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 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. [Background technology]

[0002] The GHKL family of ATPases includes the chaperone protein HSP90[1], the mismatch repair (MMR) component proteins PMS2 and MLH1[2], and the DNA gyrase-bacterial type II topoisomerase enzyme that catalyzes the supercoiling of double-stranded DNA[3]. Each member of the GHKL family contains 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 utility as anticancer drugs [5,6], and the discovery of compounds that bind to the ATP-binding site of HSP90 and displace the fluorescent probe has been reported for ATP-competitive fluorescent probes [7]. In such assays, fluorophores bound to small ligands (so-called fluorescent probe molecules) that rapidly rotate and perturb the plane of polarized light before emission result in low fluorescence polarization. Binding of the probe molecule to a slowly rotating protein such as HSP90 results in high fluorescence polarization. Displacement of the probe molecule by the test ligand results in a concentration-dependent decrease in fluorescence polarization. Hence, fluorescence polarization can be applied to directly quantitatively read out the degree of binding of test compounds to proteins such as Hsp90 [7,8] or DNA gyrase [9].

[0004] Surprisingly, it was discovered that 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) show no signal in fluorescence polarization assays performed according to the type of protocol exemplified herein and show very weak binding to the N-terminal domain of MLH1 by surface plasmon resonance. Thus, their fluorescence is not suitable for use as a fluorescence polarization screening tool.

[0005] Thus, there is a need for additional probe compounds that provide improved binding to the ATP-binding site of the GHKL family members MLH1 and PMS2 and are suitable for use as fluorescence polarization screening tools.

[0006] It is with the above in mind that the present invention has been made. Summary of the Invention

[0007] The present invention is based on the discovery of a new series of probe compounds that effectively bind to the ATP binding sites of GHKL family proteins MLH1 and PMS2. These probe compounds can be used in assays and methods for evaluating the binding affinity of test compounds to the ATP binding sites of MLH1 and PMS2. These probe compounds can also be used to determine the location and / or amount 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 aspect, the present invention provides a method of synthesizing a probe compound of formula I or formula II, or a salt thereof, as defined herein.

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

[0012] In another aspect, the present invention provides an assay for determining the binding affinity of a test molecule to an ATP-binding site of a target protein, comprising the steps of: (i) incubating a test molecule with a target protein in the presence of a probe compound of formula I, or a salt thereof, as defined herein; (ii) determining whether the probe compound of formula I is displaced from the ATP binding site of the target protein; The present invention provides an assay comprising: 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 aspect, the present invention provides a method for determining the binding affinity of a test molecule to an ATP-binding site of a target protein, comprising the steps of: (i) incubating a test molecule with a target protein in the presence of a probe compound of formula I, or a salt thereof, as defined herein; (ii) determining whether the probe compound of formula I is displaced from the ATP binding site of the target protein; The present invention provides an assay comprising:

[0014] In another aspect, the present invention provides an assay for determining the location 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, or a salt thereof, as defined herein; (ii) 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 moiety of the compound of formula I present in the sample; The present invention provides an assay comprising:

[0015] In another aspect, the present invention provides an assay for determining the location and / or amount of a target protein present in a biological sample, comprising: (i) contacting the 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, or a compound of formula Q-L2-Y as defined herein, capable of reacting with a functional group X present in a compound of formula II to form a compound of formula I in situ within the biological sample; (iii) 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 moiety of the compound of formula I present in the sample; The present invention provides an assay comprising:

[0016] In another aspect, the present invention provides a method for determining the location and / or amount of a target protein present in a biological sample, comprising the steps of: (i) contacting the biological sample with a probe compound of formula I, or a salt thereof, as defined herein; (ii) 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 moiety of the compound of formula I present in the sample; The present invention provides a method comprising:

[0017] In another aspect, the present invention provides a method for determining the location and / or amount of a target protein present in a biological sample, comprising the steps of: (i) contacting the 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, or a compound of formula Q-L2-Y as defined herein, capable of reacting with a functional group X present in a compound of formula II to form a compound of formula I in situ within the biological sample; (iii) 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 moiety of the compound of formula I present in the sample; The present invention provides a method comprising:

[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 description of the drawings]

[0019] [Figure 1] FIG. 1 shows a synthetic scheme for the preparation of (2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone (Example 6). [Diagram 2] FIG. 1 shows a synthetic scheme for the preparation of (5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride. [Diagram 3] FIG. 1 shows a synthetic scheme for the preparation of 2,4-dihydroxy-5-isopropylbenzoic acid. [Figure 4] FIG. 1 shows a synthetic scheme for the preparation of (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone. [Diagram 5] FIG. 1 shows a synthetic scheme for the preparation of benzyl 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazine-1-carboxylate (AA) and (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (AB). [Figure 6] FIG. 1 shows data from Example 1 plotted and analyzed in GraphPad Prism 8.1.0. [Figure 7] FIG. 1 shows data from Example 3 plotted and analyzed in GraphPad Prism 8.1.0. [Figure 8] FIG. 1 shows data from Example 4 plotted and analyzed in GraphPad Prism 8.1.0. [Figure 9] FIG. 1 shows data from Example 5 plotted and analyzed in GraphPad Prism 8.1.0. [Figure 10] FIG. 1 shows inhibition data for compound AB, IC50 determined using assay protocol A. [Figure 11] FIG. 1 shows inhibition data for compound AB, IC50 determined using assay protocol C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0021] "Alkyl" includes both straight-chain and branched-chain alkyl groups. References to individual alkyl groups such as "propyl" are to the straight-chain version only, and references to individual branched-chain alkyl groups such as "isopropyl" are to the branched-chain version only. 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", used alone or as a prefix, refers to any group having m through n carbon atoms.

[0023] An "alkylene" group is an alkyl group that is positioned between and serves to link two other chemical groups. For example, "(3-20C)alkylene" means a linear saturated divalent hydrocarbon radical of 1 to 3 carbon atoms or a branched saturated divalent hydrocarbon radical of 3 atoms, e.g., methylene, ethylene, propylene, etc.

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

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

[0026] "Optionally substituted" refers to either groups, structures, or molecules that are substituted and to groups, structures, or molecules that are not substituted.

[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 invention" means compounds disclosed generally and specifically herein.

[0029] [Probe compound] The present 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 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 a linker; Q is a detection moiety; L x is a linker; and X is a functional group.

