Isoindoline derivatives that bind to the ATP binding site

Abstract

The present invention relates to novel probe compounds of Formula I and Formula II as defined herein. The present invention also relates to methods for synthesizing these novel probe compounds and their use in assays and screens for determining the binding of test molecules to the ATP-binding site of target proteins, such as the mismatch repair (MMR) component proteins PMS2 and MLH1, or for determining the location and / or amount of such target proteins in biological samples.

Description

[Technical Field]

[0001] The present invention relates to novel probe molecules. The present invention also relates to methods for synthesizing these novel probe molecules, as well as their use in assays and screenings to determine the binding of test molecules to the ATP binding site of target proteins, such as mismatch repair (MMR) constituent proteins PMS2 and MLH1, or to determine the location and / or quantity of such target proteins in biological samples. [Background technology]

[0002] The GHKL family of ATPases includes the chaperone protein HSP90[1], the mismatch repair (MMR) constituent proteins PMS2 and MLH1[2], and the DNA gyrase-bacterial type II topoisomerase enzyme[3] which catalyzes the supercoiling of double-stranded DNA. Each member of the GHKL family has an ATP binding site, and ATP binding is known to be important for the function of GHKL family proteins[4].

[0003] ATP-competitive inhibitors of HSP90 have shown potential as anticancer agents [5,6], and for ATP-competitive fluorescent probes, the discovery of compounds that bind to the ATP binding site of HSP90 and replace the fluorescent probe has been reported [7]. In such assays, a fluorescent dye molecule bound to a small ligand (so-called fluorescent probe molecule) that rapidly rotates and disrupts the plane of polarization before emission results in low fluorescence polarization. Binding of the probe molecule to a slowly rotating protein such as HSP90 results in high fluorescence polarization. Replacement of the probe molecule with the test ligand results in a concentration-dependent decrease in fluorescence polarization. Therefore, fluorescence polarization can be applied to directly and quantitatively read the degree of binding of the test compound to proteins such as Hsp90 [7,8] or DNA gyrase [9].

[0004] Surprisingly, commercially available ATP-derived fluorescent probes such as BODIPY-FL-ATP-gamma-S (Invitrogen), EDA-AMPPNP-5 / 6-TAMRA (Jena Bioscience GmbH), and EDA-AMPPNP-Cy5 (Jena Bioscience GmbH) were found to show no signal in fluorescence polarization assays performed according to the types of protocols exemplified herein, and to exhibit extremely weak binding to the N-terminal domain of MLH1 via surface plasmon resonance. Therefore, these fluorescence probes are unsuitable for use as fluorescence polarization screening tools.

[0005] Thus, there is a need for further probe compounds that improve the binding of GHKL family members MLH1 and PMS2 to their ATP binding sites and are suitable for use as fluorescence polarization screening tools.

[0006] This invention was made with the above in mind. [Overview of the Initiative]

[0007] This invention relates to the discovery of a new series of probe compounds that effectively bind to the ATP binding sites of the GHKL family proteins MLH1 and PMS2. These probe compounds can be used in assays and methods to evaluate the binding affinity of test compounds to the ATP binding sites of MLH1 and PMS2. Furthermore, these probe compounds can be used to determine the location and / or quantity of MLH1 or PMS2 in biological samples.

[0008] In a first aspect, the present invention provides a probe compound of formula I or formula II as defined herein, or a salt thereof.

[0009] In another embodiment, the present invention provides a method for synthesizing probe compounds of formula I or formula II as defined herein, or salts thereof.

[0010] In another embodiment, the present invention provides the use of a probe compound of formula I, or a salt thereof, in a substitution assay for determining the binding affinity of a test molecule to the ATP binding site of a target protein. In one embodiment, the target protein is selected from MLH1, PMS2, or HSP90. In a particular embodiment, the target protein is MLH1 or PMS2.

[0011] In another embodiment, the present invention provides a probe compound of formula I, or a salt thereof, for use in a substitution assay to determine the binding affinity of a test molecule to the ATP binding site of a target protein. In one embodiment, the target protein is selected from MLH1, PMS2, or HSP90. In a particular embodiment, the target protein is MLH1 or PMS2.

[0012] In another embodiment, the present invention relates to an assay for determining the binding affinity of a test molecule to the ATP binding site of a target protein, (i) The step of incubating a test molecule with a target protein in the presence of a probe compound of formula I as defined herein, or a salt thereof, (ii) A step to determine whether the probe compound of formula I is substituted for the ATP binding site of the target protein. The assay includes the following: In one embodiment, the target protein is selected from MLH1, PMS2, or HSP90. In a particular embodiment, the target protein is MLH1 or PMS2.

[0013] In another embodiment, the present invention relates to a method for determining the binding affinity of a test molecule to the ATP binding site of a target protein, (i) The step of incubating a test molecule with a target protein in the presence of a probe compound of formula I as defined herein, or a salt thereof, (ii) A step to determine whether the probe compound of formula I is substituted for the ATP binding site of the target protein. The assay includes the following:

[0014] In another embodiment, the present invention relates to an assay for determining the location and / or amount of a target protein present in a biological sample, (i) The step of contacting a biological sample with a probe compound of formula I as defined herein, or a salt thereof, (ii) A step of determining the location and / or amount of the compound of formula I present in a biological sample by detecting the location and / or intensity of the detectable portion of the compound of formula I present in the biological sample. The assay includes the following:

[0015] In another embodiment, the present invention relates to an assay for determining the location and / or amount of a target protein present in a biological sample, (i) The step of contacting a biological sample with a probe compound of formula II as defined herein, or a salt thereof, (ii) The step of contacting a biological sample with a detection portion Q that reacts with a functional group X present in the compound of formula II to form the compound of formula I in situ within the biological sample, or with a compound of formula Q-L2-Y as defined herein, (iii) A step of determining the location and / or amount of the compound of formula I present in a biological sample by detecting the location and / or intensity of the detectable portion of the compound of formula I present in the biological sample. The assay includes the following:

[0016] In another aspect, the present invention relates to a method for determining the location and / or quantity of a target protein present in a biological sample, (i) The step of contacting a biological sample with a probe compound of formula I as defined herein, or a salt thereof, (ii) A step of determining the location and / or amount of the compound of formula I present in a biological sample by detecting the location and / or intensity of the detectable portion of the compound of formula I present in the biological sample. This provides a method that includes this.

[0017] In another aspect, the present invention relates to a method for determining the location and / or quantity of a target protein present in a biological sample, (i) The step of contacting a biological sample with a probe compound of formula II as defined herein, or a salt thereof, (ii) The step of contacting a biological sample with a detection portion Q that reacts with a functional group X present in the compound of formula II to form the compound of formula I in situ within the biological sample, or with a compound of formula Q-L2-Y as defined herein, (iii) A step of determining the location and / or amount of the compound of formula I present in a biological sample by detecting the location and / or intensity of the detectable portion of the compound of formula I present in the biological sample. This provides a method that includes this.

[0018] In one embodiment, the target protein is selected from MLH1, PMS2, or HSP90. In a particular embodiment, the target protein is MLH1 or PMS2. [Brief explanation of the drawing]

[0019] [Figure 1] This figure shows the synthetic scheme for the preparation of (2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindoline-2-yl)methanone (Example 6). [Figure 2] This figure shows the synthetic scheme for the preparation of (5-((4-(2-(2-aminoethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride. [Figure 3] This figure shows the synthetic scheme for the preparation of 2,4-dihydroxy-5-isopropylbenzoic acid. [Figure 4] This figure shows the synthetic scheme for the preparation of (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone. [Figure 5] This figure shows the synthetic scheme for the preparation of 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-carboxylate benzyl (AA) and (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazine-1-ylmethyl)isoindoline-2-yl)methanone hydrochloride (AB). [Figure 6] This figure shows the data from Example 1, plotted and analyzed using GraphPad Prism 8.1.0. [Figure 7] This figure shows the data from Example 3, plotted and analyzed using GraphPad Prism 8.1.0. [Figure 8] This figure shows the data from Example 4, plotted and analyzed using GraphPad Prism 8.1.0. [Figure 9] This figure shows the data from Example 5, plotted and analyzed using GraphPad Prism 8.1.0. [Figure 10] This figure shows the inhibition data for compound AB and IC50 determined using assay protocol A. [Figure 11] This figure shows the inhibition data for compound AB and IC50 determined using assay protocol C. [Modes for carrying out the invention]

[0020] [Definition] Terms used in this specification and in the claims have the following meanings unless otherwise specified:

[0021] The term "alkyl" includes both linear and branched alkyl groups. References to individual alkyl groups, such as "propyl," refer only to their linear versions, and references to individual branched alkyl groups, such as "isopropyl," refer only to their branched versions. For example, "(1-6C)alkyl" includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl, and t-butyl. A similar convention applies to other radicals; for example, "cycloalkyl(1-2C)alkyl" includes cyclopropyl-methyl, 1-cyclopropylethyl, 2-cyclopropylethyl, etc.

[0022] "(m~nC)" or "Cm~n" or "(m~nC) group" or "Cm~n" is used alone or as a prefix to refer to any group having m to n carbon atoms.

[0023] The "alkylene" group is an alkyl group that is located between two other chemical groups and acts as a link between them. For example, "(3-20C)alkylene" refers to a linear saturated divalent hydrocarbon radical with 1-3 carbon atoms or a branched saturated divalent hydrocarbon radical with 3 atoms, such as methylene, ethylene, and propylene.

[0024] "(m~nC)cycloalkyl" refers to a hydrocarbon ring containing m to n carbon atoms. For example, "(3~6C)cycloalkyl" refers to a hydrocarbon ring containing 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. "(m~nC)cycloalkyl" also includes non-aromatic saturated or partially saturated monocyclic carbocyclic systems.

[0025] "Halo" or "halogeno" refers to fluoro, chloro, bromo, and iodine.

[0026] "Optionally substituted" refers to either a substituted group, structure, or molecule, or an unsubstituted group, structure, or molecule.

[0027] When an optional substituent is selected from "one or more" groups, this definition is understood to include all substituents selected from one of the specified groups, or substituents selected from two or more of the specified groups.

[0028] "Compounds according to the present invention" means the compounds disclosed herein in general and specifically.

[0029] [Probe compounds] This invention relates to a probe compound of formula I or formula II, or a salt thereof:

[0030] [ka] (In the formula, R1 is a halo or (1-6C)alkyl, (3-6C)cycloalkyl, or (3-4C)cycloalkyl-(1-2C)alkyl group, each of which is optionally substituted with one or more substituents selected from halo, cyano, or hydroxyl groups; L is the linker; Q is the detection part; L x is a linker; and X is a functional group.

