PROBES FOR IMAGING HUNTINGTIN PROTEIN
20170281804 · 2017-10-05
Inventors
- Celia Dominguez (Los Angeles, CA)
- John Wityak (Carlsbad, CA)
- Jonathan Bard (New York, NY)
- Christopher John Brown (Abingdon, GB)
- Thomas Martin KRÜLLE (Oxford, GB)
- Daniel Clark-Frew (Wantage, GB)
- Sarah Hayes (Abingdon, GB)
Cpc classification
A61K51/0455
HUMAN NECESSITIES
C07D403/04
CHEMISTRY; METALLURGY
C07B2200/05
CHEMISTRY; METALLURGY
International classification
Abstract
Provided are imaging agents comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and methods of their use.
##STR00001##
Claims
1. An imaging agent comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, ##STR00042## wherein Z.sub.1, Z.sub.2, Z.sub.3, and Z.sub.4 are independently chosen from CH and N, provided that at least two of Z.sub.1, Z.sub.2, Z.sub.3, and Z.sub.4 are CH; R.sub.1 is chosen from aryl and heteroaryl, and heterocycloalkenyl, each of which is optionally substituted with one or two groups independently chosen from alkynyl, heteroaryl, cyano, optionally substituted amino, halo, lower alkyl, and lower alkyl substituted with optionally substituted amino; L.sub.1 is chosen from O and NR.sub.4; R.sub.4 is chosen from hydrogen and lower alkyl; L.sub.2 is (CH.sub.2).sub.m where m is 0, 1, or 2; and R.sub.2 is chosen from hydrogen, aryl, aryl substituted with hydroxyl or lower alkoxy, heteroaryl, and heteroaryl substituted with hydroxyl or lower alkoxy, R.sub.5 is chosen from lower alkyl, lower alkoxy, halo, and oxo (as a substituent on the heterocycloalkyl ring); and n is 0 or 1; wherein the compound of Formula I, or a pharmaceutically acceptable salt there of, is labeled with one or more positron-emitting radionuclides.
2. The imaging agent of claim 1, wherein R.sub.1 is phenyl optionally substituted with one or two groups independently chosen from cyano, optionally substituted amino, halo, lower alkyl, and lower alkyl substituted with optionally substituted amino.
3. The imaging agent of claim 2, wherein R.sub.1 is phenyl optionally substituted with one or two groups independently chosen from cyano, methyl, and methyl substituted with amino, (alkyl)amino or (dialkyl)amino.
4. The imaging agent of claim 3, wherein R.sub.1 is 2-cyanophenyl.
5. The imaging agent of claim 1, wherein R.sub.1 is heteroaryl optionally substituted with one or two groups independently chosen from alkynyl, cyano, optionally substituted amino, halo, lower alkyl, and lower alkyl substituted with optionally substituted amino.
6. The imaging agent of claim 5, wherein R.sub.1 is chosen from pyridine-4-yl, pyridine-2-yl, pyridine-3-yl, pyrimidine-4-yl, 1,2-dihydropyridin-2-one-3-yl, 1H-indazole-4-yl, and 1H-indazole-7-yl, each of which is optionally substituted with one or two groups independently chosen from alkynyl, cyano, optionally substituted amino, halo, lower alkyl, and lower alkyl substituted with optionally substituted amino.
7. The imaging agent of claim 6, wherein R.sub.1 is chosen from pyridine-4-yl, pyridine-2-yl, pyridine-3-yl, and pyrimidine-4-yl, each of which is optionally substituted with one or two groups independently chosen from alkynyl, cyano, optionally substituted amino, halo, lower alkyl, and lower alkyl substituted with optionally substituted amino.
8. The imaging agent of claim 6, wherein R.sub.1 is chosen from 5-cyano-pyrimidine-4-yl, pyridine-4-yl, 5-bromo-1,2-dihydropyridin-2-one-3-yl, 3-acetamido-pyridine-4-yl, 2-acetamido-pyridine-6-yl, 3-cyano-pyridine-4-yl, 3-cyano-pyridine-6-yl, 3-bromo-pyridine-4-yl, 3-bromo-pyridine-2-yl, 3-cyano-pyridine-2-yl, 3-fluoro-pyridine-4-yl, 2-cyano-pyridine-4-yl, 4-cyano-pyridine-3-yl, and 3-ethynyl-pyridine-4-yl.
9. The imaging agent of claim 8, wherein R.sub.1 is pyridine-4-yl, 5-cyano-pyrimidine-4-yl, or 3-cyano-pyridine-4-yl.
10. The imaging agent of claim 1, wherein R.sub.1 is heterocycloalkenyl optionally substituted with lower alkyl.
11. The imaging agent of claim 10, wherein R.sub.1 is 2,3-dihydropyridazine-6-yl optionally substituted with lower alkyl.
12. The imaging agent of claim 1, wherein L.sub.1 is O.
13. The imaging agent of claim 12, wherein m is 1.
14. The imaging agent of claim 12, wherein R.sub.2 is chosen from hydrogen, aryl, aryl substituted with hydroxyl or lower alkoxy, heteroaryl, and heteroaryl substituted with hydroxyl or lower alkoxy.
