Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
  • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6875—Nucleoproteins
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
  • G01N2333/922—Ribonucleases (RNAses); Deoxyribonucleases (DNAses)

Definitions

• the present invention relates to novel probe molecules.
• the present invention also relates to methods of synthesising these novel probe molecules and to their use in assays and screens for determining the binding of a test molecule to the ATP-binding site of a target protein, such as, for example, the Mismatch Repair (MMR) component proteins PMS2 and MLH1 , or for determining the location and/or quantity of such target proteins in a biological sample.
• MMR Mismatch Repair
• the GHKL-family of ATPases comprise the chaperone protein HSP90 [1], the Mismatch Repair (MMR) component proteins PMS2 and MLH1 [2] as well as DNA Gyrase - a bacterial type II topoisomerase enzyme that catalyzes the super-coiling of double- stranded DNA [3]
• MMR Mismatch Repair
• DNA Gyrase - a bacterial type II topoisomerase enzyme that catalyzes the super-coiling of double- stranded DNA
• Each member of the GHKL family harbors an ATP-binding site and ATP-binding is known to be important for the function of GHKL family proteins [4].
• ATP-competitive inhibitors of HSP90 have shown utility as anticancer agents [5,6] and ATP-competitive fluorescent probes have been reported for the discovery of compounds that bind to the ATP-binding site of HSP90 and displace the fluorescent probe [7].
• fluorescent dye molecules that are attached to a small ligand (so called fluorescence probe molecule) that rapidly rotates and disrupts the plane of polarized light prior to emission resulting in low fluorescence polarisation. Binding of the probe molecule to a slowly rotating protein, such as HSP90, results in high fluorescence polarisation; displacement of the probe molecule by a test ligand results in a concentration- dependent reduction in fluorescence polarisation.
• fluorescence polarisation can be applied to provide a direct quantitative readout of the extent of binding of test compounds to a protein such as Hsp90 [7, 8] or DNA Gyrase [9].
• the present invention resides in the discovery of a new series of probe compounds that effectively bind to the ATP-binding site of the GHKL-family proteins MLH1 and PMS2. These probe compounds can be used in assays and methods for assessing the binding affinity of test compounds to the ATP-binding site of MLH1 and PMS2. In addition, these probe compounds can be used to determine the location and/or quantity of MLH1 or PMS2 in a biological sample.
• the present invention provides a probe compound of formula I or II, or a salt thereof, as defined herein.
• the present invention provides a method of synthesising a probe compound of formula I or II, or a salt thereof, as defined herein.
• the present invention provides the use of a probe compound of formula I, or a salt thereof, in a displacement assay to determine the binding affinity of a test molecule to the ATP-binding site of a target protein.
• the target protein is selected from MLH1, PMS2 or HSP90.
• the target protein is MLH1 or PMS2.
• 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 for the ATP-binding site of a target protein.
• the target protein is selected from MLH1, PMS2 or HSP90.
• the target protein is MLH1 or PMS2.
• the present invention provides an assay for determining the binding affinity of a test molecule for the ATP-binding site of a target protein, the assay comprising:
• 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 for the ATP-binding site of a target protein, the assay comprising:
• the present invention provides an assay for determining the location and/or quantity of a target protein present within a biological sample, the assay comprising:
• the present invention provides an assay for determining the location and/or quantity of a target protein present within a biological sample, the assay comprising:
• the present invention provides a method for determining the location and/or quantity of a target protein present within a biological sample, the method comprising:
• the present invention provides a method for determining the location and/or quantity of a target protein present within a biological sample, the method comprising:
• the target protein is selected from MLH1, PMS2 or HSP90. In a particular embodiment, the target protein is MLH1 or PMS2.
• alkyl includes both straight and branched chain alkyl groups. References to individual alkyl groups such as “propyl” are specific for the straight chain version only and references to individual branched chain alkyl groups such as “isopropyl” are specific for the branched chain version only.
• (1-6C)alkyl includes (1-4C)alkyl, (1-3C)alkyl, propyl, isopropyl and t butyl.
• cycloalkyl(1-2C)alkyl includes cyclopropyl-methyl, 1- cyclopropylethyl, 2-cyclopropylethyl etc.
• (m-nC) or “Cm-n”, or “(m-nC) group” or “Cm-n” used alone or as a prefix, refers to any group having m to n carbon atoms.
• alkylene is an alkyl group that is positioned between and serves to connect two other chemical groups.
• (3-20C)alkylene means a linear saturated divalent hydrocarbon radical of one to three carbon atoms or a branched saturated divalent hydrocarbon radical of three atoms, for example, methylene, ethylene, propylene, and the like.