[0031] The probe compounds according to the present invention particularly include probe compounds of formula I or formula II, or salts thereof. In the formulas, R1, L, Q, L x And X are as defined herein or as defined in any one of the following paragraphs (1) to (56): (1) R1 is a halo or (1-6C)alkyl, (3-4C)cycloalkyl, or (3-4C)cycloalkyl-(1-2C)alkyl, each of which is optionally substituted with one or more halo substituents; (2) R1 is a halo or (1-6C)alkyl, (3-4C)cycloalkyl, or (3-4C)cycloalkyl-(1C)alkyl, each of which is optionally substituted with one or more halo substituents; (3) R1 is a halo or (1-6C)alkyl, (3-4C)cycloalkyl, or (3-4C)cycloalkyl-(1C)alkyl, each of which is optionally substituted with one or more fluoro substituents; (4) R1 is a fluoro, chloro, or (1-6C)alkyl, (3-4C)cycloalkyl, or (3-4C)cycloalkyl-(1C)alkyl group, each of which is optionally substituted with one or more fluoro substituents; (5) R1 is a (1-6C) alkyl, a (3-4C) cycloalkyl, or a (3-4C) cycloalkyl-(1C) alkyl group, each of which is optionally substituted with one or more fluoro substituents; (6) R1 is a (1-6C) alkyl group that is optionally substituted with one or more fluoro substituents; (7) R1 is a (1-4C) alkyl group; (8) R1 is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, and trifluoromethyl; (9) R1 is isopropyl; (10)L is a (2-20C) alkylene linker, where the alkylene chain is one or more -O-, -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -, -NR 10 The following optionally further contain C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds: 10is hydrogen or (1-2C) alkyl, and the alkylene chain is optionally substituted by one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C) alkyl, (1-2C) hydroxyalkyl, (1-2C) haloalkyl or (1-2C) alkoxy. (11) L is a (3-20C) alkylene linker, where the alkylene chain optionally further contains one or more -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or a triazole ring linkage, and R 10 is hydrogen or (1-2C) alkyl, and the alkylene chain is optionally substituted by one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C) alkyl, (1-2C) hydroxyalkyl, (1-2C) haloalkyl or (1-2C) alkoxy. (12) L is a (2-20C) alkylene linker, where the alkylene chain contains 1 to 8 -O- bonds and / or 1 to 4 -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or a triazole ring linkage; R 10 is hydrogen or (1-2C) alkyl, and the alkylene chain is optionally substituted by one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C) alkyl, (1-2C) hydroxyalkyl, (1-2C) haloalkyl or (1-2C) alkoxy. (13) L is a (3-20C) alkylene linker, where the alkylene chain contains 1 to 8 -O- bonds and / or 1 to 4 -C(O)-, -C(O)NR 10 -, -NR 10Further containing C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (14)L is a (2-18C)alkylene linker, where the alkylene chain has 1-5 -O- bonds located either within the alkylene chain or / or at its end; and / or 1-4 -C(O)-, -C(O)NR 10 -, -NR 10 It optionally further comprises a C(O)-, -C(O)O-, -OC(O)- bond or one triazole ring bond, R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (15)L is a (3-12C)alkylene linker, where the alkylene chain has 1-5 -O- bonds located either within the alkylene chain or / or at its end; and / or 1-4 -C(O)-, -C(O)NR 10 -, -NR 10 It further optionally contains C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds, R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (16)L is a (2-18C)alkylene linker, where the alkylene chain has 1-5 -O-bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further containing a C(O)-bond or one triazole ring bond; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (17)L is a (3-12C)alkylene linker, where the alkylene chain has 1-5 -O-bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further containing a C(O)-bond or one triazole ring bond; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (18)L is a (2-18C) alkylene linker, where the alkylene chain has 1-5 -O- bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (19)L is a (3-12C)alkylene linker, where the alkylene chain has 1-5 -O-bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (20)L is a (2-18C)alkylene linker, where the alkylene chain has 1-3 -O-bonds and / or 1-3 -C(O)-,-C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (21)L is a (3-12C)alkylene linker, where the alkylene chain has 1-3 -O-bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (22)L is a (2-18C) alkylene linker, where the alkylene chain has one -O- bond and / or one -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (23)L is a (3-12C)alkylene linker, where the alkylene chain has one -O- bond and / or one -C(O)-, -C(O)NR located either within the alkylene chain and / or at its end. 10 -or-NR10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (24)L is a (2-17C) alkylene linker, where the alkylene chain has 1-4 -O- bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -, -NR 10 It optionally further contains C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (25)L is a (3-10C) alkylene linker, where the alkylene chain has 1-4 -O- bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -, -NR 10 It optionally further contains C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (26)L is a (2-17C)alkylene linker, where the alkylene chain has 1-4 -O- bonds located either within the alkylene chain or / or at its end; and / or 1-3 -C(O)-, -C(O)NR 10 -, -NR 10 Further containing C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (27)L is a (3-10C)alkylene linker, where the alkylene chain has 1-4 -O- bonds located either within the alkylene chain or / or at its end; and / or 1-3 -C(O)-, -C(O)NR 10 -, -NR 10 Further containing C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (28)L is a (2-17C) alkylene linker, where the alkylene chain has 1-4 -O- bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -, -NR 10 Further containing a C(O)- or triazole ring bond; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (29)L is a (3-10C) alkylene linker, where the alkylene chain has 1-4 -O- bonds and / or 1-3 -C(O)-, -C(O)NR 10 -, -NR 10Further comprising a triazole ring bond located either within or at the end of a C(O)- or alkylene chain; R 10 The substituent is hydrogen or (1-2C)alkyl, and the alkylene chain is optionally substituted with one or more substituents independently selected from halo, hydroxy, cyano, amino, (1-2C)alkyl, (1-2C)hydroxyalkyl, (1-2C)haloalkyl, or (1-2C)alkoxy. (30)L is a (2-17C)alkylene linker, where the alkylene chain has 1-4 -O- bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 It further includes a C(O)-bond or one triazole ring bond, where R 10 This is hydrogen or (1-2C) alkyl. (31)L is a (3-10C)alkylene linker, where the alkylene chain has 1-4 -O-bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (32)L is a (2-17C)alkylene linker, where the alkylene chain has 1-3 -O-bonds and / or 1-3 -C(O)-,-C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (33)L is a (3-10C)alkylene linker, where the alkylene chain has 1-3 -O-bonds and / or 1-3 -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end.10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (34)L is a (2-17C)alkylene linker, where the alkylene chain has one -O- bond and / or one -C(O)-, -C(O)NR located either within the alkylene chain or / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (35)L is a (3-10C)alkylene linker, where the alkylene chain has one -O- bond and / or one -C(O)-, -C(O)NR located either within the alkylene chain and / or at its end. 10 -or -NR 10 Further comprising a C(O)-bond or one triazole ring bond, R 10 This is hydrogen or (1-2C) alkyl. (36)L is the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is either absent or (1-8C) alkylene; X1 does not exist, or -O-, -C(O)-, -C(O)NR 10 -, -NR 10 Selected from C(O)-, -C(O)O-, -OC(O)-, or a triazole ring; L2 is (1-10C) alkylene, or formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (where a is between 1 and 10, and b is between 2 and 4) is the basis; X2 does not exist, or -O-, -C(O)-, -C(O)NR 10 -, -NR 10Selected from -C(O)-, -C(O)O-, -OC(O)- or a triazole ring; L3 is absent or is (1-8C) alkylene; where R 10 is hydrogen or (1-2C) alkyl; provided that at least one of X1 or X2 is present.) is a linker having (37)L is of the formula: -L1-X1-L2-X2-L3- (wherein, L1 is absent or is (2-6C) alkylene; X1 is absent or is selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or a triazole ring; L2 is (1-10C) alkylene, or a group of the formula -[CH2CH2-O] a -[CH2] b - or -[CH2] b -[O-CH2CH2] a -(where a is 2-10 and b is 2-4).); X2 is absent or is selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or a triazole ring; L3 is absent or is (1-6C) alkylene; where R 10 is hydrogen or methyl; provided that at least one of X1 or X2 is present.) is a linker having (38)L is of the formula: -L1-X1-L2-X2-L3- (wherein, L1 is (2-6C) alkylene; X1 is absent or is selected from -O-, -C(O)-,-C(O)NR10 -, -NR 10 Selected from a C(O)- or triazole ring; L2 is (2-10C) alkylene, or formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (where a is between 2 and 10, and b is between 2 and 4) is the basis; X2 does not exist, or -O-, -C(O)-, -C(O)NR 10 -, -NR 10 Selected from a C(O)- or triazole ring; L3 is either absent or (1-6C) alkylene; R 10 is hydrogen or methyl; (However, this is conditional on at least one of X1 or X2 existing.) It is a linker that possesses this property. (39)L is the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is an (2-6C) alkylene; X1 does not exist, or -O-, -C(O)-, -C(O)NR 10 -, -NR 10 Selected from a C(O)- or triazole ring; L2 is (2-6C)alkylene, or formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (where a is between 2 and 10, and b is between 2 and 4) is the basis; X2 does not exist, or -O-, -C(O)-, -C(O)NR 10 -, -NR 10 Selected from a C(O)- or triazole ring; L3 is either absent or (2-6C)alkylene; R 10 is hydrogen or methyl; (However, this is conditional on at least one of X1 or X2 existing.) It is a linker that possesses this property. (40)L is formula: -L1-X1-L2-X2-L3- (In the formula, L1 does not exist; X1 does not exist; L2 is either absent, or (2-6C) alkylene, or of the formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (where a is between 2 and 10, and b is between 2 and 4) is the basis; X2 is -O-, -C(O)-, -C(O)NR 10 -, -NR 10 Selected from a C(O)- or triazole ring; L3 is either absent or (2-6C)alkylene; R 10 is hydrogen or methyl; (However, this is conditional on at least one of X1 or X2 existing.) It is a linker that possesses this property. (41)L is the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is an (2-6C) alkylene; X1 is -O-, -C(O)NR 10 -, -NR 10 Selected from a C(O)- or triazole ring; L2 is (2-6C)alkylene, or formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (where a is between 2 and 10, and b is between 2 and 4) is the basis; X2 is -O-, -C(O)NR 10 -, -NR 10Selected from a C(O)- or triazole ring; L3 is (2-6C)alkylene; R 10 is hydrogen or methyl; (However, this is conditional on at least one of X1 or X2 existing.) It is a linker that possesses this property. (42) Q is a detection portion selected from the group consisting of fluorophores, oligonucleotides, biomolecules, molecular sensors, proteins, or peptides. (43) Q is a fluorophore. (44) Q is a fluorophore selected from the group consisting of AlexaFluor dye, cyanine dye, fluorescein, BODIPY or BODIPY derivative (e.g., BODIPY TMR), TAMRA, Oregon Green dye, FITC, Ru(bpy)3, rhodamine dye, acridine orange, and Texas Red. (45) Q is a fluorophore selected from the group consisting of AlexaFluor-647, AlexaFluor-633, AlexaFluor-594, AlexaFluor-488, cyanine-5B, cyanine-3B, fluorescein, BODIPY or a BODIPY derivative (e.g., BODIPY TMR), TAMRA, Oregon Green 488, Oregon Green 514, FITC, Ru(bpy)3, rhodamine dye, acridine orange, and Texas Red. (46)X is a functional group that can react with a functional group present in the detection region so as to covalently bond the detection region to the compound of formula II. (47)X is a functional group that can react with a functional group present in the detection portion so as to covalently bond the detection portion to the compound of formula II, and the detection portion is selected from the group consisting of fluorophores, oligonucleotides, biomolecules, molecular sensors, proteins, or peptides. (48)X is a functional group X that can react with a functional group present on the fluorophore so as to covalently bond the fluorophore to the compound of formula II. (49)X is a functional group that can react with a functional group present on the fluorophore so as to covalently bond the fluorophore to the compound of formula II, and the fluorophore is as defined in paragraph (44) or (45) above. (50)X is halo, N3, or ethinyl [ka] It is a functional group selected from ). (51) X is ethinyl ( [ka] ) or N3. (52)L x This is a linker group L as defined in any one of the paragraphs (10) to (41) above. (53)L x This is a (1-8C)alkylene linker, where the alkylene chain is one or more -O-,-C(O)NR located either within the alkylene chain and / or at its end. 10 -, -NR 10 The following optionally further contain C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds: 10 This is hydrogen or (1-2C) alkyl. (54)L x This is a (1-8C)alkylene linker, where the alkylene chain has one -O-, -C(O)NR located either within the alkylene chain and / or at its end. 10 -, -NR 10 The following optionally further contain C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds: 10 This is hydrogen or (1-2C) alkyl. (55)L x This is a (1-5C) alkylene linker. (56)L x This is an (1-3C) alkylene linker.

[0032] Ideally, the halo group, if present, is fluoro and / or chloro, particularly fluoro.

[0033] More appropriately, R1 is as defined in any one of the above paragraphs (1) to (9). More appropriately, R1 is as defined in either paragraphs (8) and (9). In the group of the probe compound of formula I or formula II, in particular, R1 is isopropyl.

[0034] The linker group L may be any suitable linker portion that links the detection portion Q to the remainder of the probe compound of formula I, or the functional group X to the remainder of the probe compound of formula II. Preferably, the linker group L is 3 to 30 atomic lengths, more preferably 4 to 20 atomic lengths, and even more preferably 5 to 18 atomic lengths. In particular, for the groups of the probe compounds of formula I or formula II as defined herein, L is 5 to 12 atomic lengths.

[0035] Appropriately, L is as defined in any one of paragraphs (10) to (41) above. More appropriately, it is as defined in any one of paragraphs (24) to (35) or (39) to (41) above. In particular, for the group of the probe compound of formula I or formula II, L is as defined in paragraph (40) or (41) above.

[0036] The detection portion Q may be any portion capable of detecting and quantifying the probe compound of formula I. As described below, the probe compounds of formula I as defined herein are designed for use in substitution assays, thereby allowing the ability of a test compound to substitute the probe compound of formula I from the ATP binding site of a target protein (e.g., MLH1 or PMS2) to be used to determine the binding affinity of that test compound to the ATP binding site of the target protein. Thus, the detection portion Q may be any portion that can be readily detected and quantified. In certain circumstances, the detection portion Q may be capable of detecting and quantifying any probe compound of formula I that is substituted (i.e., “unbound”) from the ATP binding site of the target protein. In certain embodiments of the present invention, this may be achieved by recovering any substituted or “unbound” compound of formula I from a test sample and assaying the sample to determine how much of the unbound probe compound is present. This would then serve as an indicator of how much of the probe compound present in the sample is substituted by the test compound.

[0037] Thus, it will be understood that the properties of the detection portion Q are not critical as long as they can be used to determine the amount of the probe compound of formula I present in the sample. Those skilled in the art will be able to select an appropriate detection portion Q and a suitable methodology for detecting and quantifying the amount of the compound of formula I in a sample, in particular for detecting the amount of the probe compound of formula I that is substituted from the ATP binding site of the target protein by the test compound.

[0038] Appropriately, the detection portion Q is selected from the group consisting of fluorophores, oligonucleotides, biomolecules, molecular sensors, proteins, or peptides.

[0039] In embodiments where the detection portion Q is an oligonucleotide, biomolecule, molecular sensor, protein, or peptide, any suitable method known in the art for detecting and quantifying the amount of present oligonucleotide, biomolecule, molecular sensor, protein, or peptide may be used. For example, a fluorescently labeled secondary probe capable of specifically binding to the detection portion Q of the probe compound of formula I may be used, and once all excess secondary probe has been removed, the amount of the secondary probe bound to the detection portion Q of the compound of formula I can be detected and quantified, thereby allowing for the determination of the amount of the probe compound of formula I.

[0040] For example, if Q is an oligonucleotide, a secondary probe having a suitable detectable label, such as a fluorophore or radiolabel, and a complementary oligonucleotide sequence that can hybridize to Q may be used to detect and quantify the amount of the probe compound of formula I present in the sample (and more preferably, the amount of the probe compound substituted from the ATP binding site of the target protein by the test compound). Similarly, if Q is a protein or peptide, the secondary probe may be an antibody that can selectively bind to that protein or peptide and a suitable detectable label, such as a fluorophore or radiolabel.

[0041] More precisely, the detection portion Q is a fluorophore. In such cases, compounds of formula I can be used in fluorescence polarization assays. In the base of the probe compounds of formula I, in particular, the detection portion Q is a fluorophore selected from the group consisting of AlexaFluor dye, cyanine dye, fluorescein, BODIPY or a BODIPY derivative (e.g., BODIPY TMR), TAMRA, Oregon Green dye, FITC, Ru(bpy)3, rhodamine dye, acridine orange, and Texas Red. In further bases of the probe compounds of formula I, the detection portion Q is a fluorophore selected from the group consisting of AlexaFluor-647, AlexaFluor-633, AlexaFluor-594, AlexaFluor-488, cyanine-5B, cyanine-3B, fluorescein, BODIPY TMR, TAMRA, Oregon Green 488, Oregon Green 514, FITC, Ru(bpy)3, rhodamine dye, acridine orange, and Texas Red.

[0042] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (1) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0043] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (2) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0044] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (3) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0045] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (4) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0046] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (5) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0047] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (6) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0048] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (7) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0049] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (8) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0050] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in any one of paragraphs (10) to (41) above.

[0051] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (10) above.

[0052] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (11) above.

[0053] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (14) above.

[0054] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (15) above.

[0055] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (18) above.

[0056] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (19) above.

[0057] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (22) above.

[0058] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (23) above.

[0059] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (26) above.

[0060] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (27) above.

[0061] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (30) above.

[0062] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (31) above.

[0063] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (34) above.

[0064] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (35) above.

[0065] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (36) above.

[0066] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (37) above.

[0067] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (38) above.

[0068] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (39) above.

[0069] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (40) above.

[0070] In the group of the probe compound of formula I, in particular, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (41) above.

[0071] Appropriately, X is defined as in any one of the above paragraphs (46) to (51).

[0072] It will be understood that X may be any suitable functional group that reacts with the functional group Q present in the detection region, or with the compound of formula Q-L2-Y as defined herein, to covalently bond the detection region to the compound of formula II, thereby forming the compound of formula I as defined herein.

[0073] Those skilled in the art will be familiar with suitable functional groups that can be used to form a covalent bond with the appropriate detection site. For example, X may be halo, N3 or ethynyl ( [ka] ) may be selected from the following.

[0074] In certain embodiments, X is N3 or ethynyl.

[0075] Appropriately, X is a functional group that can react with a functional group present in the detection moiety to form a bond or triazole bond between the detection moiety and the compound of formula II. In certain embodiments, the bond formed is a triazole bond (which may be formed by CLICK chemistry).

[0076] Appropriately, L x This is defined in any one of the paragraphs (52) to (56) above.

[0077] In a particular embodiment of the present invention, the compound of formula I is the compound of formula II, which is the compound of formula: QL y -Y (In the formula, Q is the detection part as defined above: L y It does not exist, or the group L of the compound of formula II is absent. x When covalently bonded, it is a linker group that forms linker L of the compound of formula I; Y reacts with the functional group X present in the compound of formula II, and Q-L2 is converted to L in the compound of formula II. x It is a functional group that can be covalently bonded to it. It is formed by reacting with the group.

[0078] In one embodiment, L y This is a (1-8C)alkylene linker, where the alkylene chain is one or more -O-,-C(O)NR located either within the alkylene chain and / or at its end. 10 -, -NR 10 The following optionally further contain C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds: 10 This is hydrogen or (1-2C) alkyl.