[0031] The probe compounds according to the present invention include, in particular, probe compounds of formula I or formula II, or salts thereof, wherein R1, L, Q, L x and X are each as defined herein or in any one of paragraphs (1) to (56) below: (1) R1 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; (2) R1 is halo or a (1-6C)alkyl, (3-4C)cycloalkyl or (3-4C)cycloalkyl-(1C)alkyl group, each of which is optionally substituted by one or more halo substituents; (3) R1 is halo or a (1-6C)alkyl, (3-4C)cycloalkyl or (3-4C)cycloalkyl-(1C)alkyl group, each of which is optionally substituted by one or more fluoro substituents; (4) R1 is fluoro, chloro, or a (1-6C)alkyl, (3-4C)cycloalkyl, or (3-4C)cycloalkyl-(1C)alkyl group, each of which is optionally substituted by one or more fluoro substituents; (5) R1 is a (1-6C)alkyl, (3-4C)cycloalkyl or (3-4C)cycloalkyl-(1C)alkyl group, each of which is optionally substituted by one or more fluoro substituents; (6) R1 is a (1-6C)alkyl group 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 and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; R 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 is one or more -O-, -C(O)-, -C(O)NR located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; 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 has 1-8 -O- bonds and / or 1-4 -C(O)-, -C(O)NR located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10 further comprising a C(O)-, -C(O)O-, -OC(O)- or 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 has 1-8 -O- bonds and / or 1-4 -C(O)-, -C(O)NR located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10further comprising a C(O)-, -C(O)O-, -OC(O)- or 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. (14) L is a (2-18C) alkylene linker, where the alkylene chain has 1-5 -O- bonds located either within the alkylene chain and / or at one of its termini; and / or 1-4 -C(O)-, -C(O)NR 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- bond or one triazole ring bond; 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. (15) L is a (3-12C) alkylene linker, where the alkylene chain has 1-5 -O- bonds located either within the alkylene chain and / or at one of its termini; and / or 1-4 -C(O)-, -C(O)NR 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; 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. (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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)-bond or one triazole ring bond; 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. (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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)-bond or one triazole ring bond; 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. (18) L is a (2-18C) alkylene linker, where the alkylene chain is substituted with 1-5 -O- bonds and / or 1-3 -C(O)-, -C(O)NR, located either within the alkylene chain and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (22) L is a (2-18C) alkylene linker, where the alkylene chain is substituted with one -O- bond and / or one -C(O)-, -C(O)NR, located either within the alkylene chain and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (23) L is a (3-12C) alkylene linker, where the alkylene chain is substituted with one -O- bond and / or one -C(O)-, -C(O)NR, located either within the alkylene chain and / or at one of its termini. 10 -or-NR10 further comprising a C(O)- bond or one triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (24) L is a (2-17C) alkylene linker, where the alkylene chain is substituted with 1-4 -O- bonds and / or 1-3 -C(O)-, -C(O)NR, located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; 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. (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 and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; 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. (26) L is a (2-17C) alkylene linker, where the alkylene chain has 1 to 4 -O- bonds located either within the alkylene chain and / or at one of its termini; and / or 1 to 3 -C(O)-, -C(O)NR 10 -, -NR 10 further comprising a C(O)-, -C(O)O-, -OC(O)- or 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. (27) L is a (3-10C) alkylene linker, where the alkylene chain has 1-4 -O- bonds located either within the alkylene chain and / or at one of its termini; and / or 1-3 -C(O)-, -C(O)NR 10 -, -NR 10 further comprising a C(O)-, -C(O)O-, -OC(O)- or 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. (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 and / or at one of its termini. 10 -, -NR 10 Further containing a C(O)- or triazole ring bond; 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. (29) L is a (3-10C) alkylene linker, where the alkylene chain is 1-4 -O- bonds and / or 1-3 -C(O)-, -C(O)NR 10 -, -NR 10further comprising a triazole ring linkage located either within the C(O)- or alkylene chain and / or at one of its termini; 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. (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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond, where R 10 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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 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 and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (33) L is a (3-10C) alkylene linker, where the alkylene chain is substituted with one to three -O- bonds and / or one to three -C(O)-, -C(O)NR, located either within the alkylene chain and / or at one of its termini.10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (34) L is a (2-17C) alkylene linker, where the alkylene chain is substituted with one -O- bond and / or one -C(O)-, -C(O)NR, located either within the alkylene chain and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (35) L is a (3-10C) alkylene linker, where the alkylene chain is substituted with one -O- bond and / or one -C(O)-, -C(O)NR, located either within the alkylene chain and / or at one of its termini. 10 -OR-NR 10 further comprising a C(O)- bond or one triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (36) L is a group represented by the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is absent or (1-8C)alkylene; X1 is absent, -O-, -C(O)-, or -C(O)NR 10 -, -NR 10 selected from C(O)-, -C(O)O-, -OC(O)- or a triazole ring; L2 is a (1-10C) alkylene or a group of the formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (wherein a is 1 to 10 and b is 2 to 4); X2 is absent 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 (1-8C)alkylene; Here, R 10 is hydrogen or (1-2C) alkyl; provided that at least one of X1 or X2 is present.) is a linker having the formula: (37) L is a group represented by the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is absent or (2-6C)alkylene; X1 is absent, -O-, -C(O)-, or -C(O)NR 10 -, -NR 10 selected from C(O)-, -C(O)O-, -OC(O)- or a triazole ring; L2 is a (1-10C) alkylene or a group of the formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (wherein a is 2 to 10 and b is 2 to 4); X2 is absent or -O-, -C(O)-, -C(O)NR 10 -, -NR 10 L3 is absent or is (1-6C)alkylene; Here, R 10 is hydrogen or methyl; provided that at least one of X1 or X2 is present.) is a linker having the formula: (38) L is a group represented by the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is Does not exist, (2-6C) alkylene; X1 is absent, -O-, -C(O)-, or -C(O)NR10 -, -NR 10 C(O)- or a triazole ring; L2 is a (2-10C) alkylene or a group of the formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (wherein a is 2 to 10 and b is 2 to 4); X2 is absent or -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring; L3 is absent or is (1-6C)alkylene; R 10 is hydrogen or methyl; provided that at least one of X1 or X2 is present.) is a linker having the formula: (39) L is a group represented by the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is Does not exist, (2-6C) alkylene; X1 is absent, -O-, -C(O)-, or -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring; L2 is a (2-6C) alkylene or a group of the formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (wherein a is 2 to 10 and b is 2 to 4); X2 is absent or -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring; L3 is absent or is (2-6C)alkylene; R 10is hydrogen or methyl; provided that at least one of X1 or X2 is present.) is a linker having the formula: (40) L is a group represented by the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is absent; X1 is not present; L2 is absent or a (2-6C) alkylene or a group of the formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (wherein a is 2 to 10 and b is 2 to 4); X2 is -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring; L3 is absent or is (2-6C)alkylene; R 10 is hydrogen or methyl; provided that at least one of X1 or X2 is present.) is a linker having the formula: (41) L is a group represented by the formula: -L1-X1-L2-X2-L3- (In the formula, L1 is (2-6C)alkylene; X1 is -O-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring; L2 is a (2-6C) alkylene or a group of the formula -[CH2CH2-O] a -[CH2] b -or-[CH2] b -[O-CH2CH2] a - (wherein a is 2 to 10 and b is 2 to 4); X2 is -O-, -C(O)NR 10 -, -NR 10C(O)- or a triazole ring; L3 is (2-6C)alkylene; R 10 is hydrogen or methyl; provided that at least one of X1 or X2 is present.) is a linker having the formula: (42) Q is a detection moiety selected from the group consisting of a fluorophore, an oligonucleotide, a biomolecule, a molecular sensor, a protein, or a peptide. (43) Q is a fluorophore. (44) Q is a fluorophore selected from the group consisting of AlexaFluor dyes, cyanine dyes, fluorescein, BODIPY or BODIPY derivatives (e.g., BODIPY TMR), TAMRA, Oregon Green dyes, FITC, Ru(bpy)3, rhodamine dyes, 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 dyes, acridine orange, and Texas Red. (46) X is a functional group capable of reacting with a functional group present on the detection moiety so as to covalently attach the detection moiety to the compound of formula II. (47) X is a functional group capable of reacting with a functional group present on the detection moiety so as to covalently attach the detection moiety to the compound of formula II, the detection moiety being selected from the group consisting of a fluorophore, an oligonucleotide, a biomolecule, a molecular sensor, a protein, or a peptide. (48) X is a functional group X capable of reacting with a functional group present on a fluorophore so as to covalently attach the fluorophore to a compound of formula II. (49) X is a functional group capable of reacting with a functional group present on a fluorophore so as to covalently attach the fluorophore to a compound of formula II, the fluorophore being as defined in paragraph (44) or (45) above. (50) X is halo, N3, or ethynyl ( [ka] ) is a functional group selected from (51) X is ethynyl ( [ka] ) or N3. (52)L x is a linker group L as defined in any one of paragraphs (10) to (41) above. (53)L x is a (1-8C) alkylene linker, where the alkylene chain is comprised of one or more -O-, -C(O)NR located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (54)L x is a (1-8C) alkylene linker, where the alkylene chain is not limited to one -O-, -C(O)NR located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; R 10 is hydrogen or (1-2C) alkyl. (55)L x is a (1-5C) alkylene linker. (56)L x is a (1-3C) alkylene linker.

[0032] Suitably halo groups, when present, are fluoro and / or chloro, especially fluoro.

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

[0034] The linker group L may be any suitable linker moiety that links the detection moiety 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. Suitably, the linker group L is 3 to 30 atoms in length, more suitably 4 to 20 atoms in length, even more suitably 5 to 18 atoms in length. In the groups of probe compounds of formula I or formula II defined herein, in particular L is 5 to 12 atoms in length.

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

[0036] The detection moiety Q may be any moiety that may allow the probe compound of formula I to be detected and quantified. As described below, the probe compounds of formula I defined herein are designed to be used in a displacement assay, whereby the ability of a test compound to displace a probe compound of formula I from the ATP binding site of a target protein (e.g., MLH1 or PMS2) may be used to determine the binding affinity of that test compound to the ATP binding site of the target protein. Thus, the detection moiety Q may be any moiety that may be easily detected and quantified. In certain circumstances, the detection moiety Q allows the detection and quantification of any probe compound of formula I that has been displaced (i.e., "unbound") from the ATP binding site of the target protein. In certain embodiments of the invention, this may be achieved by recovering any displaced or "unbound" compound of formula I from the test sample and assaying the sample to determine how much unbound probe compound is present. This will then be an indication of how much of the probe compound present in the sample has been displaced by the test compound.

[0037] Thus, it will be appreciated that the nature of the detection moiety Q is not important so long as it can be used to enable the determination of the amount of the probe compound of formula I present in a sample. A person skilled in the art will be able to select a suitable detection moiety Q and a suitable methodology for detecting and quantifying the amount of the compound of formula I in a sample, and in particular for detecting the amount of the probe compound of formula I that has been displaced from the ATP binding site of the target protein by the test compound.