• (m-nC)cycloalkyl means a hydrocarbon ring containing from m to n carbon atoms, for example “(3-6C)cycloalkyl” means a hydrocarbon ring containing from 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
• (m-nC)cycloalkyl also encompasses non-aromatic saturated or partially saturated monocyclic carbocyclic ring system(s).
• halo or halogeno refers to fluoro, chloro, bromo and iodo.
• optionally substituted refers to either groups, structures, or molecules that are substituted and those that are not substituted.
• Probe compounds [0028] The present invention relates to a probe compound of formula I or II, or a salt thereof: wherein: 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 a linker
• Q is a detection moiety
• Lx is a linker; and X is a functional group.
• probe compounds of the invention include probe compounds of formula I or II, or a salt thereof, wherein R 1 , L, Q, L x and X are each as defined herein before, or are as defined in any one of the following paragraphs (1) to (56):
• R 1 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;
• R 1 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;
• R 1 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;
• R 1 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;
• R 1 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;
• R 1 is a (1-6C)alkyl group, which is optionally substituted by one or more fluoro substituents;
• R 1 is a (1-4C)alkyl group
• R 1 is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, trifluoromethyl;
• R 1 is isopropyl
• (10) L is a (2-20C)alkylene linker, wherein the alkylene chain optionally further comprises one or more -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini, wherein R 1 o is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (3-20C)alkylene linker, wherein the alkylene chain optionally further comprises one or more -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini, wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (2-20C)alkylene linker, wherein the alkylene chain further comprises one to eight -O- linkages and/or one to four -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (3-20C)alkylene linker, wherein the alkylene chain further comprises one to eight -O- linkages and/or one to four -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (2-18C)alkylene linker, wherein the alkylene chain optionally further comprises one to five -O- linkages; and/or one to four -C(O)-, -C(O)NR 10 -, - NR 10 C(O)-, -C(O)O-, -OC(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (3-12C)alkylene linker, wherein the alkylene chain optionally further comprises one to five -O- linkages; and/or one to four -C(O)-, -C(O)NR 10 -, - NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini, wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (2-18C)alkylene linker, wherein the alkylene chain further comprises one to five -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (3-12C)alkylene linker, wherein the alkylene chain further comprises one to five -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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, wherein the alkylene chain further comprises one to five -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini and wherein R 10 is hydrogen or (1-2C)alkyl.
• L is a (3-12C)alkylene linker, wherein the alkylene chain further comprises one to five -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini and wherein R 10 is hydrogen or (1-2C)alkyl.
• (20) L is a (2-18C)alkylene linker, wherein the alkylene chain further comprises one to three -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• (21) L is a (3-12C)alkylene linker, wherein the alkylene chain further comprises one to three -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• (22) L is a (2-18C)alkylene linker, wherein the alkylene chain further comprises one -O- linkage and/or one -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkage or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• (23) L is a (3-12C)alkylene linker, wherein the alkylene chain further comprises one -O- linkage and/or one -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkage or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• (24) L is a (2-17C)alkylene linker, wherein the alkylene chain optionally further comprises one to four -O- linkages and/or one to three -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (3-10C)alkylene linker, wherein the alkylene chain optionally further comprises one to four -O- linkages and/or one to three -C(O)-, -C(O)NR 10 -, - NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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, wherein the alkylene chain further comprises one to four -O- linkages; and/or one to three -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (3-10C)alkylene linker, wherein the alkylene chain further comprises one to four -O- linkages; and/or one to three -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, C(O)O-, -OC(0 )- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (2-17C)alkylene linker, wherein the alkylene chain further comprises one to four -O- linkages and/or one to three -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (3-10C)alkylene linker, wherein the alkylene chain further comprises one to four -O- linkages and/or one to three -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini; wherein R 10 is hydrogen or (1-2C)alkyl and wherein 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.