15. The imaging agent of claim 14, wherein R.sub.2 is chosen from hydrogen, phenyl, pyridine-2-yl, pyrimidine-5-yl, pyrazin-2-yl, and pyrimidin-5-yl, each of which, other than hydrogen, is optionally substituted with hydroxyl or lower alkoxy.
16. The imaging agent of claim 1, wherein Z.sub.1, Z.sub.2, Z.sub.3, and Z.sub.4 are CH.
17. The imaging agent of claim 1, wherein Z.sub.1 is N and Z.sub.2, Z.sub.3, and Z.sub.4 are CH.
18. The imaging agent of claim 1, wherein Z.sub.2 is N and Z.sub.1, Z.sub.3, and Z.sub.4 are CH.
19. The imaging agent of claim 1, wherein Z.sub.2 and Z.sub.4 are N and Z.sub.1 and Z.sub.3 are CH.
20. The imaging agent of claim 1, wherein the compound of Formula I is chosen from 2-(5-methoxy-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile; 2-{5-[(5-methoxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}pyridine-3-carbonitrile; 2-[5-(pyrimidin-5-ylmethoxy)-2,3-dihydro-1H-isoindol-2-yl]pyridine-3-carbonitrile; 4-{5-[(5-methoxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}pyridine-3-carbonitrile; 4-(5-methoxy-2,3-dihydro-1H-isoindol-2-yl)pyrimidine-5-carbonitrile; 4-{5-[(5-methoxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}pyrimidine-5-carbonitrile; 4-{5-[(5-hydroxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}pyridine-3-carbonitrile; 4-(5-methoxy-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile; 4-[5-(pyrimidin-5-ylmethoxy)-2,3-dihydro-1H-isoindol-2-yl]pyridine-3-carbonitrile; 5-[(5-methoxypyrazin-2-yl)methoxy]-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindole; 4-[5-(benzyloxy)-2,3-dihydro-1H-isoindol-2-yl]pyrimidine-5-carbonitrile; 4-{5-[(5-hydroxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}pyrimidine-5-carbonitrile; 6-{5-[(5-methoxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}-2-methyl-2,3-dihydropyridazin-3-one; and 5-[(5-methoxypyridin-2-yl)methoxy]-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindol-1-one.
21. The imaging agent of claim 1, wherein said compound contains one or more positron-emitting radionuclides selected from: .sup.11C, .sup.13N, .sup.15O, and .sup.18F.
22. A method of generating diagnostic images in an individual comprising administering an effective amount of an imaging agent of claim 1 to an individual, and generating an image of at least a part of said individual.
23. The method of claim 22, wherein generating an image of at least a part of said individual comprises generating an image to detect the presence or absence of huntingtin protein (HTT protein) monomers or aggregates in the brain of said individual; and detecting the presence or absence of a pathologic process.
24. The method of claim 23, wherein said HTT protein monomers or aggregates are present in the basal ganglia of said brain of said individual.
25. The method of claim 23, wherein the pathologic process is a neurodegenerative disease.
26. The method of claim 25, wherein the neurodegenerative disease is chosen from Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, Prion disease and spinocerebellar ataxias.
27. The method of claim 26, wherein the neurodegenerative disease is Huntington's disease (HD).
28. The method of claim 1, wherein said effective amount of said imaging agent comprises from about 0.1 to about 20 mCi.
29. The method of claim 28, wherein said effective amount of said imaging agent comprises about 10 mCi.
30. The method of claim 1, wherein said generating an image comprises positron emission tomography (PET) imaging, PET with concurrent computed tomography imaging (PET/CT), PET with concurrent magnetic resonance imaging (PET/MRI), or a combination thereof.
31. The method of claim 30, wherein said generating an image comprises PET imaging.
Description
EXAMPLES
General Experimental Details
[0164] Commercially available reagents and solvents (HPLC grade) were used without further purification. .sup.1H NMR spectra were recorded on a Bruker DRX 500 MHz spectrometer or a Bruker DPX 250 MHz spectrometer in deuterated solvents. Chemical shifts (δ) are in parts per million. SCX chromatography was performed with Biotage Isolute Flash SCX-2 loading the sample in methanol and eluting with methanol then 5% ammonia in methanol.
[0165] Analytical HPLC-MS (METCR1278), was performed on Shimadzu LCMS-2010EV systems using reverse phase Atlantis dC18 columns (3 μm, 2.1×50 mm), gradient 5-100% B (A=water/0.1% formic acid, B=acetonitrile/0.1% formic acid) over 3 minutes injection volume 3 μL, flow=1.0 mL/minute. UV spectra were recorded at 215 nm using a SPD-M20A photo diode array detector. Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2 scans per second using a LCMS2010EV. Data were integrated and reported using Shimadzu LCMS-Solutions and PsiPort software.