[0079] In another embodiment, L y This is a (1-8C)alkylene linker, where the alkylene chain has one -O-, -C(O)NR located either within the alkylene chain and / or at its end. 10 -, -NR 10 The following optionally further contain C(O)-, -C(O)O-, -OC(O)-, or triazole ring bonds: 10 This is hydrogen or (1-2C) alkyl.

[0080] In further embodiments, L y This is a (1-5C) alkylene linker.

[0081] In another embodiment, L yThis is an (1-3C) alkylene linker.

[0082] It will be understood that Y can be any suitable functional group that reacts with functional group X present in the probe compound of formula II to covalently bond the group Q-Ly to the compound of formula II, thereby forming the compound of formula I as defined herein.

[0083] Those skilled in the art will be familiar with suitable functional groups that can be used to form a covalent bond with functional group X of the compound of formula II. For example, Y may be selected from halo, N3, or ethynyl.

[0084] In certain embodiments, Y is N3 when X is ethynyl, or Y is ethynyl when X is N3.

[0085] Appropriately, Y is a functional group that can react with functional group X present in the probe compound of formula II to form a bond or a triazole bond. In certain embodiments, the bond formed is a triazole bond (which may be formed by CLICK chemistry).

[0086] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (1) above, and L x This is as defined in paragraphs (52), (53), or (54) above.

[0087] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (2) above, and L x This is as defined in paragraphs (52), (53), or (54) above.

[0088] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (3) above, and L xThis is as defined in paragraphs (52), (53), or (54) above.

[0089] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (4) above, and L x This is as defined in paragraphs (52), (53), or (54) above.

[0090] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (5) above, and L x This is as defined in paragraphs (52), (53), or (54) above.

[0091] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (6) above, and L x This is as defined in paragraphs (52), (53), or (54) above.

[0092] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (7) above, and L x This is as defined in paragraphs (52), (53), or (54) above.

[0093] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (8) above, and L x This is as defined in paragraphs (52), (53), or (54) above.

[0094] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (9) above, and L xThis is as defined in paragraphs (52), (53), or (54) above.

[0095] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (9) above, and L x This is as defined in paragraph (52) above.

[0096] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (9) above, and L x This is as defined in paragraph (53) above.

[0097] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (9) above, and L x This is as defined in paragraph (54) above.

[0098] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (9) above, and L x This is as defined in paragraph (55) above.

[0099] In the group of the probe compound of formula II, in particular, X is as defined in paragraph (50) above, R1 is as defined in paragraph (9) above, and L x This is as defined in paragraph (56) above.

[0100] The compounds according to the present invention include, in particular, any one of the following: 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-ium (Example 1); 3-(2-((1E,3E)-5-((Z)-3-(5-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-5-oxopentyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indole-1-ium-1-yl)propane-1-sulfonic acid (Example 2); 1-(6-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-ium formate (Example 3); 2-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-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)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid (Example 4); 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)ammonium benzoate (Example 5); 2,4-Dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindorin-2-yl)methanone (Example 6); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)propenamide (Example 7); 1-(3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)-3,6,9,12-tetraoxapentadecane-15-amide (Example 8); 4-(3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-5λ 4 ,6λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)butanamide (Example 9); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4-Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)propan-1-one (Example 10); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(15-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)-15-oxo-3,6,9,12-tetraoxapentadecyl)propanamide (Example 11); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-5λ 4 ,6λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(4-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)-4-oxobutyl)propanamide (Example 12); (2-(2-(4-((2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl; (5-((4-(2-(2-aminoethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride; Tert-butyl(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamate tert-butyl; (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone.

[0101] The probe compounds according to the present invention include, in particular, any one of the following: 2,4-Dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindorin-2-yl)methanone (Example 6); (2-(2-(4-((2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl; (5-((4-(2-(2-aminoethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride; (5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl) tert-butyl carbamate; (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone.

[0102] The probe compounds according to the present invention include, in particular, any one of the following: 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-ium (Example 1); 3-(2-((1E,3E)-5-((Z)-3-(5-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-5-oxopentyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indole-1-ium-1-yl)propane-1-sulfonic acid (Example 2); 1-(6-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-ium formate (Example 3); 2-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-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)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid (Example 4); 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)ammonium benzoate (Example 5); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4-Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)propenamide (Example 7); 1-(3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)-3,6,9,12-tetraoxapentadecane-15-amide (Example 8); 4-(3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-5λ 4 ,6λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)butanamide (Example 9); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)propan-1-one (Example 10); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4-Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(15-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)-15-oxo-3,6,9,12-tetraoxapentadecyl)propanamide (Example 11); 3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-5λ 4 ,6λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(4-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)-4-oxobutyl)propanamide (Example 12).

[0103] Suitable salts of the probe compound according to the present invention include, for example, acid addition salts of the compound according to the present invention that are sufficiently basic, such as acid addition with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, trifluoroacetic acid, formic acid, citric acid, methanesulfonic acid, or maleic acid. Furthermore, suitable salts of the compound according to the present invention that are sufficiently acidic include alkali metal salts, such as sodium or potassium salts, alkaline earth metal salts, such as calcium or magnesium salts, ammonium salts, or salts with organic bases.

[0104] The present invention also includes probe compounds according to the present invention as defined herein, which include one or more isotopic substitutions. For example, H is 1 H, 2 H(D) and 3 It may be any isotope type including H(T), and C is 12 C, 13 C and 14 It may be any isotope type containing C, and O is 16 O and 18 Any isotope containing O may be used, for example.

[0105] It should also be understood that the probe compound of formula (I) can exist in both solvated and non-solvated forms, such as a hydrated form.

[0106] It should be understood that the probe compound of formula (I) may exhibit polymorphism, and that the present invention encompasses all such polymorphic forms.

[0107] The probe compound of formula (I) may also exist in several different tautomerized forms, and references to the compound of formula (I) include all such forms.

[0108] [Synthesis of probe compounds] In another embodiment, the present invention provides a method for synthesizing probe compounds of formula I or formula II as defined herein, or salts thereof.

[0109] The probe compounds according to the present invention can be prepared by any suitable method known in the art. Specific processes for preparing these compounds are further described in the attached examples.

[0110] It should be understood that in the description of the synthesis methods described herein, and in any mentioned synthesis methods used to prepare the starting materials, all proposed reaction conditions, including the choice of solvent, reaction atmosphere, reaction temperature, experimental procedure, and duration of work-up procedure, can be selected by those skilled in the art.

[0111] Those skilled in the art of organic synthesis will understand that the reagents and reaction conditions must be suitable for utilizing the functionalities present in various parts of the molecule.

[0112] It will be appreciated that during the synthesis of the compounds according to the invention by the process defined herein, or during the synthesis of certain starting materials, it may be desirable to protect certain substituents in order to prevent their unwanted reactions. A skilled chemist will understand when such protection is required and how such protecting groups can be introduced and later removed.

[0113] For examples of protecting groups, reference may be made to one of the many general texts on this subject, for example, ‘Protective Groups in Organic Synthesis’ by Theodora Green; 4th Edition; 2006 (publisher: John Wiley & Sons). The protecting groups can be removed by any convenient method described in the literature or known to the skilled chemist as suitable for the removal of the protecting groups in question, such methods being selected to effect the removal of the protecting group while minimizing interference with the groups elsewhere in the molecule.

[0114] In one aspect, the invention provides a process for synthesizing a compound of formula I as defined herein, the process comprising reacting a compound of formula II as defined herein with a compound of formula Q-L2-Y as defined herein to form a compound of formula I; and optionally thereafter: (i) removing any protecting groups present; (ii) forming a salt of the compound of formula I; and / or (iii) isolating and purifying the compound of formula I is provided.

[0115] [Application of Probe Compound] In one aspect, the invention provides the use of a probe compound of formula I as defined herein, or a salt thereof, in a displacement assay for determining the binding affinity of a test molecule for the ATP binding site of a target protein.

[0116] In another aspect, the present invention provides a probe compound of Formula I, or a salt thereof, for use in a displacement assay for determining the binding affinity of a test molecule to the ATP binding site of a target protein.

[0117] In one embodiment, the target protein is selected from MLH1, PMS2 or HSP90. In certain embodiments, the target protein is MLH1 or PMS2. In certain embodiments, the target protein is MLH1. In further certain embodiments, the target protein is PMS2.

[0118] Displacement assays are known in the art. In embodiments of the present invention where Q is a fluorophore, the displacement assay is a fluorescence polarization assay. Fluorescence polarization assays are known in the art (see, for example, references [7, 8, 9] herein).

[0119] The test compound may be any biomolecule or small molecule compound to be screened for binding to the ATP binding site of a target protein (e.g., MLH1 or PMS2).

[0120] In another aspect, the present invention is an assay for determining the binding affinity of a test molecule to the ATP binding site of a target protein, comprising: (i) incubating the test molecule with the target protein in the presence of a probe compound of Formula I, or a salt thereof, as defined herein; and (ii) determining whether any of the probe compound has been displaced from the ATP binding site of the target protein. The assay is provided. [[ID=**************]]

[0121] In one embodiment, the target protein is selected from MLH1, PMS2 or HSP90. In certain embodiments, the target protein is MLH1 or PMS2. In certain embodiments, the target protein is MLH1. In further certain embodiments, the target protein is PMS2.

[0122] Appropriately, the assay is a substitution assay. Substitution assays are known in the art. In embodiments of the present invention where Q is a fluorophore, the substitution assay is a fluorescence polarization assay. Fluorescence polarization assays are known in the art (see, for example, references [7, 8, 9] herein).

[0123] The step of determining whether a given probe compound is substituted for an ATP binding site of a target protein appropriately includes the step of detecting whether the test compound results in an increase in the proportion of a formula I probe compound that is not bound to an ATP binding site. This can be achieved by detecting whether an increase in the amount of the unbound probe compound is observed (compared to a control), or, if the amount of the bound probe compound is readily detectable (e.g., by fluorescence polarization), by detecting whether a decrease in the amount of the bound probe compound is observed (again, compared to a control).

[0124] Determining the proportion of a probe compound bound to the ATP binding site of a target protein compared to the unbound amount can be achieved by methods known in the art. The specific method used will depend on the nature of the detection portion Q in the compound of formula I. Appropriately, Q is a fluorophore, and the assay is a fluorescence polarization assay. In such an assay, the difference in fluorescence resulting from the substitution of the probe compound from the ATP binding site of the target protein can be detected and used to quantify the amount of any substituted probe compound, thereby determining the binding affinity of the test compound to the ATP binding site of the target protein.

[0125] In another embodiment, the present invention relates to a method for determining the binding affinity of a test molecule to the ATP binding site of a target protein, (i) The step of incubating a test molecule with a target protein in the presence of a probe compound of formula I as defined herein, or a salt thereof, (ii) A step of determining whether any probe compound is substituted for the ATP binding site of the target protein. The assay includes the following:

[0126] In one embodiment, the target protein is selected from MLH1, PMS2, or HSP90. In a particular embodiment, the target protein is MLH1 or PMS2. In another particular embodiment, the target protein is MLH1. In yet another particular embodiment, the target protein is PMS2.

[0127] Appropriately, the method is a substitution assay. Substitution assays are known in the art. In embodiments of the present invention where Q is a fluorophore, the substitution assay is a fluorescence polarization assay. Fluorescence polarization assays are known in the art (see, for example, references [7, 8, 9] herein).

[0128] The step of determining whether an arbitrary probe compound is substituted for the ATP binding site of the target protein can be performed by the method described above.

[0129] In another embodiment, the present invention relates to an assay for determining the location and / or amount of a target protein present in a biological sample, (i) The step of contacting a biological sample with a probe compound of formula I as defined herein, or a salt thereof, (ii) A step of determining the location and / or amount of the compound of formula I present in the biological sample by detecting the location and / or intensity of the detection portion of the compound of formula I present in the biological sample. The assay includes the following:

[0130] In another embodiment, the present invention relates to an assay for determining the location and / or amount of a target protein present in a biological sample, (i) The step of contacting a biological sample with a probe compound of formula II as defined herein, or a salt thereof, (ii) contacting the biological sample with a detection moiety Q that can react with the functional group X present in the compound of formula II to form in situ the compound of formula I within the biological sample, or a compound of formula Q-L2-Y as defined herein; (iii) determining the position and / or amount of the compound of formula I present in the sample by detecting the position and / or intensity of the detection moiety of the compound of formula I present in the biological sample; to provide an assay comprising the above.

[0131] In another aspect, the present invention provides a method for determining the position and / or amount of a target protein present in a biological sample, comprising: (i) contacting the biological sample with a probe compound of formula I as defined herein, or a salt thereof; (ii) determining the position and / or amount of the compound of formula I present in the sample by detecting the position and / or intensity of the detection moiety of the compound of formula I present in the biological sample; to provide a method comprising the above.

[0132] In yet another aspect, the present invention provides a method for determining the position and / or amount of a target protein present in a biological sample, comprising: (i) contacting the biological sample with a detection moiety Q that can react with the functional group X present in the compound of formula II to form in situ the compound of formula I within the biological sample, or a compound of formula Q-L2-Y as defined herein, in contact with MLH1 or PMS2; (ii) determining the position and / or amount of the compound of formula I present in the sample by detecting the position and / or intensity of the detection moiety of the compound of formula I present in the biological sample; to provide a method comprising the above.

[0133] In one embodiment, the target protein is selected from MLH1, PMS2, or HSP90. In a particular embodiment, the target protein is MLH1 or PMS2. In another particular embodiment, the target protein is MLH1. In yet another particular embodiment, the target protein is PMS2.

[0134] Preferably, the step of contacting a biological sample with a probe compound of formula I or formula II (step (i) of the assay and method described above) involves incubating the biological sample with the probe compound under conditions that allow the probe compound to bind to the ATP binding site of a target protein (e.g., MLH1 or PMS2).

[0135] The distribution and amount of compound I in a biological sample can be determined by methods known in the art. The specific method used will depend on the properties of the detection portion Q in compound I. Appropriately, Q is a fluorophore, and the location and amount of compound I in a biological sample can be determined by fluorescence microscopy / imaging. Appropriate calibration of fluorescence intensity may be used to determine the amount of compound I at various locations within the sample.

[0136] The assays and methods according to the present invention as defined herein may optionally further include one or more washing steps performed after step (i) to remove excess probe compounds of formula I or formula II from a biological sample.

[0137] In embodiments where the probe compound is a compound of formula II, the assay and method defined herein comprises the steps of: contacting a biological sample with the compound of formula II so that it can bind to the ATP binding site of a target protein; optionally, washing the biological sample once or more times to remove any unbound probe compound of formula II; and then contacting the biological sample with a suitable detection moiety Q that can react in situ with the probe compound of formula II in the biological sample to form a compound of formula I, or a compound of formula Q-L2-Y as defined herein. The sample may optionally be washed after incubation with the detection moiety to remove any unbound / unreacted detection moieties from the sample. The detection moiety may be a group Q as defined herein, supporting a suitable functional group that can react with the functional group X of the probe compound of formula II; or the detection moiety may be a compound of formula Q-L2-Y as defined herein, which reacts with the compound of formula II to form a compound of formula I as defined herein. [Examples]

[0138] <Analysis method> For the preparation of Examples 1–6 and related intermediates, commercially available starting materials, reagents, and dry solvents were used as supplied. Flash column chromatography was performed using Merck silica gel with a mesh size of 230–400. Column chromatography was also performed using glass column chromatography or flash chromatography. Flash chromatography was performed on a CombiFlash RF Teledyne Isco instrument. Preparative TLC was performed on Merck plates. 1 ¹H NMR spectra were recorded using a Bruker Avance-400. Samples were prepared as solutions in appropriate deuterated solvents, with a suitable internal non-deuterated solvent peak or tetramethylsilane used as a reference. Chemical shifts were recorded at a low magnetic field of ppm(δ) for tetramethylsilane.

