EP0591489A1

EP0591489A1 - Substituted thiazoline-dioxetane-substrates, process for producing the same and their use

Info

Publication number: EP0591489A1
Application number: EP93907842A
Authority: EP
Inventors: Rupert Herrmann; Hans-Peter Josel; Christian Klein; Dieter Heindl
Original assignee: Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1992-04-01
Filing date: 1993-03-29
Publication date: 1994-04-13
Also published as: JPH06102669B2; US5712106A; JPH06503844A; US5994552A; WO1993020083A1; DE4210759A1; US5455357A

Abstract

Des composés répondent à la formule générale (III), dans laquelle R désigne le groupe (a) ou le groupe (b), R1 et R2, qui peuvent être identiques ou différents, désignent hydrogène ou alkyle inférieur ayant 1 à 6 atomes de carbone, à châine droite ou ramifiée, X représente un groupe clivable, au moins un des groupes R4 ou R5 représente un groupe stabilisateur de la structure en dioxetane et tout au plus un des groupes R4 ou R5 désigne de l'hydrogène. Ces composés sont utiles comme substrats dans des dosages immunologiques et dans le diagnostic par ADN se faisant à l'aide d'activateurs de la chromogenèse.Compounds correspond to the general formula (III), in which R denotes the group (a) or the group (b), R1 and R2, which may be the same or different, denote hydrogen or lower alkyl having 1 to 6 carbon atoms , straight or branched chain, X represents a cleavable group, at least one of the groups R4 or R5 represents a stabilizing group of the dioxetane structure and at the most one of the groups R4 or R5 denotes hydrogen. These compounds are useful as substrates in immunoassays and in DNA diagnostics using chromogenesis activators.

Description

Substituted thiazoline dioxetane substrates, process for their preparation and use

The invention relates to thiazoline-dioxetane substrates, processes for the production and use in enzymatic analysis processes.

The reaction of luciferin (formula I) with luciferase, oxygen and ATP produces oxiluciferin. In this reaction, light (wavelength maximum at 562 nm) is emitted as chemiluminescence. The dioxetane of formula II is presumably formed as an energy-rich intermediate (F. McCapra, Che. Commun. 155 (1968)). A large number of chemiluminescent 1,2-dioxetane compounds were developed on the basis of this postulate. To stabilize the per se unstable 1,2-dioxetanes, the adamantyl residue has been described (EP-A 0 254 051, EP-A 0 352 713, WO 91/03479, WO 90/07511 and the publications cited therein). However, none of the known compounds is the activated intermediate stage of luciferin as a base. Formula II, a thiazoline derivative.

H

^(π) The object of the present invention was to provide a 1,2-dioxetane based on the activated intermediate of thiazolines and preferably luciferin, which is stable and only decomposes by reaction with an activating agent with the formation of chemiluminescence. This object is achieved by a compound of the general formula III,

in the R either

-ÖD- ox

or

^O

and R- [and R2 are in each case identical or different and represent hydrogen or lower alkyl C 1 -C 4, straight-chain or branched, X is a cleavable group which can be cleaved off by an activating agent and at least one of the groups R4 or R5 represents a group stabilizing the dioxetane structure and at most one of the groups R4 or R5 represents hydrogen.

Acids, bases, salts, enzymes, inorganic or organic catalysts and Electron donors used.

The group -O-X can be located at any position on the phenyl ring. When R is a benzothiazole group, however, the group -O-X is preferably arranged in the 5-position. If R is a phenyl group, the group -O-X is preferably arranged in the 3-position. The same applies to the group -OR3, from which the group -O-X arises.

-O-X is preferably the hydroxy salt of an oxyacid, phosphate, aryl or alkyl carboxyl ester, alkyloxy or arylsilyloxy, sulfate, oxypyranoside.

The composition of the aryl and alkyl radicals is not critical. Any person skilled in the art can select suitable radicals X without further ado. Solubility and cleavage of X by the activating agents must only be possible.

R1 and R2 are preferably hydrogen, methyl or ethyl groups. If, for example, phosphate is used as -O-X, the chemiluminescence reaction can be induced by adding alkaline phosphatase. When using the galactoside, the chemiluminescence reaction can be induced by ß-galactosidase. If a silyloxy radical is used as -O-X, the addition of fluoride can induce chemiluminescence.

