ISOINDOLINE DERIVATIVES WHICH BIND TO AN ATP BINDING SITE
20230061002 · 2023-03-02
Inventors
- Julian Blagg (Altrincham, GB)
- Jonathon Richard Anthony Roffey (Altrincham, GB)
- Paul Colin Michael Winship (Saffron Walden, GB)
- Gareth William Langley (Saffron Walden, GB)
Cpc classification
C07D405/12
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to novel probe compounds of formulae I and II defined herein. The present invention also relates to methods of synthesising these novel probe compounds 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.
Claims
1. A probe compound of formula I or II, or a salt thereof: ##STR00014## wherein: R.sub.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; L.sub.x is a linker; and X is a functional group.
2. A probe compound according to claim 1, wherein R.sub.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.
3. A probe compound according to claim 1 or claim 2, wherein R.sub.1 is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, trifluoromethyl.
4. A probe compound according to any one of the preceding claims, wherein R.sub.1 is isopropyl.
5. A probe compound according to any one of the preceding claims, wherein L is a (2-20C)alkylene linker, wherein the alkylene chain optionally further comprises one to ten —O— linkages and/or one to four —C(O)—, —C(O)NR.sub.10—, —NR.sub.10C(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.sub.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.
6. A probe compound according to any one of the preceding claims, L is a (2-18C)alkylene linker, wherein the alkylene chain optionally further comprises one —O— linkage and/or one —C(O)—, —C(O)NR.sub.10— or —NR.sub.10C(O)— linkage or one triazole ring linkage positioned either within the alkylene chain and/or at one of its termini, and wherein R.sub.10 is hydrogen or (1-2C)alkyl.
7. A probe compound according to any one of claims 1 to 4, wherein L is a linker having the formula:
-L.sub.1-X.sub.1-L.sub.2-X.sub.2-L.sub.3- wherein L.sub.1 is absent or a (1-8C)alkylene; X.sub.1 is absent or selected from —O—, —C(O)—, —C(O)NR.sub.10—, —NR.sub.10C(O)—, —C(O)O—, —OC(O)— or a triazole ring; L.sub.2 is a (1-10C)alkylene or a group of the formula —[CH.sub.2CH.sub.2—O].sub.a—[CH.sub.2].sub.b— or —[CH.sub.2].sub.b—[—CH.sub.2CH.sub.2].sub.a—, wherein a is 1 to 10 and b is 1 to 4; X.sub.2 is absent or selected from —O—, —C(O)—, —C(O)NR.sub.10—, —NR.sub.10C(O)—, —C(O)O—, —OC(O)— or a triazole ring; L.sub.3 is absent or a (1-8C)alkylene; wherein R.sub.10 is hydrogen or (1-2C)alkyl; and with the proviso that at least one of X.sub.1 or X.sub.2 is present.
8. A probe compound according to any one of claims 1 to 4 or claim 7, wherein L is a linker having the formula:
-L.sub.1-X.sub.1-L.sub.2-X.sub.2-L.sub.3- wherein L.sub.1 is absent or a (2-6C)alkylene; X.sub.1 is absent or selected from —O—, —C(O)—, —C(O)NR.sub.10—, —NR.sub.10C(O)— or a triazole ring; L.sub.2 is a (2-6C)alkylene or a group of the formula —[CH.sub.2CH.sub.2—O].sub.a—[CH.sub.2].sub.b— or —[CH.sub.2].sub.b—[O—CH.sub.2CH.sub.2].sub.a—, wherein a is 2 to 10 and b is 2 to 4; X.sub.2 is absent or selected from —O—, —C(O)—, —C(O)NR.sub.10—, —NR.sub.10C(O)— or a triazole ring; L.sub.3 is absent or a (2-6C)alkylene; wherein R.sub.10 is hydrogen or methyl; and with the proviso that at least one of X.sub.1 or X.sub.2 is present.
9. A probe compound according to any one of claims 1 to 4 or claim 7 or claim 8, wherein L is a linker having the formula:
-L.sub.1-X.sub.1-L.sub.2-X.sub.2-L.sub.3- wherein L.sub.1 is a (2-6C)alkylene; X.sub.1 is selected from —O—, —C(O)—, —C(O)NR.sub.10—, —NR.sub.10C(O)—, —C(O)O—, —OC(O)— or a triazole ring; L.sub.2 is a (2-6C)alkylene or a group of the formula —[CH.sub.2CH.sub.2—O].sub.a—[CH.sub.2].sub.b— or —[CH.sub.2].sub.b—[O—CH.sub.2CH.sub.2].sub.a—, wherein a is 2 to 10 and b is 2 to 4; X.sub.2 is absent; L.sub.3 is absent; wherein R.sub.10 is hydrogen or methyl; and with the proviso that at least one of X.sub.1 or X.sub.2 is present.
10. A probe compound according to any one of claims 1 to 4 or claims 7 to 9, wherein L is a linker having the formula:
-L.sub.1-X.sub.1-L.sub.2-X.sub.2-L.sub.3- wherein L.sub.1 is a (2-6C)alkylene; X.sub.1 is selected from —O—, —C(O)—, —C(O)NR.sub.10—, —NR.sub.10C(O)— or a triazole ring; L.sub.2 is a (2-6C)alkylene or a group of the formula —[CH.sub.2CH.sub.2—O].sub.a—[CH.sub.2].sub.b— or —[CH.sub.2].sub.b—[O—CH.sub.2CH.sub.2].sub.a—, wherein a is 2 to 10 and b is 2 to 4; X.sub.2 is selected from —O—, —C(O)NR.sub.10—, —NR.sub.10C(O)— or a triazole ring; L.sub.3 is absent or a (2-6C)alkylene; wherein R.sub.10 is hydrogen or methyl; and with the proviso that at least one of X.sub.1 or X.sub.2 is present.
11. A probe compound according to any one of the preceding claims, wherein Q is a detection moiety is selected from the group consisting of a fluorophore, an oligonucleotide, a biomolecule, a molecular sensor, a protein, or a peptide.
12. A probe compound according to any one of the preceding claims, wherein Q is a fluorophore.
13. A probe compound according to any one of claims 1 to 4, wherein L.sub.x is a (1-8C)alkylene linker, wherein the alkylene chain optionally further comprises one or more —O—, —C(O)NR.sub.10—, —NR.sub.10C(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.sub.10 is hydrogen or (1-2C)alkyl; or L.sub.x is a (1-6C)alkylene linker optionally comprising one —O—, —C(O)NH—, —NHC(O)—, —C(O)O—, —OC(O)— or triazole ring linkages positioned either within the alkylene chain and/or at one of its termini.