[0166] Alternatively, (METCR1416) analytical HPLC-MS on Shimadzu LCMS-2010EV systems using reverse phase Water Atlantis dC18 columns (3 nm, 2.1×100 mm), gradient 5-100% B (A=water/0.1% formic acid, B=acetonitrile/0.1% formic acid) over 7 minutes, injection volume 3 μL, flow=0.6 mL/minute. UV spectra were recorded at 215 nm using a SPD-M20A photo diode array detector. Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2 scans per second using a LCMS2010EV. Data were integrated and reported using Shimadzu LCMS-Solutions and PsiPort software.
[0167] Alternatively, (MET-uHPLC-AB-101) analytical HPLC-MS were performed on a Waters Acquity UPLC system with Waters PDA and ELS detectors using a Phenomenex Kinetex-XB C-18 column, (1.7 μM, 2.1 mm×100 mm at a column temperature of 40° C., gradient 5-100% B (A=water/0.1% formic acid; B=acetonitrile/0.1% formic acid) over 5.3 minutes, then 100% B for 0.5 minute, flow=0.6 mL/minute. UV spectra were recorded at 215 nm using a Waters Acquity photo diode array. Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 5 scans per second using a Waters SQD. Data were integrated and reported using Waters MassLynx and OpenLynx software.
[0168] All example compounds display an LC purity of >95% unless stated otherwise.
Method 1
[0169] ##STR00005##
Step 1, Method 1: 5-(Chloromethyl)pyrimidine hydrochloride
[0170] To a solution of pyrimidin-5-ylmethanol (48 mg, 0.43 mmol) in dichloromethane (3 mL), thionyl dichloride (0.26 mL, 3.6 mmol) was added drop-wise at 0° C. The mixture was heated to reflux for 2 hours, then the mixture was concentrated. Dichloromethane (5 mL) was added and the mixture was concentrated (×3) to give the title compound as a yellow oil which was used directly in the next step. Tr(METCR1278)=0.90 min, (ES.sup.+) (M+H).sup.+129/131.
Step 2, Method 1: 4-(5-Methoxy-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile
[0171] 5-Methoxy-2,3-dihydro-1H-isoindole hydrochloride (500 mg, 2.69 mmol), 4-chloropyridine-3-carbonitrile (448 mg, 3.23 mmol) and diisopropylethylamine (1.4 mL, 8.08 mmol) were suspended in n-butanol (6 mL). The reaction was heated in a microwave at 140° C. for 1 hour. The reaction mixture was partitioned between ethyl acetate (50 mL) and water (30 mL) and the aqueous was extracted with ethyl acetate (2×30 mL). The combined organic extracts were washed with brine (15 mL), dried over anhydrous sodium sulphate, filtered and concentrated. Purification by FCC (silica, 20-100% ethyl acetate in heptane) gave the title compound 230 mg (34% yield) as an off white solid. Tr(METCR1278)=1.28 min, (ES.sup.+) (M+H).sup.+252.
Step 3, Method 1: 4-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile
[0172] 4-(5-Methoxy-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile (230 mg, 0.92 mmol) was dissolved in dichloromethane (15 mL) and stirred in a nitrogen atmosphere. The reaction mixture was cooled to 0° C. and 1 M boron tribromide in dichloromethane (4.58 mL, 4.58 mmol) was added slowly. The reaction mixture was allowed to warm to room temperature and was stirred for 48 hours. The reaction mixture was cooled to 0° C. and methanol (20 mL) was added slowly. The solvents were removed in vacuo to give the title compound 313 mg (quantitative yield) as a beige solid. Tr(METCR1278)=1.36 min, (ES.sup.+) (M+H).sup.+238, 90%.
Step 4, Method 1: 4-[5-(Pyrimidin-5-ylmethoxy)-2,3-dihydro-1H-isoindol-2-yl]pyridine-3-carbonitrile
[0173] 4-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile (90%, 313 mg, 1.19 mmol), 5-(chloromethyl)pyrimidine hydrochloride (crude 1.42 mmol) and potassium iodide (217 mg, 1.31 mmol) were dissolved in anhydrous N,N-dimethylformamide (3 mL) and stirred for 5 minutes at room temperature. Sodium hydride (60% in mineral oil, 142 mg, 3.56 mmol) was added and the reaction mixture was stirred at room temperature for 24 hours. Water (0.1 mL) was added and the solvents were removed in vacuo. The residue was partitioned between ethyl acetate (50 mL) and water (50 mL) and the aqueous phase was extracted with ethyl acetate (2×50 mL). The combined organic extracts were washed with water (20 mL), brine (20 mL), dried over anhydrous sodium sulphate, filtered and concentrated. Purification by preparative HPLC (acetonitrile-water-0.2% ammonium hydroxide) gave the title compound 19.5 mg (5% yield) as an off white solid.
