Granted Patent B2

US 12,576,164 · App. 18/424,207 · Granted Mar 17, 2026

Inventors: Yves-Louis Janin (Paris, FR); Eloi Paul Coutant (Montrouge, FR); Vincent Hervin (Le Perreux-sur-marne, FR); Glwadys Gagnot (Paris, FR); Yves Jacob (Maintenon, FR); Sophie Goyard (Paris, FR); Thierry Rose (Paris, FR)

Assignees: INSTITUT PASTEUR; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE

A61K49/0013C07D487/04G01N21/763G01N33/573

Abstract

Claims (113)

1 . A method to detect and/or measure an enzymatic activity of a luminogenic protein, comprising:

(a) contacting a compound of following formula (I) with a solution comprising a strong acid to obtain a deprotected form of the compound of formula (I);

wherein:

R 1 represents H or a group selected from C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, C 6 -C 10 aryl, aralkyl and C 5 -C 10 -membered heteroaryl groups, said C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, C 6 -C 10 aryl, aralkyl and C 5 -C 10 -membered heteroaryl groups optionally substituted by at least one Y 1 group;

R 2 represents a group selected from C 6 -C 10 aryl and C 5 -C 10 -membered heteroaryl groups, said C 6 -C 10 aryl and C 5 -C 10 -membered heteroaryl groups optionally substituted by at least one Y 2 group;

or R 1 and R 2 together form with the two carbon atoms to which they are respectively attached a C 5 -C 7 cycloalkene group, a C 4 -C 7 heterocycloalkene group, or a C 6 -C 10 arene, said C 5 -C 7 cycloalkene group and C 4 -C 7 heterocycloalkene groups fused with a C 6 -C 10 arene, said C 5 -C 7 cycloalkene group, C 4 -C 7 heterocycloalkene, C 6 -C 10 arene, C 5 -C 7 cycloalkene group and C 4 -C 7 heterocycloalkene groups optionally substituted by at least one Y 12 group;

R 3 represents a C 1 -C 6 alkyl, an aralkyl group, a hetaralkyl group or a heterocycloalkyl-CH 2 — group, said C 1 -C 6 alkyl, aralkyl group, hetaralkyl group and heterocycloalkyl-CH 2 — group being optionally substituted by at least one Y 3 group;

R 4 represents H or a group selected from C 1 -C 6 alkyl and C 3 -C 7 cycloalkyl groups, said C 1 -C 6 alkyl and C 3 -C 7 cycloalkyl groups optionally substituted by at least one Y 4 group;

R 5 represents a —C(═O)R a group or a —C(═O)OR a group;

wherein when R 5 is the —C(═O)R a group:

R a represents H, a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group, a C 6 -C 10 aryl, or an aralkyl group, said C 1 -C 6 alkyl group, C 3 -C 7 cycloalkyl group, C 6 -C 10 aryl, and aralkyl group optionally substituted by at least one Ya group, selected from a C 1 -C 6 alkyl, a C 3 -C 7 cycloalkyl, a C 6 -C 10 aryl, a C 5 -C 10 -membered heteroaryl group, a halogen, a —CF 3 group, a —CN group, a —OR i group, a —OSO 3 H group, a —NR i R ii group, and a guanidinyl group; and

wherein when R 5 is the —C(═O)OR a group:

(i) R a represents H, a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group optionally substituted by at least one Y a group selected from a C 1 -C 6 alkyl; a C 3 -C 7 cycloalkyl; a C 6 -C 10 aryl;

a C 5 -C 10 -membered heteroaryl group; a halogen; a —CF 3 group; a —CN group; a —OR i group; a —OSO 3 H group; a —NR i R ii group; and a guanidinyl group; or

(ii) R a represents a C 6 -C 10 aryl group optionally substituted by at least one Y a group selected from a C 1 -C 6 alkyl; a C 3 -C 7 cycloalkyl; a C 6 -C 10 aryl; a C 5 -C 10 -membered heteroaryl group; a —CF 3 group; a —CN group; a —OR i group; a —OSO 3 H group; a —NR i R ii group; and a guanidinyl group;

Z represents a C 6 -C 10 aryl, a C 5 -C 10 -membered heteroaryl groups, a C 1 -C 6 alkyl, a C 3 -C 7 cycloalkyl, a C 4 -C 7 heterocycloalkyl, said C 6 -C 10 aryl, C 5 -C 10 -membered heteroaryl groups, C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, and C 4 -C 7 heterocycloalkyl optionally substituted by at least one Y z group;

said Y 1 , Y 2 , Y 12 , Y 3 , Y 4 , Y 5 , and Y z , groups each independently selected from:

a C 1 -C 6 alkyl;

a C 3 -C 7 cycloalkyl;

a C 6 -C 10 aryl;

a C 5 -C 10 -membered heteroaryl group;

a halogen;

a —CF 3 group;

a —CN group;

a —OR i group;

a —OSO 3 H group;

a —NR i R ii group; and

a guanidinyl group;

