Aminoalkylmaleimides and hapten and antigen derivatives derived therefrom as well as conjugates with peptides or proteins

• US 5,595,741 A
• Filed: 07/21/1994
• Issued: 01/21/1997
• Est. Priority Date: 06/19/1989
• Status: Expired due to Fees

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Huber, Emasmus, Klein, Christian, Batz, Hans-Georg, Zink, Bruno
• Primary Examiner(s): Springer, David B.
• Application Number:
  US08/278,621
• Time in Patent Office: 915 Days
• Field of Search: 548/546, 424/194.1, 530/405, 530/403
• US Class Current: 424/194.1
• CPC Class Codes: C07D 207/452 with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 239/62 Barbituric acids
• CPC Class Codes: C07D 473/08 with methyl radicals in positions 1 and 3, e.g. theophylline
• CPC Class Codes: C07J 41/0016 Oximes
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

The invention concerns new aminoalkylmaleimides of the general formula I ##STR1## in which R₁ and R₂ are the same or different and represent hydrogen or a C₁ -C₄ alkyl group and A represents a straight-chain or branched, saturated or unsaturated alkylene group with 2 to 6 carbon atoms which is interrupted, if desired, by an oxygen or sulphur atom or a carbonyl group, as well as their corresponding acid addition salts.

The present invention also concerns amidoalkylmaleimide derivatives formed from compounds of the general formula I and immunological binding partners. In addition, the invention concerns immunological conjugates which can be produced by reaction of the amidoalkylmaleimide derivatives with peptides or proteins.

The subject matter of the present invention are also the corresponding processes of production of the compounds according to the present invention as well as the use of these conjugates in diagnostic methods of determination, in particular in immunoassays.

Claims

1. An amidoalkylmaleimide compound of formula II ##STR10## in which Hap is a residue formed by amidation of a hapten or antigen carrying one or several carboxyl groups or carboxyl derivatives, R₁ and R₂ are the same or different and denote a C_1-C4 alkyl group or a hydrogen atom and A is a straight-chain or branched, saturated or unsaturated alkylene group having 2-6 C atoms which is uninterrupted or is interrupted by an oxygen or a sulphur atom or a carbonyl group.

2. Amidoalkyl-maleimides of claim 1, wherein the hapten is a steroid.

3. A peptide or protein conjugate compound as produced by reaction of an amidoalkylmaleimide of formula II ##STR11## with a peptide or protein carrying at least one sulfhydryl group wherein in Formula II Hap is a hapten, R₁ and R₂ are the same or different and are C₁ -C₄ -alkyl or hydrogen and A is a straight-chain or branched, saturated or unsaturated C₂ -C₆ -alkylene, which alkylene is uninterrupted or is interrupted by an oxygen or a sulphur atom or a carbonyl.

4. The conjugate compound of claim 3 wherein the peptide is an enzyme-donor polypeptide of β

-galactoside.

5. The conjugate compound of claim 4 wherein the enzyme-donor polypeptide is ED4.

6. The conjugate compound of claim 3 wherein the peptide is β

-galactoside.

7. The conjugate compound of claim 3 wherein R₁ and R₂ in formula II are the same and are hydrogen.

8. The conjugate compound of claim 3, wherein A in formula II is --(CH₂)₂ --, --(CH₂)_m --(CH₃)--(CH₂)_o --, (CH₂)_m --C(CH₃)₂ --(CH₂)_o --, (CH₂)_m --X--(CH₂)_o or --(CH₂)_m --CH═

CH--, wherein X is O, S or CO, n is 2-6, m=o=1-4 and m +o=2-5.

9. The conjugate compound of claim 3 wherein A is --(CH₂)_n -- wherein n is 2, 3, 4 or 5, or A is --CH₂ --O--CH₂ --, CH₂ --S--CH₂ -- or --CH₂ --CO--CH₂.

10. The conjugate compound of claim 3 designated theophylline-8-acetic acid-[2-(N-maleinimido)ethylamide]-ED4, phenobarbital-1-butyric acid-[2-(N-maleinimido)ethylamide]-ED4 or cortisol-3-carboxylic acid-[2-(N-maleinimido)ethylamide]-β

-Gal.