Example 1, Method 1: 4-[5-(Pyrimidin-5-ylmethoxy)-2,3-dihydro-1H-isoindol-2-yl]pyridine-3-carbonitrile
[0174] δ.sub.H NMR (500 MHz, DMSO) 9.19 (s, 1H), 8.93 (s, 2H), 8.51 (s, 1H), 8.29 (d, J=6.3 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 7.16 (d, J=1.9 Hz, 1H), 7.05 (dd, J=8.4, 2.3 Hz, 1H), 6.77 (d, J=6.3 Hz, 1H), 5.23 (s, 2H), 5.03 (s, 2H), 4.97 (s, 2H). Tr(MET-uHPLC-AB-101)=1.4 min, (ES.sup.+) (M+H).sup.+330.
[0175] The following examples were prepared using Method 1 described above:
TABLE-US-00001 TABLE 1 Mol. Ex. Structure Weight IUPAC Name LCMS data 1
Method 2
[0176] ##STR00016##
Step 1, Method 2: Methyl 5-(methoxymethoxy)pyridine-2-carboxylate
[0177] Sodium hydride (60% in mineral oil, 144 mg, 3.59 mmol) was suspended in anhydrous N,N-dimethylformamide (5 mL) and cooled to 0° C. Methyl 5-hydroxypyridine-2-carboxylate (500 mg, 3.27 mmol) dissolved in N,N-dimethylformamide (5 mL) was added slowly to the suspension. The reaction mixture was allowed to stir under nitrogen and warm to room temperature over 30 minutes. The reaction was cooled to 0° C. and chloro(methoxy)methane (0.26 mL, 3.43 mmol) was added drop-wise over 15 minutes. The reaction was allowed to warm to room temperature and stirred for 16 hours. Water (20 mL) was added and the solvents were removed in vacuo. The mixture was partitioned between ethyl acetate and water (1:1, 100 mL) and extracted with ethyl acetate (3×60 mL). The combined organic extracts were washed with water (3×80 mL), brine (50 mL), dried over anhydrous magnesium sulphate, filtered and concentrated to give the title compound 0.6 g (89% yield) as an orange oil which solidified upon standing. Tr(METCR1278)=1.33 min, (ES.sup.+) (M+H).sup.+198.
Step 2, Method 2: [5-(Methoxymethoxy)pyridin-2-yl]methanol
[0178] Methyl 5-(methoxymethoxy)pyridine-2-carboxylate (0.39 g, 1.9 mmol) was dissolved in anhydrous tetrahydrofuran (15 mL) and cooled to 0° C. in a nitrogen atmosphere. 2.4 M lithium aluminium hydride in tetrahydrofuran (0.87 mL, 2.09 mmol) was added drop-wise over a period of 5 minutes, and the reaction was stirred at 0° C. for 1.5 hours. The reaction was cooled to 0° C. and saturated aqueous Rochelle's salt (1 mL) was added drop-wise with vigorous stirring over a period of 10 minutes. The reaction mixture was allowed to warm to room temperature for 1 hour. The resultant emulsion was filtered through glass fibre filter paper. The filter paper was washed with saturated aqueous sodium bicarbonate (10 mL), followed by ethyl acetate (3×10 mL). The phases were separated and the aqueous phase was extracted with ethyl acetate (3×10 mL). The combined organic extracts were washed with brine (10 mL), dried over anhydrous sodium sulphate, filtered, and concentrated to give the title compound 276 mg (86% yield) as an orange oil. Tr(METCR1278)=1.09 min, (ES.sup.+) (M+H).sup.+170.
Step 3, Method 2: [5-(Methoxymethoxy)pyridin-2-yl]methyl methanesulfonate
[0179] [5-(Methoxymethoxy)pyridin-2-yl]methanol (276 mg, 1.63 mmol) was dissolved in dichloromethane (5 mL), cooled to 0° C. and stirred in a nitrogen atmosphere. Triethylamine (250 μL, 1.79 mmol) was added, followed by drop-wise addition of methanesulfonyl chloride (133 μL, 1.71 mmol). The reaction was stirred for 45 minutes at 0° C. and allowed to warm to room temperature. Water (5 mL) was added and the phases separated. The aqueous layer was extracted with dichloromethane (3×15 mL); the combined organic extracts were washed with brine (10 mL), dried over anhydrous sodium sulphate, filtered, and concentrated to give the title compound, 275 mg (59% yield) as a dark red oil. Tr(METCR1278)=1.35 min, (ES.sup.+) (M+H).sup.+248.