14. A probe compound according to any one of claims 1 to 4 or claim 13, wherein X is a functional group selected from halo, N.sub.3, or ethynyl.
15. A probe compound which is selected from any one of the following: 1-(6-((2-(2-(4-((2-(2,4-Dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-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-sulfonate; 1-(6-((5-(4-((2-(2,4-Dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-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; 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 ammonium; 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.sup.4,5λ.sup.4-dipyrrolo[1,2-c:2′,1′-f][1,3,2] diazaborinin-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.sup.4,5λ.sup.4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)propanamido)-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.sup.4,6λ.sup.4-dipyrrolo[1,2-c:2′,1′-f][1,3,2] diazaborinin-3-yl)propanamido)-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.sup.4,5λ.sup.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; 3-(5,5-Difluoro-7-(1H-pyrrol-2-yl)-5H-4λ.sup.4,5λ.sup.4-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-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.sup.4,6λ.sup.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; 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.
16. The use of a probe compound of formula I, or a salt thereof, according to any one of claims 1 to 15, in a displacement assay to determine the binding affinity of a test molecule for the ATP-binding site of a target protein.
17. The use according to claim 16, wherein the target protein is MLH1 or PMS2.
18. An assay for determining the binding affinity of a test molecule for the ATP-binding site of a target protein, the assay 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 12 and 15; and (ii) determining whether any probe compound is displaced from the ATP-binding site of the target protein.
19. A method for determining the binding affinity of a test molecule for the ATP-binding site of a target protein, the assay 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 12 and 15; and (ii) determining whether any probe compound is displaced from the ATP-binding site of the target protein.
20. An assay for determining the quantity and location of a target protein in a biological sample, the assay comprising: (i) contacting the biological sample with a probe compound of formula I, or a salt thereof, according to any one of claims 1 to 12 or 15; and (ii) determining distribution and quantity of the compound of formula I within the sample by detecting and quantifying the location and distribution of the compound of formula I in the biological sample.
21. An assay for determining the quantity and location of a target protein in a biological sample, the assay comprising: (i) contacting the biological sample with a probe compound of formula II, or a salt thereof, according to any one of claims 1 to 4 or 13 to 15; and (ii) contacting the biological sample with a detection moiety that is capable of reacting with the functional group X present on the compound of formula II to form a compound of formula I according to any one of claims 1 to 12 and 15 in situ within the biological sample; (iii) determining distribution and quantity of the compound of formula I within the sample by detecting and quantifying the location and distribution of the compound of formula I in the biological sample.
22. An assay according to claim 18, 20 or 21, or a method according to claim 19, wherein the target protein is MLH1 or PMS2.
Description
FIGURES
[0317]
[0318]
[0319]
[0320]
[0321]
[0322]
[0323]
[0324]
[0325]
[0326]
[0327]
Analytical Methods
[0328] For the preparation of Examples 1 to 6 and associated 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 combi-flash RF Teledyne Isco machine. Preparative TLC was performed on Merck plates. .sup.1H NMR spectra were recorded on a Bruker Avance-400. Samples were prepared as solutions in a suitable deuterated solvent and referenced to the appropriate internal non-deuterated solvent peak or tetramethylsilane. Chemical shifts were recorded in ppm (δ) downfield of tetramethylsilane.
[0329] For the preparation of Examples 7 to 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, Wis. Mass spectra were recorded on a Thermo Fisher Vanquish™ (LC-MS) and purity (≥95%) determined by reverse-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 30×250 mm column according to Standard Method 1: Initial mobile phase: 90% aqueous (0.1% TFA in H.sub.2O), 10% acetonitrile; final mobile phase: 0% aqueous, 100% acetonitrile; 30 min linear gradient.
HPLC Method
Method-A
[0330] The sample was 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 temp., auto sampler temperature: 15° C., mobile phase A: 0.05% (v/v) tri-fluoroacetic acid in Milli-Q water (pH˜2.5), mobile phase B: 100% acetonitrile; mobile phase gradient details: t=0 min (90% A, 10% B; gradient to t=7 min (10% A, 90% B); gradient to t=9 min (0% A, 100% B); t=14 min (0% A, 100% B); gradient to 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.
LC-MS Method
Method-A
[0331] 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., auto sampler temperature: 5° C., mobile phase A: 0.1% (v/v) formic acid in ddH.sub.2O (pH=2.70), Mobile Phase B: 0.1% formic acid (v/v) in 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 to t=2.7 min (2% A, 98% B) flow: 0.8 mL/min; gradient to t=3 min (0% A, 100% B) flow: 1 mL/min; t=3.5 min (0% A, 100% B) flow: 1 mL/min; gradient to t=3.51 min (97% A, 3% B) flow: 0.8 mL/min; end of run at t=4 min (97% A, 3% B), Flow rate: 0.8 mL/min, analysis time 4 min. Mass detector parameter: ionization mode was cycled through positive and negative modes with cone voltage 10 V and 30 V and 0.8 kV capillary voltage, temperature of source and probe were 120° C. and 600° C. respectively.
Method-B
[0332] 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., auto sampler temperature: 5° C., mobile phase A: 0.1% (v/v) Formic acid in ddH.sub.2O (pH=2.70), mobile phase B: 0.1% (v/v) formic acid in 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 to t=2.7 min (2% A, 98% B) flow: 0.8 mL/min; gradient to t=3 min (0% A, 100% B) flow: 1 mL/min; t=3.5 min (0% A, 100% B) flow: 1 mL/min; gradient to t=3.51 min (97% A, 3% B) flow: 0.8 mL/min; end of run at t=4 min (97% A, 3% B), Flow rate: 0.8 mL/min, analysis time 4 min. Mass detector parameter: probe was ESI capillary probe, ionization mode was cycled through positive and negative modes with cone voltage 10 V and 30V and 0.8 kV capillary voltage, temperature of source and probe were 120° C. and 600° C. respectively.
Method-C
[0333] Waters Acquity with PDA detector and SQ Detector, column: X-bridge C18, 50*4.6 mm, 3.5 micron, column temperature: 25° C., mobile phase A: 5 mM ammonium bicarbonate in ddH.sub.2O (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 parameter: ionization mode was cycled through positive and negative mode with cone voltage 10 V and 30 V and 3.25 kV capillary voltage, temperature of source and probe were 120° C. and 600° C. respectively.
