Probes for fluorescence imaging

Patent metadata

Field	Value
Publication number	US-2025283800-A1
Application number	US-202318844896-A
Country	US
Kind code	A1
Filing date	Mar 31, 2023
Priority date	Apr 2, 2022
Publication date	Sep 11, 2025
Grant date	—

Abstract

The present disclosure provides photocleavable rhodamine probes that facilitate live- and fixed-cell immunofluorescence. The ultra-fast spirocyclization of the dye following cleavage depletes the fluorescence signal, enabling cyclic multiplexed imaging.

First claim

1 . A compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:
X 1 is selected from O, S, C(R 15 ) 2 , and Si(R 15 ) 2 ;
each R 15 is independently selected from H, C 1-6 alkyl, C 2-6 alkenylene, and C 1-6 haloalkyl; wherein said C 1-6 alkyl and C 1-6 haloalkyl are each optionally substituted with OH, SH, NH 2 , NO 2 , CN, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkylthio, C 1-3 alkoxy, or C 1-3 haloalkoxy;
or any two R 15 together with the C or Si atom to which they are attached from a 3-7 membered saturated or unsaturated carbocyclic or heterocyclic ring, which is optionally substituted with 1 or 2 substituents independently selected from halo OH, SH, NH 2 , NO 2 , CN, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkylthio, C 1-3 alkoxy, and C 1-3 haloalkoxy;
R N1 is selected from C 1-3 alkyl and C 1-3 haloalkyl, wherein said C 1-3 alkyl is optionally substituted with OH, SH, NH 2 , NO 2 , CN, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkylthio, C 1-3 alkoxy, or C 1-3 haloalkoxy;
X 2 is selected from OR N2 and N(R N2 ) 2 ;
X 3 is selected from O and NR N2 ;
X 4 is O, S; or NR N2 ;
each R N2 is independently selected from H, C 1-6 alkyl, and C 1-6 haloalkyl, wherein said C 1-6 alkyl and C 1-6 haloalkyl are each optionally substituted with OH, SH, NH 2 , NO 2 , CN, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkylthio, C 1-3 alkoxy, or C 1-3 haloalkoxy;
or any two R N2 together with the O or N atom to which they are attached from a 3-7 membered saturated or unsaturated carbocyclic or heterocyclic ring, which is optionally substituted with 1 or 2 substituents independently selected from halo, OH, SH, NH 2 , NO 2 , CN, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkylthio, C 1-3 alkoxy, and C 1-3 haloalkoxy;
R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 are each independently selected from H, halo, OH, SH, SO 3 H, NO 2 , CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-4 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, amino, C 1-6 alkylamino, di(C 1-6 alkyl)amino, C 1-6 alkylthio, carbamyl, C 1-6 alkylcarbamyl, di(C 1-6 alkyl)carbamyl, and C(═O)OH, wherein said C 1-6 alkyl and C 1-4 haloalkyl are each optionally substituted with OH, SH, NH 2 , NO 2 , SO 3 H, CN, C(═O)OH, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkylthio, C 1-3 alkoxy, or C 1-3 haloalkoxy;
or each pair of RN 2 and R 3 , R N2 and R 1 , R N2 and R 4 , R N2 and R 5 , R 1 and R 2 , and R 5 and R 6 together with the C, N, or O atoms to which they are attached, form a 5-8 membered saturated or unsaturated carbocyclic or heterocyclic ring, which is optionally substituted with 1, 2, or 3 substituents independently selected from halo, OH, SH, SO 3 H, NO 2 , CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-4 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, amino, C 1-6 alkylamino, di(C 1-6 alkyl)amino, C 1-6 alkylthio, carbamyl, C 1-6 alkylcarbamyl, di(C 1-6 alkyl)carbamyl, and C(═O)OH, wherein said C 1-6 alkyl and C 1-4 haloalkyl are each optionally substituted with OH, SH, NH 2 , NO 2 , SO 3 H, CN, C(═O)OH, C 1-3 alkylamino, di(C 1-3 alkyl)amino, C 1-3 alkylthio, C 1-3 alkoxy, or C 1-3 haloalkoxy;
each L 1 is independently selected from N(R N ), O, C(═O), S(═O) 2 , C 1-6 alkylene, —(OCH 2 CH 2 ) x —, and —(CH 2 CH 2 O) x —;
n is an integer from 1 to 10;
each R N is independently selected from H, C 1-3 alkyl, and C 1-3 haloalkyl;
each x is independently an integer from 1 to 2,000;
Y 1 is selected from NR N2 R 1A , OR 2A , C(═O)OR 3A , and a group reactive with a side chain of an amino acid of a protein.
R 1A selected from H and an amine protecting group;
R 2A is selected from H and an alcohol protecting group; and
R 2A is selected from H and a carboxylic acid protecting group.