[0139] For the preparation of Examples 7–12, solvents were purchased from Sigma Aldrich or Fisher Scientific and used without purification. NanoBRET® 590 SE, NanoBRET® 590-O4-SE, and NanoBRET® 590-C4-SE were obtained from Promega Corp. Madison, WI. Mass spectra were recorded using Thermo Fisher Vanquish® (LC-MS), and purity (≧95%) was determined by reversed-phase high-pressure liquid chromatography (RP-HPLC) using a Kinetex 5μm EVO C18 100Å LC Column 30×2.1mm column or a Phenomenex Synergi 2.5μm Max-RP 100Å LC column. The compound was purified using "Standard Method 1": initial mobile phase: 90% aqueous solution (0.1% TFA / H2O), 10% acetonitrile; final mobile phase: 0% aqueous solution, 100% acetonitrile; following a linear gradient for 30 minutes using a Waters XBridge Prep C18 OBD 30×250 mm column on a Waters LC Prep 150.

[0140] [HPLC method] (Method-A) The following conditions were used to analyze the sample: Agilent Series 1260 Infinity-II with PDA detector, column: Waters Sunfire C18, 150 × 4.6 mm, 3.5 microns, column temperature: room temperature, autosampler temperature: 15°C, mobile phase A: 0.05% (v / v) trifluoroacetic acid / Milli-Q water (pH approx. 2.5), mobile phase B: 100% acetonitrile; mobile phase gradient details: t=0 min (90% A, 10% B); gradient up to t=7 min (10% A, 90% B); gradient up to t=9 min (0% A, 100% B); t=14 min (0% A, 100% B); gradient up to t=14.01 min (90% A, 10% B); run ended at t=17 min (90% A, 10% B), flow rate: 1 mL / min, analysis time: 17 minutes.

[0141] [LC-MS method] (Method-A) Waters Acquity UPLC with binary solvent manager, PDA detector and Acquity QDA performance mass detector, Column: Ymc tri-art C18, 50 × 2.1 mm, 1.9 micron, Column temperature: 35°C, Autosampler temperature: 5°C, Mobile phase A: 0.1% (v / v) formic acid / ddH2O (pH=2.70), Mobile phase B: 0.1% formic acid (v / v) / water:acetonitrile (10:90), Mobile phase gradient details: t=0 min (97% A, 3% B) flow: 0.8 mL / min; t=0.75 min (97% A, 3% B) flow: 0.8 mL / min; Gradient up to t=2.7 min (2% A, 98% B) flow: 0.8 mL / min; Gradient up to t=3 min (0% A, 100% B) flow: 1 mL / min; t=3.5 min (0% A, 100% B) Flow: 1 mL / min; gradient up to t=3.51 min (97% A, 3% B) Flow: 0.8 mL / min; run ended at t=4 min (97% A, 3% B), flow rate: 0.8 mL / min, analysis time 4 min. Mass detector parameters: Ionization mode was periodically switched between positive and negative modes with cone voltages of 10 V and 30 V and a capillary voltage of 0.8 kV, and source and probe temperatures were 120 °C and 600 °C, respectively.

[0142] (Method-B) Waters Acquity UPLC with quaternary solvent manager, PDA detector and Acquity QDA mass detector, Column: Ymc tri-art C18, 50 × 2.1 mm, 1.9 micron, Column temperature: 35°C, Autosampler temperature: 5°C, Mobile phase A: 0.1% (v / v) formic acid / ddH2O (pH=2.70), Mobile phase B: 0.1% (v / v) formic acid / water:acetonitrile (10:90), Mobile phase gradient details: t=0 min (97% A, 3% B) flow: 0.8 mL / min; t=0.75 min (97% A, 3% B) flow: 0.8 mL / min; Gradient up to t=2.7 min (2% A, 98% B) flow: 0.8 mL / min; Gradient up to t=3 min (0% A, 100% B) flow: 1 mL / min; t=3.5 min (0% A, 100% B) Flow: 1 mL / min; gradient up to t=3.51 min (97% A, 3% B) Flow: 0.8 mL / min; run ended at t=4 min (97% A, 3% B), flow rate: 0.8 mL / min, analysis time 4 min. Mass detector parameters: The probe was an ESI capillary probe, and the ionization mode was periodically switched between positive and negative modes with cone voltages of 10 V and 30 V and a capillary voltage of 0.8 kV. The source and probe temperatures were 120 °C and 600 °C, respectively.

[0143] (Method-C) Waters Acquity with PDA and SQ detectors, column: X-bridge C18, 50×4.6mm, 3.5 microns, column temperature: 25℃, mobile phase A: 5mM ammonium bicarbonate / ddH2O (pH=7.35), mobile phase B: MeOH, mobile phase gradient details: t=0 min (92% A, 8% B); t=0.75 min (92% A, 8% B); gradient up to t=3 min (30% A, 70% B); gradient up to t=3.75 min (5% A, 95% B); gradient up to t=4.20 min (0% A, 100% B); t=5.20 min (0% A, 100% B); gradient up to t=5.21 min (92% A, 8% B); run ended at t=7 min (92% A, 8% B), flow rate: 1 mL / min, analysis time: 7 minutes. Mass detector parameters: The ionization mode was periodically switched between positive and negative modes with cone voltages of 10V and 30V and a capillary voltage of 3.25kV, and the source and probe temperatures were 120°C and 600°C, respectively.

[0144] (Method-D) Waters Acquity with PDA and SQ detectors, Column: X-bridge C18, 50 × 4.6 mm, 3.5 microns, Column temperature: 25°C, Mobile phase A: 5 mM ammonium bicarbonate / ddH2O (pH=7.35), Mobile phase B: Acetonitrile; Mobile phase gradient details: t=0 min (97% A, 3% B) flow: 0.5 mL / min; t=0.2 min (97% A, 3% B) flow: 0.5 mL / min; Gradient up to t=2.7 min (2% A, 98% B) flow: 0.5 mL / min; Gradient up to t=3 min (0% A, 100% B) flow: 0.7 mL / min; t=3.5 min (0% A, 100% B) flow: 0.7 mL / min; Gradient up to t=3.51 min (97% A, 3% B) flow: 0.5 mL / min; Run ended at t=4 min (97% A, 3% B), flow rate: 0.5 mL / min, analysis time: 4 minutes. Mass detection parameters: Ionization mode was periodically switched between positive and negative modes with cone voltages of 10V and 30V and capillary voltage of 3.25kV, and source and probe temperatures were 120°C and 600°C, respectively.

[0145] (Method-E) Waters Acquity UPLC with Binary Solvent Manager, PDA Detector and Acquity QDA Performance Mass Detector; Column: Xbridge BEH C18, 50 × 2.1 mm, 2.5 microns, Column temperature: 35°C, Autosampler temperature: 5°C; Mobile phase A: 0.1% (v / v) Formic acid / ddH2O (pH=2.70); Mobile phase B: 0.1% Formic acid (v / v) / Water:Acetonitrile (10:90); Mobile phase gradient details: t=0 min (97% A, 3% B) flow: 0.8 mL / min; t=0.75 min (97% A, 3% B) flow: 0.8 mL / min; Gradient up to t=2.7 min (2% A, 98% B) flow: 0.8 mL / min; Gradient up to t=3 min (0% A, 100% B) flow: 1 mL / min; t=3.5 min (0% A, 100% B) Flow: 1 mL / min; gradient up to t=3.51 min (97% A, 3% B) Flow: 0.8 mL / min; run ended at t=4 min (97% A, 3% B), flow rate: 0.8 mL / min, analysis time 4 min. Mass detector parameters: Ionization mode was periodically switched between positive and negative modes with cone voltages of 10 V and 30 V and a capillary voltage of 0.8 kV, and source and probe temperatures were 120 °C and 600 °C, respectively.

[0146] [Preparative HPLC method (for X)] Waters Quaternary-2545 gradient module with Waters 2489-UV / Visible wavelength detector, Column: YMC-TRI ART C18, 250×21.2mm, 5 microns, Column temperature: Room temperature, Mobile phase A: 5 mM ammonium bicarbonate / water, Mobile phase B: 100% acetonitrile; Mobile phase gradient details: T=0.01 min (50% A, 50% B); Gradient up to t=17 min (40% A, 60% B); Gradient up to t=17.01 min (2% A, 98% B); T=19 min (2% A, 98% B); Gradient up to t=19.01 min (50% A, 50% B); Run ended at t=21 min (50% A, 50% B), Flow rate: 17 mL / min, Analysis time: 21 minutes.

[0147] [Example 1] Preparation of 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-ium>

[0148] [ka] (5-((4-(2-(2-aminoethoxy)ethyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride (3.5 mg, 0.0073 mmol) was dissolved in DMF (1.0 ml) to which cyanine-5 NHS ester (CAS: 1263093-76-0, 5.1 mg, 0.0040 mmol) and N,N-diisopropylethylamine (3.8 μL, 0.22 mmol) were added. The reaction mixture was stirred in the dark at room temperature for 1 hour, and then concentrated under vacuum. Purification by silica gel chromatography (4g flash, 0-20% MeOH:CH2Cl2), followed by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H2O (0.1% HCO2H)), and subsequent lyophilization yielded the title compound as a blue solid (1.6mg, 23%). 1 1H NMR (DMSO-d 6, 400 MHz) δ 10.1 (br. s, 1H), 9.68 (s, 1H), 8.45-8.31 (m, 2H), 8.19 (s, 1H), 7.97-7.90 (m, 1H), 7.70-7.63 (m, 2H), 7.52-7.23 (m, 9H), 7.09 (s, 1H), 6.61 (t, J = 12.4 Hz, 1H), 6.46 (s, 1H), 6.36 (d, J = 14.0 Hz, 1H), 6.30 (d, J = 14.0 Hz, 1H), 4.90-4.74 (m, 4H), 4.17-4.09 (m, 2H), 3.64-3.83-3.71 (m, 2H), 3.65 (s, 3H), 3.39-3.03 (m, 9H), 2.16-2.08 (m, 2H), 1.75-1.67 (m, 12H), 1.60-1.51 (m, 2H), 1.41-1.35 (m, 2H), 1.32-1.27 (m, 2H), 1.19 (d, J = 7.2 Hz, 6H). It was estimated that 8 protons were obscured by the H2O peak; HPLC purity 97%; LC / MS R t =3.83 min;MS m / z:948.2[M+H] + .

[0149] [Example 2] Preparation of 3-(2-((1E,3E)-5-((Z)-3-(5-((2(2-(4-((2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-5-oxopentyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indoline-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indole-1-ium-1-yl)propane-1-sulfonic acid>

[0150] [ka] (5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride was dissolved in DMF (1.0 ml) (3.2 mg, 0.0066 mmol) to which AlexaFluor-647 NHS ester (5.0 mg, 0.0076 mmol) and N,N-diisopropylethylamine (2.6 μL, 0.20 mmol) were added. The reaction mixture was stirred in the dark at room temperature for 8 hours, and then diluted to a total volume of 1.5 mL with DMSO. The title compound was obtained as a blue solid (1.3 mg, 25%) by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H2O (10 mM NH4CO3)) followed by lyophilization. HPLC purity 91%; LC / MS R t =2.39min;MS m / z:662.8[M+2H] 2+ .

[0151] [Example 3] Preparation of 1-(6-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-iumformate>

[0152] [ka] (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5 mg, 0.0085 mmol) was dissolved in DMF (1.0 ml) and cyanine-5 NHS ester (CAS: 1263093-76-0, 3.9 mg, 0.0068 mmol) and N,N-diisopropylethylamine (22 μL, 0.13 mmol) were added. The reaction mixture was stirred in the dark at room temperature for 4 hours. The crude reaction mixture was purified by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H2O (0.1% HCO2H)), concentrated under vacuum, and lyophilized to obtain the title compound as a blue solid (3.0 mg, 38%). 1 1H NMR (DMSO-d 6 , 400 MHz) δ 10.89-9.55 (br. m, 1H), 8.44 (s, 1H), 8.39-8.30 (m, 2H), 7.74 (t, J = 5.6 Hz, 1H), 7.65-7.0 (m, 2H), 7.45-7.37 (m, 4H), 7.29-7.16 (m, 5H), 7.04 (s, 1H), 6.61-6.53 (m, 1H), 6.43 (s, 1H), 6.34-6.24 (m, 2H), 4.79-4.72 (m, 4H), 4.12-4.06 (m, 2H), 3.60 (s, 3H), 3.11 (quintet, J = 6.8, 1H), 3.02-2.95 (m, 2H), 2.37-2.17 (m, 10H), 2.11-2.02 (m, 2H), 1.69 (s, 12H), 1.59-1.51 (m, 3H), 1.40-1.30 (m, 6H), 1.25-1.21 (m, 3H), 1.14 (d, J = 6.8 Hz, 6H). 1×OH was not observed. Two protons were obscured by the H2O peak; LC / MS Rt = 3.86 min; MS m / z: 946.0 [M+H] + .

[0153] [Example 4] Preparation of 2-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-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)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-hydroxy-3-oxo-3H-xanthene-9-yl)benzoic acid>

[0154] [ka] (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.0 mg, 0.0085 mmol) was dissolved in DMF (1.0 ml) and NHS-fluorescein (5 / 6-carboxyfluorescein succinimidyl ester), a mixed isomer (CAS: 117548-22-8, 3.2 mg, 0.0068 mmol) and N,N-diisopropylethylamine (22 μL, 0.13 mmol) were added. The reaction mixture was stirred in the dark at room temperature for 2 hours. The crude reaction mixture was purified by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H2O (10 mM (NH4)2CO3)) and concentrated under vacuum to obtain the title compound as an orange powder (3.2 mg, 45%). 1 1H NMR (DMSO-d 6(400 MHz) δ 10.50-9.50 (m, 3H), 8.90-8.13 (m, 3H), 7.75-7.24 (m, 4H), 7.13 (s, 1H), 6.74-6.67 (m, 4H), 6.62-6.58 (m, 2H), 6.48 (s, 1H), 4.88-4.79 (m, 4H), 3.18 (quintuplet, J = 6.8, 1H), 2.50-2.24 (m, 8H), 1.69-1.28 (m, 6H), 1.22 (d, J = 6.8 Hz, 6H). Six protons were obscured by the H2O peak, and CO2H was not observed; LC / MS R t =3.32 min;MS m / z:839.7[M+H] + .

