KYNURENINE-3-MONOOXYGENASE INHIBITORS, PHARMACEUTICAL COMPOSITIONS, AND METHODS OF USE THEREOF
20190047977 ยท 2019-02-14
Inventors
- Celia Dominguez (Los Angeles, CA)
- Leticia Toledo-Sherman (Santa Monica, CA, US)
- Michael Prime (Abingdon, GB)
- William Mitchell (Market Rasen, GB)
- Naomi Went (Didcot, GB)
Cpc classification
A61P25/14
HUMAN NECESSITIES
C07D311/02
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
C07D307/79
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C07D317/50
CHEMISTRY; METALLURGY
C07D317/62
CHEMISTRY; METALLURGY
C07D263/58
CHEMISTRY; METALLURGY
C07D211/72
CHEMISTRY; METALLURGY
C07D311/04
CHEMISTRY; METALLURGY
C07D215/06
CHEMISTRY; METALLURGY
International classification
C07D317/62
CHEMISTRY; METALLURGY
C07D211/72
CHEMISTRY; METALLURGY
C07D215/06
CHEMISTRY; METALLURGY
C07D311/02
CHEMISTRY; METALLURGY
C07D311/04
CHEMISTRY; METALLURGY
C07D319/18
CHEMISTRY; METALLURGY
C07D317/50
CHEMISTRY; METALLURGY
C07D263/58
CHEMISTRY; METALLURGY
Abstract
Certain compounds, or pharmaceutically acceptable salts or prodrugs thereof, are provided herein. Also provided are pharmaceutical compositions comprising at least one compound, or pharmaceutically acceptable salt or prodrug thereof, described herein and one or more pharmaceutically acceptable vehicle. Methods of treating patients suffering from certain diseases and disorders responsive to the inhibition of KMO activity are described, which comprise administering to such patients an amount of at least one compound, or pharmaceutically acceptable salt or prodrug thereof, described herein effective to reduce signs or symptoms of the disease or disorder are disclosed. These diseases include neurodegenerative disorders such as Huntington's disease. Also described are methods of treatment include administering at least one compound, or pharmaceutically acceptable salt or prodrug thereof, described herein as a single active agent or administering at least one compound, or pharmaceutically acceptable salt or prodrug thereof, described herein in combination with one or more other therapeutic agents. Also provided are methods for screening compounds capable of inhibiting KMO activity.
Claims
1.-33. (canceled)
34. A method of treating a condition or disorder mediated by Kynurenine 3-mono-oxygenase activity in a subject in need of such a treatment which method comprises administering to the subject a therapeutically effective amount of a compound of formula: ##STR00051## or a pharmaceutically acceptable salt thereof.
35. The method of claim 34, wherein said condition or disorder involves a neurodegenerative pathology.
36. The method of claim 34, wherein said condition or disorder is Huntington's Disease.
37. A method of treating a condition or disorder mediated by Kynurenine 3-mono-oxygenase activity in a subject in need of such a treatment which method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound of formula: ##STR00052## or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
38. The method of claim 37, wherein said condition or disorder involves a neurodegenerative pathology.
39. The method of claim 37, wherein said condition or disorder is Huntington's Disease.
Description
EXAMPLES
[0224] The compounds, pharmaceutically acceptable salts and prodrugs thereof, described herein, compositions, and methods described herein are further illustrated by the following non-limiting examples.
[0225] As used herein, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.
[0226] CDI=carbonyldiimidazole
[0227] DCM=dichloromethane
[0228] DME=dimethyl ether
[0229] DMEM=Dulbecco's modified Eagle's medium
[0230] DMF=N,N-dimethylformamide
[0231] DMSO=dimethylsulfoxide
[0232] EDC.HCl=1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
[0233] EtOH=ethanol
[0234] Et.sub.2O=diethylether
[0235] EtOAc=ethyl acetate
[0236] g=gram
[0237] hr=hour
[0238] hrs=hours
[0239] HOBt=1-Hydroxybenzotriazol
[0240] LiHMDS=lithium hexamethyl-disilazide
[0241] LC/MS=liquid chromatography/mass spectrometry
[0242] mg=milligram
[0243] min=minutes
[0244] mL=milliliter
[0245] mmol=millimoles
[0246] mM=millimolar
[0247] ng=nanogram
[0248] nm=nanometer
[0249] nM=nanomolar
[0250] PBS=phosphate buffered saline
[0251] rt=room temperature
[0252] TBME=t-butyl methyl ether
[0253] THF=tetrahydrofuran
[0254] TMOF=trimethylorthoformate
[0255] L=microliter
[0256] M=micromolar
[0257] 1 g/1 ml=1 vol
Experimental
[0258] Commercially available reagents and solvents (HPLC grade) were used without further purification.
[0259] Thin-layer chromatography (TLC) analysis was performed with Kieselgel 60 F.sub.254 (Merck) plates and visualized using UV light. Microwave reactions were carried out using CEM focussed microwaves.
[0260] .sup.1H NMR spectra were recorded on a Bruker DRX 500 MHz spectrometer or Bruker DPX 250 MHz spectrometer in deuterated solvents.
[0261] Analytical HPLC-MS was performed on Agilent HP1100 and Shimadzu 2010, systems using reverse phase Atlantis dC18 columns (5 m, 2.150 mm), gradient 5-100% B (A=water/0.1% formic acid, B=acetonitrile/0.1% formic acid) over 2 or 3.5 minutes, injection volume 3 l, flow=1.0 ml/min. UV spectra were recorded at 215 nm using a Waters 2487 dual wavelength UV detector or the Shimadzu 2010 system. Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2 scans per second using Waters ZMD and over m/z 100 to 1000 at a sampling rate of 2 Hz using Electrospray ionisation, by a Shimadzu 2010 LC-MS system, or analytical HPLC-MS was performed on Agilent HP1100 and Shimadzu 2010, systems using reverse phase Water Atlantis dC18 columns (3 m, 2.1100 mm), gradient 5-100% B (A=water/0.1% formic acid, B=acetonitrile/0.1% formic acid) over 7 min, injection volume 3 l, flow=0.6 ml/min. UV spectra were recorded at 215 nm using a Waters 2996 photo diode array or on the Shimadzu 2010 system. Mass spectra were obtained over the range m/z 150 to 850 at a sampling rate of 2 scans per second using Waters ZQ and over m/z 100 to 1000 at a sampling rate of 2 Hz using Electrospray ionisation, by a Shimadzu 2010 LC-MS system. Data were integrated and reported using OpenLynx and OpenLynx Browser software or via Shimadzu PsiPort software.
Method 1:
Scheme for Method 1: (1S, 2S)-2-(7-chloro-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylic acid
[0262] ##STR00004##
Step 1, Method 1: 2-Chloro-6-(2-hydroxyethyl)phenol
[0263] Sodium periodate (8.9 g, 41.5 mmol) was added portion wise to a stirred solution of 2-chloro-6-(prop-2-en-1-yl)phenol (3.72 g, 20.7 mmol) in THF (100 mL) and water (100 mL) at 0 C. After 5 minutes, osmium tetroxide (0.1 g, 0.41 mmol) was added and stirring continued for 1.5 hours. After this time, the mixture was poured into ice and brine (100 mL) and extracted with ethyl acetate (3100 mL). The combined organic extracts were dried (Na.sub.2SO.sub.4), filtered and concentrated. The resulting residue was dissolved in methanol (100 mL) and cooled to 0 C. before being treated with sodium tetrahydroborate (1.57 g, 41.5 mmol) in small portions over 30 minutes. After this time, the reaction mixture was warmed to room temperature and stirred overnight. The resulting mixture was concentrated, treated with aqueous 1M hydrochloric acid (80 mL) and extracted with ethyl acetate (3100 mL). The combined organic extracts were dried (Na.sub.2SO.sub.4), filtered and concentrated. The resulting residue was purified by flash column chromatography (elution; 0-40% ethyl acetate in heptanes) to give the title compound (1.41 g, 36% yield) as a yellow oil. .sub.H (250 MHz, CDCl.sub.3) 7.23 (dd, J=8.0, 1.6 Hz, 1H), 7.03 (dd, J=7.5, 1.6 Hz, 1H), 6.81 (t, J=7.8 Hz, 1H), 3.95 (t, J=5.9 Hz, 2H), 2.95 (t, J=5.8 Hz, 2H).
