# US 11,078,226 B2 · Fluorogenic glycosidase substrate and associated detection method

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- Number: US 11,078,226 B2
- Document ID: US11078226B2
- Status: Granted
- Kind: B2
- Application number: 16/471,153
- Filed: 2017-12-19
- Published: 2020-01-02
- Granted: 2021-08-03
- Earliest claimed: 2016-12-19
- Assignees: Ecole Normale Superieure De Lyon; Universite Claude Bernard Lyon 1; Centre National De La Recherche Scientifique
- Applicants: Ecole Normale Superieure De Lyon; Universite Claude Bernard Lyon; Centre National De La Recherche Scientifique
- Inventors: Jens Hasserodt; Corentin Gondrand; Maxime Prost; Gaël Yvert; Gérard Triqueneaux
- Web page: https://patentgrep.com/patent/US11078226B2

## CPC

- C07H 15/20: Carbocyclic rings
- C07H 15/203: Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
- C07H 17/02: Heterocyclic radicals containing only nitrogen as ring hetero atoms
- C12Q 1/34: involving hydrolase
- G01N 33/575: for cancer
- G01N 33/6893: {related to diseases not provided for elsewhere}
- G01N 2333/924: acting on glycosyl compounds (3.2)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

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′10 and R0 as defined in claim 1.

### Claim 14 (depends on 1)

Compounds (I) according to claim 1, wherein at least one of groups R9 or R′9 represents a halogen atom, a —NO2 group, or a —CN group.

### Claim 15 (depends on 1)

Compounds (I) according to claim 1, wherein R10 and, if present, R′10 are identical and represent a hydrogen atom.

### Claim 16 (depends on 1)

Compounds (I) according to claim 1 of formula (Ia):
where R1, R2, R3, R4, R5, R6, R7, R8, R0 and V are as defined in claim 1, in the form of a mixture of optical isomers according to all proportions, or in an enriched form in an optical isomer.

### Claim 17 (depends on 1)

Compounds (I) according to claim 1 of formula (Ib):
where R1, R2, R3, R4, R9, R′9, X, Y and Z are as defined in claim 1, in the form of a mixture of optical isomers according to all proportions, or in an enriched form in an optical isomer.

### Claim 18 (depends on 1)

Compounds (I) according to claim 1 of formula (Ic):
where R0 and R1 are as defined in claim 1, in the form of a mixture of optical isomers according to all proportions, or in an enriched form in an optical isomer.

### Claim 19 (depends on 1)

Process for the preparation of a compound of formula (I) according to claim 1 comprising the following steps:
providing a compound (II) of formula
providing a compound (III) of formula
obtaining compound (IV) by addition reaction of said compound (II) to compound (III), said compound (IV) having the formula:
and
deprotecting the alcohol functions present in the R′0 group of said compound (W) in order to obtain said compound (I),
wherein in the formulas:
R1, R2, R3, R4, R5, R6, R7, R8, R9, R′9 and V are as defined in claim 1,
R12 represents a hydrogen atom, or an amine functions protecting group,
X, Y′ and Z′ are such that:
either X represents CR10, Y′ represents CR′10 and Z′ represents OR′0,
or X represents CR10, Y′ represents COR′0 and Z′ represents R′10,
or X represents CR10, Y′ represents a nitrogen atom and Z′ represents OR′0,
or X represents a nitrogen atom, Y′ represents COR′0 and Z′ represents R10,
with R′0 representing a R0 group of which all of the alcohol functions are protected by a protecting group, and R0, R10 et R′10 are as defined in claim 1,
M represents a leaving group.

### Claim 20 (depends on 5)

Compounds (I) according to claim 5, wherein
represents a (C5-C24) aryl or a (C5-C24) hetero-aryl selected from the group consisting of phenyl, naphthyl, and:
said 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, alcoxycarbonyl and aryloxycarbonyl,
with X3 which represents S, O or NRd and Rd which represents a hydrogen atom or a (C1-C4) alkyl group.

### Claim 21 (depends on 5)

Compounds (I) according to claim 5, wherein
OR1 is of the phenoxy type and corresponds to one of the following structures (A2) or (A3):
in which:
T is —NH—C(O)—, —S—, —O—, —NH—, —N((C1-C20) alkyl)- or —N(C5-C24)aryl)-,
Re is a hydrogen atom or an electron-withdrawing carbonaceous substituent, or Re is —CONRiRj, with Ri and Rj, identical or different, which represent a hydrogen atom, or an (C1-C4) alkyl group, or Re is —CF3, or a 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-benzo imidazolyl, 4-pyrimidinone-2-yl or quinazolinone-2-yl group,
Rf is a hydrogen atom, a chlorine, bromine, iodine or fluorine atom, —OH, —NH2, —NRkRl, —NHRk or —ORk, with Rk and RI, identical or different, which each, independently, represent an (C1-C4) alkyl group,
Or Re and Rf are bonded to each other to form a hydrocarbon chain comprising 4 or 5 members, saturated or unsaturated, substituted or non-substituted, possibly interrupted by one or more hetero-atoms selected from the group consisting of N, S and O,
Rg is a hydrogen, Br, Cl, I or F atom,
in which:
T′ is —NH2, —OH, an (C5-C24) aryl group, an (C1-C4) alkyl group, —SH, —NHR′g, —OR′g, —NR′gRh′ or —SR′g, R′g and Rh′, identical or different, representing an (C1-C4) alkyl or aryl group,
R′e is a hydrogen atom or an electron-withdrawing carbonaceous substituent, or R′e is —CONR′jR′k, with R′j and R′k, identical or different, which represent a hydrogen atom, or an (C1-C4) alkyl group, or R′e is —CF3, or a 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 2-benzo imidazolyl, 4-pyrimidinone-2-yl or quinazolinone-2-yl group,
R′f is a hydrogen, chlorine, bromine, iodine or fluoride atom, —OH, —NH2, —NR′IR′m or —OR′I, with R′I and R′m, identical or different, which represent an (C1-C4) alkyl group,
or Re and Rf′ are bonded to each other to form a hydrocarbon chain comprising 4 or 5 members, saturated or unsaturated, substituted or non-substituted, possibly interrupted by one or more hetero-atoms selected from the group consisting of N, S and O.