Groups which protect the dioxetane group from reactions are suitable as groups R4 and / or R5 which stabilize the dioxetane structure. This stabilization is preferably carried out by sterically shielding this group. For example, adamantanyl, phenyl, cyclohexyl, secondary and tertiary aliphatic alkyl groups (such as __. B. the t-butyl group) in substituted and unsubstituted form. R4 and R5 can be the same or different. In the case of the adamantyl residue, it is preferably bound in such a way that R4 and R5 represent parts of the ring structure and are therefore bridged (formula purple):

The compounds of general formula III are new. A synthetic method for these compounds is not yet known.

The invention accordingly also relates to a process for the preparation of the compounds of the formula III, which is characterized in that a compound of the formula IV or IVa.

in the R3 alkyl, straight-chain or branched with 1 - 6 C- Represents atoms, preferably methyl or ethyl, with a compound of formula V.

^ß 1 COOH l / (ΪL)

₂ "* -

implemented in which R1 and R2 have the meanings given above, with the exclusion of water and air in the presence of an alkyllithium compound with aldehydes or ketones, decarboxylated the reaction product, dealkylated the alkoxy group, there the chemically labile group according to methods familiar to the skilled worker -OX introduces and photo-oxygenates to the dioxetane.

The reaction of the compound of the formula IV (alkoxybenzothiazole-2-carboxylic acid nitrile) or IVa with the compound of the formula V (preferably penicillamine or cysteine) is preferably carried out in a polar solvent, such as water or water / alcohol mixtures, at room temperature with the exclusion of light.

Suitable compounds as aldehydes and ketones are those which, as radical R4 and / or R5, stabilize the dioxetane structure of the compounds of the formula III. Such stabilization can be achieved by steric shielding of the dioxetane structure. Suitable ketones and aldehydes are accordingly, for example, adamantanone, benzaldehydes, cyclohexanones, secondary and tertiary aliphatic aldehydes and ketones, such as. B. Di-t-butyl ketone.

The introduction of R3 is preferably water-free in an aprotic organic solvent such as. B. THF with exclusion of air and cooling by reacting the thiazoline carboxylic acid thus obtained with an aldehyde or ketone. It is particularly preferred to work at temperatures from -50 to -100 ° C. The alkyl lithium compound is preferably added as a solution in a hydrocarbon (pentane, hexane). Butyllithium is particularly preferably used. After the reaction has taken place, the compound obtained is precipitated from the reaction mixture, preferably by adding an aqueous salt solution at room temperature.

The decarboxylation of the resulting β-hydroxycarboxylic acid to introduce a double bond is preferably carried out under dehydrating conditions, e.g. B. with diethyl azodicarboxylate and triphenylphosphine.

The dealkylation at the 6 'position is expediently carried out by adding a dealkylating agent such as trimethyl iodosilane. It is preferred to work anhydrous.

Likewise, the thiazoline carboxylic acid can preferably be converted to a thiazolin-4-one via the intermediate stage of the amine and alcohol and then to a thiazolin-4-thione by reaction with sulfide. This thiazolin-4-thione is reacted with a hydrazone of the aldehyde or ketone, preferably with adamantanone-2-hydrazone or benzaldehyde hydrazone, which can contain a removable group (adamantanontosyl hydrazone or benzaldehyde tosyl hydrazone) to give the compounds of the formula III according to the invention.

To prepare compounds of the formula III in which R4 and / or R5 represents a phenyl radical, an aliphatic radical or cycloalkyl radical, a substituted 2- Phenyl-5,5-dialkyl-thiazolin-4-one in a Grignard reaction to the 4-substituted derivative with subsequent introduction of a double bond at the 4-position of the thiazoline. Then, as described below, the dioxetane group is introduced.

The introduction of the chemically labile group X takes place according to methods familiar to the person skilled in the art. Such methods are described, for example, in Houben-Weyl XII / 2.

The dioxetane group is expediently introduced by reacting the alkene derivative (double bond at the 2-position of the thiazoline), which is preferably dissolved in a polar organic solvent, such as methylene chloride or chloroform, at a temperature of -100-50 ° C and irradiation with visible light using a sensitizer (for example Rose Bengal, methylene blue, cf. Tetra¬ hedron Letters (1988) 3137 - 3140).

For purification, for example, it is filtered. The supernatant contains the desired compound of formula III.

Another object of the invention is a method for determining an acid, base of a salt, enzyme, inorganic or organic catalyst and electron donor by reacting this compound with a compound of general formula III and measuring the light emitted as a measure of the content of the connection to be determined.

This method is particularly preferably used for the determination of enzymes, especially marker enzymes in immunological systems or for DNA diagnostics with labeled DNA probes. Alkaline phosphatase is preferably determined where the chemically labile group X is phosphate or β-galactosidase, where X = galactoside.