• L is a (2-17C)alkylene linker, wherein the alkylene chain further comprises one to four -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• (31) L is a (3-10C)alkylene linker, wherein the alkylene chain further comprises one to four -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• L is a (2-17C)alkylene linker, wherein the alkylene chain further comprises one to three -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• (33) L is a (3-10C)alkylene linker, wherein the alkylene chain further comprises one to three -O- linkages and/or one to three -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkages or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• L is a (2-17C)alkylene linker, wherein the alkylene chain further comprises one -O- linkage and/or one -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkage or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• L is a (3-10C)alkylene linker, wherein the alkylene chain further comprises one -O- linkage and/or one -C(O)-, -C(O)NR 10 - or -NR 10 C(O)- linkage or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R 10 is hydrogen or (1-2C)alkyl.
• L is a linker having the formula:
• L 1 is absent or a (1-8C)alkylene
• X 1 is absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-,
• L 2 IS a (1-10C)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 1 to 10 and b is 2 to 4;
• X2 is absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-,
• L 3 is absent or a (1-8C)alkylene; wherein R 10 is hydrogen or (1-2C)alkyl; and with the proviso that at least one of X 1 or X2 is present.
• L is a linker having the formula:
• L 1 is absent or a (1-6C)alkylene
• X 1 is absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, - OC(O)- or a triazole ring;
• l_2 is a (1-10C)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;
• X2 is absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, - OC(0 )- or a triazole ring;
• L3 is absent or a (1-6C)alkylene; wherein R 10 is hydrogen or methyl; and with the proviso that at least one of X 1 or X2 is present.
• L is a linker having the formula:
• L 1 is absent or a (2-6C)alkylene
• X 1 is absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring;
• L 2 is a (2-10C)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 absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring;
• L 3 is absent or a (1-6C)alkylene; wherein R 10 is hydrogen or methyl; and with the proviso that at least one of X 1 or X2 is present.
• L is a linker having the formula:
• L 1 is absent or a (2-6C)alkylene
• X 1 is absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring;
• 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;
• X2 is absent or selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring;
• L 3 is absent or a (2-6C)alkylene; wherein R 10 is hydrogen or methyl; and with the proviso that at least one of X 1 or X 2 is present.
• L is a linker having the formula:
• X 1 is absent
• L 2 is absent or 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;
• X2 is selected from -O-, -C(O)-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring;
• L 3 is absent or a (2-6C)alkylene; wherein R 10 is hydrogen or methyl; and with the proviso that at least one of X 1 or X2 is present.
• L is a linker having the formula:
• L 1 is a (2-6C)alkylene
• X 1 is selected from -O-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring;
• 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;
• X2 is selected from -O-, -C(O)NR 10 -, -NR 10 C(O)- or a triazole ring;
• L 3 is a (2-6C)alkylene; wherein R 10 is hydrogen or methyl; and with the proviso that at least one of X 1 or X2 is present.
• 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.
• Q is a fluorophore.
• 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.
• 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 BODIPY derivatives (e.g. BODIPY TMR), TAMRA, Oregon Green 488, Oregon Green 514, FITC, Ru(bpy)3, Rhodamine dyes, Acridine orange, and Texas Red.
• X is a functional group that is capable of reacting with a functional group present on a detection moiety so as to covalently bind the detection moiety to the compound of formula II.
• X is a functional group that is capable of reacting with a functional group present on a detection moiety so as to covalently bind the detection moiety to the compound of formula II, wherein the detection moiety selected from the group consisting of a fluorophore, an oligonucleotide, a biomolecule, a molecular sensor, a protein, or a peptide.
• X is a functional group X that is capable of reacting with a functional group present on a fluorophore so as to covalently bind the fluorophore to the compound of formula II.
• X is a functional group that is capable of reacting with a functional group present on a fluorophore so as to covalently bind the fluorophore to the compound of formula II, and wherein the fluorophore is as defined in paragraph (44) or (45) above.
• X is a functional group selected from halo, N 3 , or ethynyl
• L x is a linker group L as defined in any one of paragraphs (10) to (41) above.
• L x is a (1-8C)alkylene linker, wherein the alkylene chain optionally further comprises one or more -O-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini, wherein R 10 is hydrogen or (1-2C)alkyl.
• L x is a (1-8C)alkylene linker, wherein the alkylene chain optionally further comprises one -O-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, wherein R 10 is hydrogen or (1-2C)alkyl.