[0038] Suitably, the detection moiety Q is selected from the group consisting of a fluorophore, an oligonucleotide, a biomolecule, a molecular sensor, a protein, or a peptide.

[0039] In the embodiment where the detection moiety Q is an oligonucleotide, a biomolecule, a molecular sensor, a protein, or a peptide, any suitable technique known in the art can be used to detect and quantify the amount of oligonucleotide, biomolecule, molecular sensor, protein, or peptide present.For example, a fluorescently labeled secondary probe can be used that can specifically bind to the detection moiety Q of the probe compound of formula I, and once all excess secondary probe is removed, the amount of binding of the secondary probe to the detection moiety Q of the compound of formula I can be detected and quantified, thereby allowing the amount of the probe compound of formula I to be determined.

[0040] For example, if Q is an oligonucleotide, a secondary probe having a suitable detectable label, such as a fluorophore or radioactive label, and a complementary oligonucleotide sequence capable of hybridizing to Q may be used to detect and quantify the amount of the probe compound of formula I present in a sample (and more appropriately, the amount of probe compound displaced 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 capable of selectively binding to the protein or peptide and a suitable detectable label, such as a fluorophore or radioactive label.

[0041] More suitably, the detection moiety Q is a fluorophore. In such a case, the compound of formula I can be used in a fluorescence polarization assay. In particular, in the group of probe compounds of formula I, the detection moiety Q is a fluorophore selected from the group consisting of AlexaFluor dyes, cyanine dyes, fluorescein, BODIPY or BODIPY derivatives (e.g. BODIPY TMR), TAMRA, Oregon Green dyes, FITC, Ru(bpy)3, rhodamine dyes, acridine orange, and Texas Red. In a further group of probe compounds of formula I, the detection moiety 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.

[0042] In particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, 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 particular, in the group of probe compounds of formula I, Q is a fluorophore, R1 is as defined in paragraph (9) above, and L is as defined in paragraph (10) above.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0072] It will be appreciated that X may be a functional group Q present on the detection moiety, or any suitable functional group that can react with a compound of formula Q-L2-Y as defined herein to covalently link the detection moiety to a compound of formula II, thereby forming a compound of formula I as defined herein.

[0073] One of skill in the art would be familiar with suitable functional groups that can be used to form a covalent bond with an appropriate detection moiety. For example, X can be halo, N3, or ethynyl ( [ka] ) may be selected from.

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

[0075] Suitably, X is a functional group capable of reacting with a functional group present on 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 is as defined in any one of paragraphs (52) to (56) above.

[0077] In certain embodiments of the invention, the compound of formula I may be prepared by reacting a compound of formula II with the compound of formula: QL y -Y (In the formula, Q is a detection moiety as defined above: L y is absent or the group L of the compound of formula II x is a linker group which, when covalently attached to, forms the linker L of the compound of formula I; Y reacts with the functional group X present in the compound of formula II to convert Q-L2 to L of the compound of formula II. x is a functional group that can be covalently bonded to The compound is formed by reacting the compound with a group represented by the formula:

[0078] In one embodiment, L y is a (1-8C) alkylene linker, where the alkylene chain is comprised of one or more -O-, -C(O)NR located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; R 10 is hydrogen or (1-2C) alkyl.

[0079] In another embodiment, L y is a (1-8C) alkylene linker, where the alkylene chain is not limited to one -O-, -C(O)NR located either within the alkylene chain and / or at one of its termini. 10 -, -NR 10 optionally further comprising a C(O)-, -C(O)O-, -OC(O)- or triazole ring bond; R 10 is hydrogen or (1-2C) alkyl.

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

[0081] In another embodiment, L yis a (1-3C) alkylene linker.

[0082] It will be appreciated that Y may be any suitable functional group that is capable of reacting with the functional group X present in the probe compound of formula II to covalently attach the group Q-Ly to the compound of formula II, thereby forming a compound of formula I as defined herein.

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

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

[0085] Suitably, Y is a functional group capable of reacting with functional group X present on 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 particular, in the group of probe compounds of formula II, X is as defined in paragraph (50) above, R1 is as defined in paragraph (1) above, and L x is as defined in paragraph (52), (53) or (54) above.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0100] Compounds according to the invention include, in particular, any one of the following: 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium (Example 1); 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 (Example 2); 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 (Example 3); 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 (Example 4); 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 ammonium (Example 5); 2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone (Example 6); 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)propenamide (Example 7); 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]diazaborin-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 (Example 8); 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 (Example 9); 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 (Example 10); 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 (Example 11); 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 (Example 12); 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.

[0101] Probe compounds according to the invention include, in particular, any one of the following: 2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone (Example 6); 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.

[0102] Probe compounds according to the invention include, in particular, any one of the following: 1-(6-((2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium (Example 1); 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 (Example 2); 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 (Example 3); 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 (Example 4); 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 (Example 5); 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)propenamide (Example 7); 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]diazaborin-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 (Example 8); 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 (Example 9); 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 (Example 10); 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 (Example 11); 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 (Example 12).

[0103] Suitable salts of the probe compounds according to the present invention are, for example, acid addition salts of the compounds according to the present invention that are sufficiently basic, for example, acid addition salts 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 compounds according to the present invention that are sufficiently acidic are alkali metal salts, for example, sodium salts or potassium salts, alkaline earth metal salts, for example, calcium salts or magnesium salts, ammonium salts or salts with organic bases.

[0104] The present invention also encompasses probe compounds according to the invention as defined herein which contain one or more isotopic substitutions. For example, H is 1 H, 2 H(D) and 3 H(T) and C may be any isotopic form, including 12 C. 13 C and 14 It may be any isotopic form including C, and O is 16 O and 18 It may be any isotopic form including O, etc.

[0105] It should also be understood that the probe compounds of formula (I) can exist in solvated, e.g., hydrated, as well as unsolvated forms.

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

[0107] The probe compounds of formula (I) may exist in a number of different tautomeric forms and a reference to a compound of formula (I) includes all such forms.

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

[0109] The probe compounds of the present invention can be prepared by any suitable technique known in the art. Particular processes for preparing these compounds are further described in the accompanying examples.

[0110] In the description of synthetic methods provided herein, and in any mentioned synthetic methods used to prepare starting materials, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of experimental and work-up procedures, can be selected by one of ordinary skill in the art.

[0111] It is understood by one skilled in organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions utilized.

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

[0113] For examples of protecting groups, see one of the many general texts on the subject, such as 'Protective Groups in Organic Synthesis' by Theodora Green; 4th Edition; 2006 (publisher: John Wiley & Sons). 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 group in question, and such methods can be selected to effect removal of the protecting group while minimizing interference of the group with other parts of the molecule.

[0114] In one aspect, the present invention provides a process for the synthesis of a compound of formula I as defined herein, comprising the steps of 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 a compound of formula I; and / or (iii) isolating and purifying the compound of formula I The present invention provides a process including:

[0115] [Application of probe compounds] In one aspect, the present invention provides the use of a probe compound of formula I, as defined herein, or a salt thereof, in a displacement assay to determine the binding affinity of a test molecule to an 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 to determine the binding affinity of a test molecule to an ATP-binding site of a target protein.

[0117] 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 a particular embodiment, the target protein is MLH1. In a further particular embodiment, the target protein is PMS2.

[0118] Displacement assays are known in the art. In an embodiment 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 (eg, MLH1 or PMS2).

[0120] In another aspect, the present invention provides an assay for determining the binding affinity of a test molecule to an ATP-binding site of a target protein, comprising the steps of: (i) incubating a test molecule with a target protein in the presence of a probe compound of formula I, or a salt thereof, as defined herein; (ii) determining whether any of the probe compounds are displaced from the ATP binding site of the target protein; The present invention provides an assay comprising:

[0121] 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 a particular embodiment, the target protein is MLH1. In a further particular embodiment, the target protein is PMS2.

[0122] Suitably, the assay is a displacement assay. Displacement assays are known in the art. In an embodiment 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).

[0123] Determining whether any probe compounds are displaced from the ATP binding site of the target protein suitably comprises detecting whether the test compound results in an increase in the proportion of probe compounds of formula I that are not bound to the ATP binding site. This can be achieved either by detecting an increase in the amount of unbound probe compound (relative to a control) or, if the amount of bound probe compound is readily detectable (e.g. by fluorescence polarization), by detecting a decrease in the amount of bound probe compound (again relative to a control).