Step 2, Method 1: 7-Chloro-2,3-dihydro-1-benzofuran
[0264] Diisopropyldiazene-1,2-dicarboxylate (1.97 ml, 9.9 mmol) was added portionwise to a stirred, cooled (0 C.) solution of 2-chloro-6-(2-hydroxyethyl)phenol (1.4 g, 7.7 mmol) and triphenylphosphine (2.62 g, 9.9 mmol) in anhydrous THF (25 ml) and the reaction mixture was stirred a nitrogen atmosphere for 15 hours. After this time the reaction mixture was concentrated and the resulting residue purified by Biotage (Isolera snap 50 g cartridge, eluting with 0-20% EtOAc in Heptanes) to give the title compound (1.46 g, 92% yield) as an orange oil. .sub.H (250 MHz, CDCl.sub.3) 7.16-7.02 (m, 2H), 6.85-6.71 (m, 1H), 4.67 (t, J=8.8 Hz, 2H), 3.29 (t, J=8.8 Hz, 2H).
Step 3, Method 1: Methyl (1S, 2S)-2-(7-chloro-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylate
[0265] Methyl (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (0.39 g, 2.43 mmol) in DCE (2 mL) was added portionwise to a cooled (0 C.), stirred solution of aluminium trichloride (0.65 g, 4.85 mmol) in DCE (4 mL) under a nitrogen atmosphere. 7-Chloro-2,3-dihydro-1-benzofuran (0.5 g, 2.43 mmol) was added dropwise over 5 minutes and the reaction mixture was stirred at 0 C. for a further 1 hour. After this time, the mixture was allowed to warm to room temperature before being stirred overnight. The reaction mixture was then cooled to 0 C. before being added portionwise to a mixture of concentrated HCl (4 mL) and ice (20 g). The resulting mixture was then extracted with DCM (350 mL), the combined organic extracts were washed with brine (30 mL), before being dried (MgSO.sub.4), filtered and concentrated. The resulting residue was purified using a Biotage Isolera, (Snap 50 g cartridge, eluting in 0-35% EtOAc in Heptanes) to give the title compound (0.14 g, 21% yield) as a white solid. .sub.H (250 MHz, CDCl.sub.3) 7.88 (d, J=1.6 Hz, 1H), 7.78 (d, J=1.5 Hz, 1H), 4.79 (t, J=8.9 Hz, 2H), 3.74 (s, 3H), 3.35 (t, J=8.8 Hz, 2H), 3.07 (ddd, J=8.6, 5.9, 3.9 Hz, 1H), 2.37 (ddd, J=8.6, 6.0, 3.8 Hz, 1H), 1.58 (ddt, J=12.0, 5.9, 2.9 Hz, 2H). Tr=2.08 min m/z (ES.sup.+) (M+H.sup.+) 281.
Step 4, Method 1: (1S, 2S)-2-(7-chloro-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylic acid
[0266] 2M NaOH (0.25 mL, 0.5 mmol) was added in one portion to a stirred solution of methyl (1 S,2S)-2-[(7-chloro-2,3-dihydro-1-benzofuran-6-yl)carbonyl]cyclopropane-1-carboxylate (0.07 g, 0.25 mmol) in dioxane (5 mL) and the resulting solution was stirred at room temperature for 18 hours. After this time the reaction mixture was acidified with 1M HCl, and the resulting suspension was extracted with EtOAc (210 mL). The combined organic extracts were washed with brine (10 mL), before being dried (MgSO4), filtered and concentrated. The resulting residue was part-dissolved in TBME (2 mL), sonicated and the resulting precipitate was collected by filtration and dried under vacuum to give the title compound (0.04 g, 54% yield) as a white powder.
Example 1, Method 1: (1S, 2S)-2-(7-chloro-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylic acid
[0267] .sub.H (500 MHz, CDCl.sub.3) 7.88 (s, 1H), 7.78 (s, 1H), 4.79 (t, J=8.9 Hz, 2H), 3.36 (t, J=8.8 Hz, 2H), 3.12 (ddd, J=9.4, 5.8, 3.9 Hz, 1H), 2.37 (ddd, J=9.3, 5.7, 3.9 Hz, 1H), 1.70-1.60 (m, 2H). Tr=2.61 min (7 minute method, low pH) m/z (ES+) (M+H+) 267.
Example 2, Method 1: (1S,2S)-2-(2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylic acid
[0268] .sub.H (500 MHz, CDCl.sub.3) 7.96-7.83 (m, 2H), 6.84 (d, J=8.78 Hz, 1H), 4.68 (t, J=8.78 Hz, 2H), 3.27 (t, J=8.76 Hz, 2H), 3.18 (ddd, J=3.84, 5.89, 9.44 Hz, 1H), 2.35 (ddd, J=3.83, 5.65, 9.20 Hz, 1H), 1.67 (ddd, J=3.50, 5.90, 9.02 Hz, 1H), 1.59 (ddd, J=3.51, 5.66, 8.96 Hz, 1H). Tr=2.21 min, m/z (ES+) (M+H+) 233.
TABLE-US-00001 TABLE 1 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 2
Scheme for Method 2: (1S,2S)-2-(8-Chloro-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclopropane-1-carboxylic acid
[0269] ##STR00007##
Step 1, Method 2: 1-Chloro-2-(prop-2-en-1-yloxy)benzene
[0270] Sodium hydride (60%, 5.6 g, 140.02 mmol) was suspended in anhydrous DMF (80 mL) in an ice-bath before 2-chlorophenol (11.9 mL, 116.7 mmol) as a solution DMF (20 mL) was added dropwise over 30 minutes. Once the addition was complete, the reaction mixture was allowed to stir for 45 minutes. After this time 3-bromoprop-1-ene (12.12 mL, 140.0 mmol) was added dropwise and the reaction was allowed to warm to room temperature and stirring was continued for a further 15 hours. After this time, aqueous saturated ammonium chloride (50 mL) was added and the mixture was extracted with ethyl acetate (3200 mL). The combined organic extracts were washed with brine (50 mL) dried (Na.sub.2SO.sub.4), filtered, and concentrated, The resulting residue was dissolved in ethyl acetate (200 mL) and washed with water (3200 mL) and brine solution (50 mL). The organics were dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound (17.5 g, 89% yield) as an orange oil which was taken on directly without further purification.
Step 2, Method 2: 2-Chloro-6-(prop-2-en-1-yl)phenol
[0271] A solution of 1-chloro-2-(prop-2-en-1-yloxy)benzene (17.5 g, 93.4 mmol) in mesitylene (150 mL) was heated under nitrogen for 48 h at 190 C. with stirring. After this time, the reaction was cooled to room temperature and concentrated. The resulting residue was purified Biotage (Snap Isolera 340 g, eluting with 100% heptanes). The purified material was treated with 2M NaOH (6 mL), diluted water (50 mL) and the starting material was extracted with TBME (100 mL). The basic aqueous layer was acidified with 6M HCl (6 mL). The aqueous layer was extracted with TBME (2100 mL), the organic extracts were combined, dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound (6.5 g, 41% yield) as a brown oil. .sub.H (250 MHz, CDCl.sub.3) 7.34 (s, 1H), 7.17 (s, 1H), 6.08-5.82 (m, 1H), 5.60 (s, 1H), 5.15 (d, J=1.2 Hz, 1H), 5.12-5.05 (m, 1H), 3.40 (d, J=6.7 Hz, 2H).
Step 3, Method 2: 2-Chloro-6-(3-hydroxypropyl)phenol
[0272] 2-Chloro-6-(prop-2-en-1-yl)phenol (1.37 g, 0.01 mol) in anhydrous THF (20 mL) at room temperature was treated dropwise with 1M borane-tetrahydrofuran (1:1 solution, 7.3 mL) and stirring was continued for 15 hours. After this time, water (0.13 mL) was added carefully over 5 minutes, before 2M NaOH (1.68 mL) was added dropwise over 15 minutes. Hydrogen peroxide (0.2 mL, 0.01 mol) was then added dropwise and the mixture stirred at room temperature for a further 1.5 hours. After this time, the mixture was treated with water (20 mL) and then partitioned between ethyl acetate (100 mL) and water (50 mL). The organic layer was separated and the aqueous extracted with ethyl acetate (2100 mL), the combined organic extracts were washed with brine (50 mL), before being dried (MgSO.sub.4) filtered and concentrated. The resulting residue was purified by flash column chromatography (elution: 0-80% EtOAc, in heptanes) to give the title compound (0.69 g, 43% yield) as a pale orange oil which was used directly.