[0155] [Example 5] Preparation of 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)ammonium benzoate)

[0156] [ka] (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (3.8 mg, 0.0064 mmol) was dissolved in DMF (1.0 ml) and 5-TAMRA SE (5-carboxytetramethylrhodamine, succinimidyl ester) (CAS: 150810-68-7, 2.7 mg, 0.0052 mmol) and N,N-diisopropylethylamine (17 μL, 0.097 mmol) were added. The reaction mixture was stirred in the dark at room temperature for 2.5 hours. The crude reaction mixture was purified by reverse-phase HPLC (Sunfire C18, 20-80% MeCN:H2O (10 mM (NH4)2CO3)) and concentrated under vacuum to obtain the title compound as a purple powder (3.9 mg, 67%). 11H NMR (DMSO-d 6 , 400 MHz) δ 10.33-10.02 (m, 2H), 9.67 (s, 1H), 8.92-8.83 (m, 1H), 8.54-8.43 (m, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.39-7.18 (m, 7H), 7.11-7.03 (m, 2H), 6.62-6.49 (m, 5H), 6.44 (s, 1H), 4.82-4.73 (m, 4H), 3.60-3.52 (m, 2H), 3.14-2.84 (m, 19H), 1.90-1.56 (m, 4H), 1.41-1.32 (m, 2H), 1.15 (d, J = 6.8 Hz, 6H). Six protons were obscured by the H2O and DMSO peaks. 0.5 equivalent NH4CO3 adduct; LC / MS R t =3.09 min;MS m / z:893.8[M+H] + .

[0157] [Example 6] <(2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindorin-2-yl)methanone>

[0158] [ka]

[0159] [Synthesis scheme (see Figure 1)] [Tert-butyl-5-methylisoindoline-2,5-dicarboxylic acid (A)] 5-Bromoisoindoline-2-carboxylate tert-butyl (CAS#201940-08-1) (10.0 g, 33.5 mmol) was added to a MeOH:DMF (9:1, 100 mL) solution with TEA (14 mL, 137.81 mmol) at room temperature. The reaction mixture was degassed using N2 for 10-15 minutes. PdCl2 (dppf) (4.91 g, 6.71 mmol) was added to the reaction mixture at room temperature. 30 kg / cm³ was added to the reaction mixture. 2 CO(ガス The mixture was heated under pressure to 120°C and stirred for 16 hours. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude product was diluted with water (200 mL) and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were washed with brine solution (200 mL), dried over sodium sulfate, and concentrated under reduced pressure to obtain the crude product. This was purified by column chromatography (eluted with silica gel, 12% ethyl acetate / hexane) to obtain 2-(tert-butyl)5-methylisoindoline-2,5-dicarboxylic acid (A) as a brown solid (10 g).

[0160] LCMS (Method B): 2.657 min, MS: ES+ 278.18 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.45 (s, 9H), 3.84 (s, 3H), 4.61(t, J = 7.6 Hz, 4H), 7.43 - 7.47 (m, 1H), 7.86 - 7.91 (m, 2H).

[0161] [5-(Hydroxymethyl)isoindoline-2-carboxylate Tert-butyl(B)] A LiAlH4 solution (1M in THF) (36 mL, 36.10 mmol) was added dropwise at 0°C to a stirred solution of 2-(tert-butyl)5-methylisoindoline-2,5-dicarboxylic acid (A) (10 g, 36.07 mmol) in THF (200 mL). The reaction mixture was stirred at 0°C for 3 hours, then poured into a cold saturated ammonium chloride aqueous solution (100 mL), and extracted in ethyl acetate (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain 5-(hydroxymethyl)isoindoline-2-carboxylic acid tert-butyl (B) as a brown solid (5 g, 56%). This substance was used in the next step without further purification. LCMS (Method B): 2.061 min, MS: ES+ 250.18 (M+1); 1 1H NMR (DMSO-d 6, 400 MHz): δ 1.45 (s, 9H), 4.48 - 4.55 (m, 6H), 5.21 (br. s, 1H), 7.19 - 7.26 (m, 3H).

[0162] [5-Formylisoindoline-2-carboxylate Tert-butyl(C)] 5-(hydroxymethyl)isoindoline-2-carboxylate tert-butyl (B) (3 g, 12.04 mmol) was dissolved in DCM (30 mL) and activated MnO2 (10.36 g, 120.40 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered through a Celite bed and washed with 10% MeOH:DCM (3 × 50 mL). The combined filtrate was concentrated under vacuum to obtain 5-formylisoindoline-2-carboxylate tert-butyl (C) as a gray solid (2.6 g, 86%). This substance was used in the next step without further purification.

[0163] LCMS (Method B): 2.387 min, MS: ES+ 248.18 (M+1); 1 H NMR (CDCl3, 400 MHz): δ 1.54 (s, 9H), 4.73 - 4.76 (m, 4H), 7.40 - 7.45 (dd, J = 8.4, 7.6 Hz, 1H), 7.77 - 7.82 (m, 2H), 10.20 (s, 1H).

[0164] [4-(prop-2-in-1-yl)piperazine-1-carboxylate Tert-butyl(D)] To a solution of piperazine-1-carboxylate tert-butyl (15 g, 80.51 mmol) in acetonitrile (300 mL), K2CO3 (22.26 g, 161.30 mmol) was added at 0°C. Propargyl bromide (80% (w / v) toluene solution) (12 mL, 80.51 mmol) was added dropwise to the reaction mixture at 0°C. The resulting reaction mixture was slowly warmed to room temperature and stirred at room temperature for 1.5 hours. The reaction mixture was filtered and washed with acetonitrile (100 mL). The combined filtrate was concentrated under vacuum, and the resulting crude substance was purified by column chromatography (elution with silica gel, 3% (v / v) MeOH / DCM) to obtain 4-(prop-2-in-1-yl)piperazine-1-carboxylate tert-butyl as a brown solid (12.5 g, 71%).

[0165] LCMS (Method A): 0.937 min, MS: ES+ 225.20 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.38 (s, 9H), 2.36 (t, J = 4.8Hz, 4H), 3.18 (t, J = 2.4Hz, 1H), 3.27 - 3.34 (m, 6H).

[0166] [1-(prop-2-in-1-yl)piperazine(E)] 4M HCl / dioxane (20 mL) was added dropwise to a solution of 4-(prop-2-in-1-yl)piperazine-1-carboxylate tert-butyl (D) (2 g, 8.93 mmol) in DCM (10 mL) at 0°C under a nitrogen atmosphere. The reaction mixture was stirred at 0°C for 10-15 minutes. The reaction mixture was slowly warmed to room temperature and stirred at room temperature for 2 hours. The resulting reaction mixture was concentrated under vacuum and neutralized using methanolic ammonia (pH 7) added dropwise. The resulting precipitate was collected by filtration and dried under high vacuum to obtain 1-(prop-2-in-1-yl)piperazine (E) as a brown oil (1.45 g). This substance was used in the next step without further purification.

[0167] LCMS (Method A): 0.295 min, MS: ES+ 125.00 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 2.36 - 2.38 (m, 4H), 2.73 (t, J = 4.8Hz, 4H), 3.16 (t, J = 2.4Hz, 1H), 3.22 (d, J = 2.4 Hz, 2H), 3.58 (br. s, 1H).

[0168] [5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindoline-2-carboxylate Tert-butyl(F)] Acetic acid (4-5 drops) was added at room temperature to a 10 mL mixture of 5-formylisoindoline-2-carboxylate tert-butyl (C) (0.996 g, 4.03 mmol) and 1-(prop-2-in-1-yl)piperazine (E) (0.5 g, 4.03 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was cooled to 0°C. NaBH(OAc)3 (2.13 g, 10.05 mmol) was gradually added to the reaction mixture at 0°C. The reaction mixture was slowly warmed to room temperature and stirred at room temperature for 24 hours. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with saturated aqueous solution of NaHCO3 (50 mL), dried over sodium sulfate, filtered, and concentrated under vacuum. The crude substance was purified by flash chromatography (elution with silica gel, 2.5% (v / v) MeOH / DCM) to obtain 5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindoline-2-carboxylate tert-butyl(F) as an off-white solid (0.550 g, 77%).

[0169] LCMS (Method A): 1.588 min, MS: ES+ 356.27 (M+1); 1 1H NMR (DMSO-d 6, 400 MHz): δ 1.45 (s, 9H), 2.32 - 2.43 (m, 8H), 3.14 (s, 1H), 3.23 (d, J = 2.4 Hz, 2H), 3.43 (s, 2H), 4.54 - 4.55 (m, 4H), 7.18 - 7.25 (3H).

[0170] [5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindoline hydrochloride (G)] 4M HCl / dioxane (5 mL) was added dropwise at 0°C to a solution of 5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindoline-2-carboxylate tert-butyl (F) (0.55 g, 15.48 mmol) in DCM (5 mL). The reaction mixture was stirred at 0°C for 10-15 minutes. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The resulting mixture was concentrated under vacuum, the crude substance was pulverized with dimethyl ether (2 × 10 mL), and the resulting solid was concentrated under high vacuum to obtain 5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindoline hydrochloride (G) as a white solid (0.55 g). This substance was used in the next step without further purification.

[0171] LCMS (Method D): 1.24 min, MS: ES+ 256.46(M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): 3.24 - 3.43 (m, 6H), 3.69 (br. s, 1H), 3.90 (br. s, 2H), 4.36 (br. s, 3H), 4.50 - 4.51 (m, 4H), 7.48 (d, J = 7.6Hz, 1H), 7.62 (d, J = 8.8Hz, 2H), 10.13 (br. s, 1H), 11.70 (br. s, 1H).

[0172] [(2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindorin-2-yl)methanone (Example 6)] To a mixture of 5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindoline hydrochloride (G) (0.058 g, 0.198 mmol) and 2,4-dihydroxy-5-isopropylbenzoic acid (0.032 g, 0.163 mmol) in DMF (2 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.063 g, 0.33 mmol), HOBt (0.033 g, 0.24 mmol), and DIPEA (0.057 mL, 0.33 mmol) were added in a microwave glass tube at room temperature. The microwave glass tube was sealed and irradiated under microwave at 120 °C for 3 hours. The resulting reaction mixture was poured into ice-cold water (30 mL) and extracted with ELISA (3 × 30 mL). The combined organic layer was dried over sodium sulfate and concentrated under vacuum. The crude substance was purified by flash chromatography (elution with silica gel, 6.8% (v / v) MeOH / DCM) to obtain (2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-in-1-yl)piperazine-1-yl)methyl)isoindorin-2-yl)methanone (Example 6) as an off-white solid (0.032 g, 43%).

[0173] LCMS (Method A): 1.340 min, MS: ES+ 434.22 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): 1.07 - 1.13 (dd, J = 7.5, 6.8Hz, 6H), 2.32 - 2.45 (m, 8H), 3.05 - 3.11 (m, 1H), 3.14 (s, 1H), 3.23 (s, 2H), 3.43 (s, 2H), 4.74 - 4.76 (m, 4H), 6.39 (s, 1H), 7.03 (s, 1H), 7.20 - 7.29 (m, 3H), 9.62 (br. s, 1H), 10.32 - 10.10 (m, 1H).

[0174] [Example 7] <3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)propanamide>

[0175] [ka] (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.4 mg, 0.011 mmol) was packed into a 5 mL amber vial and DMF (1 mL) was added. N,N-diisopropylethylamine (5.9 μL, 0.034 mmol) was added to the stirred solution and stirring was continued for 5 minutes. NanoBRET® 590-SE (5.8 mg, 0.014 mmol) was added, the vial was capped, and the mixture was stirred in the dark for 2 hours. The mixture was diluted 1:1:0.01 acetonitrile, water, and TFA and purified by reverse-phase preparative HPLC using standard method 1. The product containing the fractions was pooled and concentrated under reduced pressure to obtain a purple film, which was dissolved in acetonitrile (10 mL) and concentrated to dry in three ways to remove spurious trifluoroacetic acid. The resulting residue was dried under high vacuum to obtain the product as a purple solid (8.1 mg, 91.1%).

[0176] C 44 H 53 BF5N7O4[M+H] + We calculated an MS(ESI, m / z): 792.4 and observed 792.5.

[0177] [Example 8] <1-(3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4λ4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)-3,6,9,12-tetraoxapentadecane-15-amide>

[0178] [ka] (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.0 mg, 0.010 mmol) was packed into a 5 mL amber vial and treated with DMF (0.5 mL). N,N-diisopropylethylamine (5.4 μL, 0.031 mmol) was added to the suspension and the solution was clarified. NanoBRET® 590 PEG-O4-SE (8.4 mg, 0.012 mmol) was dissolved in 0.5 mL of DMF and added to the reaction mixture using an additional 2 × 0.5 mL of DMF to quantitatively transfer the reactive dye. The mixture was capped and left to stand with stirring in the dark for 2 hours. The mixture was diluted 1:1:0.01 acetonitrile, water, and TFA and purified by reverse-phase preparative HPLC using standard method 1. The product containing the fraction was pooled and concentrated under reduced pressure to obtain a purple film. This film was dissolved in acetonitrile (10 mL), concentrated to dryness, and pseudotrifluoroacetic acid was removed. The resulting residue was dried under high vacuum to obtain the product as a purple solid (8.3 mg, 76.4%).

[0179] C 55 H 74 BF2N8O9[M+H] + We calculated an MS(ESI, m / z): 1039.6 and observed 1039.7.

[0180] [Example 9] <9-4-(3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-5l4,6l4-dipyrrole[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)butanamide>

[0181] [ka] (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.0 mg, 0.010 mmol) was packed into a 5 mL amber vial and DMF (1 mL) was added. N,N-diisopropylethylamine (7.6 μL, 0.042 mmol) was added to the stirred solution and stirring was continued for 5 minutes. NanoBRET 590(registered trademark)-C4-SE (6.4 mg, 0.012 mmol) was added, the vial was capped, and the mixture was stirred in the dark for 2 hours. The mixture was diluted 1:1:0.01 with acetonitrile, water, and TFA and purified by reverse-phase preparative HPLC using standard method 1. The product containing the fractions was pooled and concentrated under reduced pressure to obtain a purple film, which was dissolved in acetonitrile (10 mL) and concentrated to dry in three ways to remove pseudotrifluoroacetic acid. The resulting residue was dried under high vacuum to obtain the product as a purple solid (3.6 mg, 39.4%).

[0182] C 48 H 60 BF2N8O5[M+H] + We calculated an MS(ESI, m / z): 877.5 and observed 877.6.

[0183] [Example 10] <3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4l4,5l4-dipyrrole[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-1-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)propan-1-one>

[0184] [ka] (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazine-1-ylmethyl)isoindorin-2-yl)methanone hydrochloride (5.0 mg, 0.012 mmol) was packed into a 5 mL amber vial, and DMF (1 mL) was added. N,N-diisopropylethylamine (8.1 μL, 0.046 mmol) was added to the stirred solution, and stirring was continued for 5 minutes. NanoBRET® 590-SE (5.9 mg, 0.014 mmol) was added, the vial was capped, and the mixture was stirred in the dark for 2 hours. The mixture was diluted 1:1:0.01 with acetonitrile, water, and TFA, and purified by reverse-phase preparative HPLC using standard method 1. The product containing the fractions was pooled and concentrated under reduced pressure to obtain a purple film, which was dissolved in acetonitrile (10 mL) and concentrated to dry in three ways to remove pseudotrifluoroacetic acid. The resulting residue was dried under high vacuum to obtain the product as a purple solid (3.25 mg, 39.7%).

