DE2818644C2 - 3'-Bromo-thyrocarboxylic acid derivatives labeled with radioactive iodine and process for their preparation - Google Patents

Description

in which:

R is hydrogen or Ci to d-alkyl R ^{1 is} a direct bond, -CH ₂ -, -CH ₂ -CH ₂ - or

to

15th

-CH ₂ -CH-

X is hydrogen C _{1 to C 4} alkyl or C 1 to C alkanoyl

R ^J and R ⁵ each independently of one another are hydrogen or iodine.

2. Process for the preparation of the compounds according to Claim!, Characterized in that one is a compound of the general formula

R '- COOR

with a radioactive iodide and with sodium N-chloro-4-toluose sulfonic acid amide implemented in a manner known per se.

Thyroxine (T ₄ ) is a thyroid hormone of the formula JJ

HO

₂ - CH-COOH (HT)

NH ₂

For the detection of glandular dysfunction or when monitoring the course of therapy, it is important to monitor this hormone and its metabolites, such as B. Triiodothyronine or tetraiodothyroessic acid, quantitatively in the blood of the Determine patient. The quantitative determination of thyroxine is currently mainly with the so-called Radioimmunoassay performed. A thyroxine antibody is required for this analytical method and radiolabelled thyroxine. The thyroxine

antibodies are obtained by immunizing test animals using a Tj-protem conjugate and later Obtaining blood serum from these animals.

Thyroxine metabolites can be determined by analogous methods.

The radioactively labeled thyroxine can be obtained according to Hunter (in the modification according to J. Weeke & H. Örskov, Scand. J. elin. Lab. Invest. 32, 357; 1973) through implementation of 3,5'5-triiodothyronine (Tj) of the formula

CH ₂ -CH-COOH (IV)

NH ₂

with radioactive iodide and chloramine T (Na salt from N-chloro-4-methyl-benzenesulfonamide). Corresponding radioactively labeled thyroxine metabolites can be prepared analogously.

The radioimmunoassay is based in principle on the fact that the z. B. serum sample to be analyzed for thyroxine with radioactively labeled thyroxine and thyroxine body is incubated, with the thyroxine body opposite the thyroxine present in the sample to be analyzed and the added radioactively labeled thyroxine is used in deficit. The thyroxine in the serum sample competes with the radioactively labeled one Thyroxine around binding sites on the Tj antibody. The antigen-antibody complex formed, for example by precipitation, is then not removed from the body Bound hormone separated, whereupon the radioactivity of one or the other phase is measured. With The amount of thyroxine present in the sample to be analyzed can be determined with the aid of a calibration curve

6ö be.

Also for the determination of thyroxine by other methods, e.g. B. the competitive protein binding Assay (CPBA) and for the determination of other parameters, e.g. B. of thyroxine blinding globulin, is radloaktlv labeled thyroxine required as a tracer.

Analogous statements as apply to thyroxine itself for the other metabolites formed from thyroxine in the body. such as B. 3,3 ', 5'-Trlyiod-L-thyronin (rTj).

HO

their concentration in serum or other body fluids also allow conclusions to be drawn about disease states.

It has been shown, however, that in the iodination of thyrocarboxylic acid derivatives (e.g. by the above process according to Hunter with radioactive iodide and chloramine T) in addition to the substitution also an exchange reaction existing iodine substituents takes place in the outer ring. Therefore, z. B. radioactively labeled Thyroxine under the above-mentioned iodination conditions also through iodine exchange from thyroxine get yourself. Due to this exchange reaction are z. B. in the production of radiolabeled 3 ', 5'-diiodine-substituted thyronines in the outer ring increasing amounts of doubly labeled molecules are formed, as there is partly a second radioactive iodine substituent is installed. Such compounds labeled twice as radioactive, however, are undesirable because they are decompose faster.

The invention had the object to provide radioactive thyronine carboxylic acid derivatives that do not have the mentioned disadvantage of double marking.