[0124] Determining the proportion of probe compound bound to the ATP binding site of target protein compared to the amount that is not bound can be achieved by methods known in the art.The specific method used will depend on the nature of the detection moiety Q in the compound of formula I.Suitably, Q is a fluorophore, and the assay is a fluorescence polarization assay.In such an assay, the difference in fluorescence caused by the displacement of probe compound from the ATP binding site of target protein is detected, and can be used to quantify the amount of any displaced probe compound, thereby determining the binding affinity of test compound to the ATP binding site of target protein.

[0125] In another aspect, the present invention provides a method for determining the binding affinity of a test molecule to an ATP-binding site of a target protein, comprising the steps of: (i) incubating a test molecule with a target protein in the presence of a probe compound of formula I, or a salt thereof, as defined herein; (ii) determining whether any of the probe compounds are displaced from the ATP binding site of the target protein; The present invention provides an assay comprising:

[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 a particular embodiment, the target protein is MLH1. In a further particular embodiment, the target protein is PMS2.

[0127] Suitably, the method is a displacement assay. Displacement assays are known in the art. In an embodiment 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).

[0128] The step of determining whether any of the probe compounds are displaced from the ATP binding site of the target protein can be carried out as described above.

[0129] In another aspect, the present invention provides an assay for determining the location 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, or a salt thereof, as defined herein; (ii) 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 moiety of the compound of formula I present in the sample; The present invention provides an assay comprising:

[0130] In another aspect, the present invention provides an assay for determining the location and / or amount of a target protein present in a biological sample, comprising: (i) contacting the 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, or a compound of formula Q-L2-Y as defined herein, capable of reacting with a functional group X present in a compound of formula II to form a compound of formula I in situ within the biological sample; (iii) 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 moiety of the compound of formula I present in the sample; The present invention provides an assay comprising:

[0131] In another aspect, the present invention provides a method for determining the location and / or amount of a target protein present in a biological sample, comprising the steps of: (i) contacting the biological sample with a probe compound of formula I, or a salt thereof, as defined herein; (ii) 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 moiety of the compound of formula I present in the sample; The present invention provides a method comprising:

[0132] In another aspect, the present invention provides a method for determining the location and / or amount of a target protein present in a biological sample, comprising the steps of: (i) contacting the biological sample with a detection moiety Q, or a compound of formula Q-L2-Y as defined herein, capable of reacting with a functional group X present in a compound of formula II to form a compound of formula I in situ within the biological sample; (ii) 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 moiety of the compound of formula I present in the sample; The present invention provides a method comprising:

[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 a particular embodiment, the target protein is MLH1. In a further particular embodiment, the target protein is PMS2.

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

[0135] The determination of the distribution and amount of the compound of formula I in a biological sample can be achieved by methods known in the art. The specific method used will depend on the nature of the detection moiety Q in the compound of formula I. Suitably, Q is a fluorophore, and the location and amount of the compound of formula I in a biological sample can be achieved by fluorescence microscopy / imaging. Appropriate calibration of fluorescence intensity can be used to determine the amount of the compound of formula I at various locations in the sample.

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

[0137] In embodiments where the probe compound is a compound of formula II, the assays and methods defined herein involve contacting a biological sample with a compound of formula II so that it can bind to the ATP binding site of the target protein, optionally washing the biological sample one or more times to remove any unbound probe compound of formula II, and then contacting the biological sample with a suitable detection moiety Q, capable of reacting in situ with the probe compound of formula II within the biological sample to form a compound of formula I, or a compound of formula Q-L2-Y as defined herein. The sample may be optionally washed after incubation with the detection moiety to remove any unbound / unreacted detection moiety from the sample. The detection moiety may be a group Q, as defined herein, carrying a suitable functional group capable of reacting 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 230-400 mesh size. Column chromatography was also performed using glass column chromatography or flash chromatography. Flash chromatography was performed on a Combi-Flash RF Teledyne Isco machine. Preparative TLC was performed on Merck plates. 1 H NMR spectra were recorded on a Bruker Avance-400. Samples were prepared as solutions in appropriate deuterated solvents and referenced to the appropriate internal non-deuterated solvent peak or to tetramethylsilane. Chemical shifts are reported in ppm (δ) downfield from 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 on a Thermo Fisher Vanquish™ (LC-MS) and purity (≧95%) was determined by reversed-phase high pressure liquid chromatography (RP-HPLC) using a Kinetex 5 μm EVO C18 100 Å LC Column 30×2.1 mm column or a Phenomenex Synergi 2.5 μm Max-RP 100 Å LC column. Compounds were purified on a Waters LC Prep 150 using a Waters XBridge Prep C18 OBD 30x250mm column following "Standard Method 1": initial mobile phase: 90% water (0.1% TFA / H20), 10% acetonitrile; final mobile phase: 0% water, 100% acetonitrile; 30 min linear gradient.

[0140] [HPLC method] (Method-A) The samples were analyzed using the following conditions: Agilent series 1260 Infinity-II with PDA detector; Column: Waters Sunfire C18, 150×4.6 mm, 3.5 micron; 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 until t=7 min (10% A, 90% B); gradient until t=9 min (0% A, 100% B); t=14 min (0% A, 100% B); gradient until t=14.01 min (90% A, 10% B); End of run at t=17 min (90% A, 10% B); Flow rate: 1 mL / min; Analysis time 17 min.

[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 until t=2.7 min (2% A, 98% B) flow: 0.8 mL / min; Gradient until t=3 min (0% A, 100% B) flow: 1 mL / min; t=3.5 min (0% A, 100% B) Flow: 1 mL / min; gradient (97% A, 3% B) until t=3.51 min flow: 0.8 mL / min; run end at t=4 min (97% A, 3% B), flow rate: 0.8 mL / min, analysis time 4 min. Mass detector parameters: ionization mode was cycled through positive and negative modes with cone voltages of 10 V and 30 V and 0.8 kV capillary voltage, 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 until t=2.7 min (2% A, 98% B) flow: 0.8 mL / min; Gradient until t=3 min (0% A, 100% B) flow: 1 mL / min; t=3.5 min (0% A, 100% B) Flow: 1 mL / min; gradient (97% A, 3% B) until t=3.51 min flow: 0.8 mL / min; run end 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 cycled through positive and negative modes with cone voltages of 10 V and 30 V and 0.8 kV capillary voltage. 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, 50×4.6 mm, 3.5 micron; Column temperature: 25° C.; Mobile phase A: 5 mM 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 to t=3 min (30% A, 70% B) Gradient to t=3.75 min (5% A, 95% B); Gradient to t=4.20 min (0% A, 100% B); t=5.20 min (0% A, 100% B); Gradient to t=5.21 min (92% A, 8% B); End of run at t=7 min (92% A, 8% B); Flow rate: 1 mL / min; Analysis time 7 min. Mass detector parameters: ionization mode was cycled through positive and negative modes with cone voltages of 10V and 30V and 3.25kV capillary voltage, 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 micron; 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 until t=2.7 min (2% A, 98% B) flow: 0.5 mL / min; Gradient until 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 until t=3.51 min (97% A, 3% B) flow: 0.5 mL / min; End of run at t=4 min (97% A, 3% B), flow rate: 0.5 mL / min, analysis time 4 min. Mass detection parameters: ionization mode was cycled through positive and negative modes with cone voltages of 10 V and 30 V and 3.25 kV capillary voltage, 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 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 until t=2.7 min (2% A, 98% B) flow: 0.8 mL / min; Gradient until t=3 min (0% A, 100% B) flow: 1 mL / min; t=3.5 min (0% A, 100% B) Flow: 1 mL / min; gradient (97% A, 3% B) until t=3.51 min flow: 0.8 mL / min; run end at t=4 min (97% A, 3% B), flow rate: 0.8 mL / min, analysis time 4 min. Mass detector parameters: ionization mode was cycled through positive and negative modes with cone voltages of 10 V and 30 V and 0.8 kV capillary voltage, 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, 250×21.2 mm, 5 micron; 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 until t=17 min (40% A, 60% B); gradient until t=17.01 min (2% A, 98% B); T=19 min (2% A, 98% B); gradient until t=19.01 min (50% A, 50% B); end of run at t=21 min (50% A, 50% B); Flow rate: 17 mL / min; Analysis time 21 min.