R i and R ii each independently represent H, a C 1 -C 6 alkyl group, a C 3 -C 10 cycloalkyl group or an aralkyl group; or together form with the nitrogen atom to which they are attached a C 4 -C 7 heterocycloalkyl group;

(b) contacting the deprotected form of the compound of formula (I) with a buffered media to obtain a solution comprising the deprotected form of the compound of formula (I);

(c) contacting the solution of the deprotected form of the compound of formula (I) with a luminogenic protein; and

(d) detecting a luminescence signal, so as to detect and/or measure the enzymatic activity of the luminogenic protein.

2 . The method according to claim 1 , wherein:

R 1 represents H;

R 5 represents —(C═O)Me;

R 3 represents an aralkyl group or a hetaralkyl group, optionally substituted;

R 2 represents a phenyl, optionally substituted, and/or

Z is selected from phenyl, furanyl, thiophenyl, and is optionally substituted.

3 . The method according to claim 1 , wherein the luminogenic protein is a luciferase derived from Oplophorus luciferase comprising a domain that catalyzes luminescence of native luciferase.

4 . The method of claim 3 , wherein the Oplophorus luciferase is a nanoKAZ luciferase.

5 . The method of claim 1 , wherein the buffer is PBS.

6 . The method of claim 1 , wherein the solution is at a pH of 6 to 9.

7 . A method to detect and/or measure an enzymatic activity of a luminogenic protein, comprising:

(a) contacting a compound of following formula (I) with a solution comprising a strong acid to obtain a deprotected form of the compound of formula (I);

wherein the deprotected form of the compound of formula (I) is a compound according to formula (III);

wherein:

R 1 represents H or a group selected from C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, C 6 -C 10 aryl, aralkyl and C 5 -C 10 -membered heteroaryl groups, said C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl, C 6 -C 10 aryl, aralkyl and C 5 -C 10 -membered heteroaryl groups optionally substituted by at least one Y 1 group;

R 2 represents a group selected from C 6 -C 10 aryl and C 5 -C 10 -membered heteroaryl groups, said C 6 -C 10 aryl and C 5 -C 10 -membered heteroaryl groups optionally substituted by at least one Y 2 group;

R 3 represents a C 1 -C 6 alkyl, an aralkyl group, a hetaralkyl group or a heterocycloalkyl-CH 2 — group substituted by at least one Y 3 group;

R 4 represents H or a group selected from C 1 -C 6 alkyl and C 3 -C 7 cycloalkyl groups, said C 1 -C 6 alkyl and C 3 -C 7 cycloalkyl groups optionally substituted by at least one Y 4 group;

R 5 represents a —C(═O)R a group or a —C(═O)OR a group;

wherein when R 5 is the —C(═O)R a group:

R a represents H, a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group, a C 6 -C 10 aryl, or an aralkyl group, said C 1 -C 6 alkyl group, C 3 -C 7 cycloalkyl group, C 6 -C 10 aryl, and aralkyl group optionally substituted by at least one Y a group, selected from a C 1 -C 6 alkyl, a C 3 -C 7 cycloalkyl, a C 6 -C 10 aryl, a C 5 -C 10 -membered heteroaryl group, a halogen, a —CF 3 group, a —CN group, a —OR i group, a —OSO 3 H group, a —NR i R ii group, and a guanidinyl group; and

wherein when R 5 is the —C(═O)OR a group:

(i) R a represents H, a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group optionally substituted by at least one Y a group selected from a C 1 -C 6 alkyl; a C 3 -C 7 cycloalkyl; a C 6 -C 10 aryl; a C 5 -C 10 -membered heteroaryl group; a halogen; a —CF 3 group; a —CN group; a —OR i group; a —OSO 3 H group; a —NR i R ii group; and a guanidinyl group; or

(ii) R a represents a C 6 -C 10 aryl group optionally substituted by at least one Y a group selected from a C 1 -C 6 alkyl; a C 3 -C 7 cycloalkyl; a C 6 -C 10 aryl; a C 5 -C 10 -membered heteroaryl group; a —CF 3 group; a —CN group; a —OR i group; a —OSO 3 H group; a —NR i R ii group; and a guanidinyl group;

Z represents a group of following formula:

an oxazolyl; or an oxadiazolyl;