Step 4, Method 2: 8-Chloro-3,4-dihydro-2H-1-benzopyran
[0273] Diisopropyldiazene-1,2-dicarboxylate (0.64 ml, 3 mmol) was added portionwise to a stirred, cooled (0 C.) solution of 2-chloro-6-(3-hydroxypropyl)phenol (0.49 g, 2.0 mmol) and triphenylphosphine (0.85 g, 3.0 mmol) in anhydrous THF (15 ml) and the reaction mixture was stirred a nitrogen atmosphere for 15 hours. After this time the reaction mixture was concentrated and the resulting residue purified by Biotage (Isolera snap 50 g cartridge, eluting with 0-20% EtOAc in Heptanes) to give the title compound (0.36 g, 83% yield) as a pale pink oil. .sub.H (250 MHz, CDCl.sub.3) 7.17 (d, J=7.9 Hz, 1H), 6.98-6.88 (m, 1H), 6.76 (t, J=7.7 Hz, 1H), 4.35-4.25 (m, 2H), 2.81 (t, J=6.5 Hz, 2H), 2.11-1.93 (m, 2H).
Step 5, Method 2: Methyl (1S, 2S)-2-(8-chloro-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclopropane-1-carboxylate
[0274] Methyl (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (0.19 g, 1.19 mmol) in DCE (2 mL) was added portionwise to a cooled (0 C.), stirred solution of aluminium trichloride (0.32 g, 2.37 mmol) in DCE (4 mL) under a nitrogen atmosphere. 8-Chloro-3,4-dihydro-2H-1-benzopyran (0.2 g, 1.19 mmol) was added dropwise over 5 minutes and the reaction mixture was stirred at 0 C. for a further 1 hour. After this time, the mixture was allowed to warm to room temperature before being stirred overnight. The reaction mixture was then cooled to 0 C. before being added portionwise to a mixture of concentrated HCl (4 mL) and ice (20 g). The resulting mixture was then extracted with DCM (350 mL), the combined organic extracts were washed with brine (30 mL), before being dried (MgSO.sub.4), filtered and concentrated. The resulting residue was purified using a Biotage Isolera, (Snap 50 g cartridge, eluting in 0-45% EtOAc in Heptanes) to give the title compound (0.15 g, 43% yield) as a pale yellow oil. .sub.H (250 MHz, CDCl.sub.3) 7.88 (d, J=2.1 Hz, 1H), 7.69-7.62 (m, 1H), 4.43-4.31 (m, 2H), 3.73 (s, 3H), 3.08 (ddd, J=8.5, 5.9, 3.8 Hz, 1H), 2.86 (t, J=6.4 Hz, 2H), 2.36 (ddd, J=8.5, 6.1, 3.8 Hz, 1H), 2.16-1.96 (m, 2H), 1.58 (tdd, J=7.6, 5.9, 3.4 Hz, 2H). Tr=2.00 min m/z (ES.sup.+) (M+H.sup.+) 295.
Step 6, Method 2: (1S, 2S)-2-(8-chloro-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclopropane-1-carboxylic acid
[0275] 2M NaOH (0.6 mL, 1.02 mmol) was added in one portion to a stirred solution of methyl (1S,2S)-2-[(8-chloro-3,4-dihydro-2H-1-benzopyran-6-yl)carbonyl]cyclopropane-1-carboxylate (0.15 g, 0.51 mmol) in dioxane (5 mL) and the resulting solution was stirred at room temperature for 18 hours. After this time the reaction mixture was acidified with 1M HCl, and the resulting suspension was extracted with EtOAc (210 mL). The combined organic extracts were washed with brine (10 mL), before being dried (MgSO4), filtered and concentrated. The resulting residue was part-dissolved in TBME (2 mL), sonicated and the resulting precipitate was collected by filtration and dried under vacuum to give the title compound (0.01 g, 10% yield) as a white powder.
Example 1, Method 2: (1S, 2S)-2-(8-Chloro-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclopropane-1-carboxylic acid
[0276] .sub.H (500 MHz, DMSO-d6) 12.63 (s, 1H), 7.95-7.79 (m, 2H), 4.45-4.23 (m, 2H), 3.23-3.16 (m, 1H), 2.87 (t, J=6.35 Hz, 2H), 2.06 (ddd, J=3.86, 5.82, 9.44 Hz, 1H), 1.97 (p, J=6.20 Hz, 2H), 1.44 (ddd, J=3.25, 5.85, 8.81 Hz, 1H), 1.40 (ddd, J=3.26, 5.58, 8.70 Hz, 1H). Tr=2.81 min (7 minute method, low pH) m/z (ES+) (M+H+) 281, 283.
TABLE-US-00002 TABLE 2 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 3
Scheme for Method 3: (1S,2S)-2-(4-chloro-3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-6-carbonyl)cyclopropane-1-carboxylic acid
[0277] ##STR00009##
Step 1, Method 3: 2-Amino-5-bromo-3-chlorophenol
[0278] Bromine (1.08 ml, 20.9 mmol) was added dropwise to an ice-cooled solution of 2-amino-3-chlorophenol (2.00 g, 13.9 mmol) in DCM (100 ml). The reaction mixture was stirred for 1 h after addition was complete, then filtered. Collected grey solid was washed with DCM (410 ml) and dried under suction to afford the crude product as a grey-black powder. The powder was partitioned between sat. aq. NaHCO.sub.3 (20 ml) and DCM (50 ml). The layers were separated and the aqueous extracted with DCM (350 ml). Combined DCM extracts were washed with water (25 ml) and brine (25 ml), then dried (MgSO.sub.4), filtered and concentrated to afford the title compound as a dark red-brown powder (1.7 g, 27% at ca. 50% NMR purity). .sub.H (500 MHz, DMSO) 10.13 (s, 1H), 6.90 (d, J=2.16 Hz, 1H), 6.76 (d, J=2.17 Hz, 1H), 4.81 (s, 2H). Tr=1.74 min 67% m/z 222, 224, 226 (M+H).sup.+.
Step 2, Method 3: 6-Bromo-4-chloro-2,3-dihydro-1,3-benzoxazol-2-one
[0279] 2-Amino-5-bromo-3-chlorophenol (1.55 g, 3.5 mmol) was dissolved in THF (20 ml). CDI (2.73 g, 16.9 mmol) was added and the reaction was stirred at 65 C. After 2 h the reaction was cooled to rt and concentrated to give an orange solid. The residue was redissolved in EtOAc (100 mL) and the organic phase was washed with water (50 mL), 2M HCl (350 mL), water (100 mL) and brine (20 mL) and dried (MgSO.sub.4). Filtration and concentration afforded the title compound (1.7 g, 97% yield) as a red-brown powder. .sub.H (500 MHz, DMSO-d.sub.6) 12.28 (s, 1H), 7.61 (d, J=1.60 Hz, 1H), 7.51 (d, J=1.61 Hz, 1H). Tr (3 min)=1.87 min m/z (ES.sup.) 246, 248 (MH).sup..
Intermediate 2, Step 2, Method 3: (5-Bromo-7-chloro-2,3-dihydro-1,3-benzoxazol-2-one)
[0280] .sub.H (500 MHz, DMSO-d.sub.6) 12.01 (br. s., 1H) 7.44 (d, J=1.73 Hz, 1H) 7.26 (d, J=1.73 Hz, 1H). Tr (3 min)=1.87 min m/z (ES.sup.) 246, 248 (MH).sup..
Step 3, Method 3: 6-Bromo-4-chloro-3-methyl-2,3-dihydro-1,3-benzoxazol-2-one
[0281] 6-Bromo-4-chloro-2,3-dihydro-1,3-benzoxazol-2-one (1.26 g, 3.1 mmol) was dissolved in anhydrous DMF (20 mL) and the reaction was cooled in an ice bath. Sodium hydride (60% in oil, 0.31 g, 7.7 mmol) was added portionwise and the reaction was stirred in the ice bath for 1 h. Methyl iodide (0.4 ml, 6.5 mmol) was added and the reaction was stirred at rt for 2 hours. The reaction was cooled in an ice-water bath. Water (30 mL) was added cautiously followed by EtOAc (50 mL). The layers were separated and the aqueous layer re-extracted with EtOAc (250 mL). The combined organic layers were washed with water (430 mL) and brine (230 mL) and dried (MgSO.sub.4). Filtration and concentration gave the title compound (1.3 g, 75% yield) as a brown powder. .sub.H (500 MHz, DMSO-d.sub.6) 7.68 (d, J=1.71 Hz, 1H), 7.53 (d, J=1.74 Hz, 1H), 3.54 (s, 3H).