[0147] [Example 1] <Preparation of 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>

[0148] [ka] To a solution of (5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride (3.5 mg, 0.0073 mmol) in DMF (1.0 ml) was added Cyanine-5 NHS ester (CAS: 1263093-76-0, 5.1 mg, 0.0040 mmol) and N,N-diisopropylethylamine (3.8 μL, 0.22 mmol). The reaction mixture was stirred in the dark at room temperature for 1 h and then concentrated in vacuo. Purification by silica gel chromatography (Flash 4g, 0-20% MeOH:CH2Cl2) followed by reverse phase HPLC (Sunfire C18, 5-60% MeCN:H2O (0.1% HCO2H)) followed by lyophilization afforded the title compound as a blue solid (1.6 mg, 23%). 1 H 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).Eight protons were estimated to be obscured by the HO 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-(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>

[0150] [ka] To a solution of (5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride (3.2 mg, 0.0066 mmol) in DMF (1.0 mL) was added AlexaFluor-647 NHS ester (5.0 mg, 0.0076 mmol) and N,N-diisopropylethylamine (2.6 μL, 0.20 mmol). The reaction mixture was stirred in the dark at room temperature for 8 h and then diluted to a total volume of 1.5 mL with DMSO. Purification by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H2O (10 mM NH4CO3) followed by lyophilization afforded the title compound as a blue solid (1.3 mg, 25%). 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)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>

[0152] [ka] To a solution of (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5 mg, 0.0085 mmol) in DMF (1.0 ml) was added Cyanine-5 NHS ester (CAS: 1263093-76-0, 3.9 mg, 0.0068 mmol) and N,N-diisopropylethylamine (22 μL, 0.13 mmol). The reaction mixture was stirred in the dark at room temperature for 4 h, and the crude reaction mixture was purified by reverse phase HPLC (Sunfire C18, 5-60% MeCN:H2O (0.1% HCO2H)), concentrated in vacuo, and lyophilized to give the title compound as a blue solid (3.0 mg, 38%). 1 H 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). No 1×OH was 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)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>

[0154] [ka] To a solution of (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.0 mg, 0.0085 mmol) in DMF (1.0 ml) was added NHS-fluorescein (5 / 6-carboxyfluorescein succinimidyl ester), mixed isomers (CAS: 117548-22-8, 3.2 mg, 0.0068 mmol) and N,N-diisopropylethylamine (22 μL, 0.13 mmol). The reaction mixture was stirred in the dark at room temperature for 2 h, and the crude reaction mixture was purified by reverse phase HPLC (Sunfire C18, 5-60% MeCN:H2O (10 mM (NH4)2CO3)) and concentrated in vacuo to give the title compound as an orange powder (3.2 mg, 45%). 1 H 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 (quintet, 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 no CO2H was observed; LC / MS R t =3.32 min;MS m / z:839.7[M+H] + .

[0155] [Example 5] <Preparation of 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>

[0156] [ka] To a solution of (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (3.8 mg, 0.0064 mmol) in DMF (1.0 ml) was added 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). The reaction mixture was stirred in the dark at room temperature for 2.5 h, and the crude reaction mixture was purified by reverse phase HPLC (Sunfire C18, 20-80% MeCN:H2O (10 mM (NH4)2CO3)) and concentrated in vacuo to give the title compound as a purple powder (3.9 mg, 67%). 1H 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). 6 protons obscured by H2O and DMSO peaks. 0.5 equiv. 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-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone>

[0158] [ka]

[0159] [Synthetic scheme (see Figure 1)] [Tert-butyl-5-methylisoindoline-2,5-dicarboxylic acid (A)] To a solution of tert-butyl 5-bromoisoindoline-2-carboxylate (CAS#201940-08-1) (10.0 g, 33.5 mmol) in MeOH:DMF (9:1, 100 mL) was added TEA (14 mL, 137.81 mmol) at room temperature. The reaction mixture was degassed using N2 for 10-15 min. PdCl2(dppf) (4.91 g, 6.71 mmol) was added to the reaction mixture at room temperature. The reaction mixture was pressurized with 30 kg / cm 2 CO(ガス ) pressure, heated to 120° C. and stirred for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting crude material was diluted with water (200 mL) and extracted into EtOAc (3×100 mL). The combined organic layers were washed with brine solution (200 mL), dried over sodium sulfate and concentrated under reduced pressure to give the crude material, which was purified by column chromatography (silica gel, eluted with 12% ethyl acetate / hexanes) to give 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 H 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 solution of LiAlH4 (1M in THF) (36 mL, 36.10 mmol) was added dropwise to a stirred solution of 2-(tert-butyl)5-methylisoindoline-2,5-dicarboxylic acid (A) (10 g, 36.07 mmol) in THF (200 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 3 h, then poured into cold saturated aqueous ammonium chloride solution (100 mL) and extracted into ethyl acetate (3 x 100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give tert-butyl 5-(hydroxymethyl)isoindoline-2-carboxylate (B) as a brown solid (5 g, 56%). This material was used in the next step without further purification. LCMS (Method B): 2.061 min, MS: ES+ 250.18 (M+1); 1 H 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)] To a solution of tert-butyl 5-(hydroxymethyl)isoindoline-2-carboxylate (B) (3 g, 12.04 mmol) in DCM (30 mL) was added activated MnO2 (10.36 g, 120.40 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 h. The reaction mixture was filtered through a bed of celite and washed with 10% MeOH:DCM (3 x 50 mL). The combined filtrate was concentrated in vacuo to give tert-butyl 5-formylisoindoline-2-carboxylate (C) as a grey solid (2.6 g, 86%). This material 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-yn-1-yl)piperazine-1-carboxylate tert-butyl (D)] To a solution of tert-butyl piperazine-1-carboxylate (15 g, 80.51 mmol) in acetonitrile (300 mL) was added K2CO3 (22.26 g, 161.30 mmol) at 0° C. Propargyl bromide (80% (w / v) in toluene) (12 mL, 80.51 mmol) was added dropwise to the reaction mixture at 0° C. The resulting reaction mixture was allowed to warm slowly to room temperature and stirred at room temperature for 1.5 h. The reaction mixture was filtered and washed with acetonitrile (100 mL). The combined filtrate was concentrated under vacuum and the resulting crude material was purified by column chromatography (silica gel, eluted with 3% (v / v) MeOH / DCM) to give tert-butyl 4-(prop-2-yn-1-yl)piperazine-1-carboxylate as a brown solid (12.5 g, 71%).

[0165] LCMS (Method A): 0.937 min, MS: ES+ 225.20 (M+1); 1 H 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-yn-1-yl)piperazine(E)] 4M HCl / dioxane (20 mL) was added dropwise to a solution of tert-butyl 4-(prop-2-yn-1-yl)piperazine-1-carboxylate (D) (2 g, 8.93 mmol) in DCM (10 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 10-15 min. The reaction mixture was slowly warmed to room temperature and stirred at room temperature for 2 h. The resulting reaction mixture was concentrated under vacuum and neutralized using dropwise addition of methanolic ammonia (pH 7). The resulting precipitate was collected by filtration and dried under high vacuum to give 1-(prop-2-yn-1-yl)piperazine (E) as a brown oil (1.45 g). This material was used in the next step without further purification.

[0167] LCMS (Method A): 0.295 min, MS: ES+ 125.00 (M+1); 1 H 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-yn-1-yl)piperazin-1-yl)methyl)isoindoline-2-carboxylate tert-butyl (F)] To a mixture of tert-butyl 5-formylisoindoline-2-carboxylate (C) (0.996 g, 4.03 mmol) and 1-(prop-2-yn-1-yl)piperazine (E) (0.5 g, 4.03 mmol) in DCM (10 mL) was added acetic acid (4-5 drops) at room temperature. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was cooled to 0 °C. NaBH(OAc)3 (2.13 g, 10.05 mmol) was added portionwise to the reaction mixture at 0 °C. The reaction mixture was allowed to warm slowly to room temperature and stirred at room temperature for 24 h. The reaction mixture was poured into water (50 mL) and extracted into EtOAc (3 x 50 mL). The combined organic layers were washed with saturated aqueous NaHCO3 (50 mL), dried over sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, eluted with 2.5% (v / v) MeOH / DCM) to give tert-butyl 5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (F) as an off-white solid (0.550 g, 77%).

[0169] LCMS (Method A): 1.588 min, MS: ES+ 356.27 (M+1); 1 H 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-yn-1-yl)piperazin-1-yl)methyl)isoindoline hydrochloride (G)] 4M HCl / dioxane (5 mL) was added dropwise to a solution of tert-butyl 5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (F) (0.55 g, 15.48 mmol) in DCM (5 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 10-15 min. The reaction mixture was allowed to warm slowly to room temperature and stirred for 16 h. The resulting mixture was concentrated under vacuum and the crude material was triturated with dimethyl ether (2 x 10 mL) and the resulting solid material was concentrated under high vacuum to give 5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindoline hydrochloride (G) as a white solid (0.55 g). This material was used in the next step without further purification.