This object is achieved according to the invention by creating 3'-bromothyrocarboxylic acid derivatives labeled with radioactive iodine the general formula

HO

R ¹ —COOR ίο -CH ₂ -CH-

NH ₂

and R stands for hydrogen, has a special role.

The radioactive iodine present in the compounds according to the invention can be iodine-125 or iodine-131. Iodine-125 is preferred for radioimmunoassays.

The compounds according to the invention can be in the D, L or DL form. They are preferably in the L-shape, since d>. Compounds formed by the organism also have the 'form.

It has been shown that the compounds according to the invention act against the corresponding antibodies are almost as reactive as those compounds that contain an iodine atom instead of a bromine atom contain. The ability of an antibody to react to a substance other than that against which it was generated is called "cross reactivity" and expressed as a percentage, the reactivity to the associated antigen being the same 100% is set. As a result, there are several cross-reactivities of compounds according to the invention indicated, which are each obtained with an antiserum that corresponds to the corresponding non-radioactive Compound was generated in which there is an iodine atom instead of the bromine atom.

Cross-reactivities (in%) of 3'-bromo-5 '(iodine 125) 3,5-dijodhyronintracer with T ₄ antibodies compared to

40

Br

(D

in which:

R is hydrogen or C, - to C ₄ -alkyl,

R 'is a direct bond, -CH ₂ -, -CH ₂ -CH ₂ - or

CH ₂ -CH-COOR

-CH ₂ -CH-

X is hydrogen Ci to _C4 alkyl or C, - to C ₄ alkanoyl 55 _H R ^J and R ⁵ are each independently hydrogen or iodine.

The root word "Thyro" denotes in the sense of Invention is a radical of the formula

HO-

(VI)

Because of the great importance of the determination of thyroxine in serum, the corresponding radioactive compound according to the invention of the above formula I, in which R ¹ and R "each stand for iodine. R 'for the group

/
Br R. J X NH
I. cross
reak
activity H A. X 86 50 H H 84 H methyl Confi
gura
tion 89 55 H ethyl L. 92 H Isopropyl D. 85 H Butyl L. 23 60 H Ihobutyi L. 32 H t-butyl D. 63 Acetvl ti D. 158 65 L. D. L.

continuation

R. X Confi Circular gura react- tion tivitii!

Isopropionyl Il Butyryl II Isobutyryl Il Isopropyl Il Acetyl ethyl Acetyl Isopropyl Acetyl Butyl Isobutyryl ethyl Isopropyl Isopropyl

D. 165 L. 322 L. 163 D. 138 L. 155 L. 162 L. 27 L. 157 D. 129

CH R °

Cross reactivity

COOH
CH _: C00H

As in the preparation of the compounds according to the invention because of the greater stability of the bromine-carbon bond compared to the iodine-carbon bond practically no exchange reaction, d. H. no double marking (<1 \) occurs, are those according to the invention Compounds especially at high specific activities (the theoretically possible specific activity can can be achieved) through longer shelf life and usability compared to conventional tracers excellent. For the radioimmunoassays, an increase in sensitivity is possible without the previous disadvantage of the shorter usability of tracers with higher specific activity in purchase to have to take. In the figure is the difference in the rate of decomposition of a known tracer and of the corresponding tracer according to the invention.

The compounds of the invention can be prepared by making a compound the general formula

HO

Ri_COOR

ΓΠ)

with a radioactive iodide and with ChloraminT in known catfish converts.

The invention is illustrated in more detail by the following examples.

example 1
3'-Bromo-5 '- (iodo-125) -3.5-diiodo-L-thyronine

To 0.5 mCI sodium iodide (J-125, carrier-free) in IO ul Buffers (0.5 M phosphate, pH 7.5) are 0.4 µm 3'-bromo-3,5-diiodo-L-thyronin Added in 10 µl of the same buffer. With thorough mixing, 9 ng Chloramine T In 10 ng of the same buffer added. the Reaction is started after 30 s by adding 24% sodium betasullite stopped in lOul of water. Get that; The product is purified by column chromatography. For this purpose, the product can be placed on a column of cross-linked dextran oil (e.g. Sephadex®G-25), whereupon the unreacted iodine first with 0.05 M TrIs (hydroxymethyllaminomethane / hydrochloric acid buffer (pH 9) is elulert. Elution with this buffer is continued with the addition of methanol. This will the desired product elutes.

