -aminoj-benzoesäuri, 4-carboxy-clonidine, works as a hapten and with tritiated clonidine as a tracer. Due to the - - 030035/0153

higher specific radioactivity of H-clonidine of 26.7 Ci /
mmol could increase the sensitivity of the test to 20 pg / ml
will. In addition, apart from 4-hydroxy-clonidine, there was no cross-reaction with the metabolites of clonidine.

The principle of the radioimmunoassay is specific competitive displacement $ a radioactive tracer of a limited amount | Fischer binding sites on an immune serum due to a variable or unknown amount of an antigen. The lower the amount of antigen, | which, as in the present case, can also be a drug j f \ , which still causes the tracer to be displaced, '| the more sensitive the test is. The amount of tracer and antigen must be present in approximately the same concentration in order to
to be able to achieve a quantifiable competition. The dilutability of the tracer and thus the sensitivity of the test
however, for technical reasons, it is about 5000
counts per minute (cpm) limited amount of radioactivity,
which must be added uniformly to each test tube. Ever
however, the specific radioactivity of the tracer in
; Ci / mmol, the more it can be diluted without
'■ its radioactivity below the critical limit of measurement technology
; falls. The object of the present invention is therefore to provide a: | 'To provide a new tracer for a highly specific radioimmuno test for | i clonidine, which is characterized by a particularly high sensitivity. · U

The invention relates to the new compounds N- ^ 3,5-Di-; §

chloro-4 - [(2-imidazolidinylidene) -amino] -benzoyl} -tyrosine-methyl- |]

ester and its acid addition salts and N- / 3i5-dichloro-4- |

[(2-imidazolidinylidene) -amino] -benzoylJ -diiodo-tyrosine-methyl-: (f

ester and its acid addition salts and the use of a fj

with radioactive iodine labeled compound as a tracer in one, si

Radioimmunoassay for clonidine, in which the antigen is a coupling ^; : Product made from bovine serum albumin with the hapten 3,5-dichloro-4 - [(2-imidazolidinylidene) -amino] -benzoic acid is used.

The production of the new connections takes place through implementation ■

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of 3,5-dichloro-4 - [(2-imidazolidinylidene) -amino] -benzoic acid or of its salts with tyrosyl methyl ester in the presence of a carbodiimide and further reaction of the intermediate with Iodine.

The preferred carbodiimide is N-ethyl-N- (3-dimethyl-aminopropyl) -carbodiimide used. It has also proven to be advantageous proven to carry out iodination with iodine generated in situ.

j lead by example, an alkali metal iodide in the presence of Ox / - used ■ dationsmittels. The use of sodium iodide as alkali metal iodide and sodium p-toluene isulfonic acid chloramide (chloramine T) as oxidizing agent is particularly advantageous.

; For the production of a tracer labeled with radioactive iodine : iodination is carried out with radioactive iodine, whereby

12 * 5
j found the use of -v particularly beneficial. the

j specific radioactivity of the tracer is about 500 Ci / mmol. : The manufacture, recovery, storage and titration of the antibody

; i

j was done as follows:!

The 3,5-dichloro-4 - [(2-imidazolidinylidene) amino] benzoic acid used as a hapten was obtained by reacting S-methyl-N- (4-carboxy- ■ 2,6-dichlorophenyl) thiuronium iodide with ethylenediamine in Methylglycol and customary work-up prepared. The compound has a melting point of 305 to 307 ⁰ C.

200 mg of pure bovine serum albumin were dissolved in 20 ml of an aqueous solution of the sodium salt of 3,5-dichloro-4 - [(2-imidazolidinylidene) amino] "benzoic acid (0.2 mmol, pH 7.4) of; 100 mg of N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide, the mixture was stirred for 24 hours at room temperature in the dark. I The solution was then for 2 days at 4 ^° C. against a phosphate-buffered saline solution (0.01 mol , pH 7.4) The antigen obtained was freeze-dried and stored at 4 ° C.

The antigen was in a concentration of 1 mg per ml of saline

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solution dissolved and equal volumes of antigen solution diCGsr ziiz complete Freund's adjuvant enulgiert. To produce the arrtiserura, 10 female rabbits were injected intradennally with 2 ml of freshly prepared antigen emulsion each. Animals received booster intramuscular injections every 3 or 4 weeks with the same volume of the antigen emulsion.