[0185] C 39 H 42 BF2N6O4[M+H] + The MS (ESI, m / z) was calculated and observed values ​​of 707.3 and 707.4.

[0186] [Example 11] <3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-4l4,5l4-dipyrrole[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(15-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)-15-oxo-3,6,9,12-tetraoxapentadecyl)propanamide>

[0187] [ka] (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazine-1-ylmethyl)isoindorin-2-yl)methanone hydrochloride (5.0 mg, 0.012 mmol) was packed into a 5 mL amber vial and treated with DMF (0.5 mL). N,N-diisopropylethylamine (8.1 μL, 0.046 mmol) was added to the mixture, and the resulting solution was stirred for 5 minutes. NanoBRET® 590 PEG-O4-SE (8.6 mg, 0.013 mmol) was dissolved in 0.5 mL of DMF, and this was added to the reaction mixture using an additional 2 × 0.5 mL of DMF to quantitatively transfer the reactive dye. The reaction mixture was capped and left to stand in the dark with stirring for 2 hours, then diluted 1:1:0.01 acetonitrile, water, and TFA, and purified by reverse-phase preparative HPLC using standard method 1. The product containing the fraction was pooled and concentrated under reduced pressure to obtain a purple film. This film was dissolved in acetonitrile (10 mL) and concentrated to dryness in three different ways to remove pseudotrifluoroacetic acid. The resulting residue was dried under high vacuum to obtain the product as a purple solid (4.8 mg, 43.6%).

[0188] C 50 H 63 BF2N7O9[M+H] + The MS (ESI, m / z) was calculated and observed values ​​of 954.5 and 954.6.

[0189] [Example 12] <3-(5,5-difluoro-7-(1H-pyrrole-2-yl)-5H-5l4,6l4-dipyrrole[1,2-c:2',1'-f][1,3,2]diazabolinin-3-yl)-N-(4-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)-4-oxobutyl)propanamide>

[0190] [ka] (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazine-1-ylmethyl)isoindorin-2-yl)methanone hydrochloride (5.0 mg, 0.012 mmol) was packed into a 5 mL amber vial and DMF (1 mL) was added. N,N-diisopropylethylamine (20.2 μL, 0.116 mmol) was added to the mixture and stirring was continued for 5 minutes. NanoBRET® 590-C4-SE (8.9 mg, 0.017 mmol) was added, the vial was capped, and the mixture was stirred in the dark for 2 hours. The mixture was diluted 1:1:0.01 with acetonitrile, water, and TFA and purified by reverse-phase preparative HPLC using standard method 1. The product containing the fractions was pooled and concentrated under reduced pressure to obtain a purple film, which was dissolved in acetonitrile (10 mL) and concentrated to dry in three ways to remove pseudotrifluoroacetic acid. The resulting residue was dried under high vacuum to obtain the product as a purple solid (2.3 mg, 24.7%).

[0191] C 43 H 49 BF2N7O5[M+H] + The MS (ESI, m / z) was calculated and observed values ​​of 792.4 and 792.5.

[0192] <Preparation of intermediates> Preparation of (5-((4-(2-(2-aminoethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride (Figure 2) [(2-(2-iodoethoxy)ethyl)carbamate Tert-butyl(H)] To a solution of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (CAS#139115-91-6) (5.2 g, 25.36 mmol) in DCM (60 mL), triphenylphosphine (7.98 g, 30.43 mmol) and imidazole (0.569 g, 8.36 mmol) were added at room temperature. The reaction mixture was cooled to 0°C. Iodine (8.36 g, 32.92 mmol) was gradually added to the reaction mixture at 0°C. The reaction mixture was slowly warmed to room temperature and stirred for 20 hours. The reaction mixture was poured into water (100 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. Dimethyl ether (100 mL) was added to the crude product, the resulting precipitate was filtered, and washed with dimethyl ether (3 × 50 mL). The combined filtrate was concentrated under reduced pressure, and the crude substance was purified by column chromatography (eluting with silica gel, 20% (v / v) ethyl acetate / hexane) to obtain (2-(2-iodoethoxy)ethyl)carbamate tert-butyl(H) as a colorless oil (7.8 g, 98%).

[0193] LCMS (Method B): 2.254 min, MS: ES+ 316.13 (M+1); 1 1H NMR (DMSO-d 6 ,400 MHz): δ 1.36 (s, 9H), 3.04 - 3.08 (m, 2H), 3.30 (t, J = 6.4Hz, 2H), 3.40 (t, J = 6.4Hz, 2H), 3.62 (t, J = 6.4Hz, 2H), 6.08 (s, 1H).

[0194] [4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-carboxylate benzyl(I)] To a solution of tert-butyl(H) (2.0 g, 6.35 mmol) (2-(2-iodoethoxy)ethyl)carbamate and benzylpiperazine-1-carboxylate (CAS#31166-44-6) (1.25 g, 5.72 mmol) in acetone (20 mL), K2CO3 (2.45 g, 17.75 mmol) and NaI (0.38 g, 2.54 mmol) were added at room temperature. The reaction mixture was heated at 70 °C for 16 hours. The reaction mixture was concentrated under vacuum, diluted with water (100 mL), and extracted with ethyl acetate (3 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude substance was purified by flash chromatography (elution with silica gel, 2.5% (v / v) MeOH / DCM) to obtain 4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-carboxylate benzyl(I) as a yellow oil (2.0 g, 77%).

[0195] LCMS (Method A): 1.640 min, MS: ES+ 408.32 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1. 35 (s, 9H), 2.38 (t, J = 4.8Hz, 4H), 2.44 - 2.47 (m, 2H), 3.02 - 3.06 (m, 2H), 3.33 - 3.36 (m, 6H), 3. 48 (t, J = 5.6Hz, 2H), 5.06 (s, 2H), 6.80 (t, J = 5.2Hz, 1H), 7.29 - 7.39 (m, 5H).

[0196] [(2-(2-(piperazin-1-yl)ethoxy)ethyl)carbamate tert-butyl(J)] 4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-carboxylate benzyl(I) (1.5 g, 3.68 mmol) and acetic acid (0.23 mL, 4.03 mmol) in MeOH (7 mL) were mixed with 10% Pd(OH)2 / carbon (0.45 g) at room temperature. The reaction mixture was bubbling with H2 (g) for 4 hours. The reaction mixture was filtered through a Celite bed, washed with MeOH (2 × 10 mL), and the combined filtrate was concentrated under vacuum and then freeze-dried to obtain (2-(2-(piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl(J) as oil (0.971 g, 96%). This substance was used in the next step without further purification.

[0197] 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1. 36 (s, 9H), 2.39 - 2.42 (m, 6H), 2.71 (t, J = 4.8Hz, 4H), 3.02 - 3.06 (m, 2H), 3.34 (t, J = 6.0Hz, 2H), 3.45 (t, J = 5.6 Hz, 2H), 5.76 (s, 1H), 6.80 (t, J = 5.2Hz, 1H).

[0198] [Isoindoline-5-carbaldehyde hydrochloride (K)] A 4M HCl / dioxane solution (20 mL) was added dropwise to a DCM (20 mL) solution of 5-formylisoindoline-2-carboxylate tert-butyl (C) (1.3 g, 5.26 mmol) at 0°C. The reaction mixture was slowly warmed to room temperature and stirred for 6 hours. The reaction mixture was concentrated under reduced pressure, and the crude substance was purified by grinding with dimethyl ether (3 × 20 mL). The resulting solid was dried under high vacuum to obtain isoindoline-5-carbaldehyde hydrochloride (K) as an off-white solid (0.8 g, 83%).

[0199] LCMS (Method C): 2.154 min, MS: ES+ 148.18 (M+1); 1 1H NMR (DMSO-d 6, 400 MHz) δ 4.57 (d, J = 4.0Hz, 4H), 7.62 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.6 Hz, 2H), 10.21 (s, 1H), 10.63 (s, 1H).

[0200] [5-Formylisoindoline-2-carboxylate benzyl(L)] To a solution of isoindoline-5-carbaldehyde hydrochloride (K) (0.8 g, 4.37 mmol) in THF (10 mL), TEA (2.30 mL, 13.02 mmol) was added at room temperature. The reaction mixture was cooled to 0°C. Benzyl chloroformate (0.745 g, 4.38 mmol) was added dropwise to the reaction mixture at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. The reaction mixture was concentrated under vacuum, diluted with 2 M HCl (10 mL), and extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with water (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude material was purified by column chromatography (eluted with silica gel, 25% (v / v) ethyl acetate / hexane) to obtain 5-formylisoindoline-2-carboxylate benzyl (L) as a yellow solid (1.0 g, 82%).

[0201] 1 1H NMR (DMSO-d 6 , 400 MHz) δ 4.72 - 4.80 (m, 4H), 5.15 (s, 2H), 7.32 - 7.43 (m, 5H), 7.53 - 7.58 (m, 1H), 7.84 - 7.88 (m, 2H), 9.99 (d, J = 1.6Hz, 1H).

[0202] [5-((4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate benzyl(M)] To a solution of 5-formylisoindoline-2-carboxylate benzyl(L) (1.0 g, 3.56 mmol) and acetic acid (0.1 mL) in DCM (20 mL), tert-butyl(J) (2-(2-(piperazin-1-yl)ethoxy)ethyl)carbamate (0.971 g, 3.56 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 5 hours. NaBH(OAc)3 (4.52 g, 21.35 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was poured into a saturated aqueous solution of NaHCO3 (50 mL) and extracted in DCM (2 × 150 mL). The combined organic layers were dried over sodium sulfate and concentrated under vacuum. The crude substance was purified by flash chromatography (elution with silica gel, 7% (v / v) MeOH / DCM) to obtain 5-((4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate benzyl (M) as a brown oil (0.9 g, 48%).

[0203] LCMS (Method C): 4.34 min, MS: ES+ 539.64 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1. 34 (s, 9H), 2.32 - 2.43 (m, 8H), 3.02 - 3.04 (m, 2H), 3.31 - 3.34 (m, 2H), 3.42 - 3.46 (m, 4H), 4.48 - 4.49 (m, 1H), 5.22 (t, J = 5.6Hz, 1H), 4.62 - 4.68 (m, 4H), 5.14 (s, 2H), 6.72 - 6.77 (t, J = 5.2Hz, 1H), 7.20 - 7.42 (m, 8H).

[0204] [(2-(2-(4-(isoindoline-5-ylmethyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl(N)] 5-((4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate benzyl(M) (0.1 g, 0.186 mmol) was added to a solution of EtOH (5 mL) with 10% Pd / C (50% water) (0.020 g) at room temperature. The reaction mixture was bubbling with H2 (g) for 12 hours. The reaction mixture was filtered through a Celite bed and washed with MeOH (2 × 5 mL). The combined filtrate was concentrated under vacuum to obtain (2-(2-(4-(isoindoline-5-ylmethyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl(N) as a brown oil (0.076 g, 100%). This substance was used in the next step without further purification.

[0205] LCMS (Method A): 1.036 min, MS: ES+ 405.32 (M+1)

[0206] [(2-(2-(4-((2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl(O)] To a mixture of (2-(2-(4-(isoindoline-5-ylmethyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl(N) (0.076 g, 0.187 mmol) and 2,4-dihydroxy-5-isopropylbenzoic acid (0.030 g, 0.153 mmol) in DMF (2 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.059 g, 0.307 mmol), HOBt (0.020 g, 0.148 mmol), and DIPEA (0.05 mL, 0.305 mmol) were added in a microwave glass tube at room temperature. The tube was sealed and irradiated under microwave at 120°C for 1 hour. The resulting reaction mixture was poured into ice-cold water (20 mL) and extracted with ELISA (5 × 30 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to obtain the crude product, which was purified by flash chromatography (eluting with silica gel, 8% (v / v) MeOH / DCM) to obtain (2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl(O) as a brown oil (0.020 g, 18%).

[0207] LCMS (Method A): 1.728 min, MS: ES+ 583.5 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.12 (d, J = 6 Hz, 6H), 1.34 (s, 9H), 2.23 - 2.33 (m, 10H), 3.04 (s, 4 H), 3.46 (s, 4H), 4.75 (s, 4H), 6.39 (s, 1H), 6.78(br. s, 1H), 7.02 (br. s, 1H), 7.20 - 7.28 (m, 4H), 9.63 (s, 1H), 10.09 (d, J = 24.4Hz, 1H).

[0208] [(5-((4-(2-(2-aminoethoxy)ethyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride] To a solution of (2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)carbamate tert-butyl(O) (0.015 g, 0.0257 mmol) in DCM (1 mL), 4 M HCl / dioxane (0.2 mL) was added at 0°C under a nitrogen atmosphere. The reaction mixture was stirred at 0°C for 4 hours. The resulting reaction mixture was concentrated under vacuum to obtain a crude substance, which was pulverized with dimethyl ether (2 × 10 mL) and dried under high vacuum to obtain (5-((4-(2-(2-aminoethoxy)ethyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride as a brown oil (0.012 g, 90%).

[0209] LCMS (Method B): 1.456 min, MS: ES+ 483.5 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.12 (d, J = 6.8Hz, 6H), 1.29 - 1.30 (m, 4H), 2.99 - 3.07 (m, 4H), 3.25 - 3.30 (m, 4H), 3.62 - 3.63 (m, 3H), 3.76 - 3.77 (m, 2H), 4.32 - 4.37 (br. s, 2H), 4.75 - 4.80 (m, 4H), 6.43 (s, 1H), 7.01 (s, 1H), 7.38 - 7.52 (m, 3H), 8.10 (br. s, 3H), 9.68 (bs, 1H), 10.03 (bs, 1H).

[0210] <Preparation of 2,4-dihydroxy-5-isopropylbenzoic acid (Figure 3)> [2,4-Dihydroxybenzoate Methyl (P)] To a stirred solution of 2,4-dihydroxybenzoic acid (100 g, 649.35 mmol) in methanol (400 mL), concentrated H2SO4 (50 mL) was added at room temperature. The reaction mixture was heated to 100 °C and stirred for 12 hours. The resulting solution was concentrated under vacuum and poured into water (500 mL). The resulting precipitate was collected by filtration and dried under high vacuum to obtain methyl 2,4-dihydroxybenzoate (P) as an off-white solid (170 g, 78%). This substance was used in the next step without further purification.

[0211] LCMS (Method C): 4.24 min, MS: ES+ 169. 19(M+1); 1 H NMR (CDCl3, 400 MHz) δ 3.39 (s, 3H), 5.72 (br. s, 1H), 6.38 - 6.42 (m, 2H), 7.74 (d, J = 8.8Hz, 1H), 11.02 (s, 1H).