Instead of column chromatography, layer chromatography on paper or silica gel can also be carried out will.

The product (and also the product of the following examples) was determined by thin layer chromatography Kieselgel 60 (Merck. Darmstadt) characterized. The localization was carried out using a thin-layer scanner.

R, = 0.33

Mobile phase: chloroform / methanol / 25% ammonia (60/30/3 x. Parts by volume)

The starting material used in the above process can be obtained as follows:

To 5.25 g (10 mmol) of 3,5-diiodo-L-thyronine in 70 ml Glacial acetic acid and 1 ml of concentrated hydrochloric acid are mixed with 0.51 ml of UOmMoI) Beom in at room temperature 30 ml of glacial acetic acid were added dropwise over 30 minutes. After a further 30 min, 100 ml of water and a little blush are added up to Decolorization added.

The pH is slowly adjusted to approx. 4 with 10% sodium hydroxide solution. The precipitate that forms is sucked off and washed out well with water. Dry in a desiccator over phosphorus pentoxide yields 5.4 g (89%) of 3'-bromo-3,5-dliodo-L-thyronine as a colorless product with a melting point of 248 ° (decomposition). Further purification of the product can be carried out by tipping it over or by Column chromaCography is carried out. This enables a product to be produced which, according to thin-layer chromatography or is uniform according to high pressure liquid chromatography.

Example 2
3'-Bromo-5 '- (iodine-125) -3-iodine-L-thyronin

To 0.5 mCi sodium iodide (J-125, carrier-free) in 10 μg phosphate buffer (0.5 M, pH 7.5), 0.32 μg 3'-bromo-3-iodo-L-thyronine in 10 μg of the same buffer admitted. Then 9 ig chloramine T in 10μΙ of the same buffer are added with vigorous mixing. After 30 s, the reaction is stopped by adding 24 μg of sodium metabisulphite in 10 μ! Water stopped.

The product obtained is subjected to column chromatography cleaned. For this purpose, a column (10x0 learning) can be networked! hydroxypropylated dextran gel (Sees phadcx® LH-20) can be used. As an elution medium can be a mixture of ethyl acetate / methanol / 2 N ammonia (20/5/2; volume) serve. With the help of a fraction collector, approx. 0.5 ml fraction

15th

Honing collected. Those containing the desired product Fractions are merged.

Instead of column chromatography can also be Paper and / or Schlchtchromatographle (silica gel) for Separation of excess radioactive iodine can be carried out.

R, = 0 17

Runner: ethyl acetate / methanol / 2N ammonia (5/2/1; parts by volume)

The starting compound 3'-bromo-3-iodo-L-lhyronin was according to the instructions of M. V. Musett. R. Pitt Rivers. Metab. ClIn. and I-xptl. 6. 18 (1957).

Example 3

3'-Bromo-5 '- (iodo-125) -3,5-dliodothyroacetic acid
To 0.5 ml sodium iodide (J-125. Carrier-free) in 10 μΙ

ηΐ. _η . _η ί _π ι ^ .. Γ ^. in cw "11 ~ i ;> ι λ at * η

ι iiu3 | <iftu | 'uiit.i \ u, .i ι · ι, μιι i.ji vn.iui.il u, t μ $ J -υιυιιι- 3.5-dljodthvroessigsiiure In 10 ng of the same buffer added. 9 ng of chloramine T in 10 µl of the same buffer are then added with thorough mixing. 30 s afterwards, 24 ng of sodium metablsulflt in ΙΟμΙ water are added to stop the reaction.