Step 4, Method 2: 4-(5-{[5-(Methoxymethoxy)pyridin-2-yl]methoxy}-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile
[0180] 4-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile (330 mg, 1.66 mmol prepared by Method 1), [5-(methoxymethoxy)pyridin-2-yl]methyl methanesulfonate (532 mg, 1.66 mmol) and potassium iodide (62 mg, 0.37 mmol) were dissolved in anhydrous N,N-dimethylformamide (4 mL). Sodium hydride (60% in mineral oil, 27 mg, 0.16 mmol) was added and the mixture was stirred at room temperature for 44 hours. The reaction mixture was quenched with methanol (2 mL) and partitioned between ethyl acetate (100 mL), saturated aqueous sodium bicarbonate (50 mL) and brine (50 mL). After separation the aqueous layer was extracted with ethyl acetate (3×100 mL). The organic extracts were combined, washed with brine (30 mL), dried over sodium sulphate and concentrated. The residue was triturated with ethyl acetate:heptane (1:1) to give the title compound 269 mg (39% yield) as a brown solid. δ.sub.H NMR (500 MHz, DMSO) 8.49 (s, 1H), 8.33 (d, J=2.6 Hz, 1H), 8.27 (d, J=6.3 Hz, 1H), 7.49 (d, J=2.7 Hz, 1H), 7.48 (s, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.09 (d, J=1.9 Hz, 1H), 6.98 (dd, J=8.4, 2.3 Hz, 1H), 6.74 (d, J=6.3 Hz, 1H), 5.26 (s, 2H), 5.12 (s, 2H), 4.96 (d, J=22.4 Hz, 4H), 3.38 (s, 3H).
Step 5, Method 2: 4-{5-[(5-Hydroxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}pyridine-3-carbonitrile
[0181] To a solution of 4-(5-{[5-(methoxymethoxy)pyridin-2-yl]methoxy}-2,3-dihydro-1H-isoindol-2-yl)pyridine-3-carbonitrile (269 mg, 0.69 mmol) in tetrahydrofuran (40 mL) was added 3 M hydrochloric acid (4.6 mL) and the mixture was stirred at 60° C. for 8 hours. The mixture was stirred overnight at room temperature. The volatiles were removed in vacuo and the remaining residue was diluted with water. Solid sodium bicarbonate was added portion-wise until the pH was approximately 8. The solid was collected by filtration, washed with water (2×10 mL) and dried under vacuum. Purification by FCC (silica, 0-60% tetrahydrofuran in heptane) gave the title compound 101 mg (42% yield) as a white solid.
Example 4, Method 2: 4-{5-[(5-Hydroxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}pyridine-3-carbonitrile
[0182] δ.sub.H NMR (500 MHz, DMSO) 10.01 (s, 1H), 8.49 (s, 1H), 8.27 (d, J=6.3 Hz, 1H), 8.11 (d, J=2.5 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.18 (dd, J=8.4, 2.9 Hz, 1H), 7.08 (d, J=2.0 Hz, 1H), 6.97 (dd, J=8.4, 2.4 Hz, 1H), 6.75 (d, J=6.3 Hz, 1H), 5.05 (s, 2H), 4.98 (s, 2H), 4.94 (s, 2H). Tr(MET-uHPLC-AB-101)=1.28 min, (ES.sup.+) (M+H).sup.+345.
[0183] The following examples were prepared using Method 2 described above:
TABLE-US-00002 TABLE 2 Mol. Ex. Structure Weight IUPAC Name LCMS data 1
Method 3
[0184] ##STR00020##
Step 1, Method 3: Methyl 5-methoxypyrazine-2-carboxylate
[0185] To methyl 5-chloropyrazine-2-carboxylate (2.00 g, 11.6 mmol) under nitrogen, was added a 0.5 M solution of sodium methoxide in methanol (27.8 mL, 13.9 mmol). The mixture was refluxed at 90° C. for 15 minutes. The mixture was then dissolved with water (80 mL) and extracted with ethyl acetate (2×100 mL). The combined organic extracts were dried over sodium sulphate, filtered and concentrated to give the title compound 1.68 g (79% yield) as a white powder. δ.sub.H NMR (500 MHz, Chloroform) 8.88 (d, J=1.2 Hz, 1H), 8.28 (d, J=1.2 Hz, 1H), 4.05 (s, 3H), 4.00 (s, 3H). Tr(METCR1278)=1.23 min, (ES.sup.+) (M+H).sup.+169.
Step 2, Method 3: (5-Methoxypyrazin-2-yl)methanol
[0186] Sodium borohydride (270 mg, 7.14 mmol) was added to a stirred solution of methyl 5-methoxypyrazine-2-carboxylate (200 mg, 1.19 mmol) in anhydrous tetrahydrofuran (8 mL) under nitrogen. The mixture was refluxed at 65° C. for 15 minutes, after which methanol (1.59 mL, 39.2 mmol) was added slowly. The reaction was refluxed at 65° C. for 1.5 hours. The mixture was quenched with water (0.5 mL), then diluted with further water (15 mL), extracted with ethyl acetate (2×25 mL) then 20% 2-propanol in dichloromethane (25 mL). The combined organic extracts were dried over sodium sulphate, filtered and concentrated to give the title compound 115 mg (69% yield) as a white crystalline solid. δ.sub.H NMR (500 MHz, DMSO) 8.28-8.16 (m, 2H), 5.41 (t, J=5.8 Hz, 1H), 4.54 (d, J=5.6 Hz, 2H), 3.90 (s, 3H). Tr(METCR1278)=0.74 min, (ES.sup.+) (M+H).sup.+141.