Method-D
[0334] Waters Acquity with PDA detector and SQ Detector, column: X-bridge C18, 50*4.6 mm, 3.5 micron, column temperature: 25° C., mobile phase A: 5 mM ammonium bicarbonate in ddH.sub.2O (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 to t=2.7 min (2% A, 98% B) flow: 0.5 mL/min; gradient to t=3 min (0% A, 100% B) flow: 0.7 mL/min; t=3.5 min (0% A, 100% B) flow: 0.7 mL/min; gradient to 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 parameter: ionization mode was cycled through positive and negative mode with cone voltage 10 V and 30 V and 3.25 kV capillary voltage, temperature of source and probe were 120° C. and 600° C. respectively.
Method E
[0335] 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., auto sampler temperature: 5° C.; mobile phase A: 0.1% (v/v) formic acid in ddH.sub.2O (pH=2.70); mobile Phase B: 0.1% formic acid (v/v) in 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 to t=2.7 min (2% A, 98% B) flow: 0.8 mL/min; gradient to t=3 min (0% A, 100% B) flow: 1 mL/min; t=3.5 min (0% A, 100% B) flow: 1 mL/min; gradient to t=3.51 min (97% A, 3% B) flow: 0.8 mL/min; end of run at t=4 min (97% A, 3% B), Flow rate: 0.8 mL/min, analysis time 4 min. Mass detector parameter: ionization mode was cycled through positive and negative modes with cone voltage 10 V and 30 V and 0.8 kV capillary voltage, temperature of source and probe were 120° C. and 600° C. respectively.
Preparatory HPLC Method (for X)
[0336] Waters Quaternary-2545 gradient module with Waters 2489-UV/Visible wave-length detector, Column: YMC-TRI ART C18, 250*21.2 mm, 5 micron, column temperature: room temp., mobile phase A: 5 mM ammonium bicarbonate in water, mobile phase B: 100% acetonitrile; mobile phase gradient details: T=0.01 min (50% A, 50% B; gradient to T=17 min (40% A, 60% B); gradient to T=17.01 min (2% A, 98% B); T=19 min (2% A, 98% B); gradient to 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.
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
[0337] ##STR00002##
[0338] 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 at room temperature in the dark for 1 h before being concentrated in vacuo.
[0339] Purification by chromatography on silica gel (Flash 4 g, 0-20% MeOH:CH.sub.2Cl.sub.2), then reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H.sub.2O (0.1% HCO.sub.2H)), followed by freeze drying, afforded the title compound as a blue solid (1.6 mg, 23%). .sup.1H NMR (DMSO-d.sup.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). 8 protons assumed to be obscured by H.sub.2O peak; HPLC purity 97%; LC/MS R.sub.t=3.83 min; MS m/z: 948.2 [M+H].sup.+.
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-sulfonate
[0340] ##STR00003##
[0341] 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 at room temperature in the dark for 8 h before being diluted with DMSO to a total volume of 1.5 mL. Purification by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H.sub.2O (10 mM NH.sub.4CO.sub.3) followed by freeze drying, afforded the title compound as a blue solid (1.3 mg, 25%). HPLC purity 91%; LC/MS R.sub.t=2.39 min; MS m/z: 662.8 [M+2H].sup.2+.
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
[0342] ##STR00004##
[0343] 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 at room temperature in the dark for 4 h and the crude reaction mixture was purified by reverse-phase HPLC (Sunfire C18, 5-60% MeCN:H.sub.2O (0.1% HCO.sub.2H)), concentrated in vacuo and freeze dried to give the title compound as a blue solid (3.0 mg, 38%). .sup.1H NMR (DMSO-d.sup.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 (quin, J=6.8, 1H), 3.02-2.95 (m, 2H), 2.37-2.17 (m, 10H), 2.11-2.02 (m, 2H), 1.69 (s, 12H), 1.59-1.51 (m, 3H), 1.40-1.30 (m, 6H), 1.25-1.21 (m, 3H), 1.14 (d, J=6.8 Hz, 6H). 1×OH not observed. 2 protons obscured by H.sub.2O peak; LC/MS Rt=3.86 min; MS m/z: 946.0 [M+H]+.
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
[0344] ##STR00005##
[0345] 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 isomer (CAS: 117548-22-8, 3.2 mg, 0.0068 mmol) and N,N-diisopropylethylamine (22 μL, 0.13 mmol). The 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.sub.2O (10 mM (NH.sub.4).sub.2CO.sub.3)) and concentrated in vacuo to give the title compound as an orange powder (3.2 mg, 45%). .sup.1H NMR (DMSO-d.sup.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 (quin, J=6.8, 1H), 2.50-2.24 (m, 8H), 1.69-1.28 (m, 6H), 1.22 (d, J=6.8 Hz, 6H). 6 protons obscured by H.sub.2O peak, CO.sub.2H not observed; LC/MS R.sub.t=3.32 min; MS m/z: 839.7 [M+H].sup.+.
Example 5—Preparation 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 ammonium
[0346] ##STR00006##
[0347] 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 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.sub.2O (10 mM (NH.sub.4).sub.2CO.sub.3)), and concentrated in vacuo to give the title compound as a purple powder (3.9 mg, 67%). .sup.1H NMR (DMSO-d.sup.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 H.sub.2O and DMSO peaks. 0.5 eq NH.sub.4CO.sub.3 adduct; LC/MS R.sub.t=3.09 min; MS m/z: 893.8 [M+H].sup.+.
Example 6—(2,4-Dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone
[0348] ##STR00007##
Synthetic Scheme (See FIG. 1)
Tert-butyl-5-methyl isoindoline-2,5-dicarboxylate (A)
[0349] 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 N.sub.2 for 10-15 min. PdCl.sub.2(dppf) (4.91 g, 6.71 mmol) was added to the reaction mixture at room temperature. The reaction mixture was subject to 30 kg/cm.sup.2 of CO.sub.(gas) pressure, heated to 120° C. and stirred for 16 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The obtained crude material was diluted with water (200 mL) and extracted in EtOAc (3×100 mL). The combined organic layer was washed with brine solution (200 mL), dried over sodium sulphate and concentrated under reduced pressure to give crude material which was purified by column chromatography (silica gel, eluting with 12% ethyl acetate in hexane) yielding 2-(tert-butyl) 5-methyl isoindoline-2,5-dicarboxylate (A) as a Brown solid (10 g).
[0350] LCMS (Method B): 2.657 min, MS: ES+ 278.18 (M+1); .sup.1H NMR (DMSO-d.sup.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).
