Rhodol-based thallium sensors for high-throughput screening of potassium channels

Patent metadata

Field	Value
Publication number	US-11519859-B2
Application number	US-201916379100-A
Country	US
Kind code	B2
Filing date	Apr 9, 2019
Priority date	Apr 9, 2018
Publication date	Dec 6, 2022
Grant date	Dec 6, 2022

Abstract

Provided are novel rhodol-based compounds, the preparation method thereof, and use thereof in Tl+ flux assays. The fluorescent thallium ion sensor compounds have a rhodol fluorophore attached to an amino dicarboxylic acid metal binding moiety. The compounds are prepared in the “pro-dye” form, with the rhodol oxygen and the carboxylic acids of the metal binding unit masked by protecting groups. The disclosed compounds may be used as more red-shifted and less pH-sensitive variants of Thallos.

First claim

We claim:

1. A compound of the following formula,

or a salt thereof,
wherein:
Z is O, CR x R y , SiR x R y , or SO 2 ;
R 1 is C 1-10 alkyl-C(O)—;
—NR 2 R 3 is —NHC(O)CH 3 , —N(CH 3 ) 2 , —N(CH 2 CH 3 ) 2 , —N(CH 2 CH 2 CH 3 ) 2 , —N(CH 2 CH 2 CH 2 CH 3 ) 2 , —NH-cyclohexyl, —NH—CH 2 CH 2 CH 2 CH 3 , —NH-phenyl, azetidin-1-yl, 3,3-difluoroazetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl;
R 4 is C 1-10 alkyl;
R 5 and R 6 are each independently C 1-6 alkyl, —C 1-4 -alkylene-O—R z , —C 1-4 -alkylene-O—C(O)—R z , or —C 1-4 -alkylene-O—C(O)—O—R z ;
R x and R y are each independently C 1-4 alkylene; and
R z , at each occurrence, is independently C 1-6 alkyl, C 3-10 cycloalkyl, C 6-20 aryl, or a 4 to 8-membered heterocycle, wherein the C 3-10 cycloalkyl, C 6-20 aryl, and 4 to 8-membered heterocycle are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C 1-4 haloalkyl, —CN, C 1-4 alkyl-O—, C 1-4 alkyl-C(O)—NH—, and C 1-4 alkyl-NH—C(O)—.

2. The compound of claim 1 , or a salt thereof, wherein Z is O.

3. The compound of claim 1 , or a salt thereof, wherein R 1 is C 1-4 alkyl-C(O)—.

4. The compound of claim 1 , or a salt thereof, wherein R 4 is C 1-4 alkyl.

5. The compound of claim 1 , or a salt thereof, wherein —NR 2 R 3 is —N(CH 3 ) 2 .

6. The compound of claim 1 , or a salt thereof, wherein R 5 and R 6 are independently —C 1-4 -alkylene-O—C(O)—R z , and each R z is independently C 1-6 alkyl.

7. The compound of claim 1 , or a salt thereof, wherein each of R 5 and R 6 is —CH 2 —O—C(O)—CH 3 .

8. The compound of claim 1 , having a structure of formula (I-a), or a salt thereof,

wherein —NR 2 R 3 , R 5 , and R 6 are as defined in claim 1 .

9. The compound of claim 1 , selected from the group consisting of
bis(acetoxymethyl) 2,2′-((4-(3′-acetamido-6′-acetoxy-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(diethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dipropylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dibutylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′((4-(3′-acetoxy-6′-(cyclohexylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′((4-(3′-acetoxy-6′-(butylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(phenylamino)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(azetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′((4-(3′-acetoxy-6′-(3,3-difluoroazetidin-1-yl)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate;
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(pyrrolidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate; and
bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-3-oxo-6′-(piperidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthen]-5-ylcarboxamido)-2-methoxyphenyl)azanediyl)diacetate,
or a salt thereof.

10. The compound of claim 9 that is bis(acetoxymethyl) 2,2′-((4-(3′-acetoxy-6′-(dimethylamino)-3-oxo-3H-spiro[isobenzofuran-1,9′-xanthene]-5-carboxamido)-2-methoxyphenyl)azanediyl)diacetate, or a salt thereof.

11. A method of preparing the compound of claim 1 , or a salt thereof, the method comprising reacting a compound of the following formula

with a compound of the following formula

to provide the compound of claim 1 , or salt thereof.

12. The method of claim 11 , further comprising converting a compound of the following formula

to the compound of formula

13. The method of claim 11 , wherein Z is O.

14. A method of detecting flux of a metal ion into a cell, the method comprising
(a) contacting the cell with a compound of the following formula:

or a salt thereof,
wherein:
Z is O;
R 1 is C 1-10 alkyl-C(O)—;
—NR 2 R 3 is —N(CH 3 ) 2 , —N(CH 2 CH 3 ) 2 , —N(CH 2 CH 2 CH 3 ) 2 , —N(CH 2 CH 2 CH 2 CH 3 ) 2 , —NH-cyclohexyl, —NH—CH 2 CH 2 CH 2 CH 3 , azetidin-1-yl,3,3-difluoroazetidin-1-yl, pyrrolidin-1-yl, or piperidin-1-yl;
R 4 is C 1-10 alkyl;
R 5 and R 6 are each independently C 1-6 alkyl, —C 1-4 -alkylene-O—R z , —C 1-4 -alkylene-O—C(O)—R z , or —C 1-4 -alkylene-O—C(O)—O—R z ; and
R z , at each occurrence, is independently C 1-6 alkyl, C 3-10 cycloalkyl, C 6-20 aryl, or a 4 to 8-membered heterocycle, wherein the C 3-10 cycloalkyl, C 6-20 aryl, and 4 to 8-membered heterocycle are optionally substituted with one or more substituents independently selected from the group consisting of halogen, C 1-4 haloalkyl, —CN, C 1-4 alkyl-O—, C 1-4 alkyl-C(O)—NH—, and C 1-4 alkyl-NH—C(O)—;
(b) subsequently contacting the cell with the metal ion; and
(c) subsequently measuring fluorescence produced by the cell.

15. The method of claim 14 , wherein the metal ion is Tl + .

16. The method of claim 14 , wherein the cell comprises at least one ion channel.

17. The method of claim 16 wherein the at least one ion channel comprises a potassium channel.

18. The method of claim 14 , further comprising measuring fluorescence produced by the cell after step (a) and before step (b).

Assignees

• Univ Vanderbilt

Inventors

• Weaver Charles David
• Dutter Brendan F
• Sulikowski Gary A

Classifications

• C07D493/10 Primary

  Spiro-condensed systems · CPC title

• G01N33/84

  involving inorganic compounds or pH · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N33/6872

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• C09B11/245

  Phthaleins having both OH and amino substituent(s) on aryl ring · CPC title