Step 3, Method 3: (5-Methoxypyrazin-2-yl)methyl methanesulfonate
[0187] To a stirred solution of (5-methoxypyrazin-2-yl)methanol (73 mg, 0.52 mmol) in dichloromethane (1 mL) under nitrogen, was added triethylamine (0.08 mL, 0.73 mmol) followed by methanesulfonyl chloride (0.042 mL, 0.55 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was then partitioned between dichloromethane (10 mL) and water (10 mL). The organic extract was dried over sodium sulphate, filtered and concentrated to afford the title compound 59 mg (52% yield) as a yellow oil. Tr(METCR1278)=1.25 min, (ES.sup.+) (M+H).sup.+219.
Step 4, Method 3: 5-[(5-Methoxypyrazin-2-yl)methoxy]-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindole
[0188] 2-(Pyridin-4-yl)-2,3-dihydro-1H-isoindol-5-ol (87%, 289 mg, 1.18 mmol, prepared by Method 1), (5-methoxypyrazin-2-yl)methyl methanesulfonate (310 mg, 1.42 mmol) and potassium iodide (197 mg, 1.18 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL) and stirred for 5 minutes at room temperature. Sodium hydride (60% in mineral oil, 142 mg, 3.55 mmol) was added and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 40 hours. The solvents were removed in vacuo and purification by preparative HPLC (acetonitrile-water-0.2% ammonium hydroxide) gave the title compound, 30.1 mg (8% yield) as a beige solid.
Example 1, Method 3: 5-[(5-Methoxypyrazin-2-yl)methoxy]-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindole
[0189] δ.sub.H NMR (500 MHz, DMSO) 8.38 (d, J=1.1 Hz, 1H), 8.34 (d, J=1.3 Hz, 1H), 8.17 (d, J=6.3 Hz, 2H), 7.32 (d, J=8.3 Hz, 1H), 7.10 (d, J=2.0 Hz, 1H), 7.01 (dd, J=8.4, 2.3 Hz, 1H), 6.57 (d, J=6.4 Hz, 2H), 5.17 (s, 2H), 4.62 (s, 2H), 4.58 (s, 2H), 3.92 (s, 3H). Tr(MET-uHPLC-AB-101)=1.77 min, (ES.sup.+) (M+H).sup.+335.
[0190] The following example was prepared using Method 3 described above:
TABLE-US-00003 TABLE 3 Mol. Ex. Structure Weight IUPAC Name LCMS data 1
Method 4
[0191] ##STR00023##
Step 1, Method 4: 6-(5-Methoxy-2,3-dihydro-1H-isoindol-2-yl)-2-methyl-2,3-dihydropyridazin-3-one
[0192] 5-Methoxy-2,3-dihydro-1H-isoindole hydrochloride (170 mg, 0.92 mmol), 6-bromo-2-methyl-2,3-dihydropyridazin-3-one (182 mg, 0.96 mmol) and dicaesium carbonate (895.06 mg, 2.75 mmol) were suspended in anhydrous toluene (3 mL) and sonicated under a flow of nitrogen for 5 minutes. Palladium(II) acetate (20.56 mg, 0.09 mmol) and 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene (53 mg, 0.09 mmol) were added and the reaction was heated to 100° C. for 16 hours. The reaction mixture was cooled to room temperature and filtered through celite. The celite was washed with 10% methanol in dichloromethane and the filtrate was concentrated in vacuo and purified by FCC (silica, 20-100% ethyl acetate in heptane, 0-10% methanol in dichloromethane), SCX and HPLC (acetonitrile/water +0.2% formic acid) to give the title compound, 34 mg (14% yield) as a beige solid. δ.sub.H NMR (500 MHz, DMSO) 7.30 (d, J=9.8 Hz, 1H), 7.27 (d, J=8.3 Hz, 1H), 6.96 (d, J=2.0 Hz, 1H), 6.92-6.84 (m, 2H), 4.63 (s, 2H), 4.59 (s, 2H), 3.76 (s, 3H), 3.53 (s, 3H). Tr(MET-uHPLC-AB-101)=2.56 min, (ES.sup.+) (M+H).sup.+258.
Step 2, Method 4: 6-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)-2-methyl-2,3-dihydropyridazin-3-one
[0193] 6-(5-Methoxy-2,3-dihydro-1H-isoindol-2-yl)-2-methyl-2,3-dihydropyridazin-3-one (96 mg, 0.37 mmol) was dissolved in anhydrous dichloromethane (5 mL) and 1 M tribromoborane in dichloromethane (0.6 mL) was added and the reaction was stirred at room temperature for 16 hours. 1 M tribromoborane in dichloromethane (0.2 mL) was added and the reaction was stirred at room temperature for 2 hours. Saturated sodium bicarbonate solution (15 mL) was added and the reaction mixture was stirred vigorously for 20 minutes. The reaction mixture was filtered and the precipitate was co-distilled from methanol (5 mL) and toluene (2×10 mL) to give the title compound, 84 mg (90% yield) as a beige solid. δ.sub.H NMR (500 MHz, DMSO) 7.29 (d, J=9.8 Hz, 1H), 7.14 (d, J=8.2 Hz, 1H), 6.88 (d, J=9.8 Hz, 1H), 6.75 (s, 1H), 6.69 (dd, J=8.2, 2.1 Hz, 1H), 4.58 (s, 2H), 4.55 (s, 2H), 3.52 (s, 3H). Tr(METCR1673)=0.94 min, (ES.sup.+) (M.sup.+H).sup.+244.