Tert-butyl 5-(hydroxymethyl)isoindoline-2-carboxylate (B)
[0351] LiAlH.sub.4 solution (1M in THF) (36 mL, 36.10 mmol) was added dropwise to a stirred solution of 2-(tert-butyl) 5-methyl isoindoline-2,5-dicarboxylate (A) (10 g, 36.07 mmol) in THF (200 mL) at 0° C. The reaction mixture was stirred at 0° C. for 3 h and then poured into cold saturated aqueous ammonium chloride solution (100 mL) and extracted into ethyl acetate (3×100 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under vacuum yielding tert-butyl 5-(hydroxymethyl)isoindoline-2-carboxylate (B) as 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); .sup.1H NMR (DMSO-d.sup.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).
Tert-butyl 5-formylisoindoline-2-carboxylate (C)
[0352] To a solution of tert-butyl 5-(hydroxymethyl)isoindoline-2-carboxylate (B) (3 g, 12.04 mmol) in DCM (30 mL) was added activated MnO.sub.2 (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 celite bed, washed with 10% MeOH:DCM (3×50 mL). The combined filtrate was concentrated under vacuum yielding tert-butyl-5-formylisoindoline-2-carboxylate (C) as a grey solid (2.6 g, 86%). This material was used in the next step without any further purification.
[0353] LCMS (Method B): 2.387 min, MS: ES+ 248.18 (M+1); .sup.1H NMR (CDC.sub.3, 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).
Tert-butyl 4-(prop-2-yn-1-yl)piperazine-1-carboxylate (D)
[0354] To a solution of tert-butyl piperazine-1-carboxylate (15 g, 80.51 mmol) in acetonitrile (300 mL) was added K.sub.2CO.sub.3 (22.26 g, 161.30 mmol) at 0° C. Propargyl bromide (80% (w/v) solution in toluene) (12 mL, 80.51 mmol) was added dropwise to the reaction mixture at 0° C. The resulting reaction mixture was slowly warmed to room temperature and stirred at room temperature for 1.5 h. The reaction mixture was filtered and washed with acetonitrile (100 mL). The combined filtrate was concentrated under vacuum and the resultant crude material was purified by column chromatography (silica gel, eluting with 3% (v/v) MeOH in DCM) yielding tert-butyl 4-(prop-2-yn-1-yl)piperazine-1-carboxylate as a brown solid (12.5 g, 71%).
[0355] LCMS (Method A): 0.937 min, MS: ES+ 225.20 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 1.38 (s, 9H), 2.36 (t, J=4.8 Hz, 4H), 3.18 (t, J=2.4 Hz, 1H), 3.27-3.34 (m, 6H).
1-(Prop-2-yn-1-yl)piperazine (E)
[0356] 4M HCl in 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 a nitrogen atmosphere. The reaction mixture was stirred 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 yielding 1-(prop-2-yn-1-yl)piperazine (E) as a brown oil (1.45 g). This material was used for next step without any further purification.
[0357] LCMS (Method A): 0.295 min, MS: ES+ 125.00 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 2.36-2.38 (m, 4H), 2.73 (t, J=4.8 Hz, 4H), 3.16 (t, J=2.4 Hz, 1H), 3.22 (d, J=2.4 Hz, 2H), 3.58 (br. s, 1H).
Tert-butyl 5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (F)
[0358] To a mixture of tert-butyl 5-formylisoindoline-2-carboxylate (C) (0.996 g, 4.03 mmole) 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).sub.3 (2.13 g, 10.05 mmol) was added portion-wise to the reaction mixture at 0° C. The reaction mixture was slowly warmed to room temperature and stirred at room temperature for 24 h. The reaction mixture was poured into water (50 mL) and extracted in EtOAc (3×50 mL). The combined organic layer was washed with sat. aq. NaHCO.sub.3 (50 mL), dried over sodium sulphate, filtered and concentrated under vacuum. The crude material was purified by flash chromatography (silica gel, eluting with 2.5% (v/v) MeOH in 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%).
[0359] LCMS (Method A): 1.588 min, MS: ES+ 356.27 (M+1); .sup.1H NMR (DMSO-d.sup.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).
5-((4-(Prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindoline hydrochloride (G)
[0360] 4M HCl in 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 slowly warmed to room temperature and stirred for 16 h. The resulting mixture was concentrated under vacuum and the crude material was triturated with diethyl ether (2×10 mL); the resulting solid material was concentrated under high vacuum yielding 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 for next step without any further purification.
[0361] LCMS (Method D): 1.24 min, MS: ES+ 256.46 (M+1); .sup.1H NMR (DMSO-d.sup.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=7.6 Hz, 1H), 7.62 (d, J=8.8 Hz, 2H), 10.13 (br. s, 1H), 11.70 (br. s, 1H).
(2,4-Dihydroxy-5-isopropylphenyl)(5-((4-(prop-2-yn-1-yl)piperazin-1-yl)methyl)isoindolin-2-yl)methanone (Example 6)
[0362] 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) at room temperature in a microwave glass tube. The microwave glass tube was sealed and irradiated under microwave at 120° C. for 3 h. The resulting reaction mixture was poured into ice cold water (30 mL) and extracted in EtOAc (3×30 mL). The combined organic layer was dried over sodium sulphate and concentrated under vacuum; the crude material was purified by flash chromatography (silica gel, eluting with 6.8% (v/v) MeOH in DCM) yielding (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%).
[0363] LCMS (Method A): 1.340 min, MS: ES+ 434.22 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): 1.07-1.13 (dd, J=7.5, 6.8 Hz, 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).
Example 7—3-(5,5-Difluoro-7-(1H-pyrrol-2-yl)-5H-4λ.SUP.4.,5λ.SUP.4.-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)propanamide
[0364] ##STR00008##
[0365] (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 into a 5 mL amber vial and DMF (1 mL) was added. To the stirred solution was added N,N-diisopropylethylamine (5.9 μL, 0.034 mmol) and stirring was continued for 5 min. NanoBRET® 590-SE (5.8 mg, 0.014 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 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.
[0366] MS (ESI, m/z) calcd. for C.sub.44H.sub.53BF.sub.5N.sub.7O.sub.4 [M+H].sup.+: 792.4, found 792.5.
Example 8—1-(3-(5,5-Difluoro-7-(1H-pyrrol-2-yl)-5H-4λ.SUP.4.,5λ.SUP.4.-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)propanamido)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)-3,6,9,12-tetraoxapentadecan-15-amide
[0367] ##STR00009##
[0368] (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 was treated with DMF (0.5 mL). To the suspension was added N,N-diisopropylethylamine (5.4 μL, 0.031 mmol) which rendered the solution clear. NanoBRET® 590 PEG-O4-SE (8.4 mg, 0.012 mmol) was dissolved in 0.5 mL DMF and was added to the reaction mixture using an additional 2×0.5 mL portions of DMF to quantitatively transfer the reactive dye.
