Quinone methide analog signal amplification

Patent metadata

Field	Value
Publication number	US-10168336-B2
Application number	US-201615246430-A
Country	US
Kind code	B2
Filing date	Aug 24, 2016
Priority date	Feb 24, 2014
Publication date	Jan 1, 2019
Grant date	Jan 1, 2019

Abstract

Disclosed herein are novel quinone methide analog precursors and embodiments of a method and a kit of using the same for detecting one or more targets in a biological sample. The method of detection comprises contacting the sample with a detection probe, then contacting the sample with a labeling conjugate that comprises an enzyme. The enzyme interacts with a quinone methide analog precursor comprising a detectable label, forming a reactive quinone methide analog, which binds to the biological sample proximally to or directly on the target. The detectable label is then detected. In some embodiments, multiple targets can be detected by multiple quinone methide analog precursors interacting with different enzymes without the need for an enzyme deactivation step.

First claim

The invention claimed is:

1. A compound, having a formula

or a salt or solvate thereof, wherein:
Z is O, S or NR a , and R 1 is selected from the group consisting of phosphate, phosphodiester, amide, nitro, urea, sulfate, methyl, ester, alpha-glucose, beta-glucose, beta-lactam, alpha-galactose, beta-galactose, alpha-lactose, beta-lactose, alpha-glucuronic acid, and beta-glucuronic acid;
or ZR 1 is —OP(O)(OH) 2 , NO 2 , —NHC(O)R, —OC(O)CH 3 , —OC(O)CH 2 CH 3 , —NHC(O)NH 2 , —OS(O) 2 OH, OCH 3 or a salt thereof;
R 8 is —C(LG)(R 5 )(R 3 R 4 ), —R 3 R 4 or —C(LG)(R 5 )(R 6 );
R 9 , R 11 and R 12 are each independently hydrogen, halo, cyano, aliphatic, alkoxy, NO 2 , N(R c ) 2 , aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c , —C(O)N(R c ) 2 , —R 3 R 4 or two adjacent groups together form an aliphatic ring or aryl ring;
R 10 is hydrogen, halo, cyano, aliphatic, alkoxy, NO 2 , N(R c ) 2 , aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c , —C(O)N(R c ) 2 , —R 3 R 4 , —C(LG)(R 5 )(R 6 ) or with one of R 9 or R 11 form an aliphatic ring or aryl ring;
each LG is independently a halide, alkoxy, carboxylate, inorganic ester, thiolate, amine, carboxylate, azide, sulfate ester, aryloxy, phenoxide or —N(R b ) 3 + where each R b is independently hydrogen or a lower alkyl or two R b moieties together form a heteroaliphatic ring or ZR 1 and LG together form a phosphodiester;
each R 3 is independently —(CH 2 ) n NH—, —O(CH 2 ) n NH—, —N(H)C(O)(CH 2 ) n NH—, —C(O)N(H)(CH 2 ) n NH—, —(CH 2 ) n O—, —O(CH 2 ) n O—, —O(CH 2 CH 2 O) n —, —N(H)C(O)(CH 2 ) n O—, —C(O)N(H)(CH 2 ) n O—, —C(O)N(H)(CH 2 CH 2 O) n —, —(CH 2 ) n S—, —O(CH 2 ) n S—, —N(H)C(O)(CH 2 ) n S—, —C(O)N(H)(CH 2 ) n S—, —(CH 2 ) n NH—, —C(O)N(H)(CH 2 CH 2 O) n CH 2 CH 2 NH, —C(O)(CH 2 CH 2 O) n CH 2 CH 2 NH—, —C(O)N(H)(CH 2 ) n NHC(O)CH(CH 3 )(CH 2 ) n NH— or —N(H)(CH 2 ) n NH—, where each n independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;
each R 4 is independently a chromogen, a fluorophore, a luminophore, or a hapten;
each R 5 is independently hydrogen, halo, cyano, lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c or —C(O)N(R c ) 2 ;
each R 6 is independently hydrogen, halo, cyano, lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c or —C(O)N(R c ) 2 ;
R a is hydrogen or aliphatic;
each R c independently is hydrogen, aryl, aliphatic or heteroaliphatic, or two R c moieties together form a heteroaliphatic ring;
at least one of R 8 and R 10 comprises LG, and at least one of R 8 and R 10 comprises R 3 R 4 ; and
if LG is halide, then R 5 and R 6 are not halide.

2. The compound of claim 1 , wherein the compound has a formula

3. The compound of claim 1 , wherein the compound has a formula selected from

4. The compound of claim 1 , wherein R 1 or ZR 1 is a phosphate, amide, nitro, urea, sulfate, methyl, ester, or beta-lactam; or LG and ZR 1 together form a phosphodiester.

5. The compound of claim 4 , wherein ZR 1 is —OP(O)(OH) 2 or a salt thereof.

6. The compound of claim 1 , wherein R 3 is —C(O)N(H)(CH 2 ) n NH—.

7. The compound of claim 1 , wherein R 8 is —C(LG)(R 5 )(R 3 R 4 ).

8. The compound of claim 7 , wherein LG is a halide.

9. The compound of claim 7 , wherein R 3 is —C(O)N(H)(CH 2 ) n NH—.

10. The compound of claim 9 , wherein the compound has a structure selected from the group consisting of:

11. The compound of claim 10 , wherein the compound has the structure

12. The compound of claim 1 , wherein R 3 is CH 2 CH 2 NH—, —OCH 2 CH 2 NH—, —NHCO(CH 2 ) 5 NH—, —CONH(CH 2 ) 5 NH—, —NHCO(CH 2 ) 6 NH—, CONH(CH 2 ) 6 NH—, —CONH(CH 2 ) 2 NH—, —(CH 2 CH 2 O) 4 —, —(CH 2 CH 2 O) 8 —, —C(O)N(H)(CH 2 CH 2 O) 2 CH 2 CH 2 NH—, —CO(CH 2 CH 2 O) 4 CH 2 CH 2 NH—, —CO(CH 2 CH 2 O) 8 CH 2 CH 2 NH—or —C(O)N(H)(CH 2 ) 6 NHC(O)CH(CH 3 )(CH 2 ) 4 NH—.

13. The compound of claim 2 , wherein R 3 is —C(O)N(H)(CH 2 ) n NH—, and n is 4, 5, 6, or 7.

Assignees

• Ventana Med Syst Inc

Inventors

• Bieniarz Christopher
• Ashworth-Sharpe Julia
• Kelly Brian D
• Polaske Nathan

Classifications

• C07F9/65583

  each of the hetero rings containing nitrogen as ring hetero atom · CPC title

• C07F9/65335

  condensed with carbocyclic rings or carbocyclic ring systems · CPC title

• C07D209/14

  Radicals substituted by nitrogen atoms, not forming part of a nitro radical · CPC title

• C07D403/06

  linked by a carbon chain containing only aliphatic carbon atoms · CPC title

• C07F9/6561 Primary

  containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings · CPC title