Step 3, Method 4: 6-{5-[(5-Methoxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}-2-methyl-2,3-dihydropyridazin-3-one
[0194] (5-Methoxypyridin-2-yl)methanol (54 mg, 0.37 mmol) was dissolved in anhydrous dichloromethane (3 mL), thionyl dichloride (0.25 ml, 3.35 mmol) was added and the reaction was heated to 50° C. in a nitrogen atmosphere for 2 hours. The reaction mixture was cooled to room temperature, concentrated in vacuo from dichloromethane (3×10 mL), to give a brown oil which was used crude in the subsequent step. 6-(5-Hydroxy-2,3-dihydro-1H-isoindol-2-yl)-2-methyl-2,3-dihydropyridazin-3-one (97%, 84 mg, 0.33 mmol), potassium iodide (61 mg, 0.37 mmol) and 2-(chloromethyl)-5-methoxypyridine (58 mg, 0.37 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL), sodium hydride (60%, 40.19 mg, 1 mmol) was added and the reaction was stirred for 1 hour at room temperature in a nitrogen atmosphere. Sodium hydride (60%, 40 mg, 1 mmol) was added and the reaction was heated to 70° C. for 3 hours. The reaction mixture was cooled to room temperature, quenched with the addition of water (1 mL) and the solvents were removed in vacuo and the stood for 16 hours. The residue was partitioned between ethyl acetate (50 mL) and water (50 mL), the phases were separated and the aqueous layer was further extracted with ethyl acetate (2×50 mL), the combined organics were washed with brine (10 mL), dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated in vacuo. Purification by FCC (silica, 20-100% ethyl acetate in heptane, 10% methanol in dichloromethane) gave the title compound, 32 mg (26% yield) as a beige solid.
Example 1, Method 4: 6-{5-[(5-Methoxypyridin-2-yl)methoxy]-2,3-dihydro-1H-isoindol-2-yl}-2-methyl-2,3-dihydropyridazin-3-one
[0195] δ.sub.H NMR (500 MHz, DMSO) 8.28 (d, J=2.8 Hz, 1H), 7.47 (d, J=8.6 Hz, 1H), 7.42 (dd, J=8.6, 2.9 Hz, 1H), 7.30 (d, J=9.8 Hz, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.05 (d, J=2.0 Hz, 1H), 6.95 (dd, J=8.4, 2.3 Hz, 1H), 6.89 (d, J=9.8 Hz, 1H), 5.11 (s, 2H), 4.62 (s, 2H), 4.59 (s, 2H), 3.83 (s, 3H), 3.53 (s, 3H). Tr(MET-uHPLC-AB-101)=2.56 min, (ES.sup.+) (M+H).sup.+365.
[0196] The following example was prepared using Method 4 described above:
TABLE-US-00004 TABLE 4 Mol. Ex. Structure Weight IUPAC Name LCMS data 1
Method 5
[0197] ##STR00025##
Step 1, Method 5: 5-Methoxy-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindol-1-one
[0198] 5-Methoxy-2,3-dihydro-1H-isoindol-1-one (100 mg, 0.61 mmol), 4-iodopyridine (126 mg, 0.61 mmol), (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane) (53 mg, 0.09 mmol) and dicaesium carbonate (300 mg, 0.92 mmol) were suspended in dry dioxane (1 mL) and the mixture was degassed. (1E,4E)-1,5-Diphenylpenta-1,4-dien-3-one-palladium (3:2) (28 mg, 0.03 mmol) was added and the mixture heated in a microwave at 140° C. for 90 minutes. The mixture was diluted with ethyl acetate (10 mL) and water (5 mL) and filtered to give the title compound 28 mg (20% yield) as a brown powder. δ.sub.H NMR (500 MHz, DMSO) 8.58-8.47 (m, 2H), 7.91-7.82 (m, 2H), 7.74 (d, J=8.5 Hz, 1H), 7.23 (d, J=1.9 Hz, 1H), 7.11 (dd, J=8.5, 2.3 Hz, 1H), 4.98 (s, 2H), 3.89 (s, 3H). Tr(MET-uHPLC-AB-101)=1.27 min, (ES.sup.+) (M+H).sup.+241.