• L x is a (1-5C)alkylene linker.
• L x is a (1-3C)alkylene linker.
• a halo group when present, is fluoro and/or chloro, particularly fluoro.
• R 1 is as defined in any one paragraphs (1) to (9) above. More suitably,
• R 1 is as defined in either of paragraphs (8) and (9) above. In a particular group of probe compounds of formula I or II, R 1 is isopropyl.
• the linker group L may be any suitable linker moiety that connects 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.
• the linker group L is 3 to 30 atoms in length, more suitably, 4 to 20 atoms in length and even more suitably 5 to 18 atoms in length.
• L is 5 to 12 atoms in length.
• L is as defined in any one paragraphs (10) to (41) above. More suitably as defined in either of paragraphs (24) to (35) or (39) to (41) above. In a particular group of probe compounds of formula I or II, L is as defined in paragraph (40) or (41) above.
• the detection moiety Q can be any moiety that can enable the probe compound of formula I to be detected and quantified.
• the probe compounds of formula I defined herein are designed to be used in displacement assays, whereby the ability of a test compound to displace the probe compound of formula I from the ATP-binding site of target protein (e.g. MLH1 or PMS2) can be used to determine the binding affinity of that test compound for the ATP-binding site of the target protein.
• the detection moiety Q can be any moiety that can be readily detected and quantified. In certain circumstances, the detection moiety Q enables any probe compounds of formula I that have been displaced (i.e.
• the ATP-binding site of the target protein is “unbound”) from the ATP-binding site of the target protein to be detected and quantified. In certain embodiments of the invention, this may be achieved by collecting 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 in turn give an indication of how much of the probe compound present in the sample has been displaced by the test compound.
• detection moiety Q is not critical as long as it can be used to enable the amount of the probe compound of formula I present in a sample to be determined.
• 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, in particular to detect the amount of the probe compound of formula I that has been displaced from the ATP binding site of a target protein by a test compound.
• 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.
• the detection moiety Q is an oligonucleotide, a biomolecule, a molecular sensor, a protein, or a peptide.
• any suitable technique known in the art for detecting and quantifying the amount of the oligonucleotide, biomolecule, molecular sensor, protein, or peptide present may be utilised.
• a fluorescently labelled secondary probe may be used that is capable of specifically binding to the detection moiety Q of the probe compound of formula I and, once any excess of the secondary probe has been removed, the amount of binding of the secondary probe to detection moiety Q of the compound for formula I can be detected and quantified, thereby enabling the amount of the probe compound of formula I to be determined.
• a secondary probe having a suitable detectable label e.g. a fluorophore or radio-label, and a complimentary oligonucleotide sequence capable of hybridising to Q can be used to detect and quantify the amount of the probe compound of formula I present in sample (and suitably the amount of the probe compound displaced from the ATP-binding site of the target protein by a test compound).
• a secondary probe may be an antibody capable of selectively binding to that protein or peptide and a suitable detectable label, e.g. a fluorophore or radio-label.
• the detection moiety Q is a fluorophore.
• the compound for formula I can be used in a fluorescence polarisation assay.
• 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.
• 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.
• 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.
• Q is a fluorophore
• R 1 is as defined in paragraph (1) above
• L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (2) above
• L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (3) above and L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (4) above and L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (5) above
• L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (6) above
• L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (7) above
• L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (8) above
• L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in any one of paragraphs (10) to (41) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (10) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (11) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (14) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (15) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (18) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (19) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above and L is as defined in paragraph (22) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above and L is as defined in paragraph (23) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (26) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (27) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (30) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (31) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (34) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (35) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (36) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (37) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (38) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (39) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (40) above.