The invention is illustrated by the following examples:

example 1

Synthesis of 2- (6'-methoxy-2'-benzothiazolyl) -5,5-dimethyl-A ² thiazoline-4-carboxylic acid (1):

2 g of 6-methoxybenzthiazole-2-carbonitrile are dissolved in about 100 ml of methanol and a solution of 1.62 g of penicillamine in 50 ml of H20 is added. The mixture is stirred at room temperature for 3 hours with the exclusion of light, the solution is then acidified and filtered off.

Yield: 1.3 g TLC: Rf (chloroform: methanol: glacial acetic acid

9: 1: 0.1) 0.74

Example 2

Synthesis of 2- (6'-methoxy-2'-benzothiazolyl) -5,5-dimethyl-4- (2'-hydroxy-2'-adamantylA -thiazoline-4-carboxylic acid (2):

A double molar excess of butyllithium-hexane solution is added dropwise to 1 g of compound (1), dissolved in about 100 ml of dry THF, under N2 at -78 ° C. and the mixture is stirred for 1 hour. Then the corresponding amount of 2-adamantanone dissolved in THF is added at -78 ° C., the mixture is stirred for 3 hours at -78 ° C. and the reaction mixture is then added to 100 ml of a saturated aqueous sodium chloride solution which contains 20 mmol of HCl. The residue is recrystallized from chloroform / ether.

Yield: 0.5 g

Example 3

Synthesis of 2- (6'-methoxy-2'-benzothiazolyl) -5,5-dimethyl-4-

(2'-adamantanylidene) Δ -thiazoline (3):

The decarboxylation of beta-hydroxy-carboxylic acid with introduction of the double bond is achieved by reaction with equimolar amounts of P (CC6Hs) 3 and diethyl azodicarboxylate in THF at room temperature (see, for example, Mulzer, Angev. Chemie 1977).

Synthesis of 2- (6'-hydroxy-2'-benzothiazolyl) -5,5-dimethyl-4- (2'-adamantanylidene) Δ ² -thiazoline (4):

1 g of compound (3) are dissolved in 10 ml of dry CHCL3 and 50% excess trimethyl-iodosilane is added. The mixture is heated to about 70 ° C. for 48 hours, methanol is added to the reaction mixture and the solution is rotated in.

The residue is taken up in ethyl acetate and shaken out with 5% NaHC03 solution, dried and in vacuo. evaporated.

Yield: 0.25 g Example 4

Synthesis of 2- (6'-phosphoryl-2'-benzothiazolyl) -5,5-dimethyl- 4- (2'-adamantanylidene) A ² -thiazoline (5):

The phosphorylation of (4) is achieved using the usual literature procedures. S.z.B. Houben-Weyl XII / 2.

Example 5

Synthesis of 1 [2 '- (6 "-phosphoryl-2" -benzthiazolyl) -5', 5'-dimethyl-A ² -thiazolinyl -] - 2- (2 "-spiroadamantyl) -dioxetane (6):

For the photooxygenation of (5), 10 mg of the alkene, dissolved in 10 ml of methylene chloride, with the addition of immobilized Rose Bengal (Polysσience) are irradiated for 1 h at -78 ° C. with a 1000 watt lamp. The implementation is quantitative. The Rose Bengal Sensitizer can be easily removed by filtration.

Example 6

2-alkoxyphenyl-5,5-dimethyl-thiazoline-4-carboxylic acid

The production takes place analogously to Chem. Ztg. 1987, 111, 357.

33.5 mmol of penicillamine, 33.5 mmol of alkoxynitrile and 16.7 mmol of potassium carbonate were refluxed in a mixture of 50 ml of methanol with 25 ml of water for 12 hours. After cooling, the mixture was concentrated to half in vacuo. The mixture was washed once with 50 ml of ether. With conc. The separated aqueous phase became hydrochloric acid to pH 4 set. A pale yellow precipitate separated out, which was filtered off with suction, washed once with a little water and dried at 60 ° C. in vacuo.

Educ. 50 - 60%

Example 7

2 alkoxyphenyl-5,5-dimethyl-thiazoline-4-carboxamide

4 mmol of oxalyl chloride were added at 0 ° C. to a suspension of 4 mmol of alkoxyphenylthiazolinecarboxylic acid in 10 ml of methylene chloride. After stirring at this temperature for 30 minutes, a weak stream of ammonia was introduced at -20 ° C. for 5 minutes. After warming to room temp. the suspension was diluted with 20 ml of methylene chloride. The mixture was washed once with 10 ml of 1N sodium hydroxide solution and then twice with 20 ml of water. The organic phase was dried over sodium sulfate. After the solvent had been distilled off, an oil remained which was recrystallized from a little ethyl acetate (yield: 60-80%).