The antibody titer of the immunized animals was determined every week. After sufficient titers were achieved, the animals were bled, the serum separated from the blood, dried, and stored at -80 ⁰ C.

The following examples illustrate the production of the radioactive .Tracers.

example 1

; N-1 3.5-dichloro-4-r (2-imidazolidinyldene) -amlno1-benzoylJ -tyrosine-

ι methyl ester

120 mg of 3,5-dichloro-4 - [(2-imidazolidinylidene) -amino! -Benzoic acid

I were dissolved in 7 ml of absolute dimethylformamide. After another 9.24 mg of 1-hydroxy-1H-benzotriazole, dissolved in 1 ml of dimethylformamide and 15.44 mg of L-tyrosyl methyl ester hydrochloride, were dissolved 'added in 1 ml of dimethylformamide. After the mixture had ^{cooled to 0 0} C, 11.5 mg of N-ethyl-Ni- (3-dinethyl-aminopropyl) -carbodiimide hydrochloride were added, for one hour at ^{0 0} C • hold and then for hours under the light in the case of I stirred temperature. The light yellow clear coupling product had a thin-layer chromatographic RF value of 0.68, in comparison to an RF value of 0.096 of 3,5-dichloro-4 - [(2-imidazolidinylidene) -amino] -benzoic acid (mobile phase: 1 hour in toluene: isopropanol: ammonia 50:42:10).

The yellow product was taken up clutch after distilling off the solvents' ■ means in a high vacuum in methanol and purified by multiple "each other by means of preparative Dünnschichtchromatographic. The individual fractions were clearly visible under UV light. The imido

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Dazol-containing bands could be stained on analytical layer plates placed in parallel for comparison with Schüttler's reagent. The coupling product was in each case scraped off the layered plate, taken up in methanol and filtered several times through a G 3 glass filter to remove the silica gel. The product was 95 % pure and its structure was identified using the nuclear magnetic resonance spectrum:

COOCH,

H-NMR spectrum

Frequency: 100.1 MHz
Temperature: 28 ° C
Solvent: CD, OD

Calibration: Tetramethylsilane (TMS) internal »0 ppm Spectral width: 1000 H - 10 ppm

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2305883

Assignment of the spectrum: Singlet m) Protons
I. Doublet
(8.6 H ₂ ) Singlet TH H Intensity Multiplizi Doublet
(8.6 H ₂ ) Singlet 2. H _a 2H X part 3. H ^ 2H A3 part_
(ABX syste 4. -CH-CO
N 2H 5. -CH ₂ -aryl IH 6. OCH ₃ 2H 7. CH ₂ -CH ₂ 3H (5-ring) 4H

7.76 ppm. 7.07 ppm.

t, 71 ppm.

4.72 ppm. · J 2.86-3.32 ppm. j f

3.74 ppm. 3.52 ppm. I.

; As preliminary radioimmunological tests showed, this coupling \

product capable of tritiated clonidine completely from: |

to displace specific immune serum. Thus the immunological $

Reactivity of the coupling product proved to be a pre-; ffj

. Settlement for its use as a tracer in a radioimmuno test: fi

füi · is clonidine. j f | L

^! S.

Example 2 : j

N- C. 3.5-Ittchlor-4-r (2-imidazolidinvliden) -amino'l-benzovll -dl-.1od-tyrosln-methvlester ^;

To a solution of 0.05 ml of that prepared according to Example 1
Tyrosylmethyl peptide (0.1 mmol, dissolved in 0.1 molar phosphate

"""""""03 0 0 3 5 I 0 1 5 3

• ti *

- 10 -

buffer, pH 7.4) 0.05 ml 0.1 molar phosphate buffer, pH 7.4 and 0.002 ml sodium metabisulphite solution in a concentration of 3 mg per 5 ml 0.1 molar phosphate buffer pH 7.4 were added.

adds. 0.025 ml Na activity of 100 mCi / ml were added to the batch.

J solution with a radio

To release the iodine, 0.001 ml each of a solution of sodium p-toluenesulfonic acid chloramide in a concentration of 4.6 mg each 5 ml 0.1 molar phosphate buffer pH 7.4 were pipetted in 20 times in succession. Finally were. 0.1 ml of a 3 mg sodium metabisulphite solution was added to each 5 ml 0.1 molar phosphate buffer pH 7.4.