• Q is a fluorophore
• R 1 is as defined in paragraph (9) above
• L is as defined in paragraph (41) above.
• X is as defined in any one of paragraphs (46) to (51) above.
• X may be any suitable functional group that is capable of reacting with a functional group present on a detection moiety Q, or a compound of the formula Q-L 2 -Y as defined herein, to covalently bind the detection moiety to the compound of formula II and thereby form a compound of formula I as defined herein.
• a person skilled in the art will be familiar with appropriate functional groups that can be used to form covalent linkages with a suitable detection moiety.
• X may be selected from halo, N 3 or ethynyl
• X is N 3 or ethynyl.
• X is a functional group that is capable of reacting with a functional group present on the detection moiety to form a bond or triazole linkage between the detection moiety and the compound of formula II.
• the linkage formed is a triazole linkage (which can be formed by CLICK chemistry).
• L x is as defined in any one of paragraphs (52) to (56) above.
• a compound of formula I is formed by reacting a compound of formula II with a group of the formula:
• Q is a detection moiety as defined hereinbefore:
• L y is absent or a linker group which, when covalently bound to the group L x of the compound of formula II forms a linker L of the compound of formula I;
• Y is a functional group capable of reacting with the functional group X present on the compound of formula II to covalently link Q-L 2 to L x of the compound of formula II.
• L y is a (1-8C)alkylene linker, wherein the alkylene chain optionally further comprises one or more -O-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini, wherein R 10 is hydrogen or (1-2C)alkyl.
• L y is a (1-8C)alkylene linker, wherein the alkylene chain optionally further comprises one -O-, -C(O)NR 10 -, -NR 10 C(O)-, -C(O)O-, -OC(O)- or triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, wherein
• R 10 is hydrogen or (1-2C)alkyl.
• L y is a (1-5C)alkylene linker.
• L y is a (1-3C)alkylene linker.
• Y may be any suitable functional group that is capable of reacting with a functional group X present on a probe compound of formula II to covalently bind the group Q-L y to the compound of formula II and thereby form a compound of formula I as defined herein.
• a person skilled in the art will be familiar with appropriate functional groups that can be used to form covalent linkages with a functional group X on the compound of formula II.
• Y may be selected from halo, N 3 or ethynyl.
• Y is N 3 when X is ethynyl or Y is ethynyl when X is N 3 .
• Y is a functional group that is capable of reacting with a functional group X present on the probe compound of formula II to form a bond or triazole linkage.
• the linkage formed is a triazole linkage (which can be formed by CLICK chemistry).
• X is as defined in paragraph (50) above
• R 1 is as defined in paragraph (2) above
• L x is as defined in paragraph (52), (53) or (54) above.
• X is as defined in paragraph (50) above, R 1 is as defined in paragraph (8) above and L x is as defined in paragraph (52), (53) or (54) above.
• X is as defined in paragraph (50) above, R 1 is as defined in paragraph (9) above and L x is as defined in paragraph (52), (53) or (54) above.
• Particular compounds of the invention include any one of the following:
• Particular probe compounds of the invention include 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);
• Particular probe compounds of the invention include any one of the following:
• a suitable salt of a probe compound of the invention is, for example, an acid-addition salt of a compound of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoroacetic, formic, citric methane sulfonate or maleic acid.
• a suitable salt of a compound of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base.
• the present invention also encompasses probe compounds of the invention as defined herein which comprise one or more isotopic substitutions.
• H may be in any isotopic form, including 1 H, 2 H (D) and 3 H (T);
• C may be in any isotopic form, including 12 C, 13 C and 14 C;
• O may be in any isotopic form, including 16 Oand 18 0; and the like.
• probe compounds of the formula (I) may exist in solvated as well as unsolvated forms such as, for example, hydrated forms.
• probe compounds of formula (I) may also exist in a number of different tautomeric forms and references to compounds of the formula (I) include all such forms.
• the present invention provides a method of synthesising a probe compound of formula I or II, or a salt thereof, as defined herein.
• the probe compounds of the present invention can be prepared by any suitable technique known in the art. Particular processes for the preparation of these compounds are described further in the accompanying examples.
• protecting groups see one of the many general texts on the subject, for example, ‘Protective Groups in Organic Synthesis’ by Theodora Green; 4 th Edition; 2006 (publisher: John Wiley & Sons).