In order to separate off the excess radioactive iodine, the reaction mixture is subjected to descending paper chromatography subjected to an approx. 10 cm wide strip. A solvent can advantageously be used as a solvent Mixture of n-hexane / tert-amyl alcohol / 2N ammonia (1/5/6; parts by volume) can be used.

The ;. _u s product containing radioactive zone is cut by means of locating a thin film scanner and eluted with dil. methanollschen ammonia.

The product can also be purified by column chromatography.

R. = 0.57

Mobile phase: ethyl acetate / methanol / 2N ammonia (5/2/1; volume point)

The starting product was according to the instructions of R. Cereljo-Santalo et al .. Endocrinology 69, 422 (1961) or O. Thlbault, Arch. Sci. Physiol. 13, 1 (1959).

Example 4
3'-Bromo-5 '- (iodine-125) -3,5-dliodo-D-thyronin

25th

30th

35

40

45

3'-Bromo-3,5-diiodo-D-thyronin is necessary as a starting product for these and other compounds prepared analogously to the L compound given in Example 1. Colorless product, melting point 237 ° C (decomp.).

The radioactive labeling is carried out analogously to Example 1. The separation is carried out by means of thin layer chromatography (silica gel 60; Merck, Darmstadt), mobile phase: chloroform / methanol / 25% ammonia (60/30/3; parts by volume). After localization with a thin-layer scanner, the zone 2.7 to 4.5 cm (R ₇ = 0.28) is cut out and eluted with methanolic ammonia.

Example 5

N-acyl derivatives of 3'-bromo-3,5-diiodo-L- ^M

(or D) thyronins

500 mg (0.82 mmol) 3'-bromo-3,5-diiodo-L- (or D) -thyronine are absolute in 10 to 20 ml. Dimethylformamide and I to 2 ml of Triäthylamln and dissolved with 0.2 up to 0.4 ml of the acid anhydride are added. After 1 to 4 hours at room temperature, the mixture is poured onto ice, acidified and possibly somewhat concentrated in a vacuum rotary evaporator. The colorless precipitate is suctioned off, Washed well with water and dried over calcium chloride in a deslcator.

Any additional 4'-0-acyl group that may be present can be removed by treatment with methanol. Ammonia can be split off at room temperature, or the separation of the N-acyl from the ON-dlacyl compounds can be carried out by means of preparative thin-layer chromatography.
This process was used to produce:
The respective data is given in the following order: bulge; R / value (silica gel 60; solvent system I = chloroform 70 / methanol 15 / formic acid 15, volume; solvent system H = ethyl acetate 70 / methanol 20/2 N ammonia 10, volume); MS (70 eV).

N-acetyl-3'-bromo-3,5-dliodo-L-lhyronin

93%; Rf (I) = 0.56; m / e = 647, 645 ΙΜΊ N-i5upfü [iiüfiyl-3'-uiüfu-3,5-uijüu-D-iiiyruMiri

<J4%; R, (I) = 0.57; m / e = 661.659 (M ¹ I N -butyryl-3'-bromo-3,5-d! iodo-L-thyronine

59%; R (I) = 0.65; m / e = 675, 673 (M ") N-isobutyryl-3'-bromo-3,5-dliodo-L-thyronine

45%; R, (I) = 0.68; m / e = 675, 673 (M ')

Example 6
Ester of 3'-bromo-3,5-dliodo-L- (or DMhyronine

604 mg (1 mmol) 3'-bromo-3,5-dliodo-L- (or DHhyronln are with 1.33 g (7 mmol) of p-toluenesulfonic acid in 200 ml of toluene with 25 ml of the corresponding alcohol for 1 to 10 h under reflux and heated with a water separator. After cooling, with three times 40 ml of water, 40 ml saturated sodium carbonate solution and extracted twice 40 ml of water each time; the organic phase is with Magnesium sulfate dried and concentrated in vacuo on a rotary evaporator. The colorless product will Aspirated and dried in a vacuum exslkator over calcium chloride or phosphorus pentoxide.