2 . The compound of claim 1 , wherein the compound of Formula (I) has formula:

or a pharmaceutically acceptable salt thereof.

3 . The compound of claim 2 , wherein:
R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently selected from H, halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, and di(C 1-3 alkyl)amino, wherein said C 1-3 alkyl is optionally substituted with halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, or di(C 1-3 alkyl)amino, R 7 , R 8 , R 9 , and R 10 are each independently selected from H, halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, and di(C 1-3 alkyl)amino, wherein said C 1-3 alkyl is optionally substituted with halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, or di(C 1-3 alkyl)amino; and RN 1 is selected from C 1-3 alkyl and C 1-3 haloalkyl.

4 . The compound of claim 3 , wherein:
R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently selected from H, halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 haloalkoxy, wherein said C 1-3 alkyl is optionally substituted with OH, SO 3 H, C(═O)OH, CN, C 1-3 alkoxy, and C 1-3 haloalkoxy; R 7 , R 8 , R 9 , and R 10 are each independently selected from H, halo, OH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, and C 1-3 haloalkoxy; and RN 1 is C 1-3 alkyl.

5 . The compound of claim 1 , wherein the compound of Formula (I) has formula:

or a pharmaceutically acceptable salt thereof.

6 . The compound of claim 5 , wherein:
R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently selected from H, halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, and di(C 1-3 alkyl)amino, wherein said C 1-3 alkyl is optionally substituted with halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, or di(C 1-3 alkyl)amino, R 7 , R 8 , R 9 , and R 10 are each independently selected from H, halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, and di(C 1-3 alkyl)amino, wherein said C 1-3 alkyl is optionally substituted with halo, OH, SH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkoxy, C 1-3 haloalkoxy, amino, C 1-3 alkylamino, or di(C 1-3 alkyl)amino; and RN 1 is selected from C 1-3 alkyl and C 1-3 haloalkyl.

7 . The compound of claim 6 , wherein:
R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently selected from H, halo, OH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, and C 1-3 haloalkoxy, wherein said C 1-3 alkyl is optionally substituted with OH, SO 3 H, C(═O)OH, CN, C 1-3 alkoxy, and C 1-3 haloalkoxy; R 7 , R 8 , R 9 , and R 10 are each independently selected from H, halo, OH, SO 3 H, C(═O)OH, NO 2 , CN, C 1-3 alkyl, C 1-3 haloalkyl, C 1-3 alkoxy, and C 1-3 haloalkoxy; and RN 1 is C 1-3 alkyl.

8 . The compound of claim 1 , wherein n is an integer from 1 to 5, and each L 1 is selected from NH, O, C(═O), and C 1-6 alkylene.

9 . The comp

Assignees

• Massachusetts Gen Hospital

Inventors

• Weissleder Ralph
• Rosillo Elias Arturo Halabi

Classifications

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C09B26/06

  Triazene dyes (triazene-azo dyes C09B56/20) · CPC title

• G01N2015/1006

  for cytology · CPC title

• C09K2211/1018

  Heterocyclic compounds · CPC title

• C09K2211/1014

  bridged by heteroatoms, e.g. N, P, Si or B · CPC title