[0212] [2,4-Dihydroxy-5-isopropylmethyl benzoate (Q)] To a solution of methyl 2,4-dihydroxybenzoate (80 g, 475.76 mmol) in DCM (1200 mL), anhydrous AlCl3 (126.87 g, 951.48 mmol) was added, and then isopropyl bromide (351.09 g, 2854.62 mmol) was added in three equal parts (117.03 g each) at 50°C every 6 hours. After the addition of isopropyl bromide was complete, the reaction was heated at 50°C for 6 hours. The total reaction time was 24 hours. The reaction mixture was concentrated under vacuum, the crude product was diluted with water (1000 mL), and extracted with ethyl acetate (4 × 500 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum. The obtained crude substance was purified three times by column chromatography (eluting with silica gel, 3% (v / v) Âxane) to obtain methyl 2,4-dihydroxy-5-isopropylbenzoate (Q) as a yellow solid (25.5 g, 26%).

[0213] LCMS (Method A): 2.069 min, MS: ES+ 211.11 (M+1);1 H NMR (DMSO, 400 MHz) δ 1.13 (d, J = 7.6Hz, 6H), 3.03 - 3.16 (m, 1H), 3.83 (s, 3H), 6.35 (s, 1H), 7.49 (s, 1H), 10.48 (s, 1H), 10.57 (s, 1H).

[0214] [2,4-Dihydroxy-5-isopropylbenzoic acid] To a solution of methyl 2,4-dihydroxy-5-isopropylbenzoate (Q) (23.0 g, 109.40 mmol) in MeOH:water (2:1, 300 mL), NaOH (21.88 g, 547.00 mmol) was added at room temperature. The reaction mixture was heated at 70 °C for 4 hours. The reaction mixture was cooled to room temperature, diluted with ice-cold water (500 mL), and acidified with 1 M HCl (pH 5). The resulting precipitate was collected by filtration and dried under high vacuum to obtain 2,4-dihydroxy-5-isopropylbenzoic acid as a white solid (13 g, 61%).

[0215] LCMS: (Method B) 2.044 min, MS: ES+ 197.22 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.02 (d, J = 6.8Hz, 6H), 3.02 - 3.09 (m, 1H), 6.34 (s, 1H), 7.48 (s, 1H), 10.44 (s, 1H), 11.24 (s, 1H), 13.33 (br. s, 1H).

[0216] Preparation of (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (Figure 4) [(5-hydroxypentyl)carbamate tert-butyl(R)] A mixture of 5-aminopentan-1-ol (CAS#2508-29-4) (5g, 48.54 mmol) in DCM (50 mL) was cooled to 0°C. Anhydrous Boc (11.16 mL, 48.46 mmol) was added to the reaction mixture at 0°C. The resulting reaction was warmed to room temperature and stirred for 16 hours. The reaction mixture was poured into water (150 mL) and extracted in DCM (2 × 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude product was purified by flash chromatography (eluted with silica, 18% (v / v) ethyl acetate / hexane) to obtain (5-hydroxypentyl)carbamate tert-butyl(R) as a pale yellow oil (15g, 76%).

[0217] 1 H NMR (CDCl3, 400 MHz): δ 1.34 - 1.40 (m, 2H), 1.43 (s, 9H), 1.46 - 1.51 (m, 2H), 1.54 - 1.61 (m, 2H), 1.97 (br. s, 1H), 3.10 - 3.11 (m, 2H), 6.63 (t, J = 6.40 Hz, 2H), 4.63 (bs, 1H).

[0218] [(5-bromopentyl)carbamate tert-butyl(S)] To a mixture of (5-hydroxypentyl)carbamate tert-butyl(R) (8 g, 39.41 mmol) in THF (80 mL), triphenylphosphine (15.48 g, 59.11 mmol) and CBr4 (19.60 g, 59.11 mmol) were added at 0°C. The resulting reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was poured into water (150 mL) and extracted with ethyl acetate (3 × 150 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude material was purified by column chromatography (eluting with silica gel, 10% (v / v) ethyl acetate / hexane) to obtain (5-bromopentyl)carbamate tert-butyl(S) as a colorless oil (9 g, 86%).

[0219] LCMS (Method A): 2.150 min, MS: ES+ 210.1, 212.1 (M-56); 1 H NMR (CDCl3, 400 MHz): δ 1.32 - 1.36 (m, 13H), 1.75 - 1.79 (m, 2H), 2.88 - 2.90 (m, 2H), 3.51 (t, J = 6.8 Hz, 2H), 6.81 (t, J = 5.6 Hz, 1H).

[0220] [4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-carboxylate benzyl(T)] To a mixture of benzylpiperazine-1-carboxylate (CAS#31166-44-6) (4.135 g, 18.79 mmol) and acetone (50 mL), K2CO3 (7.78 g, 56.38 mmol) and NaI (1.40 g, 9.39 mmol) were added at room temperature. The reaction was stirred at room temperature for 15 minutes. (5-bromopentyl)carbamate tert-butyl(S) (5 g, 18.79 mmol) was added to the reaction mixture at room temperature. The reaction mixture was heated at 70 °C for 16 hours. The resulting reaction mixture was cooled to room temperature, diluted with water (150 mL), and extracted with ethyl acetate (3 × 150 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain the crude product, which was purified by flash chromatography (elution with silica gel, 3% (v / v) MeOH / DCM) to obtain 4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-carboxylate benzyl (T) as a brown oil (6.5 g, 85%).

[0221] LCMS (Method A): 1.492 min, MS: ES+ 406.42 (M+1); 1 1H NMR (DMSO-d 6, 400 MHz): δ 1.21 - 1.23 (m, 2H), 1.35 (s, 11H), 2.28 - 2.32 (m, 6H), 2.85 - 2.90 (m, 2H), 3.32 - 3.34 (m, 6H), 5.06 (m, 2H), 6.77 (s, 1H), 7.31 - 7.38 (m, 5H).

[0222] [(5-(piperazin-1-yl)pentyl)carbamate tert-butyl(U)] To a solution of 4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-carboxylate benzyl(T) (6.4 g, 15.80 mmol) in MeOH (70 mL), 10% Pd / C (50% water) (0.640 g, 10% w / w) was added at room temperature. The reaction mixture was purged with H2 (g) for 5 hours. The reaction mixture was filtered through a Celite bed, washed with MeOH (2 × 10 mL), and the combined filtrate was concentrated under reduced pressure to obtain (5-(piperazine-1-yl)pentyl)carbamate tert-butyl(U) as a brown oil (4 g, 93%). This substance was used in the next step without further purification.

[0223] MS: ES+ 272.20 (M+1). 1 H NMR (CDCl3, 400 MHz): δ 1.28 - 1.35 (m, 2H), 1.43 (s, 9H), 1.43 - 1.50 (m, 2 H), 2.14 (br. s, 3H), 2.29 - 2.33 (m, 2H), 2.42 (bs, 4H), 2.90 - 2.92 (m, 3H), 3.10 - 3.11 (m, 2H), 3.47 (s, 1H), 4.61 (bs, 1H).

[0224] [5-((4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate benzyl(V)] Acetic acid (0.2 mL) was added at room temperature to a mixture of benzyl 5-formylisoindoline-2-carboxylic acid (L) (1.6 g, 5.69 mmol) and (5-(piperazin-1-yl)pentyl)carbamate tert-butyl (U) (1.54 g, 5.69 mmol) in DCM (50 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0°C. NaBH(OAc)3 (3.618 g, 17.078 mmol) was added to the reaction mixture at 0°C. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was poured into water (50 mL) and extracted in DCM (2 × 150 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum. The crude substance was purified by flash chromatography (elution with silica gel, 3% (v / v) MeOH / DCM) to obtain 5-((4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate benzyl(V) as a brown oil (2.2g, 72%).

[0225] LCMS (Method A): 1.552 min, MS: ES+ 537.41 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.15 - 1.23(m, 2H), 1.36 (s, 11H), 1.51 (br. s, 2H), 1.90 (s, 1H), 2.32 - 2.39 (m, 2H), 2.88 - 2.95 (m, 7H), 3.53 (br. s, 2H), 4.63 - 4.69 (m, 4H), 5.14 (s, 2H), 6.82 (br. s, 1H), 7.23 - 7.42 (m, 8H), 10.04 (br. s, 1H), 11.99 (br. s, 1H).

[0226] [(5-(4-(isoindoline-5-ylmethyl)piperazine-1-yl)pentyl)carbamate tert-butyl(W)] To a solution of 5-((4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate benzyl(V) (2.1 g, 3.92 mmol) in EtOH (60 mL), 10% Pd / C (50% water) (0.420 g, 20% w / w) was added at room temperature. The reaction mixture was purged with H2 (g) for 5 hours. The reaction mixture was filtered through a Celite bed, washed with MeOH (2 × 20 mL), and the combined filtrate was concentrated under vacuum to obtain (5-(4-(isoindoline-5-ylmethyl)piperazine-1-yl)pentyl)carbamate tert-butyl(W) as a brown oil (1.7 g, quantified). This substance was used without further purification.

[0227] LCMS (Method A): 0.757 min, MS: ES+ 403.37 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.19 - 1.22 (m, 2H), 1.36 (s, 13H), 2.19 - 2.21 (m, 2H), 2.23 - 2.27 (m, 3H), 2.28 - 2.32 (m, 3H), 2.86 - 2.87 (m, 2H), 2.96 - 2.97 (m, 1H), 3.40 - 3.43 (m, 4H), 4.32 - 4.33 (m, 4H), 6.77 - 6.79 (m, 1H), 7.15 - 7.29 (m, 3H).

[0228] [(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)pentyl)carbamate tert-butyl(X)] To a mixture of (5-(4-(isoindoline-5-ylmethyl)piperazine-1-yl)pentyl)carbamate tert-butyl (W) (0.504 g, 1.253 mmol) and 2,4-dihydroxy-5-isopropylbenzoic acid (0.205 g, 1.046 mmol) in DMF (8 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.399 g, 2.081 mmol), HOBt (0.141 g, 1.042 mmol), and DIPEA (0.428 mL, 2.502 mmol) were added in a microwave glass tube at room temperature. The microwave glass tube was sealed and irradiated under microwave at 120 °C for 3 hours. The resulting reaction mixture was poured into cold water (50 mL) and extracted with SiO2 (3 × 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under vacuum to obtain the crude substance (270 mg). 180 mg of the crude substance was purified by HPLC (5 mM ammonium bicarbonate in water / MeCN). The isolated fraction was freeze-dried to obtain (5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamate tert-butyl(X) as an off-white solid (0.032 g, 5%).

[0229] LCMS (Method A): 1.728 min, MS: ES+ 583.5 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.12 (d, J = 6 Hz, 6H), 1. 20 - 1.23 (m, 4H), 1.34 (s, 9H), 2.33 - 2.50 (m, 10H), 3.03 - 3.04 (m, 4H), 3.35 - 3.46 (m, 4H), 4.74 - 4.75 (m, 4H), 6.39 (s, 1H), 6.78 (s, 1H), 7.07 (s, 1H), 7.20 - 7.28 (m, 3H), 9.63 (s, 1H), 10.03 - 10.09 (d, J = 24.4 Hz, 1H).

[0230] [(5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindoline-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone] (5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamate tert-butyl(X) (0.060 g, 0.103 mmol) was added to a solution of DCM (2 mL) with 4 M HCl / dioxane (0.6 mL) at 0°C. The reaction mixture was stirred at 0°C for 1 hour. The resulting reaction mixture was concentrated under vacuum, and the crude product was pulverized with dimethyl ether (2 × 5 mL). The resulting solid was freeze-dried to obtain (5-((4-(5-aminopentyl)piperazine-1-yl)methyl)isoindorin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone as a brown viscous solid (0.043 g, 80%).

[0231] LCMS (Method B): 1.625 min, MS: ES+ 481.50 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.13 (d, J = 6.8 Hz, 6H), 1.32 - 1.34 (m, 2H), 1.52 - 1.57 (m, 2H), 1.70 - 1.72 (m, 2H), 2.81 - 2.83 (m, 2H), 3.20 - 3.30 (m, 5H), 3.40 - 3.50 (m, 3H), 4.35 (m, 2H), 4.77 - 4.80 (m, 4H), 6.45 (s, 1H), 7.02 (s, 1H), 7.40 - 7.54 (m, 3H), 7.93 (br. s, 3H), 9.68 (bs, 1H), 10.38 (br. s, 1H), 11.78 - 12.09 (br. s, 2H).

[0232] Preparation of 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-carboxylate benzyl (AA) and (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazine-1-ylmethyl)isoindorin-2-yl)methanone hydrochloride (AB) (Figure 5) [5-((4-((benzyloxy)carbonyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate Tert-butyl(Y)] A mixture of 5-formylisoindoline-2-carboxylate tert-butyl(C) (see Example 6) (3.0 g, 12.13 mmol) and benzylpiperazine-1-carboxylate (CAS: 31166-44-6) (3.2 g, 14.54 mmol) in MeOH (10 mL) was evaporated to dryness at 40°C (to remove condensed water). The residue was dissolved in dichloromethane (50 mL) and evaporated to dryness (to remove trace amounts of methanol). This process was repeated once to complete the removal of methanol. The resulting residue was dissolved in DCM (90 mL), placed under a nitrogen atmosphere, and cooled to 0°C. NaBH(OAc)3 (3.8 g, 17.93 mmol) was gradually added to the reaction mixture at 0°C. The reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The previous preparation was carried out in two parallel batches, and the combined reaction mixture was poured into water (200 mL) and extracted into DCM (3 × 250 mL). The combined organic layer was washed with saturated sodium bicarbonate aqueous solution (2 × 100 mL), dried over sodium sulfate, filtered, and concentrated under vacuum to obtain the crude product, which was purified by flash chromatography (eluted with silica gel, 25% (v / v) siRNA / n-hexane) to obtain 5-((4-((benzyloxy)carbonyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate tert-butyl (Y) as a brown oil (5.0 g, 46%).

[0233] LCMS (Method A): 1.631 min, MS: ES+ 452.32 (M+1); 1 1H NMR (DMSO-d 6, 400 MHz): δ 1.45 (s, 9H), 2.30 - 2.33 (m, 4H), 3.33 - 3.39 (m, 4H), 3.48 (s, 2H), 4.56 (d, J = 8.8 Hz, 4H), 5.07 (s, 2H), 7.22 - 7.37 (m, 8H).

[0234] [4-(isoindoline-5-ylmethyl)piperazine-1-carboxylate benzyl hydrochloride (Z)] To a solution of 5-((4-((benzyloxy)carbonyl)piperazine-1-yl)methyl)isoindoline-2-carboxylate tert-butyl (Y) (1.0 g, 2.21 mmol) in DCM (20 mL), 4 M HCl / dioxane (5.0 mL) was added at 0°C. The reaction mixture was stirred at 0°C for 2 hours, then concentrated under vacuum, and the crude product was pulverized with dimethyl ether (3 × 10 mL). The resulting solid was dried under high vacuum to obtain 4-(isoindoline-5-ylmethyl)piperazine-1-carboxylate benzyl hydrochloride (Z) as a green solid (0.99 g, quantified).

[0235] LCMS (Method E): 0.865 min, MS: ES+ 352.22 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 3.02 - 3.04 (m, 2H), 3.26 - 3.29 (m, 2H), 3.36 - 3.46 (m, 2H), 3.60 - 3.62 (m, 4H), 4.06 - 4.10 (m, 2H), 4.32 - 4.34 (m, 2H), 4.50 - 4.52 (m, 4H), 5.11 (s, 2H), 7.32 - 7.40 (m, 5H), 7.48 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 8 Hz, 1H), 10.23 (br. s, 2H), 11.71 (br s, 1H).

