Fluorogenic glycosidase substrate and associated detection method

Patent metadata

Field	Value
Publication number	US-11078226-B2
Application number	US-201716471153-A
Country	US
Kind code	B2
Filing date	Dec 19, 2017
Priority date	Dec 19, 2016
Publication date	Aug 3, 2021
Grant date	Aug 3, 2021

Abstract

The invention relates to novel glycosidase substrates of formula (I), wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R′9, V, X, Y and Z are as defined in claim 1, and a method for detecting the presence of a catalytically active glycosidase by means of one of said substrates.

First claim

The invention claimed is:

1. Compounds of formula (I):

in which:
R1 is such that HOR1, obtained after cleavage of the —C(O)—OR1 bond present in formula (I), belongs to the class of fluorophores leading to an intramolecular proton transfer in an excited state, called ESIPT,
R3 is an (C1-C4) alkyl or a hydrogen atom and R2 and R4 are bonded together and form, with the carbon and nitrogen atoms to which they are bonded, an aliphatic heterocycle which can be substituted by a water-solubilizing group,
R5 and R6 are identical or different and represent, independently of each other, a hydrogen atom, an (C1-C4) alkyl, or an (C5-C10)aryl,
R7 is selected from the group consisting of hydrogen atom, a (C1-C4) alkyl and (C1-C4) alkoxy,
R8 represents a hydrogen atom or a (C1-C10) alkyl group, non-substituted or substituted by one or more substituents selected from the group consisting of chlorine, bromine, iodine, fluorine, cyano, alkyl, trifluoralkyl, trifluoromethyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, heterocyclo-alkyl, amino, alkylamino, diaklyamino, hydroxy, alkoxy, aryloxy, alkoxycarbonyl and aryloxycarbonyl,
or R8 represents a -D1-D2-D3 group with:
D1 representing a triazolyl or —CH2-triazolyl group,
D2 representing an (C1-C10) alkylene, (C1-C10) alkenylene or (C1-C10) alkynylene group, said groups possibly being interrupted by one or more selected from the group consisting of O, N, a divalent glycosyl group, an —O—(CHR—CHR′)n-, —N—(CHR—CHR′—O)n- group, n being an integer varying from 1 to 20, R and R′, identical or different, representing H or CH3 upon condition that R and R′ are not simultaneously CH3, an amino acid or a peptide, and a combination of these groups,
D3 representing a maleimidocaproyl motif, amino acid, peptide, folic acid, antibody or antibody fragment bonded to D2, by a carboxylic acid function comprised in it, forming an ester or amide bond,
R9 and R′9, identical or different, represent a hydrogen atom, an electron-withdrawing group, or a —NH—C(O)—CH2-Ab group, with Ab representing an antibody,
V represents an oxygen atom or a sulfur atom,
X, Y and Z are such that:
either X represents CR10, Y represents CR′ 10 and Z represents OR0,
or X represents CR10, Y represents COR0 and Z represents R′ 10,
or X represents CR10, Y represents a nitrogen atom and Z represents OR0,
or X represents a nitrogen atom, Y represents COR0 and Z represents R10 with:
R0 representing a glycosyl group bound by its anomeric carbon atom to the rest of the molecule of formula (I), and
R10 and R′ 10, identical or different, representing a hydrogen atom or an electron-donating group,

in the form of a mixture of optical isomers according to all proportions, or in an optical isomer enriched form.

2. Compounds (I) according to claim 1 , wherein R3 is a hydrogen atom or an (C1-C4) alkyl, and R2 and R4 are bonded to each other and form a —(CH2)m- chain with m=3, 4 or 5.

3. Compounds (I) according to claim 1 , wherein R3 is a hydrogen atom or an (C1-C4) alkyl, and R2 and R4 are bonded to each other and form a —CH2CH2-NR11-CH2- chain in the direction of R2 toward R4, R11 representing a hydrogen atom or -(L)n-GP with n which is equal to 0 or 1, L a linking arm and GP a water-solubilizing group.

4. Compounds (I) according to claim 1 , wherein R1 is an aromatic group comprising one or more aromatic rings, which rings can comprise one or more hetero-atoms selected from the group consisting of nitrogen, oxygen, sulfur, and/or one or more carbon atoms in the form of a C═O carbonyl, and said aromatic rings being not substituted or substituted by one or more substituents selected from the group consisting of chlorine, bromine, iodine, fluorine, cyano, alkyl, trifluoroalkyl, trifluoromethyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, heterocyclo-alkyl, amino, alkylamino, diaklyamino, hydroxy, alkoxy, aryloxy, alkoxycarbonyl and aryloxycarbonyl.