This process was used to produce:

3'-Bromo-3,5-dliodo-L-thyronin methyl ester

79%; R, (I) = 0.55; m / e = 619, 617 (M *) 3'-Bromo-3,5-diiodo-L-thyronine ethyl ester

83%, R, (D = 0.54; m / e = 633, 631 (M *) 3'-Bromo-3,5-d! Iodo-D-thyronin isopropyl ester

76%; R, (D = 0.55; m / e = 647.645 (M *) 3'-Bromo-3,5-diiodo-D-thyronone butyl ester

52%; R, - (II) = 0.93; m / e = 661,659 (M *) 3'-Bromo-3,5-diiodo-L-thyronine! Sobutyl ester

46%; R, (I) = 0.60; m / e = 661, 659 (M *)

Example 7
3'-Bromo-3,5-dliodo-D-thyronine tert-butyl ester

604 mg (1 mmol) of 3'-bromo-3,5-diiodo-D-thyronine are tert with 1.2 g (6.3 mmol) of p-toluenesulfonic acid in 60 ml of chloroform and 8 ml. Buthanol heated under reflux for 1 h with a water separator. The precipitate is filtered off with suction and purified by means of preparative layer chromatography (mobile phase: ethyl acetate 70 / methanol 20 / ammonia 10, parts by volume). Yield 28%. R _r (1) = 0.61; MS (70 eV): m / e = 661.659 (M ⁺ ).

Example 8

N-Acyl derivatives of the 3'-bromo-3,5-diiodo-L- (or D) -thyronlnester

1.1 mmol N-acyl-3'-bromo-3,5-diiodo-L- (or D) thyronine with 0.8 mmol of p-toluene sulfonic acid in 50 ml

Toluene and 3 ml of the corresponding alcohol were heated under reflux for 1 to 4 h with a water separator. After cooling, it is extracted with three times 10 ml of water, 10 ml of saturated sodium bicaronate solution and twice with 10 ml of water and dried with magnesium sulfate. It is concentrated in vacuo on a rotary evaporator, the precipitate is suctioned off and geirock.net is used in a vacuum exslkator.
According to this regulation, the following were produced:

N-AcetylO'-bromO.S-dliodo-L-thyronine isopropyl ester

21%; R, (I) = 0.74; m / e = 689, 687 (M *) N -AcetylO'-bromO.S-dljod-L-thyronine buty lesler

68%; R, (0 / 0.81; m / e = 703, 701 (M ') N -isobutyryl-S'-bromoO.S-dliodo-L-thyroninethyl ester

63; R, (I) = 0.79; m / e = 703, 701 (M ')

Example 9
N-Äceιyi-3'-bΓoπl-3, J-üijuU-L · -uiyfüiundihyίcsieΓ

632 mg (0.1 mmol) of J'-BromO.S-dliodo-L-thyronin ethyl ester are dissolved in 0.8 ml of ethanol and 0.1 ml of triethylamine. After adding 30 μΐ acetic anhydride is Stirred for 1 h at room temperature. Pour on water, suck off the precipitate, wash out with water and Dry in a vacuum deslcator over calcium chloride. Yield 72%.

R, (1): 0.71; MS (70 eV): m / e = 675, 673 (M ⁺ ).

Example 10

N-Isopropyl ^ -bromo, S-dliodo-D-thyronone isopropyl ester

65 mg (0.1 mmol) of S'-bromo-S.S-dliodo-D-thyronine isopropyl ester In 10 ml of isopropanol with 6 mg (0.1 mmol) of acetone, 6.3 mg (0.1 mmol) of sodium cyanoborohydride and 23 mg (0.12 mmol) p-toluenesulfonic acid 6 h be! about 40 ° C stirred. MU water the product is precipitated, Sucked off and dried in a vacuum exslkator over phosphorus pentoxide. Yield 79%.

R, (I) = 0.84. MS (70 c V): m / e = 689.687 (M ').