[0369] 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.
[0370] MS (ESI, m/z) calcd. for C.sub.55H.sub.74BF.sub.2N.sub.8O.sub.9 [M+H].sup.+: 1039.6, found 1039.7.
Example 9—4-(3-(5,5-Difluoro-7-(1H-pyrrol-2-yl)-5H-5λ.SUP.4.,6λ.SUP.4.-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-3-yl)propanamido)-N-(5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl) isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)butanamide
[0371] ##STR00010##
[0372] (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. To the stirred 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, 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 (3.6 mg, 39.4%) as a purple solid.
[0373] MS (ESI, m/z) calcd. for C.sub.48H.sub.60BF.sub.2N.sub.8O.sub.5 [M+H].sup.+: 877.5, found 877.6.
Example 10—3-(5,5-Difluoro-7-(1H-pyrrol-2-yl)-5H-4λ.SUP.4.,5λ.SUP.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
[0374] ##STR00011##
[0375] (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 stirred solution was added N,N-diisopropylethylamine (8.1 μL, 0.046 mmol) and stirring was continued for 5 min. NanoBRET® 590-SE (5.9 mg, 0.014 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 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.
[0376] MS (ESI, m/z) calcd. for C.sub.39H.sub.42BF.sub.2N.sub.8O.sub.4 [M+H].sup.+: 707.3, found 707.4.
Example 11—3-(5,5-Difluoro-7-(1H-pyrrol-2-yl)-5H-4λ.SUP.4.,5λ.SUP.4.-dipyrrolo[1,2-c:2′,1′-f][1,3,2]diazaborinin-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
[0377] ##STR00012##
[0378] (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 was treated with DMF (0.5 mL). To the mixture was added N,N-diisopropylethylamine (8.1 μL, 0.046 mmol) 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 was added to the reaction mixture using an additional 2×0.5 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.
[0379] MS (ESI, m/z) calcd. for C.sub.50H.sub.63BF.sub.2N.sub.7O.sub.9 [M+H].sup.+: 954.5, found 954.6.
Example 12—3-(5,5-Difluoro-7-(1H-pyrrol-2-yl)-5H-5λ.SUP.4.,6λ.SUP.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
[0380] ##STR00013##
[0381] (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 μL, 0.116 mmol) and stirring was continued for 5 min. 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.
[0382] MS (ESI, m/z) calcd. for C.sub.43H.sub.49BF.sub.2N.sub.7O.sub.5 [M+H].sup.+: 792.4, found 792.5.
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 (FIG. 2)
Tert-butyl (2-(2-iodoethoxy)ethyl)carbamate (H)
[0383] 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) at room temperature was added triphenyl phosphine (7.98 g, 30.43 mmol) and imidazole (0.569 g, 8.36 mmole). The reaction mixture was cooled to 0° C. Iodine (8.36 g, 32.92 mmol) was added portion-wise to the reaction mixture at 0° C. The reaction mixture was slowly warmed 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 layer was dried over sodium sulphate, filtered and concentrated under reduce pressure. The crude material was added diethyl ether (100 mL) 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, eluting with 20% (v/v) ethyl acetate in hexane) yielding tert-butyl (2-(2-iodoethoxy)ethyl)carbamate (H) as a colorless oil. (7.8 g, 98%).
[0384] LCMS (Method B): 2.254 min, MS: ES+ 316.13 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 1.36 (s, 9H), 3.04-3.08 (m, 2H), 3.30 (t, J=6.4 Hz, 2H), 3.40 (t, J=6.4 Hz, 2H), 3.62 (t, J=6.4 Hz, 2H), 6.08 (s, 1H).
Benzyl 4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-carboxylate (1)
[0385] To a solution of tert-butyl (2-(2-iodoethoxy)ethyl)carbamate (H) (2.0 g, 6.35 mmol) and benzyl piperazine-1-carboxylate (CAS #31166-44-6) (1.25 g, 5.72 mmol) in acetone (20 mL) were added K.sub.2CO.sub.3 (2.45 g, 17.75 mmole) and NaI (0.38 g, 2.54 mmol) at room temperature. The reaction mixture was heated at 70° C. for 16 h. The reaction mixture was concentrated under vacuum, diluted with water (100 mL) and extracted with ethyl acetate (3×100 mL). The combined organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under vacuum; the crude material was purified by flash chromatography (silica gel, eluting with 2.5% (v/v) MeOH in DCM) yielding benzyl 4-(2-(2-((tert-butoxycarbonyl)amino) ethoxy)ethyl)piperazine-1-carboxylate (1) as a yellow oil (2.0 g, 77%).
[0386] LCMS (Method A): 1.640 min, MS: ES+ 408.32 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 1.35 (s, 9H), 2.38 (t, J=4.8 Hz, 4H), 2.44-2.47 (m, 2H), 3.02-3.06 (m, 2H), 3.33-3.36 (m, 6H), 3.48 (t, J=5.6 Hz, 2H), 5.06 (s, 2H), 6.80 (t, J=5.2 Hz, 1H), 7.29-7.39 (m, 5H).
Tert-butyl (2-(2-(piperazin-1-yl)ethoxy)ethyl)carbamate (J)
[0387] To a mixture of benzyl 4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazine-1-carboxylate (1) (1.5 g, 3.68 mmol) and acetic acid (0.23 mL, 4.03 mmol) in MeOH (7 mL) was added 10% Pd(OH).sub.2 on carbon (0.45 g) at room temperature. The reaction mixture was bubbled with H.sub.2(g) for 4 h. The reaction mixture was filtered through a celite bed, washed with MeOH (2×10 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.
[0388] .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 1.36 (s, 9H), 2.39-2.42 (m, 6H), 2.71 (t, J=4.8 Hz, 4H), 3.02-3.06 (m, 2H), 3.34 (t, J=6.0 Hz, 2H), 3.45 (t, J=5.6 Hz, 2H), 5.76 (s, 1H), 6.80 (t, J=5.2 Hz, 1H).
Isoindoline-5-carbaldehyde hydrochloride (K)
[0389] 4M HCl in 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) 0° C. The reaction mixture was slowly warmed to room temperature and stirred for 6 h. The reaction mixture was concentrated under reduce pressure and the crude material purified by triturating with diethyl ether (3×20 mL). The resultant solid material was dried under high vacuum yielding isoindoline-5-carbaldehyde hydrochloride (K) as an off white solid (0.8 g, 83%).