Intermediate 2, Step 3, Method 3: (5-bromo-7-chloro-3-methyl-2,3-dihydro-1,3-benzoxazol-2-one)
[0282] .sub.H (500 MHz, CDCl.sub.3) 7.30 (d, J=1.73 Hz, 1H) 7.03 (d, J=1.73 Hz, 1H) 3.41 (s, 3H); Tr (3 min)=1.97 min m/z (ES.sup.+) No ionisation.
Step 4, Method 3: 4-Chloro-3-methyl-6-(trimethylstannyl)-2,3-dihydro-1,3-benzoxazol-2-one
[0283] 6-Bromo-4-chloro-3-methyl-2,3-dihydro-1,3-benzoxazol-2-one (0.65 g, 1.19 mmol) and lithium chloride (55 mg, 1.31 mmol) were dissolved in anhydrous dioxane (25 ml) and deoxygenated with nitrogen for 1 min. Hexamethyldistannane (246 l, 1.19 mmol) and Pd(PPh.sub.3).sub.4 (137 mg, 0.12 mmol), were added and the reaction was stirred at 100 C. for 18 h. The reaction mixture was cooled to rt and concentrated. The resulting residue was purified by flash column chromatography (elution: 10% ethyl acetate, 90% heptanes) to give the title compound (210 mg, 44% yield) as a red-orange solid. .sub.H (500 MHz, CDCl.sub.3) 7.23-7.17 (m, 1H), 7.16-7.10 (m, 1H), 3.69 (s, 3H), 0.41-0.25 (m, 9H).
Intermediate 2, Step 4, Method 3: 7-Chloro-3-methyl-5-(trimethylstannyl)-2,3-dihydro-1,3-benzoxazol-2-one
[0284] .sub.H (500 MHz, CDCl.sub.3) 7.17 (s, 1H), 6.91 (s, 1H), 3.43 (s, 3H), 0.35 (s, 9H); Tr (3 min)=2.61 min m/z (ES.sup.+) 344, 346, 348.
Step 5, Method 3: Methyl (1S,2S)-2-(4-chloro-3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-6-carbonyl)cyclopropane-1-carboxylate
[0285] 4-Chloro-3-methyl-6-(trimethylstannyl)-2,3-dihydro-1,3-benzoxazol-2-one (210 mg, 0.52 mmol) and methyl (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (85 mg, 0.52 mmol) were dissolved in anhydrous toluene (5 ml) and de-oxygenated with a stream of nitrogen for 1 min. PdCl.sub.2(PPh.sub.3).sub.2 (18 mg, 0.02 mmol) was added and the reaction was stirred at 110 C. under nitrogen for 1 h. The reaction mixture was cooled to rt, concentrated and the resulting residue purified by flash column chromatography (elution: 25% ethyl acetate, 75% heptanes) to give the title compound (101 mg, 59% yield) as a light orange powder. .sub.H (500 MHz, CDCl.sub.3) 7.23-7.17 (m, 1H), 7.16-7.10 (m, 1H), 3.69 (s, 3H), 0.41-0.25 (m, 9H). Tr (3 min)=2.11 min m/z (ES.sup.+) (M+H.sup.+) 310, 312.
Intermediate 2, Step 5, Method 3: (1S,2S)-2-(7-chloro-3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-5-carbonyl)cyclopropane-1-carboxylate
[0286] .sub.H (500 MHz, DMSO-d.sub.6) 8.00 (d, J=1.53 Hz, 1H), 3.71-3.65 (m, 3H), 3.47-3.38 (m, 4H), 2.27 (ddd, J=8.6, 5.9, 3.8 Hz, 1H), 1.57 (ddd, J=8.9, 5.8, 3.4 Hz, 1H), 1.50 (ddd, J=8.7, 5.5, 3.5 Hz, 1H); Tr (3 min)=1.94 min m/z (ES.sup.+) (M+H.sup.+) 310, 312.
Steps 6 & 7, Method 3: (1S,2S)-2-[3-Chloro-5-hydroxy-4-(methylamino)benzoyl]cyclopropane-1-carboxylic acid
[0287] Methyl (1S,2S)-2-(4-chloro-3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-6-carbonyl)cyclopropane-1-carboxylate (80 mg, 0.25 mmol) was dissolved in dioxane (5 ml) and treated with 2M NaOH (0.5 ml, 1.0 mmol). The reaction mixture was stirred at room temperature for 2 h. Further 2M NaOH (0.5 ml, 1.0 mmol) was added and the reaction mixture stirred at room temperature for 64 h. The reaction mixture was neutralised with 1M HCl and extracted with DCM (210 ml). The aqueous layer was adjusted to pH3 with 1M HCl and extracted with IPA-CHCl.sub.3 (1:1; 210 ml). IPA-CHCl.sub.3 extracts were dried (MgSO.sub.4), filtered and concentrated to afford a yellow oil. The aqueous was adjusted to pH7 with 2M NaOH and again extracted with IPA-CHCl.sub.3 (1:1; 210 ml). IPA-CHCl.sub.3 extracts were dried (MgSO.sub.4), filtered and concentrated. The resulting residue was re-dissolved in THF (5 ml), CDI (73 mg, 0.46 mmol) was added and the mixture heated to 65 C. for 2 h. The reaction mixture was cooled to room temperature and concentrated to give a dark red solid. The crude product was purified by reverse phase acidic preparative HPLC (H.sub.2O/MeCN/0.1% formic acid) to afford the title compound (24 mg, 54% yield) as a white powder.
Example 1, Method 3: (1S,2S)-2-(4-Chloro-3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-6-carbonyl)cyclopropane-1-carboxylic acid
[0288] .sub.H (500 MHz, DMSO) 12.62 (br. s, 1H), 8.01 (d, J=1.41 Hz, 1H), 7.98 (d, J=1.42 Hz, 1H), 3.61 (s, 3H), 3.29-3.25 (part. obsc. m, 1H), 2.11 (ddd, J=3.87, 5.92, 9.55 Hz, 1H), 1.49 (ddd, J=3.29, 5.95, 8.93 Hz, 1H), 1.43 (ddd, J=3.31, 5.50, 8.70 Hz, 1H). Tr=2.40 min 97% m/z 296, 298 (M+H).sup.+.
Example 2, Method 3: (1S,2S)-2-(7-Chloro-3-methyl-2-oxo-2,3-dihydro-1,3-benzoxazole-5-carbonyl)cyclopropane-1-carboxylic acid
[0289] 1H NMR (500 MHz, DMSO-d6) 12.73 (s, 1H), 7.99 (s, 1H), 7.93 (s, 1H), 3.50-3.41 (part. obsc. m, 4H), 2.19-2.09 (m, 1H), 1.47 (dt, J=36.9, 8.8 Hz, 2H). Tr=2.45 min 100% m/z (M+H).sup.+296, 298.
TABLE-US-00003 TABLE 3 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 4 (Modified Stannane Formation)
Scheme for Method 4
[0290] ##STR00012##
Step 1, Method 4: 2,3-Dihydro-1,4-benzodioxin-6-yltrimethylstannane
[0291] n-Butyllithium (3.1 ml of a 1.6M solution in hexane, 4.98 mmol) was added dropwise under nitrogen to a stirred solution of 6-bromo-2,3-dihydro-1,4-benzodioxine (1.00 g, 4.65 mmol) in dry THF (20 ml) at 78 C. After 45 min, trimethyltin chloride (5.0 ml of a 1.0M solution in THF, 5 mmol) was added dropwise over 5 min. After 20 min the reaction mixture was allowed to warm to room temperature and left overnight. The reaction mixture was poured into brine (100 ml), extracted with ethyl acetate (380 ml) and the combined, dried (Na.sub.2SO.sub.4) organic extracts were evaporated in vacuo to give the title compound (1.363 g, 98%). as a colourless oil. .sub.H (500 MHz, CDCl.sub.3) 6.78 (d, J=1.1 Hz, 1H), 6.74 (dd, J=7.7, 1.1 Hz, 1H), 6.67 (d, J=7.7 Hz, 1H), 4.06 (s, 4H), 0.06 (s, 9H). Tr=2.42 min; no ionisation.
