Isoindoline derivatives that bind to the ATP binding site

By developing novel probe compounds that can effectively bind ATP binding sites of MLH1 and PMS2, the problem of insufficient signal in fluorescence polarization assays has been solved, and efficient evaluation of test molecule binding affinity and quantitative analysis of target proteins is achieved.

CN115397811BActive Publication Date: 2025-05-23NEOPHORE LTD
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Patent Information

Application Number
CN202180023267.0
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Priority Date
2020-03-11
Filing Date
2021-01-20
Publication Date
2025-05-23
Estimated Expiration
2041-01-20

AI Technical Summary

Technical Problem

Existing ATP-derived fluorescent probes do not show signals in fluorescence polarization assays and their binding to the N-terminal domain of MLH1 is very weak and cannot be effectively used as a fluorescence polarization screening tool.

Method used

A series of new probe compounds have been developed that are able to effectively bind the ATP binding sites of GHKL family proteins MLH1 and PMS2 to evaluate the binding affinity of the test compounds and determine the location and number of target proteins.

Benefits of technology

These novel probe compounds are able to effectively bind ATP binding sites of MLH1 and PMS2, providing an efficient fluorescence polarization screening tool for determining the binding affinity of test molecules to target proteins and quantitatively analyzing the location and number of target proteins in biological samples.

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Abstract

The present invention relates to novel probe compounds of formula I and II as defined herein. The present invention also relates to methods for synthesizing these novel probe compounds, and their use in assays and screening for determining the binding of a test molecule to an ATP binding site of a target protein (e.g., mismatch repair (MMR) component proteins PMS2 and MLH1), or for determining the location and / or amount of such a target protein in a biological sample.
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Claims

1. A probe compound of formula I or II or a salt thereof: in: R 1 is halogen or (1-6C)alkyl, (3-6C)cycloalkyl or (3-4C)cycloalkyl-(1-2C)alkyl, each of which is optionally substituted by one or more substituents selected from halogen, cyano or hydroxy; L is a linker having the formula: -L 1 -X 1 -L 2 -X 2 -L 3 - in L 1 Absent or (2-6C)alkylene; X 1 Not present or selected from -O-, -C(O)-, -C(O)NR 10 -, or -NR 10 C(O)-; L 2 is (2-6C)alkylene or -[CH 2 CH 2 -O] a -[CH 2 ] b -or-[CH 2 ] b -[O-CH 2 CH 2 ] a -, wherein a is 2-10, and b is 2-4; X 2 Not present or selected from -O-, -C(O)-, -C(O)NR 10 -, or -NR 10 C(O)-; L 3 Absent or (2-6C)alkylene; Where R 10 is hydrogen or methyl; and The condition is X 1 or X 2 At least one of them exists; Q is the fluorophore; L X is a (1-3C) alkylene linker; as well as X is selected from halogen, N 3 or an ethynyl functional group.

2. The probe compound according to claim 1, wherein R 1 is halogen or (1-6C)alkyl, (3-4C)cycloalkyl or (3-4C)cycloalkyl-(1-2C)alkyl, each of which is optionally substituted by one or more halogen substituents.

3. The probe compound according to claim 1 or 2, wherein R 1 Selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, trifluoromethyl.

4. The probe compound according to claim 1 or 2, wherein R 1 is isopropyl.

5. The probe compound according to claim 1 or 2, wherein L is a linker having the formula: -L 1 -X 1 -L 2 -X 2 -L 3 - in L 1 is (2-6C)alkylene; X 1 Selected from -O-, -C(O)-, -C(O)NR 10 -, or -NR 10 C(O)-; L 2 is a (2-6C) alkylene group or a group of the formula -[CH 2 CH 2 -O] a -[CH 2 b - or -[CH 2 b -[O-CH 2 CH 2 a -, where a is 2-10 and b is 2-4;​​​ X 2 Selected from -O-, -C(O)NR 10 -, or -NR 10 C(O)-; L 3 Absent or (2-6C)alkylene; Where R 10 is hydrogen or methyl; and The condition is X 1 or X 2 At least one of exists.

6. The probe compound according to claim 1 or 2, wherein L is a linker having the formula: -L 1 -X 1 -L 2 -X 2 -L 3 - in L 1 is (2-6C)alkylene; X 1 Selected from -O-, -C(O)NR 10 -, or -NR 10 C(O)-; L 2 is (2-6C)alkylene or -[CH 2 CH 2 -O] a -[CH 2 ] b -or-[CH 2 ] b -[O-CH 2 CH 2 ] a -, wherein a is 2-10, and b is 2-4; X 2 Selected from -O-, -C(O)NR 10 -, or -NR 10 C(O)-; L 3 is (2-6C)alkylene; Where R 10 is hydrogen or methyl; and The condition is X 1 or X 2 At least one of exists.