Example 11

N-Isopropyl-3'-bromo-3,5-dliodo-D-thyronine 20 mg (0.03 mmol) of N-isopropyl-S'-bromo-S.S-dliodo-D-thyronine ester are kept in 5 ml of aqueous methanol with 0.5 ml of 1N sodium hydroxide solution for 2 h at room temperature, then weakly acidified with HCI. The precipitate is filtered off with suction, washed with water and placed in a vacuum desiccator dried over phosphorus pentoxide. Yield 71%.

R, (I) = 0.73. MS (70 eV): m / e = 647.645 (M ').

Example 12

Radioactive yodelling of 3'-bromo-3.5-d! Iodine-l .- (or D) -thyroninderivate

0.2 to 0.4 ng of the 3'-bromo-3,5-dliodo-L- (or DHhyronine derivative in 10 μΐ methanol, 10 μΙ buffer (0.2 to 0.5 M phosphate, pH 6.2 to 7.5) and 0.5 mCi sodium iodide (J-125, carrier-free) are mixed thoroughly with 10 μg of N-ChloM-toluenesulfonic acid amd sodium (in

ίο 10μ1 buffer) offset. After 20 to 90s the reaction will by adding 20 to 40 ng sodium tablet stopped. The reaction mixture is I. a. by thin layer chromatography (Kleselgel 60; Merck. Darmstadt) separated; as the mobile phase are those in the above Examples given or similar solvent mixtures used. The means·' Thin-film scanner localized product zone is made with methanol. Ammonia elutes.

Well <-! I of this regulation were made:

3'-Bromo-5 '- (iodo-125) -3,5-dliodo-L-thyronine methyl ester

R, (D = 0.61 3'-Bromo-5 '- (iodo-125) -3.5-dliodo-L-thyroninethyl ester

R. (D = 0.61

3'-Bromo-5 '- (iodo-125) -3,5-dliodo-D-thyronine isopropyl ester R, (D = 0.63 3'-Bromo-5 '- (iodine-125) -3,5-diiodo-D-thyronine butyl ester

R, (111 = 0.84 3'-Bromo-5 '- (iodo-125) -3,5-dliodo-L-thyronine isobutyl ester

R, (D = 0.68

3'-Bromo-5 '- (iodine-125) -3,5-dliodo-D-thyronintert. butyl ester

R, (D = 0.70 N-acetyl-3'-bromo-5 '(iodo-125) -3,5-diiodo-L-thyronine

R, (I) = 0.63

N-Isoproplonyl-3'-bromo-5 '(iodine-125) -3,5-diiodo-D-thyronine R, (D = 0.65 N-butyryl-3'-bromo-5 '(iodine-125) -3,5-diiodo-L-thyronin

R, (D = 0.7! N-isobutyryl-3'-bromo-5 '(iodine-125) -3,5-dliodo-L-thyronine

R, (D = 0.74 N-acetyl-3'-bromo-5 '(iodine-125) -3.5-diiodo-

L-thyronine ethyl ester F., (D = 0.78

N-acetyl-3'-bromo-5 '(iodine-125) -3,5-diiodo-

L-thyronin isopropyl alcohol R, (D = 0.74

N-acetyl-3'-bromo-5 '(iodine-125) -3,5-dliodo-

L-thyronine butyl ester R, (I) = 0.81

N-isobutyryl-3'-bromo-5 '(iodine-125) -3.5-diiodine

L-thyronylnäthylester R, (D = 0.91

N-Isopropyl-3'-bromo-5 '(iodine-125) -3,5-diiodine

D-thyronin isopropyl ester R, (D = 0.94

N-isopropyl-3'-bromo-5 '(iodine-125) -3,5-dliodo-D-thyronine

K _f (I) = 0.81

1 sheet of drawings

Claims (1)

Claims: 1. 3'-bromothyro- labeled with radioactive iodine carboxylic acid derivatives of the general formula J * R ⁵ HO R ¹ -COOR