Intermediate 2, Step 1, Method 4: 2-Methyl-6-(trimethylstannyl)-1,3-benzothiazole
[0292] .sub.H NMR (500 MHz, Chloroform-d) 8.00-7.85 (m, 2H), 7.61-7.48 (m, 1H), 2.84 (s, 3H), 0.43-0.25 (m, 9H). Tr=2.47 min m/z (ES.sup.+) (M+H.sup.+) 314/316.
Intermediate 3, Step 1, Method 4: 3,4-Dihydro-2H-1-benzopyran-6-yltrimethylstannane
[0293] .sub.H NMR (500 MHz, Chloroform-d) 7.21 (d, J=7.9 Hz, 1H), 7.15 (s, 1H), 6.81 (d, J=7.9 Hz, 1H), 4.21-4.17 (m, 2H), 2.80 (t, J=6.6 Hz, 2H), 2.05-2.00 (m, 2H), 0.26 (s, 9H). Tr=2.59 min, 62% pure, compound doesn't ionise).
Step 2, Method 4: Methyl (1S,2S)-2-(2,3-dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylate
[0294] A mixture of 2,3-dihydro-1,4-benzodioxin-6-yltrimethylstannane (700 mg, 2.34 mmol), (1S,2S)-2-(methoxycarbonyl)cyclopropane-1-carboxylic acid (571 mg, 3.51 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (82 mg, 0.12 mmol), and toluene (8 mL) was de-gassed by bubbling a stream of nitrogen through the mixture for 15 min, and then stirred at 110 C. for 2 h. The reaction was cooled, and then absorbed onto silica gel (Merck 9385, 8 mL). The resultant silica was purified on a Biotage machine (100 g cartridge of silica gel) eluting with ethyl acetate-heptane (5% EtOAc, 1CV; 5% to 40% EtOAc 10 CV; 40% EtOAc, 2 CV), to give the desired product (373 mg, 58%) as pale yellow oil. .sub.H (500 MHz, CDCl.sub.3) 7.61-7.57 (m, 2H), 7.00-6.86 (m, 1H), 4.33 (ddd, J=20.0, 5.8, 2.6 Hz, 4H), 3.13 (ddd, J=9.4, 5.8, 3.9 Hz, 1H), 2.36 (ddd, J=9.5, 5.8, 3.9 Hz, 1H), 1.59 (dddd, J=25.1, 9.1, 5.8, 3.4 Hz, 2H).). Tr=1.86 min; 100% m/z (ES+) 263 (M+H.sup.+).
Intermediate 2, Step 2, Method 4: Methyl (1S,2S)-2-(2-methyl-1,3-benzothiazole-6-carbonyl)cyclopropane-1-carboxylate
[0295] .sub.H NMR (500 MHz, DMSO-d6) 8.93 (d, J=1.6 Hz, 1H), 8.10 (dd, J=8.6, 1.8 Hz, 1H), 8.02 (d, J=8.5 Hz, 1H), 3.68 (s, 3H), 3.46-3.36 (m, 1H), 2.86 (s, 3H), 2.26 (ddd, J=8.6, 5.9, 3.9 Hz, 1H), 1.56 (dtd, J=11.0, 5.8, 2.9 Hz, 2H). Tr=1.88 min m/z (ES.sup.+) 276 (M+H).sup.+.
Intermediate 3, Step 2, Method 4: 3,4-Dihydro-2H-1-benzopyran-6-yltrimethylstannane
[0296] .sub.H NMR (500 MHz, Chloroform-d) 7.80 (dd, J=8.5, 2.2 Hz, 1H), 7.77 (d, J=2.0 Hz, 1H), 6.85 (d, J=8.5 Hz, 1H), 4.29-4.23 (m, 2H), 3.74 (s, 3H), 3.14 (ddd, J=8.8, 5.8, 3.9 Hz, 1H), 2.85 (t, J=6.4 Hz, 2H), 2.36 (ddd, J=8.7, 5.8, 3.8 Hz, 1H), 2.09-2.01 (m, 2H), 1.58 (dddd, J=27.0, 9.0, 5.8, 3.4 Hz, 2H). Tr=1.90 min m/z (ES+) 261 (M+H).sup.+.
Step 3, Method 4: (1S,2S)-2-(2,3-Dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylic acid
[0297] A solution of methyl (1S,2S)-2-(2,3-dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylate (347 mg, 1.32 mmol) in 1,4-dioxane (8 ml) was treated with aqueous 2M sodium hydroxide (595 l, 1.19 mmol) at room temperature, and stirred for 22 h under nitrogen. The reaction mixture was evaporated in vacuo, treated with water (25 ml), extracted with ether (330 ml), and the ethereal extracts were discarded. The aqueous phase was filtered through a PTFE frit (0.45 M). The aqueous solution was freeze-dried to give a foam (300 mg). A solution of the foam in DMSO (3 ml) was treated with aqueous 2M hydrochloric acid (0.6 ml) and purified by low pH HPLC. The resultant gum was further dried in vacuo at 40 C. to give the title compound (104 mg, 31%) as a colourless gum.
Example 1, Method 4: (1S,2S)-2-(2,3-Dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylic acid
[0298] .sub.H (500 MHz, CDCl.sub.3) 7.62-7.56 (m, 2H), 6.99-6.93 (m, 1H), 4.33 (ddd, J=20.6, 5.8, 2.6 Hz, 4H), 3.18 (ddd, J=9.5, 5.9, 3.8 Hz, 1H), 2.37 (ddd, J=9.3, 5.7, 3.8 Hz, 1H), 1.65 (dddd, J=33.1, 9.1, 5.8, 3.5 Hz, 2 Hz). LCMS Tr=2.16 min; 99% m/z (ES+) 249 (M+H.sup.+).
Example 2, Method 4: (1S,2S)-2-(3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclopropane-1-carboxylic acid
[0299] .sub.H NMR (500 MHz, DMSO-d6) 7.85 (d, J=2.0 Hz, 1H), 7.79 (dd, J=8.6, 2.2 Hz, 1H), 6.86 (d, J=8.6 Hz, 1H), 4.27-4.20 (m, 2H), 3.16 (ddd, J=8.9, 5.6, 3.9 Hz, 1H), 2.83 (t, J=6.4 Hz, 2H), 2.06 (ddd, J=9.4, 5.8, 3.9 Hz, 1H), 1.96 (dt, J=11.5, 6.3 Hz, 2H), 1.42 (dddd, J=14.1, 8.8, 5.7, 3.2 Hz, 2H). Tr=2.47 min m/z (ES+) 247 (M+H.sup.+).
TABLE-US-00004 TABLE 4 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 5
Scheme for Method 5
[0300] ##STR00015##
Step 1, Method 5: (1S,2S)-2-(2-methyl-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylate
[0301] Aluminium chloride (1.68 g, 6.32 mmol) was dissolved in 1,2-dichloroethane (25 mL) and cooled to 0 C. under nitrogen. A solution of methyl (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (1.027 g, 6.32 mmol) in 1,2-dichloroethane (10 mL) was added to the reaction mixture. 2-Methyl-2,3-dihydro-1-benzofuran (0.848 g, 6.32 mmol) in 1,2-dichloroethane (10 mL) was injected dropwise in to the reaction mixture over 2 min and the reaction mixture was kept at 00 for 30 min, then allowed to warm to room temperature and stirred overnight. The reaction mixture was poured into ice (250 mL) containing conc hydrochloric acid (25 mL), and the organic layer separated. The aqueous was further extracted with dichloromethane (2100 mL) and the combined dried (Na.sub.2SO.sub.4) organic extracts were evaporated in vacuo. The residual oil was absorbed from ethyl acetate onto silica gel (Merck 9385, 12 mL), and the resultant silica purified on a Biotage machine (100 g KPSIL cartridge). Gradient elution with ethyl acetate-heptane (1% EtOAc, 1CV; 1 to 20% EtOAC over 10CV; 20% EtOAc, 2CV) afforded an oil. This was purified by reverse phase low pH HPLC to give the title compound (265 mg, 16%). 1H NMR (500 MHz, Chloroform-d) 8.00-7.77 (m, 2H), 6.81 (d, J=8.4 Hz, 1H), 5.12-4.96 (m, 1H), 3.74 (s, 3H), 3.38 (dd, J=15.6, 8.9 Hz, 1H), 3.13 (ddd, J=9.5, 5.8, 3.9 Hz, 1H), 2.86 (dd, J=15.6, 7.4 Hz, 1H), 2.36 (ddd, J=9.5, 5.8, 3.9 Hz, 1H), 1.61 (ddd, J=8.9, 5.8, 3.4 Hz, 1H), 1.55 (ddd, J=9.0, 5.8, 3.4 Hz, 1H), 1.50 (d, J=6.3 Hz, 3H). LCMS Tr=1.97 min; m/z (ES+) 261 (M+H).sup.+
Intermediate 2, Step 1, Method 5: Methyl (1S,2S)-2-(2,2-dimethyl-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylate
[0302] .sub.H NMR (500 MHz, Chloroform-d) 7.89 (dd, J=8.4, 1.9 Hz, 1H), 7.87-7.83 (m, 1H), 6.78 (d, J=8.4 Hz, 1H), 3.73 (s, 3H), 3.13 (ddd, J=8.8, 5.8, 3.9 Hz, 1H), 2.35 (ddd, J=8.7, 5.8, 3.8 Hz, 1H), 1.61 (ddd, J=8.9, 5.8, 3.4 Hz, 1H), 1.54 (ddd, J=9.0, 5.8, 3.4 Hz, 1H), 1.51 (s, 6H). LCMS Tr=2.10 min; m/z 275 (M+H).sup.+.