[0390] LCMS (Method C): 2.154 min, MS: ES+ 148.18 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz) δ 4.57 (d, J=4.0 Hz, 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).
Benzyl 5-formylisoindoline-2-carboxylate (L)
[0391] 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 2M HCl (10 mL) and extracted with ethyl acetate (3×30 mL). The combined organic layer was washed with water (100 mL), dried over anhydrous sodium sulphate, filtered and concentrated under vacuum. The crude material was purified by column chromatography (silica gel, eluting with 25% (v/v) ethyl acetate in hexane) yielding benzyl 5-formylisoindoline-2-carboxylate (L) as a yellow solid (1.0 g, 82%).
[0392] .sup.1H NMR (DMSO-d.sup.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=1.6 Hz, 1H).
Benzyl-5-((4-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)piperazin-1-yl)methyl) isoindoline-2-carboxylate (M)
[0393] 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).sub.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 poured into sat. aq. NaHCO.sub.3 (50 mL) and extracted in DCM (2×150 mL). The combined organic layer was dried over sodium sulphate and concentrated under vacuum; the crude material was purified by flash chromatography (silica gel, eluting with 7% (v/v) MeOH in DCM) yielding 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%).
[0394] LCMS (Method C): 4.34 min, MS: ES+ 539.64 (M+1); .sup.1H NMR (DMSO-d.sup.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=5.6 Hz, 1H), 4.62-4.68 (m, 4H), 5.14 (s, 2H), 6.72-6.77 (t, J=5.2 Hz, 1H), 7.20-7.42 (m, 8H).
Tert-butyl (2-(2-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)ethoxy)ethyl)carbamate (N)
[0395] 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% moisture) (0.020 g) at room temperature. The reaction mixture was bubbled with H.sub.2(g) for 12 h. The reaction mixture was filtered through a celite bed, washed with MeOH (2×5 mL). The combined filtrate was concentrated under vacuum to give tert-butyl (2-(2-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)ethoxy)ethyl)carbamate (N) as brown oil (0.076 g, 100%). This material was used in the next step without any further purification.
[0396] LCMS (Method A): 1.036 min, MS: ES+ 405.32 (M+1)
Tert-butyl (2-(2-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)ethoxy)ethyl) carbamate (O)
[0397] 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) was added 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) at room temperature in a microwave glass tube; the tube was sealed and irradiated under microwave at 120° C. for 1 h. The resulting reaction mixture was poured into ice cold water (20 mL) and extracted in EtOAc (5×30 mL). The combined organic layer was dried over sodium sulphate, filtered and concentrated under vacuum to give crude material which was purified by flash chromatography (silica gel, eluting with 8% (v/v) MeOH in DCM) to yield 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%).
[0398] LCMS (Method A): 1.728 min, MS: ES+ 583.5 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 1.12 (d, J=6 Hz, 6H), 1.34 (s, 9H), 2.23-2.33 (m, 10H), 3.04 (s, 4H), 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=24.4 Hz, 1H).
(5-((4-(2-(2-aminoethoxy)ethyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone hydrochloride
[0399] 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 4M HCl in 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 crude material which was triturated with diethyl ether (2×10 mL) and dried under high vacuum yielding (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%).
[0400] LCMS (Method B): 1.456 min, MS: ES+ 483.5 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 1.12 (d, J=6.8 Hz, 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).
Preparation of 2,4-Dihydroxy-5-isopropylbenzoic acid (FIG. 3)
Methyl 2,4-dihydroxybenzoate (P)
[0401] To a stirred solution of 2,4-dihydroxybenzoic acid (100 g, 649.35 mmol) in methanol (400 mL) was added concentrated H.sub.2SO.sub.4 (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 yielding methyl 2,4-dihydroxybenzoate (P) as an off white solid (170 g, 78%). This material was used for the next step without any further purification.
[0402] LCMS (Method C): 4.24 min, MS: ES+ 169.19 (M+1); .sup.1H NMR (CDC.sub.3, 400 MHz) δ 3.39 (s, 3H), 5.72 (br. s, 1H), 6.38-6.42 (m, 2H), 7.74 (d, J=8.8 Hz, 1H), 11.02 (s, 1H).
Methyl 2,4-dihydroxy-5-isopropylbenzoate (Q)
[0403] To a solution of methyl 2,4-dihydroxybenzoate (80 g, 475.76 mmol) in DCM (1200 mL) were added anhydrous AlCl.sub.3 (126.87 g, 951.48 mmol) followed by isopropyl bromide (351.09 g, 2854.62 mmol added in 3 equal portions (117.03 g each) every 6 h) at 50° C. After completion of the addition of isopropyl bromide, the reaction was heated at 50° C. for 6 h; the total reaction time was 24 h. The reaction mixture was concentrated under vacuum and the crude material diluted with water (1000 mL) and extracted with ethyl acetate (4×500 mL). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum. The obtained crude material was purified three times by column chromatography (silica gel, eluting with 3% (v/v) EtOAc in hexane) yielding methyl 2,4-dihydroxy-5-isopropylbenzoate (Q) as yellow solid (25.5 g, 26%).
[0404] LCMS (Method A): 2.069 min, MS: ES+ 211.11 (M+1); .sup.1H NMR (DMSO, 400 MHz) δ 1.13 (d, J=7.6 Hz, 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).
2,4-Dihydroxy-5-isopropylbenzoic acid
[0405] 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 1M HCl (pH 5). The resulting precipitates were collected by filtration and dried under high vacuum yielding 2,4-dihydroxy-5-isopropylbenzoic acid as a white solid (13 g, 61%)
[0406] LCMS: (Method B) 2.044 min, MS: ES+ 197.22 (M+1); .sup.1H NMR (DMSO-d.sup.6, 400 MHz): δ 1.02 (d, J=6.8 Hz, 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).
Preparation of (5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone (FIG. 4)
Tert-butyl (5-hydroxypentyl)carbamate (R)
[0407] 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 warmed to room temperature and stirred for 16 h. The reaction mixture was poured in water (150 mL) and extracted in DCM (2×100 mL). The combined organic layer dried over anhydrous sodium sulphate, filtered and concentrated under vacuum. The obtained crude material was purified by flash chromatography (silica, eluting with 18% (v/v) ethyl acetate in hexane) yielding tert-butyl (5-hydroxypentyl)carbamate (R) as a pale yellow oil (15 g, 76%).
[0408] .sup.1H NMR (CDCl.sub.3, 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).