Example 8

4-amino-2-alkoxyphenyl-5,5-dimethylthiazoline

4.8 mmol of bromine were dissolved in 8 ml of 3N sodium hydroxide solution at 0 ° C. 4.4 mmol of 2-alkoxyphenyl-5,5-dimethyl-thiazoline-carboxamide were added to this mixture at 0 ° C. and the mixture was stirred at this temperature for 30 min and at 70 ° C. for 20 min. After dilution with 20 ml of water, it is shaken out with 40 ml of ethyl acetate. The organic phase was washed twice with 15 ml of water each time and dried over sodium sulfate. After distilling off the Solvent remained an oil that from ethanol / water

I: 1 was recrystallized (yield 70-85%).

Example 9

4-hydroxy-2-alkoxyphenyl-5,5-dimethylthiazoline

To a solution of 14.5 mmol amino-2-alkoxyphenyl-5,5-dimethyl-thiazoline in 32 ml half-conc. 15 mmol of sodium nitrite were added at 0 ° C. and the mixture was stirred at this temperature for 20 min. After neutralization with 3N sodium hydroxide solution, the mixture was extracted twice with 30 ml of ethyl acetate each time. The organic phase was washed once with 20 ml of water and dried over sodium sulfate. After the solvent had been distilled off, an oil remained which was separated by column chromatography (silica gel, ethyl acetate / petroleum ether 1: 1) (yield 75-80%).

Example 10

2-alkoxyphenyl-5,5-dimethyl-thiazolin-4-one

II mmol of 4-hydroxy-2-alkoxyphenyl-5,5-dimethyl-thiazoline and 34 mmol of cycloheptanone were dissolved in 160 ml of toluene. After 40 ml of toluene had been distilled off, 9 mmol of aluminum triisopropylate were added and the mixture was heated to boiling under reflux for 3 h. After cooling to room temp. 100 ml of ethyl acetate were added and the mixture was washed twice with 50 ml of water each time. The combined aqueous phases were extracted once with 50 ml of ethyl acetate. After the combined organic phases had been dried over sodium sulfate, the solvents were distilled off. The remaining oil became the thiazolinone separated with column chromatography (silica gel, ethyl acetate / petroleum ether 1: 4).

Example 11

2-alkoxyphenyl-5,5-dimethylthiazolin-4-thione

5 mmol of 2-alkoxyphenyl-5,5-dimethylthiazolin-4-one and 11 mmol of 2,4- (4-bis-methoxyphenyl) -1,3-dithia-2,4-diphosphetane-2,4-disulfide heated to boiling under reflux in 10 ml of toluene for 8 h. After cooling, the reaction mixture was separated by column chromatography (silica gel, ethyl acetate / petroleum ether 1: 6) (yield: 30-40%).

Example 12

Adamantane-2-tosylhydrazone

3.4 iruTtol tosyl hydrazide and 3.3 mmol adamantanone and a drop of conc. Sulfuric acid was refluxed in 10 ml of ethanol for 4 hours. After cooling to RT, the mixture is concentrated in vacuo to a volume of 1 ml. It was diluted with 20 ml of water and neutralized with 2N sodium hydroxide solution. The mixture was extracted twice with 20 ml of ethyl acetate. The combined organic phases were washed once with 20 ml of water and then dried over sodium sulfate. The solution was concentrated to a volume of 4 ml and left at 4 ° C for 15 h. Colorless needles precipitated out (yield: 40%). Example 13

4- (2'-Adamantanylidene) alkoxypheny1-5,5-dimethylthiazoline

To 10 ml of a methanolic 0.2 M sodium methylate solution was added 2 mmol of adamantantosyl hydrazone and then was diluted with 20 ml of diglyme. After the methanol had been distilled off, the mixture was heated to 120 ° C. until no more tosylhydrazone can be seen in the TLC. At this temperature, 2 mmol of 2-alkoxyphenyl-5,5-dimethyl-thiazoline-4-thione were added and the mixture was stirred for 15 minutes. Then 1 g of copper powder was added and the mixture was stirred at 150 ^° C. for a further 20 min. After cooling, 100 ml of water were added and the mixture was extracted three times with 50 ml of ethyl acetate. The combined organic phases were washed once with 30 ml of water and then dried over sodium sulfate. According to Abdest. the solvent remained an oil, which was separated by column chromatography (silica gel, ethyl acetate / petroleum ether 1: 6) (yield 20%).