The approach was carried out at room temperature and immediately applied after the reaction to a Sephadex G 25 column (fine) (60 cm χ 0.9 cm in equilibrium with 0.1 molar citrate buffer pH 6.0). The column was eluted with the citrate buffer at a rate of 12 drops per minute. The radioactivity and their immunological reactivity with the specific antibody against clonidine was determined for each fraction of 3 ml. at In the above-described iodination approach, the radioactive iodine was covalently bound almost quantitatively to the immunologically reactive tyrosylmethyl peptide of 3,5-dichloro-4 {(2-imidazolidinylidene) -amino! -benzoic acid. The radioactivity is calculated from this at 500 Ci / mmol.

Those fractions of the Sephadexs & cannula containing the höhsten immunologically reactive radioactivity measurements were pooled, mixed and stored to avoid radiolytic decomposition with addition of η Prapanol and 0.1% human serum albumin at -20 ⁰ C.

Example 3

The radioimmuno test is carried out as follows:

tt
• ■ ·

• ·

2905383

- li -

The following are pipetted one after the other:

1. 0.4 ml phosphate-buffered (0.05 M; pH 7.5) physiological saline solution.

2. 0.2 ml clonidine standard solution (dissolved in human blood plasma) of the following concentrations:

i 5 ng / ml; 2.5 ng / ml; 1 ng / ml; 0.5 ng / ml; 0.1 ng / ml; 0.05 ng / ml; > 0.01 ng / ml; 0.005 ng / ml.

3. 0.1 ml iodine tracer solution (dilution in 0.05 molar phosphate buffer; pH 7.5) at 5000 cpm.

4. 0.1 ml immune serum dilution (1: 3000 in human blood plasma).

The tubes were ^{incubated at 4 °} C. for 16 hours. Then 0.4 ml of dextran-coated activated charcoal suspension was added for 30 minutes. After centrifugation, the antibody-bound iodine tracer activity of the supernatant was measured in a V-radiation meter. The calibration curves and the unknown plasma samples were evaluated with the aid of a computer.

The sensitivity of the radioimmunoassay for clonidine in this test arrangement is 10 pg / ml or 2 pg per batch. This method is therefore 250 times more sensitive than the method described in DE-OS 27 39 038. This method is still twice as sensitive as the radioimount test described in the abstracts of the 7th International Congress of Pharmacology using a tritiated clonidine as a tracer. In the method according to the invention, there is only a 17% cross-reaction with 4-hydroxy-clonidine, a metabolite of clonidine, whereas no cross-reaction whatsoever can be observed with any of the other clonidine metabolites ¹ ;

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Claims (10)

P ₌ A_T_E ₌ N_T_A_N ₌ S ₌ P_R_Ü ₌ C ₌ H ₌ E ₌ i N-0,5-dichloro-4 - [(2-imidazolidinylidene) amino] benzoyl / tyrosine methyl ester and its acid addition salts. 2. N- { 3,5-dichloro-4 - [(2-imidazolidinylidene) -amino] -benzoylj / diiodo-tyrosine methyl ester and its acid addition salts. 3. Use of a radioactive iodine labeled compound according to claim 2 as a tracer in a radioimmunoassay for clonidine. $ 4. Process for the preparation of a compound according to claim 2, characterized in that 3,5-dichloro-4-t (2-imidazolidinylidene) amino! -Benzoic acid or its salts with tyrosine j converts methyl ester in the presence of a carbodiimide and that ^! the product obtained is reacted with iodine and, if appropriate, the end product is converted into acid addition salts. !8th. Process according to claim 7, characterized in that one Sodium iodide and sodium p-toluenesulfonic acid chloramide are used . 9. The method according to any one of claims 4 to 8, characterized in that that radioactive iodine is used. 030035/0153 I5. The method according to claim 4, characterized in that as I carbodiimide N-ethyl-N- (3-dimethyl-aminopropyl) -carbodiimide a | _t puts. I |] : 6. Process according to Claim 4, characterized in that the 'i's • Performs iodination with iodine generated in situ · I I I ι i 7. The method according to claim 5 _f, characterized in that the i ·> j ! Iodine from an alkali iodide by reaction with an oxidation H releases agent. 10. The method according to claim 9, characterized in that 125 used. i'li Ü30035 / 01 53