Tert-butyl (5-bromopentyl) carbamate (S)
[0409] To a mixture of tert-butyl (5-hydroxypentyl)carbamate (R) (8 g, 39.41 mmol) in THF (80 mL) were added triphenyl phosphine (15.48 g, 59.11 mmol) and CBr.sub.4 (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 (3×150 mL). The combined organic layer was dried over sodium sulphate, filtered and concentrated under reduce pressure. The crude material was purified by column chromatography (silica gel, eluting with 10% (v/v) ethyl acetate in hexane) yielding tert-butyl (5-bromopentyl) carbamate (S) as a colorless oil. (9 g, 86%).
[0410] LCMS (Method A): 2.150 min, MS: ES+ 210.1, 212.1 (M-56); .sup.1H NMR (CDCl.sub.3, 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).
Benzyl 4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-carboxylate (T)
[0411] To a mixture of benzyl piperazine-1-carboxylate (CAS #31166-44-6) (4.135 g, 18.79 mmol) in acetone (50 mL), was added K.sub.2CO.sub.3 (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 min. 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 h. 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 layer was dried over anhydrous sodium sulphate, filtered and concentrated under vacuum to give crude material that was purified by flash chromatography (silica gel, eluting with 3% (v/v) MeOH in DCM) yielding benzyl 4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazine-1-carboxylate (T) as a brown oil (6.5 g, 85%).
[0412] LCMS (Method A): 1.492 min, MS: ES+ 406.42 (M+1); .sup.1H NMR (DMSO-d.sup.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).
Tert-butyl (5-(piperazin-1-yl)pentyl)carbamate (U)
[0413] 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% moisture) (0.640 g, 10% w/w) at room temperature. The reaction mixture was purged with H.sub.2(g) for 5 h. The reaction mixture was filtered through a celite bed, washed with MeOH (2×10 mL) and the combined filtrate concentrated under reduced pressure to give tert-butyl (5-(piperazin-1-yl)pentyl)carbamate (U) as brown oil (4 g, 93%). This material was used for next step without further purification.
[0414] MS: ES+ 272.20 (M+1). .sup.1H NMR (CDCl.sub.3, 400 MHz): δ 1.28-1.35 (m, 2H), 1.43 (s, 9H), 1.43-1.50 (m, 2H), 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).
Benzyl-5-((4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (V)
[0415] 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).sub.3 (3.618 g, 17.078 mmol) was added to the reaction mixture at 0° C. The reaction mixture was slowly warmed to room temperature and stirred for 16 h. The reaction mixture poured into water (50 mL) and extracted in DCM (2×150 mL). The combined organic layer was dried over sodium sulphate, filtered and concentrated under vacuum; the crude material was purified by flash chromatography (silica gel, eluting with 3% (v/v) MeOH in DCM) yielding benzyl 5-((4-(5-((tert-butoxycarbonyl)amino)pentyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (V) as a brown oil (2.2 g, 72%).
[0416] LCMS (Method A): 1.552 min, MS: ES+ 537.41 (M+1); .sup.1H NMR (DMSO-d.sup.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).
Tert-butyl (5-(4-(isoindolin-5-ylmethyl)piperazin-1-yl)pentyl)carbamate (W)
[0417] 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% moisture) (0.420 g, 20% w/w) at room temperature. The reaction mixture was purged with H.sub.2(g) for 5 h. The reaction mixture was filtered through a celite bed, washed with MeOH (2×20 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.
[0418] LCMS (Method A): 0.757 min, MS: ES+ 403.37 (M+1); .sup.1H NMR (DMSO-d.sup.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).
Tert-butyl (5-(4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazin-1-yl)pentyl)carbamate (X)
[0419] 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) were added 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) at room temperature in microwave glass tube. The microwave glass tube was sealed and irradiated under microwave at 120° C. for 3 h. The resulting reaction mixture was poured into cold water (50 mL) and extracted in EtOAc (3×50 mL). The combined organic layer was dried over sodium sulphate, filtered and concentrated under vacuum to give crude material (270 mg). 180 mg crude material was purified by HPLC (5 mM Ammonium bicarbonate in water/MeCN). The isolated fractions were lyophilized yielding 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%).
[0420] LCMS (Method A): 1.728 min, MS: ES+ 583.5 (M+1); .sup.1H NMR (DMSO-d.sup.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).
(5-((4-(5-aminopentyl)piperazin-1-yl)methyl)isoindolin-2-yl)(2,4-dihydroxy-5-isopropylphenyl)methanone
[0421] 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 4M HCl in 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 diethyl ether (2×5 mL). The obtained solid was dried under lyophilization yielding (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%).
[0422] LCMS (Method B): 1.625 min, MS: ES+ 481.50 (M+1); .sup.1H NMR (DMSO-d.sup.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).
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) (FIG. 5)
Tert-butyl-5-((4-((benzyloxy)carbonyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (Y)
[0423] A mixture of tert-butyl-5-formylisoindoline-2-carboxylate (C) (see Example-6) (3.0 g, 12.13 mmol) and benzyl piperazine-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 water of condensation). The residue was dissolved in dichloromethane (50 mL) and evaporated to dryness (to remove traces of methanol); this process was repeated once to complete removal of methanol. The obtained residue was dissolved in DCM (90 mL), placed under a nitrogen atmosphere and cooled to 0° C. NaBH(OAc).sub.3 (3.8 g, 17.93 mmol) was added portion wise to the reaction mixture at 0° C. The reaction mixture was slowly warmed to room temperature and stirred for 16 h. The previous preparation was conducted in 2 parallel batches and the combined reaction mixtures poured into water (200 mL) and extracted in DCM (3×250 mL). The combined organic layer was washed with saturated aqueous sodium bicarbonate solution (2×100 mL) and dried over sodium sulphate, filtered and concentrated under vacuum to give crude material that was purified by flash chromatography (silica gel, eluting with 25% (v/v) EtOAc in n-hexane) yielding tert-butyl 5-((4-((benzyloxy)carbonyl)piperazin-1-yl)methyl)isoindoline-2-carboxylate (Y) as a brown oil (5.0 g, 46%).
[0424] LCMS (Method A): 1.631 min, MS: ES+ 452.32 (M+1); .sup.1H NMR (DMSO-d.sup.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).
Benzyl 4-(isoindolin-5-ylmethyl)piperazine-1-carboxylate hydrochloride (Z)
[0425] 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 in dioxane (5.0 mL) at 0° C. The reaction mixture was stirred at 0° C. for 2 h and then concentrated under vacuum and the crude material triturated with diethyl ether (3×10 mL). The obtained solid was dried under high vacuum yielding benzyl 4-(isoindolin-5-ylmethyl)piperazine-1-carboxylate hydrochloride (Z) as a green solid (0.99 g, quantitative).