[0171] LCMS (Method D): 1.24 min, MS: ES+ 256.46(M+1); 1 H 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-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone (Example 6)] To a mixture of 5-((4-(prop-2-yn-1-yl)piperazin-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) was added 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) in a microwave glass tube at room temperature. The microwave glass tube was sealed and irradiated at 120°C under microwave for 3 h. The resulting reaction mixture was poured into ice-cold water (30 mL) and extracted into EtOAc (3 x 30 mL). The combined organic layers were dried over sodium sulfate and concentrated under vacuum. The crude material was purified by flash chromatography (silica gel, eluted with 6.8% (v / v) MeOH / DCM) to give (2,4-dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-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 H 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-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>

[0175] [ka] (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.4 mg, 0.011 mmol) was charged to a 5 mL amber vial and DMF (1 mL) was added. N,N-diisopropylethylamine (5.9 μL, 0.034 mmol) was added to the stirring solution and stirring was continued for 5 min. NanoBRET® 590-SE (5.8 mg, 0.014 mmol) was added and the vial was capped and left to stir in the dark for 2 h. The mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and subjected to reverse phase preparative HPLC purification using standard method 1. Product containing fractions were pooled and concentrated under reduced pressure to give a purple film which was dissolved in acetonitrile (10 mL) and concentrated to dryness in triplicate to remove spurious trifluoracetic acid. The resulting residue was dried under high vacuum to give the product as a purple solid (8.1 mg, 91.1%).

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

[0177] [Example 8] <1-(3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-4λ4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-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>

[0178] [ka] (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.0 mg, 0.010 mmol) was charged to 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 to clarify the solution. NanoBRET® 590 PEG-O4-SE (8.4 mg, 0.012 mmol) was dissolved in 0.5 mL DMF and added to the reaction mixture using an additional 2×0.5 mL portion of DMF to quantitatively transfer the reactive dye. The mixture was capped and left to stir in the dark for 2 h. The mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and subjected to reverse phase preparative HPLC purification using standard method 1. Product containing fractions were pooled and concentrated under reduced pressure to give a purple film, which was dissolved in acetonitrile (10 mL) and concentrated to dryness to remove pseudotrifluoroacetic acid, and the resulting residue was dried under high vacuum to give the product as a purple solid (8.3 mg, 76.4%).

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

[0180] [Example 9] <9-4-(3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-514,614-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborin-3-yl)propanamide)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)butanamide>

[0181] [ka] (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (5.0 mg, 0.010 mmol) was charged to a 5 mL amber vial and DMF (1 mL) was added. To the stirring solution was added N,N-diisopropylethylamine (7.6 μL, 0.042 mmol) and stirring was continued for 5 min. NanoBRET 590®-C4-SE (6.4 mg, 0.012 mmol) was added and the vial was capped and left to stir in the dark for 2 h. The mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and subjected to reverse phase preparative HPLC purification using standard method 1. Product containing fractions were pooled and concentrated under reduced pressure to give a purple film which was dissolved in acetonitrile (10 mL) and concentrated to dryness in triplicate to remove pseudotrifluoroacetic acid. The resulting residue was dried under high vacuum to give the product as a purple solid (3.6 mg, 39.4%).

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

[0183] [Example 10] <3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-414,514-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>

[0184] [ka] (2,4-Dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (5.0 mg, 0.012 mmol) was charged to a 5 mL amber vial and DMF (1 mL) was added. N,N-diisopropylethylamine (8.1 μL, 0.046 mmol) was added to the stirring solution and stirring was continued for 5 min. NanoBRET® 590-SE (5.9 mg, 0.014 mmol) was added and the vial was capped and left to stir in the dark for 2 h. The mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and subjected to reverse phase preparative HPLC purification using standard method 1. Product containing fractions were pooled and concentrated under reduced pressure to give a purple film which was dissolved in acetonitrile (10 mL) and concentrated to dryness in triplicate to remove pseudotrifluoroacetic acid. The resulting residue was dried under high vacuum to give the product as a purple solid (3.25 mg, 39.7%).

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

[0186] [Example 11] <3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-414,514-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>

[0187] [ka] (2,4-Dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (5.0 mg, 0.012 mmol) was charged to 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 min. NanoBRET® 590 PEG-O4-SE (8.6 mg, 0.013 mmol) was dissolved in 0.5 mL DMF and added to the reaction mixture using an additional 2×0.5 mL portion of DMF to quantitatively transfer the reactive dye. The reaction mixture was capped and left to stir in the dark for 2 h, diluted with 1:1:0.01 acetonitrile, water, TFA, and subjected to reverse phase preparative HPLC purification using standard method 1. Product containing fractions were pooled and concentrated under reduced pressure to give a purple film, which was dissolved in acetonitrile (10 mL) and concentrated to dryness in triplicate to remove pseudotrifluoroacetic acid, and the resulting residue was dried under high vacuum to give 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 to be 954.5 and 954.6.

[0189] [Example 12] <3-(5,5-difluoro-7-(1H-pyrrol-2-yl)-5H-514,614-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>

[0190] [ka] (2,4-Dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (5.0 mg, 0.012 mmol) was charged to 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 min. NanoBRET® 590-C4-SE (8.9 mg, 0.017 mmol) was added and the vial was capped and left to stir in the dark for 2 h. The mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and subjected to reverse phase preparative HPLC purification using standard method 1. Product containing fractions were pooled and concentrated under reduced pressure to give a purple film which was dissolved in acetonitrile (10 mL) and concentrated to dryness in triplicate to remove pseudotrifluoroacetic acid. The resulting residue was dried under high vacuum to give 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 to be 792.4 and 792.5.

[0192] <Preparation of intermediates> <Preparation of (5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride (Figure 2)> [(2-(2-iodoethoxy)ethyl)carbamic acid 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) was added triphenylphosphine (7.98 g, 30.43 mmol) and imidazole (0.569 g, 8.36 mmol) at room temperature. The reaction mixture was cooled to 0° C. Iodine (8.36 g, 32.92 mmol) was added portionwise to the reaction mixture at 0° C. The reaction mixture was allowed to warm slowly to room temperature and stirred for 20 h. 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 material and the resulting precipitate was filtered and washed with diethyl ether (3×50 mL). The combined filtrate was concentrated under reduced pressure and the crude material was purified by column chromatography (silica gel, eluted with 20% (v / v) ethyl acetate / hexanes) to give tert-butyl (2-(2-iodoethoxy)ethyl)carbamate (H) as a colorless oil (7.8 g, 98%).

[0193] LCMS (Method B): 2.254 min, MS: ES+ 316.13 (M+1); 1 H 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 (2-(2-iodoethoxy)ethyl)carbamate (H) (2.0 g, 6.35 mmol) and benzylpiperazine-1-carboxylate (CAS#31166-44-6) (1.25 g, 5.72 mmol) in acetone (20 mL) was added K2CO3 (2.45 g, 17.75 mmol) and NaI (0.38 g, 2.54 mmol) at room temperature. The reaction mixture was heated to 70° C. for 16 h. The reaction mixture was concentrated in vacuo, 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 in vacuo. The crude material was purified by flash chromatography (silica gel, eluted with 2.5% (v / v) MeOH / DCM) to give benzyl 4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-carboxylate (I) as a yellow oil (2.0 g, 77%).

[0195] LCMS (Method A): 1.640 min, MS: ES+ 408.32 (M+1); 1 H 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)tert-butylcarbamate (J)] To a mixture of benzyl 4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-carboxylate (I) (1.5 g, 3.68 mmol) and acetic acid (0.23 mL, 4.03 mmol) in MeOH (7 mL) was added 10% Pd(OH)2 / carbon (0.45 g) at room temperature. The reaction mixture was bubbled with H2(g) for 4 h. The reaction mixture was filtered through a bed of Celite, washed with MeOH (2 x 10 mL), and the combined filtrate was concentrated under vacuum and then lyophilized to give tert-butyl (2-(2-(piperazin-1-yl)ethoxy)ethyl)carbamate (J) as an oil (0.971 g, 96%). This material was used in the next step without further purification.

[0197] 1 H 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 solution of tert-butyl 5-formylisoindoline-2-carboxylate (C) (1.3 g, 5.26 mmol) in DCM (20 mL) at 0° C. The reaction mixture was allowed to warm slowly to room temperature and stirred for 6 h. The reaction mixture was concentrated under reduced pressure and the crude material was purified by trituration with diethyl ether (3×20 mL). The resulting solid material was dried under high vacuum to give 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 H 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) was added TEA (2.30 mL, 13.02 mmol) 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 min. 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 (silica gel, eluted with 25% (v / v) ethyl acetate / hexane) to give benzyl 5-formylisoindoline-2-carboxylate (L) as a yellow solid (1.0 g, 82%).