Intermediate 3, Step 1, Method 5: Methyl (1S,2S)-2-(2,3-dihydro-1,4-benzoxathiine-6-carbonyl)cyclopropane-1-carboxylate
[0303] LCMS Tr=3.16 min; m/z 279 (M+H).sup.+.
Intermediate 4, Step 1, Method 5: Methyl (1S,2S)-2-(2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylate
[0304] .sub.H NMR (500 MHz, Chloroform-d) 7.89 (dd, J=8.4, 1.9 Hz, 1H), 7.87-7.83 (m, 1H), 6.78 (d, J=8.4 Hz, 1H), 3.73 (s, 3H), 3.13 (ddd, J=8.8, 5.8, 3.9 Hz, 1H), 2.35 (ddd, J=8.7, 5.8, 3.8 Hz, 1H), 1.61 (ddd, J=8.9, 5.8, 3.4 Hz, 1H), 1.54 (ddd, J=9.0, 5.8, 3.4 Hz, 1H), 1.51 (s, 6H). LCMS Tr=1.9 min; m/z (ES+) 249 (M+H).sup.+.
Step 2, Method 5: (1S,2S)-2-(2-methyl-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylic acid
[0305] 2M NaOH (0.15 mL, 0.31 mmol) is added in one portion to a stirred solution of methyl (1 S,2S)-2-(2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylate (0.08 g, 0.28 mmol) in dioxane (5 mL) and the resulting solution is stirred at room temperature for 18 hours. After this time the reaction mixture is acidified with 1M HCl, and the resulting suspension is extracted with EtOAc (210 mL). The combined organic extracts are washed with brine (10 mL), before being dried (MgSO4), filtered and concentrated. The resulting residue is purified by prep HPLC to give the title compound (0.03 g, 39% yield) as a white solid.
Example 1, Method 5: (1S,2S)-2-(2-methyl-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylic acid
[0306] .sub.H (500 MHz, Chloroform-d) 7.92-7.85 (m, 2H), 6.81 (d, J=8.4 Hz, 1H), 5.05 (dq, J=13.2, 6.5 Hz, 1H), 3.38 (dd, J=15.6, 8.9 Hz, 1H), 3.18 (ddd, J=9.3, 5.9, 3.9 Hz, 1H), 2.86 (dd, i=15.6, 7.3 Hz, 1H), 2.36 (ddd, J=9.0, 5.6, 3.9 Hz, 1H), 1.68 (ddd, J=8.9, 5.9, 3.5 Hz, 1H), 1.60 (ddd, J=9.0, 5.7, 3.5 Hz, 1H), 1.50 (d, J=6.2 Hz, 2H). LCMS Tr=2.50 min; m/z (ES+) 247 (M+H).sup.+.
Example 2, Method 5: (1S,2S)-2-(2,2-Dimethyl-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylic acid
[0307] .sub.H (500 MHz, Chloroform-d) 7.90 (dd, J=8.4, 1.8 Hz, 1H), 7.86 (m, 1H), 6.79 (d, J=8.4 Hz, 1H), 3.19 (ddd, J=9.5, 5.9, 3.8 Hz, 1H), 3.06 (s, 2H), 2.36 (ddd, J=9.2, 5.7, 3.8 Hz, 1H), 1.68 (ddd, J=8.9, 5.9, 3.5 Hz, 1H), 1.60 (ddd, J=9.0, 5.7, 3.5 Hz, 1H), 1.52 (s, 6H). Tr=2.74 min; m/z (ES+) 261 (M+H).sup.+.
Example 3, Method 5: (1S,2S)-2-(2,3-dihydro-1,4-benzoxathiine-6-carbonyl)cyclopropane-1-carboxylic acid
[0308] .sub.H (500 MHz, Chloroform-d) 7.78 (d, J=2.2 Hz, 1H), 7.69 (dd, J=8.6, 2.2 Hz, 1H), 6.90 (d, J=8.6 Hz, 1H), 4.53-4.49 (m, 2H), 3.19-3.12 (m, 3H), 2.37 (ddd, J=9.2, 5.7, 3.8 Hz, 1H), 1.70-1.57 (m, 2H). 13C NMR (500 MHz, Chloroform-d) 194.4, 176.4, 155.6, 130.5, 128.7, 126.3, 118.5, 118.3, 65.8, 26.3, 23.2, 24.9, 18.2. Tr=2.47 min m/z (ES+) 265 (M+H).sup.+.
Example 4, Method 5: (1S,2S)-2-(2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylic acid
[0309] .sub.H NMR (500 MHz, DMSO-d6) 7.76 (dd, J=8.2, 1.7 Hz, 1H), 7.50 (d, J=1.6 Hz, 1H), 7.08 (d, J=8.2 Hz, 1H), 6.17 (s, 2H), 3.16 (ddd, J=9.0, 5.6, 3.9 Hz, 1H), 2.07 (ddd, J=9.5, 5.8, 3.9 Hz, 1H), 1.44 (dtd, J=10.9, 5.6, 2.5 Hz, 2H). Tr=2.13 min m/z (ES+) 235 (M+H).sup.+.
TABLE-US-00005 TABLE 5 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 6
Scheme for Method 6
[0310] ##STR00020##
Step 1, Method 6:4-Bromo-2-chloro-1-[(2-methylprop-2-en-1-yl)oxy]benzene
[0311] Potassium carbonate (33.31 g, 241 mmol) was added to a solution of 4-bromo-2-chlorophenol (25 g, 121 mmol) and 3-bromo-2-methylprop-1-ene (14.6 mL, 145 mmol) in anhydrous DMF (100 mL) was stirred at 80 C. under an atmosphere of nitrogen for 2 hours. The reaction mixture was allowed to cool to room temperature and partitioned between water (250 mL) and EtOAc (250 mL). The organic layer was separated, washed with water (2250 mL), brine (250 mL), dried (MgSO4), filtered and concentrated under reduced pressure to afford the title compound as a pale yellow oil (31.6 g, 99% yield), which was used in the next step without purification. .sub.H NMR (500 MHz, Chloroform-d) 7.50 (d, J=2.4 Hz, 1H), 7.29 (dd, J=8.8, 2.4 Hz, 1H), 6.78 (d, J=8.8 Hz, 1H), 5.12 (s, 1H), 5.02 (s, 1H), 4.48 (s, 2H), 1.84 (s, 3H). Tr=2.45 min, no mass ion.