• Protecting groups may be removed by any convenient method described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with the minimum disturbance of groups elsewhere in the molecule.
• the present invention provides a process for synthesising a compound of formula I defined herein, the process comprising reacting a compound of formula II as defined herein with a compound of the formula Q-L 2 -Y as defined herein to form a compound of formula I; and optionally thereafter:
• the present invention provides the use of a probe compound of formula I, or a salt thereof, as defined herein in a displacement assay to determine the binding affinity of a test molecule for the ATP-binding site of a target protein.
• 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 for the ATP-binding site of a target protein.
• the target protein is selected from MLH1, PMS2 or HSP90.
• the target protein is MLH1 or PMS2.
• the target protein is MLH1.
• the target protein is PMS2.
• Displacement assays are well known in the art.
• the displacement assay is a fluorescence polarisation assay.
• Fluorescence polarisation assays are well known in the art (see, for example, references [7, 8, 9] herein).
• the test compound may be any biologic or small molecule compound that is to be screened for binding to the ATP-binding site of the target protein (e.g. MLH1 or PMS2).
• the present invention provides an assay for determining the binding affinity of a test molecule for the ATP-binding site of a target protein, the assay comprising:
• the target protein is selected from MLH1, PMS2 or HSP90.
• the target protein is MLH1 or PMS2.
• the target protein is MLH1.
• the target protein is PMS2.
• the assay is a displacement assay.
• Displacement assays are well known in the art.
• the displacement assay is a fluorescence polarisation assay. Fluorescence polarisation assays are well known in the art (see, for example, references [7, 8, 9] herein).
• the step of determining whether any probe compound is 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 compound of formula I that is unbound to the ATP-binding site. This can be achieved by either detecting whether there is an increase in the amount of unbound probe compound (relative to a control) or, if the amount of bound probe compound can be readily detected (e.g. by fluorescence polarisation) then detecting whether there is a decrease in the amount of bound probe compound (again relative to a control).
• the determination of the proportion of the probe compound that is bound to the ATP-binding site of the target protein relative to the amount that is unbound can be achieved by techniques known in the art. The particular techniques utilised will depend on the nature of the detection moiety Q in the compound of formula I.
• Q is a fluorophore and the assay is a fluorescence polarisation assay.
• the difference in the fluorescence caused by the displacement of probe compound from the ATP-binding site of the target protein can be detected and used to quantify the amount of any displaced probe compound - and thereby determine the binding affinity of the test compound for the ATP binding site of the target protein.
• the present invention provides a method for determining the binding affinity of a test molecule for the ATP-binding site of a target protein, the assay comprising:
• the target protein is selected from MLH1, PMS2 or HSP90.
• the target protein is MLH1 or PMS2.
• the target protein is MLH1.
• the target protein is PMS2.
• the method is a displacement assay.
• Displacement assays are well known in the art.
• the displacement assay is a fluorescence polarisation assay. Fluorescence polarisation assays are well known in the art (see, for example, references [7, 8, 9] herein).
• the step of determining whether any probe compound is displaced from the ATP- binding site of the target protein can be conducted in the manner described above.
• the present invention provides an assay for determining the location and/or quantity of a target protein present within a biological sample, the assay comprising:
• the present invention provides an assay for determining the location and/or quantity of a target protein present within a biological sample, the assay comprising:
• the present invention provides a method for determining the location and/or quantity of a target protein present within a biological sample, the method comprising:
• the present invention provides a method for determining the location and/or quantity of a target protein present within a biological sample, the method comprising:
• the target protein is selected from MLH1, PMS2 or HSP90.
• the target protein is MLH1 or PMS2.
• the target protein is MLH1.
• the target protein is PMS2.
• the step of contacting the biological sample with a probe compound of formula I or II involves incubating the biological sample with the probe compound under conditions that enable it to bind to the ATP-binding site of a target protein (e.g. MLH1 or PMS2).
• a target protein e.g. MLH1 or PMS2.
• the determination of the distribution and quantity of the compound of formula I within the biological sample can be achieved by techniques known in the art. The particular techniques utilised will depend on the nature of the detection moiety Q in the compound of formula I.
• Q is a fluorophore and the determination of location and quantity of the compound of formula I within the sample can be achieved by fluorescence microscopy/imaging. Appropriate calibration of the fluorescence intensity can be used to determine the quantity of the compound of formula I at various locations within the sample.