[0201] 1 H 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)piperazin-1-yl)methyl)isoindoline-2-carboxylate benzyl (M)] To a solution of benzyl 5-formylisoindoline-2-carboxylate (L) (1.0 g, 3.56 mmol) and acetic acid (0.1 mL) in DCM (20 mL) was added tert-butyl (2-(2-(piperazin-1-yl)ethoxy)ethyl)carbamate (J) (0.971 g, 3.56 mmol) at room temperature. The reaction mixture was stirred at room temperature for 5 h. 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 h. The reaction mixture was poured into saturated aqueous NaHCO3 (50 mL) and extracted into DCM (2 x 150 mL). The combined organic layers were dried over sodium sulfate and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, eluting with 7% (v / v) MeOH / DCM) to give benzyl 5-((4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (M) as a brown oil (0.9 g, 48%).

[0203] LCMS (Method C): 4.34 min, MS: ES+ 539.64 (M+1); 1 H 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-(isoindolin-5-ylmethyl)piperazin-1-yl)ethoxy)ethyl)tert-butylcarbamate(N)] To a solution of benzyl 5-((4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (M) (0.1 g, 0.186 mmol) in EtOH (5 mL) was added 10% Pd / C (50% wet) (0.020 g) at room temperature. The reaction mixture was bubbled with H2(g) for 12 h. The reaction mixture was filtered through a bed of celite and washed with MeOH (2 x 5 mL). The combined filtrates were concentrated in vacuo to give tert-butyl (2-(2-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)ethoxy)ethyl)carbamate (N) as a brown oil (0.076 g, 100%). This material 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-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)carbamate tert-butyl(O)] To a mixture of tert-butyl (2-(2-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)ethoxy)ethyl)carbamate (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 at 120° C. for 1 h under microwave. The resulting reaction mixture was poured into ice-cold water (20 mL) and extracted into EtOAc (5×30 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to give a crude material which was purified by flash chromatography (silica gel, eluted with 8% (v / v) MeOH / DCM) to give tert-butyl (2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)carbamate (O) as a brown oil (0.020 g, 18%).

[0207] LCMS (Method A): 1.728 min, MS: ES+ 583.5 (M+1); 1 H 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)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride] To a solution of tert-butyl (2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)carbamate (O) (0.015 g, 0.0257 mmol) in DCM (1 mL) was added 4 M HCl / dioxane (0.2 mL) at 0° C. under nitrogen atmosphere. The reaction mixture was stirred at 0° C. for 4 h. The resulting reaction mixture was concentrated under vacuum to give the crude material which was triturated with dimethyl ether (2×10 mL) and dried under high vacuum to give (5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindolin-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 H 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)> [Methyl 2,4-dihydroxybenzoate (P)] To a stirred solution of 2,4-dihydroxybenzoic acid (100 g, 649.35 mmol) in methanol (400 mL) was added concentrated H2SO4 (50 mL) at room temperature. The reaction mixture was heated to 100° C. and stirred for 12 h. 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 give methyl 2,4-dihydroxybenzoate (P) as an off-white solid (170 g, 78%). This material 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-isopropylbenzoic acid methyl ester (Q)] To a solution of methyl 2,4-dihydroxybenzoate (80 g, 475.76 mmol) in DCM (1200 mL) was added anhydrous AlCl3 (126.87 g, 951.48 mmol), followed by isopropyl bromide (351.09 g, 2854.62 mmol) in three equal portions (117.03 g each) at 50° C. for 6 h. After completion of the isopropyl bromide addition, the reaction was heated at 50° C. for 6 h. Total reaction time was 24 h. The reaction mixture was concentrated under vacuum and the crude material 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 resulting crude material was purified three times by column chromatography (silica gel, eluted with 3% (v / v) EtOAc / hexanes) to give 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) was added NaOH (21.88 g, 547.00 mmol) at room temperature. The reaction mixture was heated to 70° C. for 4 h. The reaction mixture was cooled to room temperature, diluted with ice-cold water (500 mL) and acidified using 1 M HCl (pH 5). The resulting precipitate was collected by filtration and dried under high vacuum to give 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 H 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)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (Figure 4)> [(5-hydroxypentyl)carbamic acid tert-butyl (R)] A mixture of 5-aminopentan-1-ol (CAS#2508-29-4) (5 g, 48.54 mmol) in DCM (50 mL) was cooled to 0° C. Boc anhydride (11.16 mL, 48.46 mmol) was added to the reaction mixture at 0° C. The resulting reaction was allowed to warm to room temperature and stirred for 16 h. The reaction mixture was poured into water (150 mL) and extracted into DCM (2×100 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting crude material was purified by flash chromatography (silica, eluted with 18% (v / v) ethyl acetate / hexanes) to afford tert-butyl (5-hydroxypentyl)carbamate (R) as a pale yellow oil (15 g, 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)carbamic acid tert-butyl (S)] To a mixture of tert-butyl (5-hydroxypentyl)carbamate (R) (8 g, 39.41 mmol) in THF (80 mL) was added triphenylphosphine (15.48 g, 59.11 mmol) and CBr4 (19.60 g, 59.11 mmol) at 0°C. The resulting reaction mixture was slowly warmed to room temperature and stirred for 16 h. The reaction mixture was poured into water (150 mL) and extracted with ethyl acetate (3 x 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 (silica gel, eluted with 10% (v / v) ethyl acetate / hexane) to give tert-butyl (5-bromopentyl)carbamate (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) in acetone (50 mL) was added K2CO3 (7.78 g, 56.38 mmol) and NaI (1.40 g, 9.39 mmol) at room temperature. The reaction was stirred at room temperature for 15 minutes. tert-Butyl (5-bromopentyl)carbamate (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 in vacuo to give the crude material which was purified by flash chromatography (silica gel, eluted with 3% (v / v) MeOH / DCM) to give benzyl 4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-carboxylate (T) as a brown oil (6.5 g, 85%).

[0221] LCMS (Method A): 1.492 min, MS: ES+ 406.42 (M+1); 1 H 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)tert-butylcarbamate (U)] To a solution of benzyl 4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-carboxylate (T) (6.4 g, 15.80 mmol) in MeOH (70 mL) was added 10% Pd / C (50% wet) (0.640 g, 10% w / w) at room temperature. The reaction mixture was purged with H2(g) for 5 h. The reaction mixture was filtered through a bed of Celite, washed with MeOH (2 x 10 mL) and the combined filtrate was concentrated under reduced pressure to give tert-butyl (5-(piperazin-1-yl)pentyl)carbamate (U) as a brown oil (4 g, 93%). This material 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)piperazin-1-yl)methyl)isoindoline-2-carboxylate benzyl (V)] To a mixture of benzyl 5-formylisoindoline-2-carboxylate (L) (1.6 g, 5.69 mmol) and tert-butyl (5-(piperazin-1-yl)pentyl)carbamate (U) (1.54 g, 5.69 mmol) in DCM (50 mL) was added acetic acid (0.2 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 h. 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 allowed to warm slowly to room temperature and stirred for 16 h. The reaction mixture was poured into water (50 mL) and extracted into DCM (2×150 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by flash chromatography (silica gel, eluting with 3% (v / v) MeOH / DCM) to give benzyl 5-((4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (V) as a brown oil (2.2 g, 72%).

[0225] LCMS (Method A): 1.552 min, MS: ES+ 537.41 (M+1); 1 H 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-(isoindolin-5-ylmethyl)piperazin-1-yl)pentyl)tert-butylcarbamate (W)] To a solution of benzyl 5-((4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (V) (2.1 g, 3.92 mmol) in EtOH (60 mL) was added 10% Pd / C (50% wet) (0.420 g, 20% w / w) at room temperature. The reaction mixture was purged with H2(g) for 5 h. The reaction mixture was filtered through a bed of Celite, washed with MeOH (2 x 20 mL) and the combined filtrates were concentrated in vacuo to give tert-butyl (5-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)pentyl)carbamate (W) as a brown oil (1.7 g, quantitative). This material was used without further purification.

[0227] LCMS (Method A): 0.757 min, MS: ES+ 403.37 (M+1); 1 H 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)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)tert-butylcarbamate (X)] To a mixture of tert-butyl (5-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)pentyl)carbamate (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 at 120 °C under microwave for 3 h. The resulting reaction mixture was poured into cold water (50 mL) and extracted into EtOAc (3 x 50 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated in vacuo to give crude material (270 mg). 180 mg crude material was purified by HPLC (5 mM ammonium bicarbonate in water / MeCN). The isolated fraction was lyophilized to give tert-butyl (5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamate (X) as an off-white solid (0.032 g, 5%).