5. Compounds (I) according to claim 1 , wherein R1 is an aromatic group with —OR1 according to formula (A1):

in which:
either X2 is an oxygen atom and X1 is a —NH2, —OH, —SH, (C1-C20) alkyl, (C5-C24) aryl, —O—(C1-C20) alkyl, —O-phenyl, —NH—(C1-C20) alkyl or —NH-phenyl, —S—(C1-C20) alkyl or —S—(C5-C24) aryl group, said alkyl and phenyl groups being non-substituted or substituted by one or more substituents selected from the group consisting of chlorine, bromine, iodine, fluorine, cyano, alkyl, trifluoroalkyl, trifluoromethyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, heterocyclo-alkyl, amino, alkylamino, diaklyamino, hydroxy, alkoxy, aryloxy, alkoxycarbonyl and aryloxycarbonyl,
Or X2 represents a nitrogen atom and is bound to X1 which then represents CH, O, S, N or, NH to form a (C5-C24) hetero-aryl, not substituted or substituted by one or more substituents selected from the group consisting of chlorine, bromine, iodine, fluorine, cyano, alkyl, trifluoroalkyl, trifluoromethyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, heterocyclo-alkyl, amino, alkylamino, diaklyamino, hydroxy, alkoxy, aryloxy, alkoxycarbonyl and aryloxycarbonyl,

represents an (C5-C24) aryl or a (C5-C24) hetero-aryl, non-substituted or substituted by one or more substituents selected from the group consisting of chlorine, bromine, iodine, and fluorine, cyano, alkyl, trifluoroalkyl, trifluoromethyl, alkenyl, alkynyl, cycloalkyl, aryl, hetero-aryl, heterocyclo-alkyl, amino, alkylamino, diaklyamino, hydroxy, alkoxy, aryloxy, alkoxycarbonyl and aryloxycarbonyl.

6. Compounds (I) according to claim 1 , wherein R1 is an aromatic group with —OR1 according to one of the following formulas (A4) or (A5):

7. Compounds (I) according to claim 1 , wherein R0 is cleavable from the rest of compound (I) by the catalytic action of a glycosidase.

8. Compounds (I) according to claim 1 , wherein R0 is a group that is cleavable under the action of a glycosidase, selected from the group consisting of N-acetyl-β-galactosaminidase; N-acetyl-β-glucosaminidase; α-amylase; α-arabinofuranosidase, α-arabinosidase; β-cellobiosidase; β-chitobiosidase; α-galactosidase; β-galactosidase; α-glucosidase; β-glucosidase; β-glucuronidase; α-maltosidase; α-mannosidase; β-mannosidase; β-xylosidase; β-D-fucosidase; α-L-fucosidase, β-L-fucosidase; L-iduronidase and cellulase; and R0 is a mono-glycosylated group bound by its anomeric carbon atom, selected from the group consisting of galactosyl, glucosyl, mannosyl, gulosyl, allosyl, altrosyl, idosyl, talosyl, fucosyl, fructosyl, arabinosyl, lyxosyl, ribosyl, xylosyl, glucuronyl and N-acetyl-hexosaminyl, and a polyglycosylated group constituted of several of these monoglycosylated groups, identical or different.

9. Compounds (I) according to claim 1 , wherein R5 and R6 are identical and represent a hydrogen atom.

10. Compounds according to claim 1 , wherein R7 represents a hydrogen atom or an (C1-C4) alkyl group.

11. Compounds (I) according to claim 1 , wherein R8 represents a hydrogen atom.

12. Compounds (I) according to claim 1 , wherein V represents an oxygen atom.

13. Compounds (I) according to claim 1 , wherein X, Y and Z are such that:
either X represents CR10, Y represents CR′10 and Z represents OR0,
or X represents CR10, Y represents COR0 and Z represents R′10, with R10, R

Assignees

• Ecole Normale Superieure Lyon
• Univ Claude Bernard Lyon
• Centre Nat Rech Scient

Inventors

• Hasserodt Jens
• Gondrand Corentin
• Prost Maxime
• Yvert Gaël
• Triqueneaux Gérard

Classifications

• G01N33/575

  for cancer · CPC title

• G01N33/6893

  related to diseases not provided for elsewhere · CPC title

• G01N2333/924

  acting on glycosyl compounds (3.2) · CPC title

• C07H17/02

  Heterocyclic radicals containing only nitrogen as ring hetero atoms · CPC title

• C07H15/20 Primary

  Carbocyclic rings · CPC title