• the assays and methods of the invention defined herein may optionally further comprise one or more washing steps conducted after step (i) to remove excess probe compound of formula I or II from the biological sample.
• the assay and method defined herein involve contacting the biological sample with the compound of formula II to allow it to bind to the ATP binding site of a 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, or a compound of the formula Q-L 2 -Y as defined herein, that is capable of reacting with the probe compound of formula II in situ within the biological sample to form a compound of formula I.
• the sample may optionally be 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 bearing a suitable functional group that can react with the functional group X on the probe compound of formula II.
• the detection moiety may be a compound of the formula Q-L 2 -Y as defined herein, which reacts with the compound of formula II to form a compound of formula I as defined herein.
• Figure 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).
• Figure 2 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.
• Figure 3 shows a synthetic scheme for the preparation of 2,4-Dihydroxy-5- isopropylbenzoic acid.
• Figure 4 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.
• Figure 5 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-(piperazine-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (AB).
• Figure 6 shows data of Example 1 plotted and analysed in GraphPad Prism 8.1.0.
• Figure 7 shows data of Example 3 plotted and analysed in GraphPad Prism 8.1.0.
• Figure 8 shows data of Example 4 plotted and analysed in GraphPad Prism 8.1.0.
• Figure 9 shows data of Example 5 plotted and analysed in GraphPad Prism 8.1.0.
• Figure 10 shows inhibition data for compound AB, the IC50 determined using Assay Protocol A.
• Figure 11 shows inhibition data for compound AB, the IC50 determined using Assay Protocol C,
• NanoBRET® 590 SE, NanoBRET® 590-O4-SE, and NanoBRET® 590-C4-SE were obtained from Promega Corp. Madison, Wl. Mass spectra were recorded on a Thermo Fisher VanquishTM (LC- MS) and purity (395 %) determined by reverse-phase high pressure liquid chromatography (RP-HPLC) using a Kinetex 5 pm EVO C18 100 A LC Column 30 x 2.1 mm column or a Phenomenex Synergi 2.5 pm Max-RP 100 A LC column.
• the sample was analyzed using the following conditions: Agilent Series 1260 Infinity-ll with PDA detector, Column: Waters Sunfire C18, 150*4.6 mm, 3.5 micron,
• 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
• 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-((1 E,3E)-5-((E )-1.3.3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H- indol-1-ium formate.
• reaction mixture was stirred at room temperature in the dark for 2 h and the crude reaction mixture was purified by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H 2 O (10 mM (NH4)2CC>3)) and concentrated in vacuo to give the title compound as an orange powder (3.2 mg, 45%).
• reaction mixture was stirred at room temperature in the dark for 2.5 h and the crude reaction mixture was purified by reverse-phase HPLC (Sunfire C18, 20-80% MeCN: H 2 O (10 mM (NH 4 ) 2 CO 3 )), and concentrated in vacuo to give the title compound as a purple powder (3.9 mg, 67%).
• Example 7 3-(5.5-Difluoro-7-(1H-pyrrol-2-vn-4 ⁇ 4 5 ⁇ 4 -dipyrrolo[1,2-c:2',1'-f] 1,32] diazaborinin-3-vl)-N-(5-(4-((2-(2,4-dihvdroxv-5-isopropvlbenzovl)isoindolin-5- yl)methyl)piperazin-1-yl)pentyl)propanamide
• the mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and was subjected to reverse-phase prep HPLC purification using Standard Method 1.
• Product containing fractions were pooled and concentrated under reduced pressure to give a purple film that 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 (8.1 mg, 91.1%) as a purple solid.
• NanoBRET® 590 PEG-04-SE (8.4 mg, 0.012 mmol) was dissolved in 0.5 mL DMF and was added to the reaction mixture using an additional 2 x 0.5 mL portions of DMF to quantitatively transfer the reactive dye. The mixture was capped and allowed to stir for 2 h in the dark. The mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and was 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 that 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 (8.3 mg, 76.4%) as a purple solid.
• Example 9 4-(3-(5.5-Difluoro-7-(1H-pyrrol-2-yl)5H-4 ⁇ 4 5 ⁇ 4 -dipyrrolo[1.2-c:2'.1'- flH.3,21 _ diazaborinin-3-yl)propanamido)-N-(5-(4-((2-(2,4-dihydroxy-5- isopropylbenzoyl) isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)butanamide [00169] (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 into a 5 mL amber vial and DMF (1 mL ) was added.
• Example 10 3-(5.5-Difluoro-7-(1H-pyrrol-2-vn-5H-4 ⁇ 4 5 ⁇ 4 -dipyrrolo[1.2-c:2'.1'- f][1,3,2]diazaborinin-3-yl)-1-(4-((2-(2.4-dihydroxy-5-isopropylbenzoyl)isoindolin-5- yl)methyl)piperazin-1-yl)propan-1-one
• the mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and was subjected to reverse-phase prep HPLC purification using Standard Method 1.
• Product containing fractions were pooled and concentrated under reduced pressure to give a purple film that 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 (3.25 mg, 39.7%) as a purple solid.