[0229] LCMS (Method A): 1.728 min, MS: ES+ 583.5 (M+1); 1 H 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)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone] To a solution of tert-butyl (5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamate (X) (0.060 g, 0.103 mmol) in DCM (2 mL) was added 4 M HCl / dioxane (0.6 mL) at 0° C. The reaction mixture was stirred at 0° C. for 1 h. The resulting reaction mixture was concentrated under vacuum and the crude material was triturated with dimethyl ether (2×5 mL). The resulting solid was dried by lyophilization to give (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone as a brown sticky solid (0.043 g, 80%).

[0231] LCMS (Method B): 1.625 min, MS: ES+ 481.50 (M+1); 1 H 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)isoindolin-5-yl)methyl)piperazine-1-carboxylate benzyl (AA) and (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (AB) (FIG. 5)> [5-((4-((benzyloxy)carbonyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate tert-butyl (Y)] A mixture of tert-butyl 5-formylisoindoline-2-carboxylate (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 traces of methanol). This process was repeated once to complete the removal of methanol. The resulting residue was dissolved in DCM (90 mL), placed under nitrogen atmosphere, and cooled to 0° C. NaBH(OAc)3 (3.8 g, 17.93 mmol) was added portionwise to the reaction mixture at 0° C. The reaction mixture was allowed to warm slowly to room temperature and stirred for 16 h. The previous preparation was performed in two parallel batches, and the combined reaction mixture was poured into water (200 mL) and extracted into DCM (3 x 250 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate (2 x 100 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give the crude material, which was purified by flash chromatography (silica gel, eluted with 25% (v / v) EtOAc / n-hexane) to give tert-butyl 5-((4-((benzyloxy)carbonyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (Y) as a brown oil (5.0 g, 46%).

[0233] LCMS (Method A): 1.631 min, MS: ES+ 452.32 (M+1); 1 H 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-(isoindolin-5-ylmethyl)piperazine-1-carboxylate benzyl hydrochloride (Z)] To a solution of tert-butyl 5-((4-((benzyloxy)carbonyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (Y) (1.0 g, 2.21 mmol) in DCM (20 mL) was added 4M HCl / dioxane (5.0 mL) at 0° C. The reaction mixture was stirred at 0° C. for 2 h, then concentrated in vacuo and the crude material was triturated with diethyl ether (3×10 mL). The resulting solid was dried under high vacuum to give benzyl 4-(isoindolin-5-ylmethyl)piperazine-1-carboxylate hydrochloride (Z) as a green solid (0.99 g, quantitative).

[0235] LCMS (Method E): 0.865 min, MS: ES+ 352.22 (M+1); 1 H 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)isoindolin-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-(isoindolin-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 microwave at 120 °C for 1 h. The resulting reaction mixture was poured into cold water (60 mL) and a solid material precipitated, which was isolated and dried under vacuum to give 0.8 g crude material. Purification by flash chromatography (silica gel, eluting with 1.5% (v / v) MeOH / DCM) afforded benzyl 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazine-1-carboxylate as an off-white solid (0.395 g, 30%).

[0237] LCMS (Method E): 1.567 min, MS: ES+ 530.22 (M+1); 1 H 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-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (AB)] To a solution of 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)-piperazine-1-carboxylate benzyl (AB) (0.30 g, 0.56 mmol) in EtOH (20 mL) was added AcOH (0.6 mL) and 10% Pd / C (50% wet) (0.20 g, 20% w / w) at room temperature. The reaction mixture was cooled to 10-15 °C and purged with H2(g) for 2 h. The reaction mixture was filtered through a bed of celite and washed with 10% MeOH:DCM (200 mL) to give the crude material (0.40 g), which was purified by preparative HPLC using (0.05% HCl / water and acetonitrile). The resulting fractions were lyophilized to give (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-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 H 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> An 11 point 1 in 2 serial dilution of the proteins (either recombinant N-terminal PMS2 (residues 1-365) or 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 constructed in clear polypropylene plates (Greiner Bio-One, product no. 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 2x protein and 20 μL was added to a Black Fluotrac 200 384-well media binding plate (Greiner Bio-One, product no. 781076). Plates were centrifuged at 250×g for 1 min and incubated at room temperature for 30 min before adding 20 μL of 2x (either 5 nM or 2 nM) probe in assay buffer (prepared from 100 μM DMSO stock) with a MultiDrop Combi (ThermoFisher).

[0242] Compound plates were centrifuged at 250 x g for 1 min and incubated at room temperature for 1 h before being read on a PheraStar FSX (fitted with a 384-well aperture spoon and appropriate FP module). Before reading each plate, gain and focus were adjusted so that the polarization of the no protein control was equal to 35 mP. Data were plotted and analyzed in GraphPad Prism 8.1.0 (Figures 6-9).

[0243] Assay Protocol (Assay A: Fluorescence polarization assay of MLH1) Test compounds as 10 mM DMSO stocks were dispensed into a Black Fluotrac 200 384-well media binding plate (Greiner Bio-One, product no. 781076) using a Labcyte Echo acoustic liquid handler. Test compounds were added to wells in columns 1-22, while DMSO was added to wells in columns 23 and 24 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 in columns 1-23, and 20 μL assay buffer was added to all wells in column 24. Plates were centrifuged at 250×g for 1 min and incubated at room temperature for 30 min before adding 20 μL of 2× (10 nM) of 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 (Example 1) in 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)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 (Example 1) was 5 nM.Compound plates were centrifuged at 250×g for 1 min and incubated at room temperature for 1 h before being read on a PheraStar FSX (fitted with a 384-well aperture spoon and a 590 675 675 FP optical module). Before reading each plate, gain and focus were adjusted so that the polarization of the no enzyme control (column 24) was equal to 35 mP. Data were normalized to the no inhibitor control (column 23) and no enzyme control (column 24).

[0244] (Assay B: Fluorescence polarization assay of MLH1) Test compounds as 10 mM DMSO stocks were dispensed into Black Fluotrac 200 384-well media binding plates (Greiner Bio-One, product no. 781076) using a Labcyte Echo acoustic liquid handler. For single point screening, test compounds were added to wells in columns 1-22, while DMSO was added to wells in columns 23 and 24 to normalize the plate. For potency determination, serial dilutions of test compounds were added to wells in columns 3-22, with DMSO volumes normalized across the 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 in columns 2-23 for potency determination or columns 1-23 for single point screening. 20 μL assay buffer was added to all wells in columns 1 and 24 (only column 24 for single point screening) using a MultiDrop Combi (ThermoFisher). Plates were centrifuged at 250×g for 1 min and incubated at room temperature for 30 min before adding 20 μL of 2× (10 nM) of 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)benzoic acid (Example 5) in 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)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)benzoic acid (Example 5) was 5 nM.

[0246] Compound plates were centrifuged at 250×g for 1 min and incubated at room temperature for 15 min before being read on a PheraStar FSX (fitted with a 384-well aperture spoon and a 540 590 590 FP optical module). Before reading each plate, gain and focus were adjusted so that the polarization of the no enzyme control (column 24) was equal to 35 mP. Data were normalized to the no inhibitor control (column 23) and no enzyme control (column 24).

[0247] (Assay C: Fluorescence polarization assay of PMS2) Test compounds as 10 mM DMSO stocks were dispensed into Black Fluotrac 200 384-well media binding plates (Greiner Bio-One, product no. 781076) using a Labcyte Echo acoustic liquid handler. For single point screening, test compounds were added to wells in columns 1-22, while DMSO was added to wells in columns 23 and 24 to normalize the plate. For potency determination, serial dilutions of test compounds were added to wells in columns 3-22, with DMSO volumes normalized across the 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 in columns 2-23 for potency determination or columns 1-23 for single point screening. 20 μL assay buffer was added to all wells in columns 1 and 24 (only column 24 for single point screening) using a MultiDrop Combi (ThermoFisher). Plates were centrifuged at 250×g for 1 min and incubated at room temperature for 30 min before adding 20 μL of 2× (10 nM) of 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)benzoic acid (Example 5) in 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)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamoyl)-2-(6-(dimethylamino)-3-(dimethylimino)-3H-xanthen-9-yl)benzoic acid (Example 5) was 5 nM.

[0249] Compound plates were centrifuged at 250×g for 1 min and incubated at room temperature for 1 h before being read on a PheraStar FSX (fitted with a 384-well aperture spoon and a 540 590 590 FP optical module). Before reading each plate, gain and focus were adjusted so that the polarization of the no enzyme control (column 24) was equal to 35 mP. Data were normalized to the no inhibitor control (column 23) and no enzyme control (column 24).

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