Example 14

4-benzyl-4-hydroxyalkoxyphenyl-5,5-dimethyl-thiazoline

5 mmol of 2-alkoxyphenyl-5,5-dimethyl-thiazolin-4-one (Example 10) were added to 50 ml of an ethereal 0.2 M benzylmagnesium chloride solution and the mixture was heated to boiling under reflux for 8 h. After 20 ml of water had been added, the organic phase was separated off, washed once with 20 ml of water and then dried over sodium sulfate. After the solvent had been distilled off, an oil remained which was separated by column chromatography (silica gel? Ethyl acetate / petroleum ether 1: 5) (yield 30%). Example 15

4-benzylidene alkoxyphenyl-5,5-dimethylthiazoline

A solution of 2 mmol of 4-benzyl-4-hydroxy-alkoxyphenyl-5,5-dimethyl-thiazoline (Example 14) in 20 ml of ethyl acetate was mixed with 20 ml of semi-conc. Shaken hydrochloric acid for 5 minutes. After neutralization with 2N sodium hydroxide solution, the organic phase was separated off, washed once with 20 ml of water and then dried over sodium sulfate. After the solvent had been distilled off, an oil remained which was separated by column chromatography (silica gel, ethyl acetate, petroleum ether 1: 4) (yield 60%).

Claims

1. Compounds of the general formula III,

in the R

or

and in which ^ and R2 are the same or different and represent hydrogen or lower alkyl Ci - C, straight-chain or branched, X is a cleavable group and at least one of the groups R4 or R5 is a group stabilizing the dioxetane structure and at most one of the groups R4 or R5 represents hydrogen.

2. Compounds according to claim 1, characterized in that the dioxetane structure stabilizing group is an adamantyl, phenyl, cyclohexyl secondary or tertiary alkyl radical.

3. Compounds according to claim 1 or 2, characterized in that -O-X represents the hydroxy salt, an oxyacid, phosphate, aryl or alkyl carboxy ester, alkyloxy, trialkyloxy or arylsilyloxy, sulfate, oxypyranoside.

4. A process for the preparation of compounds of the general formula III, characterized in that a compound of the formula IV or IVa

in which R3 is alkyl, straight-chain or branched with 1-6 C atoms, with a compound of the formula V.

ß <C OH

in which R 1 and R 2 are the same or different and represent hydrogen or lower alkyl C - Cg, straight-chain or branched, in the presence of an alkyl lithium compound with aldehydes or ketones, the reaction product is decarboxylated, the alkoxy radical dealkylated and there introduces a cleavable group -OX and photooxygenates. 4. The method according to claim 3, characterized in that a compound of formula IV or IVa is used in which 3 is methyl or ethyl.

5. The method according to claim 3 or 4, characterized in that the introduction of the aldehyde or ketone residue is carried out in an aprotic organic solvent with exclusion of air and cooling at temperatures between -50 and - 100 ° C.

6. A process for the preparation of compounds of the general formula III, characterized in that a compound of the formula IV or IVa in which R3 is alkyl is straight-chain or branched with 1-6 C atoms, with a compound of the formula V implemented, in which Rl and R2 are each the same or different and represent hydrogen or alkyl Cl - C6 straight-chain or branched, the thiazoline carboxylic acid obtained is reduced to a thiazolin-4-one, converted into a thiazolin-4-thione with sulfide and this with an R4 and / or R5-substituted hydrazone to the compounds of the formula III according to the invention, alkylates the alkoxy radical, introduces a cleavable group -OX there and photooxygenates.

7. A process for the preparation of compounds of the formula III in which R4 and / or R5 is phenyl, characterized in that a compound of the formula IV or IVa in which R3 is alkyl is straight-chain or branched with 1-6 C atoms, reacted with a compound of formula V in which Rl and R2 are the same or different and represent hydrogen or alkyl Cl - C6 straight-chain or branched, the thiazoline carboxylic acid obtained is reduced to a thiazolin-4-one, in one Grignard reaction introduces the radical R4 and / or R5, introduces a double bond at the 4-position of the thiazoline ring, dealkylates the alkoxy radical, introduces a cleavable group there into 0-X and photooxygenates, thus obtaining the compounds of the formula III according to the invention.

8. A method for determining an acid, base, salt, enzyme, inorganic or organic catalyst or electron donor by reacting this analyte with a compound of general formula III, splitting off group X and measuring the light emitted as a measure of the content of the determining connection.

9. Use of the compounds according to claims 1 and 2 in immunological assays or for DNA diagnostics.