7. The probe compound according to claim 1 or 2, wherein Q is a fluorophore selected from the group consisting of AlexaFluor dye, cyanine dye, fluorescein, BODIPY or a BODIPY derivative, TAMRA, Oregon Green dye, FITC, Ru(bpy)3, rhodamine dye, acridine orange and Texas Red.

8. The probe compound according to claim 1 or 2, wherein Q is a fluorophore selected from AlexaFluor-647, AlexaFluor-633, AlexaFluor-594, AlexaFluor-488, cyanine-5B, cyanine-3B, fluorescein, BODIPYTMR, TAMRA, Oregon Green 488, Oregon Green 514, FITC, Ru(bpy)3, rhodamine dye, acridine orange and Texas Red.

9. The probe compound according to claim 1 or 2, wherein X is ethynyl or N 3 .

10. A probe compound of formula I or a salt thereof, wherein the probe compound is selected from any one of the following: 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-butadien-1-yl)-3H-indol-1-ium; 3-(2-((1E,3E)-5-((Z)-3-(5-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl))isoindolyl)-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)amino)-5-oxopentyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indolyl-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1-sulfonate; 1-(6-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-carboxylate; 2-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-5-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid and 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid; 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)ammonium benzoate; 3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H- 4 λ 4,5 λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborindan-3-yl)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)propanamide; 3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H- 4 λ 4,5 λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborindan-3-yl)propionamido)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)-3,6,9,12-tetradecanopentadecan-15-amide; 4-(3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H- 5 λ 4,6 λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborindan-3-yl)propionamido)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)butanamide; 3-(5,5-difluoro-7-(1H-pyrrol-2-yl)- 5H-4 λ 4,5 λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborindan-3-yl)-1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)propan-1-one; 3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H- 4 λ 4,5 λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborindan-3-yl)-N-(15-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)-15-oxo-3,6,9,12-tetraoxapentadecyl)propanamide; 3-(5,5-difluoro-7-(1H-pyrrol-2-yl)- 5H-5 λ 4,6 λ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborindan-3-yl)-N-(4-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)-4-oxobutyl)propanamide.

11. A probe compound of formula I or a salt thereof, wherein the probe compound is selected from any one of the following: 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-butadien-1-yl)-3H-indol-1-ium; 1-(6-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-carboxylate; 2-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-5-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid and 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid; 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)ammonium benzoate.

12. A probe compound of formula II or a salt thereof, wherein the probe compound is: (2,4-Dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone.

13. A compound or a salt thereof, wherein the compound is selected from any one of the following: tert-Butyl (2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)carbamate; (5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride; (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone.

14. Use of a probe compound of formula I or a salt thereof according to any one of claims 1 to 8 or claim 10 or claim 11 in a displacement assay for determining the binding affinity of a test molecule to an ATP binding site of a target protein.

15. The use according to claim 14, wherein the target protein is MLH1 or PMS2.

16. An assay for determining the binding affinity of a test molecule to an ATP binding site of a target protein, the assay include: (i) incubating the test molecule with the target protein in the presence of a probe compound of formula I according to any one of claims 1 to 8 or claim 10 or claim 11, or a salt thereof; and (ii) Determining whether any probe compound is displaced from the ATP binding site of the target protein.

17. A method for determining the binding affinity of a test molecule to an ATP binding site of a target protein, the determination include: (i) incubating the test molecule with the target protein in the presence of a probe compound of formula I according to any one of claims 1 to 8 or claim 10 or claim 11, or a salt thereof; and (ii) Determining whether any probe compound is displaced from the ATP binding site of the target protein.

18. An assay for determining the amount and location of a target protein in a biological sample, the assay include: (i) contacting the biological sample with a probe compound of formula I or a salt thereof according to any one of claims 1 to 8 or claim 10 or claim 11; and (ii) determining the distribution and amount of the compound of formula I in a biological sample by detecting and quantifying the location and distribution of the compound of formula I in the sample.

19. An assay for determining the amount and location of a target protein in a biological sample, the assay include: (i) contacting the biological sample with a probe compound of formula II or a salt thereof according to any one of claims 1 to 4 or claim 9 or claim 12; and (ii) contacting the biological sample with a detection moiety capable of reacting with a functional group X present on the compound of formula II to form in situ in the biological sample a compound of formula I according to any one of claims 1 to 8 or claim 10 or claim 11; (iii) determining the distribution and amount of the compound of formula I in the biological sample by detecting and quantifying the location and distribution of the compound of formula I in the sample.

20. The assay of claim 16, 18 or 19, or the method of claim 17, wherein the target protein is MLH1 or PMS2.

Citation Information

Patent Citations

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