Step 2, Method 6:4-Bromo-2-chloro-6-(2-methylprop-2-en-1-yl)phenol
[0312] N,N-Diethylaniline (17.2 mL, 108 mmol) was added to a solution of 4-bromo-2-chloro-1-[(2-methylprop-2-en-1-yl)oxy]benzene (99%, 28.52 g, 108 mmol) in mesitylene (250 mL). The reaction mixture was stirred at 190 C. for 18.5 hours, allowed to cool to room temperature, diluted with EtOAc (500 mL), washed with 1N HCl (2200 mL), brine (200 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a brown oil (m=28.0 g), which was purified by column chromatography (5% EtOAc in heptane). Mixed fractions containing product were combined and concentrated under reduced pressure to leave a yellow (m=13.49 g), which was purified again by column chromatography (Biotage, 1-10% EtOAc in heptane). Pure fractions from both columns were combined and concentrated under reduced pressure to afford the title compound as a pale yellow oil (13.75 g, 46% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.35 (d, J=2.4 Hz, 1H), 7.17 (d, J=2.3 Hz, 1H), 5.61 (s, 1H), 4.88 (s, 1H), 4.73 (s, 1H), 3.35 (s, 2H), 1.73 (s, 3H). Tr=2.27 min, no mass ion.
Step 3, Method 6: 4-Bromo-2-chloro-6-(2-hydroxypropyl)phenol
[0313] 4-Bromo-2-chloro-6-(2-methylprop-2-en-1-yl)phenol (94%, 7.18 g, 27.5 mmol) was dissolved in THF (150 mL) and water (150 mL) and the reaction mixture was cooled to 0 C. (ice/water bath). Sodium periodate (11.74 g, 54.9 mmol) was added in one portion. After 5 minutes, osmium tetroxide (3.5 ml, 4 wt % solution in water, 0.55 mmol) was added and the reaction mixture was stirred at 0 C. for 30 minutes. The reaction mixture was poured in ice-cold brine (150 mL) and extracted with EtOAc (3150 mL). The combined organic extracts were washed with brine (150 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a dark orange oil (m=8.40 g). This was taken-up in MeOH (150 mL) and the mixture cooled to 0 C. (ice-water bath). Sodium borohydride (2.08 g, 54.9 mmol) was added portionwise over 15 minutes at such a rate as to maintain temperature below 15 C. Upon addition, the reaction mixture turned from orange to dark green. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between 1N HCl (100 mL) and EtOAc (100 mL). The aqueous layer was separated and extracted further with EtOAc (2100 mL). The combined organic extracts were washed with brine (100 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a dark brown oil (8.20 g). Purification by column chromatography (Biotage, 7-60% EtOAc in heptane, Rf=0.31 in 30% EtOAc in heptane) afforded the title compound as an off-white solid (4.78 g, 98% purity, 64% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.97 (s, 1H), 7.38 (d, J=2.4 Hz, 1H), 7.11 (d, J=2.3 Hz, 1H), 4.23 (ddq, J=9.4, 6.3, 3.1 Hz, 1H), 2.86 (dd, J=14.4, 3.1 Hz, 1H), 2.75 (dd, J=14.4, 7.4 Hz, 1H), 2.18 (s, 1H), 1.28 (d, J=6.2 Hz, 3H). Tr=1.91 min m/z (ES.sup.) (MH.sup.) 263, 265, 267.
Step 4, Method 6: 5-Bromo-7-chloro-2-methyl-2,3-dihydro-1-benzofuran
[0314] Diisopropyl azodicarboxylate (4.61 mL, 23.4 mmol) was added to a cold (0 C.) solution of 4-bromo-2-chloro-6-(2-hydroxypropyl)phenol (98%, 4.78 g, 18 mmol) and triphenylphosphine (6.14 g, 23.4 mmol) in anhydrous THF (100 mL) under an atmosphere of nitrogen. The reaction mixture was stirred at 0 C. for 2 hours and concentrated under reduced pressure to leave a yellow oil. Purification by column chromatography (Biotage, 1-10% EtOAc in heptane, Rf=0.38 in 5% EtOAc in heptane) afforded the title compound as a pale yellow oil (m=4.18 g, 93% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.25 (d, J=1.8 Hz, 1H), 7.16-7.13 (m, 1H), 5.10-4.97 (m, 1H), 3.37 (dd, J=15.8, 8.8 Hz, 1H), 2.88 (dd, J=15.8, 7.8 Hz, 1H), 1.51 (d, J=6.3 Hz, 3H). Tr=2.30 min, no mass ion.
Step 5, Method 6: (7-Chloro-2-methyl-2,3-dihydro-1-benzofuran-5-yl)trimethylstannane
[0315] n-Butyllithium (11.5 mL of a 1.6M solution in hexanes, 18.4 mmol) was added dropwise over 15 minutes to a cold (78 C.) solution of 5-bromo-7-chloro-2-methyl-2,3-dihydro-1-benzofuran (99%, 4.18 g, 16.72 mmol) in anhydrous THF (50 mL) under a nitrogen atmosphere. The reaction mixture was stirred for 45 minutes and chloro(trimethyl)stannane (1M solution in THF, 18.4 mL, 18.4 mmol) was added dropwise over 15 minutes. The reaction mixture was allowed to warm slowly to room temperature and stirred for 22 hours. The reaction mixture was poured in brine (50 mL). The aqueous layer was separated and extracted further with EtOAc (250 mL). The combined organic extracts were washed with brine (50 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a yellow oil (5.28 g). Purification by column chromatography (Biotage, 2-20% EtOAc in heptane, Rf=0.51 in 10% EtOAc in heptane) afforded the title compound as a pale yellow oil (3.80 g, 59% yield), which was used without further purification. .sub.H NMR (500 MHz, Chloroform-d) 7.18-7.16 (m, 1H), 7.13 (d, J=0.9 Hz, 1H), 5.08-4.91 (m, 1H), 3.37 (dd, J=15.5, 8.8 Hz, 1H), 2.88 (dd, J=15.5, 7.7 Hz, 1H), 1.51 (d, J=6.3 Hz, 3H), 0.27 (s, 9H). Tr=2.58 min, no mass ion.
Step 6, Method 6: (1S,2S)-2-(7-chloro-2-methyl-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylate
[0316] A solution of (7-chloro-2-methyl-2,3-dihydro-1-benzofuran-5-yl)trimethylstannane (86%, 890 mg, 2.31 mmol) and methyl (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (560 mg, 3.47 mmol) in anhydrous toluene (10 mL) was degassed for 20 minutes by bubbling nitrogen. PdCl.sub.2(PPh.sub.3).sub.2 (81 mg g, 0.12 mmol) was added and the reaction mixture was stirred at 110 C. under an atmosphere of nitrogen for 3 hours. The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure to leavean orange oil. Purification by column chromatography (Biotage, 5-40% EtOAc in heptane, Rf=0.24 in 20% EtOAc in heptane) afforded the title compound as a thick yellow oil (520 mg, 73% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.87 (d, J=1.5 Hz, 1H), 7.74 (d, J=1.4 Hz, 1H), 5.20-5.09 (m, 1H), 3.74 (s, 3H), 3.45 (dd, J=15.7, 8.9 Hz, 1H), 3.07 (ddd, J=9.4, 5.8, 3.8 Hz, 1H), 2.93 (dd, J=15.7, 7.6 Hz, 1H), 2.36 (ddd, J=9.3, 5.8, 3.8 Hz, 1H), 1.63-1.50 (m, 5H). Tr=1.91 min m/z (ES.sup.+) (M+H.sup.+) 295, 297.
Step 7, Method 6: (1S,2S)-2-(7-chloro-2-methyl-2,3-dihydro-1-benzofuran-5-carbonyl)-cyclo-propane-1-carboxylic acid
[0317] 2M Sodium hydroxide (1.7 mL, 3.4 mmol) was added to a solution of methyl (1S,2S)-2-(7-chloro-2-methyl-2,3-dihydro-1-benzofuran-5-carbonyl)cyclopropane-1-carboxylate (96%, 520 mg, 1.7 mmol) in dioxane (5 mL) and the reaction mixture was stirred at room temperature for 5.5 hours. The reaction mixture was concentrated under reduced pressure and the residue taken up in water (20 mL). The aqueous mixture was acidified to pH=2 with 2N HCl and extracted with EtOAc (320 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a yellow oil (453 mg). Purification by acidic preparative HPLC afforded a white solid (m=234 mg). This was re-purified by basic preparative HPLC. Fractions containing product were concentrated under reduced pressure. The residue was taken up in 1N HCl (10 mL) and extracted with diethyl ether (310 mL). The combined organic extracts were washed with brine (10 mL), dried (MgSO4), filtered and concentrated under reduced pressure to afford the title compound as a white solid (155 mg, 32% yield).