[0426] LCMS (Method E): 0.865 min, MS: ES+ 352.22 (M+1); .sup.1H NMR (DMSO-d.sup.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).
Benzyl 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazine-1-carboxylate (AA)
[0427] To a mixture of 2,4-dihydroxy-5-isopropylbenzoic acid (0.205 g, 1.046 mmol) in DMF (10 mL) was added 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) at room temperature in a microwave glass tube. Benzyl-4-(isoindolin-5-ylmethyl)piperazine-1-carboxylate hydrochloride (Z) (0.980 g, 2.52 mmol) was added to the reaction at room temperature. The microwave glass tube was sealed and microwave irradiated at 120° C. for 1 h. The resulting reaction mixture was poured into cold water (60 mL) whereupon a solid material precipitated, isolation and drying under vacuum gave 0.8 g crude material. Purification by flash chromatography (silica gel, eluting with 1.5% (v/v) MeOH in DCM) yielded benzyl 4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)piperazine-1-carboxylate as an off white solid (0.395 g, 30%).
[0428] LCMS (Method E): 1.567 min, MS: ES+ 530.22 (M+1); .sup.1H NMR (DMSO-d.sup.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).
(2,4-dihydroxy-5-isopropylphenyl)(5-(piperazin-1-ylmethyl)isoindolin-2-yl)methanone hydrochloride (AB)
[0429] To a solution of benzyl-4-((2-(2,4-dihydroxy-5-isopropylbenzoyl)isoindolin-5-yl)methyl)-piperazine-1-carboxylate (AB) (0.30 g, 0.56 mmol) in EtOH (20 mL) was added AcOH (0.6 mL) and 10% Pd/C (50% moisture) (0.20 g, 20% w/w) at room temperature. The reaction mixture was cooled to between 10° C. and 15° C. and purged with H.sub.2(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% HCl 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%).
[0430] LCMS (Method E): 1.072 min, MS: ES+ 396.27 (M+1); .sup.1H NMR (DMSO-d.sup.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).
Protein Dependence of Fluorescence Polarisation
[0431] An 11 point 1 in 2 serial dilution of protein (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 MgCl.sub.2, 0.01% Triton X-100, 5 mM Dithiothreitol) was constructed in a clear polypropylene plate (Greiner Bio-One, item number 784201). The top concentration of the serial dilution was 2 μM. The 12.sup.th concentration point was buffer only (no protein control).
[0432] The serial dilution was used as the 2× protein and 20 μL was added to a Black Fluotrac 200 384-well medium binding plate (Greiner Bio-One, item number 781076). Plates were centrifuged for 1 minute at 250×g and were incubated at room temperature for 30 minutes prior to the addition of 20 μL of 2× (either 5 nM or 2 nM) of probe in assay buffer (prepared from a 100 μM DMSO stock) with a MultiDrop Combi (ThermoFisher).
[0433] Compound plates were centrifuged for 1 minute at 250×g for 1 minute and were incubated at room temperature for 1 hour before being read on a PheraStar FSX (fitted with 384-well aperture spoon and appropriate FP module). The gain and focus were adjusted before each plate was read so that the polarisation of the no protein control was equal to 35 mP. Data was plotted and analysed in GraphPad Prism 8.1.0 (
Assay Protocols
Assay A: Fluorescence Polarisation Assay for MLH1
[0434] 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. 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 MgCl.sub.2, 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 using a MultiDrop Combi (ThermoFisher). Plates were centrifuged for 1 minute at 250×g and were incubated at room temperature for 30 minutes prior to the addition of 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 a 1 mM DMSO stock) with 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 for 1 minute at 250×g for 1 minute and were incubated at room temperature for 1 hour before being read on a PheraStar FSX (fitted with 384-well aperture spoon and 590 675 675 FP optic module). The gain and focus were adjusted before each plate was read so that the polarisation of a no enzyme control (column 24) was equal to 35 mP. Data were normalised against the no inhibitor controls (column 23) and no enzyme controls (column 24).
Assay B: Fluorescence Polarisation Assay for MLH1
[0435] 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. For single point screening, 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. For potency determination, serial dilutions of test compounds were added to wells in columns 3-22 and DMSO volume was normalised across the plate.
[0436] 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 MgCl.sub.2, 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 (column 24 only for single point screening) using a MultiDrop Combi (ThermoFisher). Plates were centrifuged for 1 minute at 250×g and were incubated at room temperature for 30 minutes prior to the addition of 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-(dimethyliminio)-3H-xanthen-9-yl)benzoate (Example 5) in assay buffer (prepared from a 100 μM DMSO stock) with 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-(dimethyliminio)-3H-xanthen-9-yl)benzoate (Example 5) was 5 nM.
[0437] Compound plates were centrifuged for 1 minute at 250×g and were incubated at room temperature for 15 minutes before being read on a PheraStar FSX (fitted with 384-well aperture spoon and 540 590 590 FP optic module). The gain and focus were adjusted before each plate was read so that the polarisation of a no enzyme control (column 24) was equal to 35 mP. Data were normalised against the no inhibitor controls (column 23) and no enzyme controls (column 24).
Assay C: Fluorescence Polarisation Assay for PMS2
[0438] 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. For single point screening, 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. For potency determination, serial dilutions of test compounds were added to wells in columns 3-22 and DMSO volume was normalised across the plate.
[0439] 20 μL of a 2× solution (20 nM) of recombinant N-terminal PMS2 (residues 1-365) in assay buffer (25 mM HEPES, pH 7.5, 250 mM NaCl, 10 mM MgCl.sub.2, 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 (column 24 only for single point screening) using a MultiDrop Combi (ThermoFisher). Plates were centrifuged for 1 minute at 250×g and were incubated at room temperature for 30 minutes prior to the addition of 20 μL of 2× (20 nM) 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) in assay buffer (prepared from a 100 μM DMSO stock) with 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-(dimethyliminio)-3H-xanthen-9-yl)benzoate (Example 5) was 5 nM.
[0440] Compound plates were centrifuged for 1 minute at 250×g and were incubated at room temperature for 1 hour before being read on a PheraStar FSX (fitted with 384-well aperture spoon and 540 590 590 FP optic module). The gain and focus were adjusted before each plate was read so that the polarisation of a no enzyme control (column 24) was equal to 35 mP. Data were normalised against the no inhibitor controls (column 23) and no enzyme controls (column 24).
[0441] The above assays can be used to screen for compounds that bind to MLH1 and/or PMS2. For example, the IC.sub.50 for compound AB is determined as 10.7 μM using Assay Protocol A (
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