R, R′, R″ and R′″ each independently represent H or a group selected from:

a C 1 -C 6 alkyl group;

a C 3 -C 7 cycloalkyl group;

a halogen;

a —OR i group; and

a —CF 3 group; and

X represents O or S;

said Y 1 , Y 2 , Y 3 and Y 4 , groups each independently selected from:

a C 1 -C 6 alkyl group;

a C 3 -C 7 cycloalkyl group;

a halogen; and

a —OR ii group; and

R i and R ii each independently represent H, a C 1 -C 6 alkyl group, a C 3 -C 10 cycloalkyl group or an aralkyl group;

provided that:

when Z═Z1:

at least one of R, R′ and R″ does not represent H; or

when R, R′ and R″ represent H, then R 2 represents a group selected from C 6 -C 10 aryl and C 5 -C 10 -membered heteroaryl groups substituted by at least one halogen group; or R 3 represents a group selected from C 6 -C 10 aralkyl groups substituted by at least one halogen group;

when Z═Z2:

at least one of R, R′, R″ and R″ does not represent H;

when R, R′, R″ and R′″ represent H; then R 2 represents a group selected from C 6 -C 10 aryl and C 5 -C 10 -membered heteroaryl groups substituted by at least one halogen group; or R 3 represents a group selected from C 6 -C 10 aralkyl groups substituted by at least one halogen group;

when R, R′ and R′″ represent H and R″ represents a C 1 -C 6 alkyl group or a —OR i group; then R 2 does not represent a 4-HO-Ph- group; or R 3 represents a group selected from C 6 -C 10 aralkyl groups substituted by at least one halogen group;

(b) contacting the deprotected form of the compound of formula (I) with a buffered media to obtain a solution comprising the deprotected form of the compound of formula (I);

(c) contacting the solution of the deprotected form of the compound of formula (I) with a luminogenic protein; and

(d) detecting a luminescence signal, so as to detect and/or measure the enzymatic activity of the luminogenic protein.

8 . A method to detect and/or measure an enzymatic activity of a luminogenic protein, the method comprising:

(a) contacting a compound of formula (I) with a solution comprising a strong acid to produce a deprotected form of compound of formula (I); or providing the deprotected form of the compound of formula (I);

wherein:

R 1 represents H, a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group, a C 6 -C 10 aryl group, a aralkyl group or a C 5 -C 10 -membered heteroaryl group;

R 2 represents one of the following groups, optionally, substituted by at least one Y 2 group: a C 6 -C 10 aryl or a C 5 -C 10 -membered heteroaryl group;

R 3 represents H; or one of the following groups substituted by at least one Y 3 group: a C 1 -C 6 alkyl group, an aralkyl group, a hetaralkyl group or a heterocycloalkyl-CH 2 — group;

R 4 represents H, a C 1 -C 6 alkyl group, or a C 3 -C 7 cycloalkyl group;

R 5 represents a —C(═O)R a group or a —C(═O)OR a group;

R a represents H, a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group, or an aralkyl group;

Z represents a C 6 -C 10 aryl group, a C 5 -C 10 -membered heteroaryl group, a C 1 -C 6 alkyl group, a C 3 -C 7 cycloalkyl group, or a C 4 -C 7 heterocycloalkyl group;

Y 2 and Y 3 are each independently selected from:

a C 1 -C 6 alkyl;

a C 3 -C 7 cycloalkyl;

a C 6 -C 10 aryl;

a C 5 -C 10 -membered heteroaryl group;

a halogen;

a —CF 3 group;

a —CN group;

a —OR i group;

a —OSO 3 H group;

a —NR i R ii group; and

a guanidinyl group; and

R i and R ii each independently represent H, a C 1 -C 6 alkyl group, a C 3 -C 10 cycloalkyl group or an aralkyl group; or together with the nitrogen atom to which they are attached form a C 4 -C 7 heterocycloalkyl group;

(b) contacting the deprotected form of the compound of formula (I) with a buffered media to obtain a solution comprising the deprotected form of the compound of formula (I);

(c) contacting the solution of the deprotected form of the compound of formula (I) with a luminogenic protein; and

(d) detecting a luminescence signal, wherein the luminescence signal correlates to the presence of enzymatic activity of the luminogenic protein.

9 . The method of claim 8 , wherein the deprotected form of the compound of formula (I) is a compound according to formula (III);

wherein:

Z represents a C 6 -C 10 aryl or a C 5 -C 10 -membered heteroaryl group and R 1 , R 2 , R 3 , and R 4 are as defined in claim 8 .

10 . The method of claim 8 , wherein the buffer is phosphate-buffered saline (PBS).

11 . The method of claim 8 , wherein steps (c) and (d) are performed in vivo.

Assignments (2)

Priority Claims (1)

Continuity (2)

References Cited (18)