Step 2, Method 5: 5-Hydroxy-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindol-1-one
[0199] To 5-methoxy-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindol-1-one (37 mg, 0.15 mmol in 1,2-dichloroethane (10 mL) was added tribromoborane 1 M in dichloromethane (1.54 mL 1.54 mmol) and the mixture heated to reflux overnight. Tribromoborane 1 M in dichloromethane (1.54 mL 1.54 mmol) and the mixture heated to reflux for 2 days the stood at room temperature for 2 days. The mixture was poured onto a 1:1 ice:saturate sodium bicarbonate mixture (50 mL) and stirred for 1 hour. The mixture was filtered and washed with water (5 mL) and dichloromethane then dried in a vacuum oven to give the title compound 19 mg (55% yield) as a tan powder. δ.sub.H NMR (250 MHz, DMSO, 353 K) 8.72 (d, J=7.4 Hz, 2H), 8.30 (d, J=7.4 Hz, 2H), 7.73 (d, J=8.3 Hz, 1H), 7.01 (dd, J=11.3, 3.0 Hz, 2H), 5.05 (s, 2H). Tr(METCR0990)=0.85 min, (ES.sup.+) (M+H).sup.+227.
Step 3, Method 5: 5-[(5-Methoxypyridin-2-yl)methoxy]-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindol-1-one
[0200] (5-Methoxypyridin-2-yl)methanol (95%, 18 mg, 0.13 mmol) was dissolved in anhydrous dichloromethane (3 mL), thionyl dichloride (61 μl, 0.84 mmol) was added and the reaction was heated to 50° C. under a nitrogen atmosphere for 2 hours. The reaction mixture was cooled to room temperature, concentrated in vacuo to give a brown oil which was used crude in the following step. 5-Hydroxy-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindol-1-one (19 mg, 0.08 mmol) and 2-(chloromethyl)-5-methoxypyridine (20 mg, 0.13 mmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL), sodium hydride (60%, 40.19 mg, 1 mmol) was added and the reaction was stirred overnight at room temperature under a nitrogen atmosphere. The reaction mixture was quenched with the addition of water (5 mL) and filtered, washed with water (3 mL), heptane (5 mL) and methanol (2 mL) and dried in a vacuum oven to give the title compound 7 mg (24% yield) as a brown powder.
Example 1 Method 5: 5-[(5-Methoxypyridin-2-yl)methoxy]-2-(pyridin-4-yl)-2,3-dihydro-1H-isoindol-1-one
[0201] δ.sub.H NMR (500 MHz, DMSO) 8.56-8.48 (m, 2H), 8.31 (d, J=2.9 Hz, 1H), 7.90-7.85 (m, 2H), 7.74 (d, J=8.5 Hz, 1H), 7.52 (d, J=8.6 Hz, 1H), 7.44 (dd, J=8.6, 2.9 Hz, 1H), 7.31 (d, J=1.6 Hz, 1H), 7.19 (dd, J=8.5, 2.1 Hz, 1H), 5.24 (s, 2H), 4.97 (s, 2H), 3.84 (s, 3H). Tr(MET-uHPLC-AB-101)=1.59 min, (ES.sup.+) (M+H).sup.+348.
[0202] The following example was prepared using Method 5 described above:
TABLE-US-00005 TABLE 5 Mol. Ex. Structure Weight IUPAC Name LCMS data 1
Biology Examples
Q46 Radioligand Binding Assay
[0203] For radioligand binding assays (RBA) GST-Q46 protein was generated based on a previous publication (Scherzinger et al. Cell, Vol. 90, 549-558, Aug. 8, 1997). For experiments 33 μM GST-Q46 was incubated with 150 μg/ml thrombin in assay buffer (150 mM NaCl, 50 mM Tris pH 8.0) and 2 mM CaCl.sub.2 for 16 hr at 37° C. Aggregated Q46 was pelleted by centrifugation for 5 min at 13,000 rpm in a bench top centrifuge and re-dissolved in the same volume of assay buffer. Test compounds were prepared by titration in DMSO at 11 concentrations from 33 μM to 1 nM. For the RBA, Q46 protein aggregates and test compounds were pre-incubated in assay buffer for 20 min at room temperature, in 140 μL/well in a 96-well plate (pp, round bottom). Then, ligand was added in 10 μL/well and incubated for 60 minutes at 37° C. Final assay concentrations were 1 μM to 30 pM test compound, 5 μM Q46 protein (equivalent monomer concentration) and 10 nM ligand [.sup.3H.sub.3]MK-3328 (Harrision et al., ACS Med. Chem. Lett., 2 (2011), pp 498-502). Samples were transferred onto GF/B filter plates and washed 2× with 200 μL PBS using a Filtermate Harvester. After drying filter plates for 1 hour at 37° C., the back of the plates was sealed with foil and 30 μL/well scintillation fluid (Packard MicroScint 40) was added, incubated for 15 min in the dark and counted in a TopCount reader. For analysis, replicate data from independent assay plates were normalized towards 0% and 100% inhibition using control wells of vehicle (0% inhibition) and 3 μM unlabelled MK-3328 (100% inhibition). IC.sub.50 values were determined with a sigmoidal inhibition model with four variables (top, bottom, slope, IC.sub.50) in a global fit using the normalized replicate data.
TABLE-US-00006 Structure IUPAC Name Activity
[0204] Various modifications, additions, substitutions, and variations to the illustrative examples set forth herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.