[0236] [4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-carboxylate benzyl(AA)] To a mixture of 2,4-dihydroxy-5-isopropylbenzoic acid (0.205 g, 1.046 mmol) in DMF (10 mL), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (0.97 g, 5.06 mmol), HOBt (0.340 g, 2.51 mmol), and DIPEA (0.65 g, 5.03 mmol) were added in a microwave glass tube at room temperature. 4-(isoindoline-5-ylmethyl)piperazine-1-carboxylate benzyl hydrochloride (Z) (0.980 g, 2.52 mmol) was added to the reaction at room temperature. The microwave glass tube was sealed and irradiated with microwaves at 120°C for 1 hour. The resulting reaction mixture was poured into cold water (60 mL), and as soon as the solid precipitated, it was isolated and dried under vacuum to obtain 0.8 g of crude substance. Purification by flash chromatography (elution with silica gel, 1.5% (v / v) MeOH / DCM) yielded 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-carboxylate benzyl as an off-white solid (0.395 g, 30%).

[0237] LCMS (Method E): 1.567 min, MS: ES+ 530.22 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.10 - 1.19 (m, 6H), 2.30 - 2.32 (m, 4H), 3.06 - 3.13 (m, 1H), 3.39 - 3.43 (m, 4H), 3.48 (s, 2H), 4.66 - 4.76 (m, 4H), 5.07 (s, 2H), 6.39 (s, 1H), 7.04 (s, 2H), 7.22 - 7.28 (m, 2H), 7.30 - 7.39 (m, 5H), 9.61 (s, 1H), 10.06 (s, 1H).

[0238] [(2,4-dihydroxy-5-isopropylphenyl)(5-(piperazine-1-ylmethyl)isoindoline-2-yl)methanone hydrochloride (AB)] 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)-piperazine-1-carboxylate benzyl(AB) (0.30 g, 0.56 mmol) was dissolved in EtOH (20 mL), to which AcOH (0.6 mL) and 10% Pd / C (50% water) (0.20 g, 20% w / w) were added at room temperature. The reaction mixture was cooled to 10°C-15°C and purged with H2 (g) for 2 hours. The reaction mixture was filtered through a Celite bed and washed with 10% MeOH:DCM (200 mL) to obtain the crude product (0.40 g), which was purified by preparative HPLC using (0.05% HCl / water and acetonitrile). The obtained fraction was freeze-dried to obtain (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazine-1-ylmethyl)isoindorin-2-yl)methanone hydrochloride as an off-white solid (0.20 g, 82%).

[0239] LCMS (Method E): 1.072 min, MS: ES+ 396.27 (M+1); 1 1H NMR (DMSO-d 6 , 400 MHz): δ 1.12 - 1.14 (m, 6H), 3.07 - 3.10 (m, 1H), 3.40 - 3.47 (m, 8H), 4.36 - 4.37 (m, 2H), 4.77 - 4.49 (m, 4H), 6.49 (s, 1H), 7.01 (s, 1H), 7.38 - 7.59 (m, 3H), 9.73 (s, 1H), 9.92 (s, 1H), 10.05 (s, 1H).

[0240] <Protein dependence of fluorescence polarization> Eleven 1 / 2 serial dilutions (11 points 1 in 2 serial dilutions) of the protein (either recombinant N-terminal PMS2 (residues 1-365) or recombinant N-terminal MLH1 (residues 15-340)) were constructed in assay buffer (25 mM HEPES, pH 7.5, 250 mM NaCl, 10 mM MgCl2, 0.01% Triton X-100, 5 mM dithiothreitol) on a clear polypropylene plate (Greiner Bio-One, product number 784201). The highest concentration in the serial dilution was 2 μM. The 12th concentration point was buffer only (no protein control).

[0241] Serial dilutions were used as 2× protein, and 20 μL was added to a Black Fluotrac 200 384-well medium-bound plate (Greiner Bio-One, t. 781076). The plate was centrifuged at 250×g for 1 minute and incubated at room temperature for 30 minutes. Then, 20 μL of 2× (either 5 nM or 2 nM) probe was added to assay buffer (prepared from 100 μM DMSO stock) using a MultiDrop Combi (ThermoFisher).

[0242] Compound plates were centrifuged at 250 × g for 1 minute, incubated at room temperature for 1 hour, and then read using a PheraStar FSX (equipped with a 384-well aperture spoon and an appropriate FP module). Before reading each plate, the gain and focus were adjusted so that the polarization without protein control was equal to 35 mP. The data were plotted and analyzed with GraphPad Prism 8.1.0 (Figures 6–9).

[0243] <Assassination Protocol> (Assay A: MLH1 fluorescence polarization assay) The test compound, as a 10 mM DMSO stock, was dispensed into a Black Fluotrac 200 384-well medium-conjugated plate (Greiner Bio-One, t. 781076) using a Labcyte Echo acoustic liquid handler. The test compound was added to wells 1-22 of column, while DMSO was added to wells 23 and 24 of column to normalize the plate. Using a MultiDrop Combi (ThermoFisher), 20 μL of a 2× solution (200 nM) of recombinant N-terminal MLH1 (residues 15-340) in assay buffer (25 mM HEPES, pH 7.5, 250 mM NaCl, 10 mM mgCl2, 0.01% Triton X-100, 5 mM dithiothreitol) was added to all wells of column 1-23, and 20 μL of assay buffer was added to all wells of column 24. The plate was centrifuged at 250 × g for 1 minute and incubated at room temperature for 30 minutes. Then, 2 × (10 nM) 20 μL of 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-ium (Example 1) was added to the assay buffer (prepared from 1 mM DMSO stock) using a MultiDrop Combi (ThermoFisher). The final concentration of N-terminal MLH1 was 100 nM, and the final concentration of 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindoline-5-yl)methyl)piperazine-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindoline-2-ylidene)penta-1,3-dien-1-yl)-3H-indole-1-ium (Example 1) was 5 nM.Compound plates were centrifuged at 250 × g for 1 minute, incubated at room temperature for 1 hour, and then read using a PheraStar FSX (equipped with a 384-well aperture spoon and a 590 675 675 FP optical module). Before reading each plate, the gain and focus were adjusted so that the polarization without enzyme control (column 24) was equal to 35 mP. Data were normalized to the levels without inhibitor control (column 23) and without enzyme control (column 24).

[0244] (Assay B: MLH1 fluorescence polarization assay) The test compound, as a 10 mM DMSO stock, was dispensed into a Black Fluotrac 200 384-well medium-conjugated plate (Greiner Bio-One, t. 781076) using a Labcyte Echo acoustic liquid handler. For single-point screening, the test compound was added to wells 1-22 of column 1, while DMSO was added to wells 23 and 24 of column 2 to normalize the plate. For efficacy determination, serial dilutions of the test compound were added to wells 3-22 of column 1, and the DMSO volume was normalized across the entire plate.

[0245] 20 μL of a 2× solution (200 nM) of recombinant N-terminal MLH1 (residues 15-340) in assay buffer (25 mM HEPES, pH 7.5, 250 mM NaCl, 10 mM mgCl2, 0.01% Triton X-100, 5 mM dithiothreitol) was added to all wells of columns 2-23 for efficacy testing, or columns 1-23 for single-point screening. 20 μL of assay buffer was added to all wells of columns 1 and 24 (column 24 only for single-point screening) using a MultiDrop Combi (ThermoFisher). The plate was centrifuged at 250 × g for 1 minute and incubated at room temperature for 30 minutes. Then, 2 × (10 nM) 20 μL of 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoic acid (Example 5) was added to the assay buffer (prepared from 100 μM DMSO stock) using a MultiDrop Combi (ThermoFisher). The final concentration of N-terminal MLH1 was 100 nM, and the final concentration of 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoic acid (Example 5) was 5 nM.

[0246] Compound plates were centrifuged at 250 × g for 1 minute, incubated at room temperature for 15 minutes, and then read using a PheraStar FSX (equipped with a 384-well aperture spoon and a 540 590 590 FP optical module). Before reading each plate, the gain and focus were adjusted so that the polarization without enzyme control (column 24) was equal to 35 mP. Data were normalized to the levels without inhibitor control (column 23) and without enzyme control (column 24).

[0247] (Assay C: PMS2 fluorescence polarization assay) The test compound, as a 10 mM DMSO stock, was dispensed into a Black Fluotrac 200 384-well medium-conjugated plate (Greiner Bio-One, t. 781076) using a Labcyte Echo acoustic liquid handler. For single-point screening, the test compound was added to wells 1-22 of column 1, while DMSO was added to wells 23 and 24 of column 2 to normalize the plate. For efficacy determination, serial dilutions of the test compound were added to wells 3-22 of column 1, and the DMSO volume was normalized across the entire plate.

[0248] 20 μL of a 2× solution (200 nM) of recombinant N-terminal PMS2 (residues 1-365) in assay buffer (25 mM HEPES, pH 7.5, 250 mM NaCl, 10 mM mgCl2, 0.01% Triton X-100, 5 mM dithiothreitol) was added to all wells of columns 2-23 for efficacy testing, or columns 1-23 for single-point screening. 20 μL of assay buffer was added to all wells of columns 1 and 24 (column 24 only for single-point screening) using a MultiDrop Combi (ThermoFisher). The plate was centrifuged at 250 × g for 1 minute and incubated at room temperature for 30 minutes. Then, 2 × (10 nM) 20 μL of 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoic acid (Example 5) was added to the assay buffer (prepared from 100 μM DMSO stock) using a MultiDrop Combi (ThermoFisher). The final concentration of N-terminal PMS2 was 10 nM, and the final concentration of 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindorin-5-yl)methyl)piperazine-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthene-9-yl)benzoic acid (Example 5) was 5 nM.

[0249] Compound plates were centrifuged at 250 × g for 1 minute, incubated at room temperature for 1 hour, and then read using a PheraStar FSX (equipped with a 384-well aperture spoon and a 540 590 590 FP optical module). Before reading each plate, the gain and focus were adjusted so that the polarization without enzyme control (column 24) was equal to 35 mP. Data were normalized to the levels without inhibitor control (column 23) and without enzyme control (column 24).

[0250] The above assay can be used to screen compounds that bind to MLH1 and / or PMS2. For example, IC of compound AB. 50 The IC of compound AB was determined to be 10.7 μM using assay protocol A (Figure 10). 50 This is determined to be 0.179 μM using assay protocol C (Figure 11).

[0251] [Table 1]

Claims

1. A probe compound represented by the following formula I or II, or a salt thereof: 【Chemistry 1】 (In the formula, R １ is halo or a (1-6C)alkyl, (3-6C)cycloalkyl or (3-4C)cycloalkyl-(1-2C)alkyl group, each of which is optionally substituted by one or more substituents selected from halo, cyano or hydroxy; L is of the formula: -L 1 -X 1 -L 2 -X 2 -L 3 - (In the formula, L 1 is absent or is (2-6C)alkylene; X 1 is absent or selected from -O-, -C(O)-, -C(O)NR 10 - or -NR 10 C(O)-; L 2 is a (2-6C)alkylene 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 to 10 and b is 2 to 4; X 2 is absent or selected from -O-, -C(O)-, -C(O)NR 10 - or -NR 10 C(O)-; L 3 is absent or is (2-6C)alkylene; R 10 is hydrogen or methyl; provided that at least one of X 1 or X 2 is present. is a linker having the formula: Q is a fluorophore; L ｘ is a (1-3C)alkylene linker; X is halo, N 3 , or ethynyl ( 【Chemistry 2】 ).

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

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

4. R １ The probe compound according to any one of claims 1 to 3, wherein is isopropyl.

5. L is of the formula: -L １ -X １ -L ２ -X ２ -L ３ - (In the formula, L １ is (2-6C)alkylene; X １ is -O-, -C(O)-, -C(O)NR １０ - or -NR １０ C(O)-; L ２ is a (2-6C) alkylene 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 - (wherein a is 2 to 10 and b is 2 to 4); X ２ is selected from -O-, -C(O)NR10-, or -NR10C(O)-; L ３ is absent or is (2-6C)alkylene; R １０ is hydrogen or methyl; However, X １ or X ２ (Provided that at least one of the following is present.) The probe compound according to any one of claims 1 to 4, wherein the linker has the formula:

6. L is of the formula: -L 1 -X 1 -L 2 -X 2 -L 3 - (In the formula, L 1 is (2-6C)alkylene; X 1 is selected from -O-, -C(O)NR 10 -, or -NR 10 C(O)-; L 2 is a (2-6C)alkylene 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 to 10 and b is 2 to 4; X 2 is selected from -O-, -C(O)NR 10 -, or -NR 10 C(O)-; L 3 is (2-6C)alkylene; R 10 is hydrogen or methyl; provided that at least one of X 1 or X 2 is present. The probe compound according to any one of claims 1 to 5, which is a linker having the formula:

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

8. The probe compound according to claim 1, wherein Q is a fluorophore selected from the group consisting of AlexaFluor-647, AlexaFluor-633, AlexaFluor-594, AlexaFluor-488, cyanine-5B, cyanine-3B, fluorescein, BODIPY TMR, TAMRA, Oregon Green 488, Oregon Green 514, FITC, Ru(bpy)3, rhodamine dyes, acridine orange, and Texas Red.

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

10. 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-dien-1-yl)-3H-indol-1-ium; 3-(2-((1E,3E)-5-((Z)-3-(5-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)amino)-5-oxopentyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-3H-indol-1-ium-1-yl)propane-1-sulfonic acid; 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-indol-1-ium formate; 2-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-5-(6-hydroxy-3-oxo-3H-xanthen-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-xanthen-9-yl)benzoic acid; Ammonium 5-((5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)benzoate; 2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone; 3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-4λ ４ , 5λ ４ -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)propanamide; 1-(3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-4λ ４ , 5λ ４ -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)-3,6,9,12-tetraoxapentadecan-15-amide; 4-(3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-5λ ４ , 6λ ４ -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)propanamide)-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-4λ ４ , 5λ ４ -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-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-4λ ４ , 5λ ４ -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-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-5λ ４ , 6λ ４ -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)-N-(4-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)-4-oxobutyl)propanamide; (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; tert-Butyl (5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamate; (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone; or a salt thereof.

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

12. The use according to claim 11, wherein the target protein is MLH1 or PMS2.

13. 1. An assay to determine the binding affinity of a test molecule to an ATP binding site of a target protein, comprising: (i) incubating said test molecule with a target protein in the presence of a probe compound of formula I, or a salt thereof, according to any one of claims 1 to 8 or 10; (ii) determining whether any of the probe compounds are displaced from the ATP binding site of the target protein; The assay comprising:

14. 1. A method for determining the binding affinity of a test molecule to an ATP-binding site of a target protein, comprising: (i) incubating a test molecule with the target protein in the presence of a probe compound of formula I, or a salt thereof, according to any one of claims 1 to 8 or 10; (ii) determining whether any of the probe compounds are displaced from the ATP binding site of the target protein; The method includes:

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

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

17. 17. The assay of claim 13, 15 or 16, or the method of claim 14, wherein the target protein is MLH1 or PMS2.

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