• Example 11 3-(5,5-Difluoro-7-(1 ⁇ -pyrro-2-y ⁇ )-5H-4 ⁇ 4 5 ⁇ 4 -dipyrrolo[1.2-c:2'.1'- f][1,3,2]diazaborinin-3-yl)-N-(15-(4-((2-(2.4-dihydroxv-5-isopropvlbenzoyl)isoindolin-
• NanoBRET® 590 PEG- 04-SE (8.6 mg, 0.013 mmol) was dissolved in 0.5 mL DMF and was added to the reaction mixture using an additional 2 x 0.5 mL portions of DMF to quantitatively transfer the reactive dye.
• the reaction mixture was capped and allowed to stir for 2 h in the dark, diluted with 1:1:0.01 acetonitrile, water, TFA and was 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 that 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 (4.8 mg, 43.6%) as a purple solid.
• Example 12 3-(5.5-Difluoro-7-(1 H-rnp ⁇ oI-2-nh-5H-5 ⁇ 4 6 ⁇ 4 -dipyrrolo[1.2-c:2'.1'- f][1,3,2]diazaborinin-3-yl)-N-(4-(4-((2-(2.4-dihydroxy-5-isopropylbenzoyl)isoindolin-5- yl)methyl)piperazin-1-yl)-4-oxobutyl)propanamide [00175] (2,4-dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2- yl)methanone hydrochloride (5.0 mg, 0.012 mmol) was charged into a 5 mL amber vial and DMF (1 mL ) was added. To the mixture was added N ,N -diisopropylethylamine (20.2 ⁇ L,
• NanoBRET® 590-C4-SE 8.9 mg, 0.017 mmol was added, the vial was capped and allowed to stir for 2 h in the dark.
• the mixture was diluted with 1:1:0.01 acetonitrile, water, TFA and was 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 that 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 (2.3 mg, 24.7%) as a purple solid.
• reaction mixture was filtered through a celite bed, washed with MeOH (2 x 10 mL ) and the combined filtrate concentrated under vacuum followed by lyophilization yielding 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.
• reaction mixture was filtered through a celite bed, washed with MeOH (2 x 20 mL ) and the combined filtrate concentrated under vacuum to yield tert- butyl (5-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)pentyl)carbamate (W) as brown oil (1.7 g, Quantitative). This material was used without further purification.
• reaction mixture was cooled to between 10°C and 15°C and purged with H2(g) for 2 h.
• the reaction mixture was filtered through a celite bed, washed with 10% MeOH:DCM (200 mL) to give crude material (0.40 g) which was purified by Prep. HPLC using (0.05% HCI in water and acetonitrile).
• the obtained fractions were lyophilized yielding (2,4-dihydroxy-5- isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride as an off white solid (0.20 g, 82%).
• Test compounds as 10 mM DMSO stocks, were dispensed into a Black Fluotrac 200 384-well medium binding plate (Greiner Bio-One, item number 781076) using a Labcyte Echo acoustic liquid handler. Test compounds were added to wells in columns 1- 22 whilst DMSO was added to wells in columns 23 and 24 in order to normalise the plate.
• N-terminal MLH1 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.
• Test compounds as 10 mM DMSO stocks, were dispensed into a Black Fluotrac 200384 well medium binding plate (Greiner Bio-One, item number 781076) using a Labcyte Echo acoustic liquid handler.
• test compounds were added to wells in columns 1-22 whilst DMSO was added to wells in columns 23 and 24 in order to normalise the plate.
• serial dilutions of test compounds were added to wells in columns 3-22 and DMSO volume was normalised across the plate.
• N-terminal MLH1 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-(dimethyliminio)-3H- xanthen-9-yl)benzoate (Example 5) was 5 nM.
• Test compounds as 10 mM DMSO stocks, were dispensed into a Black Fluotrac 200384 well medium binding plate (Greiner Bio-One, item number 781076) using a Labcyte Echo acoustic liquid handler.
• test compounds were added to wells in columns 1-22 whilst DMSO was added to wells in columns 23 and 24 in order to normalise the plate.
• serial dilutions of test compounds were added to wells in columns 3-22 and DMSO volume was normalised across the plate.
• N-terminal PMS2 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-(dimethyliminio)-3H- xanthen-9-yl)benzoate (Example 5) was 5 nM.
• the above assays can be used to screen for compounds that bind to MLH1 and/or PMS2.
• the IC 50 for compound AB is determined as 10.7 mM using Assay Protocol A ( Figure 10); the IC 50 for compound AB is determined as 0.179 mM using Assay Protocol C, ( Figure 11).

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Urology & Nephrology (AREA)
• Biomedical Technology (AREA)
• Hematology (AREA)
• Cell Biology (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Plural Heterocyclic Compounds (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Nitrogen Condensed Heterocyclic Rings (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Priority Applications (6)

Applications Claiming Priority (4)

Publications (1)

Family

ID=74285509

Family Applications (1)

Country Status (7)

Cited By (3)

Families Citing this family (2)

Citations (4)

Family Cites Families (19)

• 2021
  • 2021-01-20 AU AU2021211925A patent/AU2021211925A1/en active Pending
  • 2021-01-20 WO PCT/GB2021/050122 patent/WO2021148786A1/en not_active Ceased
  • 2021-01-20 EP EP21702065.0A patent/EP4093731A1/en active Pending
  • 2021-01-20 CA CA3168495A patent/CA3168495A1/en active Pending
  • 2021-01-20 JP JP2022569300A patent/JP7671308B2/ja active Active
  • 2021-01-20 US US17/793,912 patent/US12517130B2/en active Active
  • 2021-01-20 CN CN202180023267.0A patent/CN115397811B/zh active Active

Patent Citations (4)

Non-Patent Citations (10)

Also Published As

Similar Documents

Legal Events