Example 1, Method 6: (1S,2S)-2-(7-chloro-2-methyl-2,3-dihydro-1-benzofuran-5-carbonyl)-cyclo-propane-1-carboxylic acid
[0318] .sub.H NMR (500 MHz, Chloroform-d) 7.88 (d, J=1.5 Hz, 1H), 7.74 (d, J=1.3 Hz, 1H), 5.20-5.11 (m, 1H), 3.45 (dd, J=15.8, 8.9 Hz, 1H), 3.12 (ddd, J=9.4, 5.9, 3.8 Hz, 1H), 2.94 (dd, J=15.7, 7.5 Hz, 1H), 2.37 (ddd, J=9.1, 5.7, 3.8 Hz, 1H), 1.66 (ddd, J=8.9, 5.8, 3.6 Hz, 1H), 1.62 (ddd, J=9.1, 5.7, 3.5 Hz, 1H), 1.55 (d, J=6.3 Hz, 3H). Tr=2.68 min m/z (ES) (MH).sup. 279, 281.
TABLE-US-00006 TABLE 6 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 7
Scheme for Method 7
[0319] ##STR00022##
Step 1, Method 7: 4-Bromo-5-chloro-2-(3-hydroxy-2-methylpropyl)phenol
[0320] Borane.THF (1M solution in THF, 25.8 mL, 25.8 mmol) was added dropwise over 10 minutes to a solution of 4-bromo-2-chloro-6-(2-methylprop-2-en-1-yl)phenol (94%, 7.18 g, 25.81 mmol) in anhydrous THF (50 mL) and the reaction mixture was stirred at room temperature for 4 hours. Water (1.55 mL, 25.8 mmol), 3M sodium hydroxide (3.96 ml, 11.9 mmol) and hydrogen peroxide (30% w/w solution in water, 2.7 mL, 25.8 mmol) were added slowly and the reaction mixture was stirred at room temperature for 45 minutes. Water (50 mL) was added and the mixture was extracted with EtOAc (350 mL). The combined organic extracts were washed with brine (50 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a yellow oil (8.73 g). Purification by column chromatography (Biotage, 7-60% EtOAc in heptane, Rf=0.23 in 30% EtOAc in heptane) afforded the title compound as an off-white solid (7.16 g, 97% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.35 (d, J=2.4 Hz, 1H), 7.14 (d, J=2.4 Hz, 1H), 3.53 (dd, J=10.7, 4.8 Hz, 1H), 3.41 (dd, J=10.7, 6.3 Hz, 1H), 2.74 (dd, J=13.8, 7.0 Hz, 1H), 2.58 (dd, J=13.8, 6.2 Hz, 1H), 2.06-1.94 (m, 1H), 0.98 (d, J=6.9 Hz, 3H). Tr=1.89 min m/z (ES.sup.) (MH.sup.) 277, 279, 281.
Step 2, Method 7: 6-Bromo-8-chloro-3-methyl-3,4-dihydro-2H-1-benzopyran
[0321] Diisopropyl azodicarboxylate (6.4 mL, 32.6 mmol) was added to a cold (0 C.) solution of 4-bromo-5-chloro-2-(3-hydroxy-2-methylpropyl)phenol (98%, 7.16 g, 25.1 mmol) and triphenylphosphine (8.56 g, 32.6 mmol) in anhydrous THF (70 mL) under an atmosphere of nitrogen. The reaction mixture was stirred at 0 C. for 2.5 hours and concentrated under reduced pressure to leave a yellow oil. Purification by column chromatography (Biotage, 1-10% EtOAc in heptane, Rf=0.42 in 5% EtOAc in heptane) afforded the title compound as a pale yellow solid (6.13 g, 92% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.31 (d, J=2.3 Hz, 1H), 7.08-7.04 (m, 1H), 4.30 (ddd, J=10.7, 3.4, 2.0 Hz, 1H), 3.81-3.70 (m, 1H), 2.81 (ddd, J=16.4, 5.1, 1.8 Hz, 1H), 2.43 (dd, J=16.4, 9.7 Hz, 1H), 2.15 (dtdd, J=12.8, 8.4, 6.7, 3.4 Hz, 1H), 1.05 (d, J=6.8 Hz, 3H). Tr=2.36 min, no mass ion.
Step 3, Method 7: (8-Chloro-3-methyl-3,4-dihydro-2H-1-benzopyran-6-yl)trimethylstannane
[0322] n-Butyllithium (15.8 mL of a 1.6M solution in hexanes, 25.3 mmol) was added dropwise over 20 minutes to a cold (78 C.) solution of 6-bromo-8-chloro-3-methyl-3,4-dihydro-2H-1-benzopyran (98%, 6.13 g, 23.0 mmol) in anhydrous THF (50 mL) under a nitrogen atmosphere. The reaction mixture was stirred for 45 minutes and chloro(trimethyl)stannane (1M solution in THF, 25.3 mL, 25.3 mmol) was added dropwise over 20 minutes. The reaction mixture was allowed to warm to room temperature slowly and stirred for 22 hours. The reaction mixture was poured into brine (100 mL). The aqueous layer was separated and further extracted with EtOAc (2100 mL). The combined organic extracts were washed with brine (100 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a pale yellow sticky solid (7.82 g). Purification by column chromatography (Biotage, 1-10% EtOAc in heptane, Rf=0.50 in 5% EtOAc in heptane) afforded the title compound as an off-white solid (5.49 g, 55% yield). .sup.1H NMR (500 MHz, Chloroform-d) 7.24 (s, 1H), 7.02-6.99 (m, 1H), 4.38-4.22 (m, 1H), 3.83-3.68 (m, 1H), 2.85 (ddd, J=16.2, 5.1, 1.6 Hz, 1H), 2.45 (dd, J=16.2, 9.8 Hz, 1H), 2.16 (dddp, J=12.9, 9.8, 6.5, 3.0 Hz, 1H), 1.06 (d, J=6.8 Hz, 3H), 0.27 (s, 9H). Tr=2.63 min, no mass ion.
Step 4, Method 7: Methyl (1S,2S)-2-(8-Chloro-3-methyl-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclopropane-1-carboxylate
[0323] A solution of (8-chloro-3-methyl-3,4-dihydro-2H-1-benzopyran-6-yl)trimethylstannane (80%, 1.00 g, 2.32 mmol) and (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (560 mg, 3.47 mmol) in anhydrous toluene (10 mL) was degassed for 20 minutes by bubbling nitrogen. PdCl.sub.2(PPh.sub.3).sub.2 (81 mg g, 0.12 mmol) was added and the reaction mixture was stirred at 110 C. under an atmosphere of nitrogen for 3 hours. The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure to leavean orange oil. Purification by column chromatography (Biotage, 5-40% EtOAc in heptane, Rf=0.27 in 20% EtOAc in heptane) afforded the title compound as a thick colourless oil (477 mg, 62% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.88 (d, J=2.1 Hz, 1H), 7.69-7.61 (m, 1H), 4.39 (ddd, J=10.7, 3.5, 2.0 Hz, 1H), 3.92-3.80 (m, 1H), 3.74 (s, 3H), 3.08 (ddd, J=9.4, 5.8, 3.8 Hz, 1H), 2.94-2.86 (m, 1H), 2.51 (dd, J=16.2, 9.8 Hz, 1H), 2.39-2.33 (m, 1H), 2.24-2.13 (m, 1H), 1.62-1.54 (m, 2H), 1.09 (d, J=6.8 Hz, 3H). Tr=2.11 min m/z (ES.sup.+) (M+H.sup.+) 309, 311.
Step 5, Method 7: (1S,2S)-2-(8-Chloro-3-methyl-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclo-propane-1-carboxylic acid
[0324] 2M Sodium hydroxide (1.4 mL, 2.8 mmol) was added to a solution of methyl (1S,2S)-2-(8-Chloro-3-methyl-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclopropane-1-carboxylate (93%, 477 mg, 1.44 mmol) in dioxane (5 mL) and the reaction mixture was stirred at room temperature for 5.5 hours. The reaction mixture was concentrated under reduced pressure and the residue taken up in water (20 mL). The aqueous mixture was acidified to pH=2 with 2N HCl and extracted with EtOAc (320 mL). The combined organic extracts were washed with brine (20 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a yellow oil (574 mg). Purification by acidic preparative HPLC afforded the title compound as a white solid (221 mg, 52% yield).
Example 1, Method 7: (1S,2S)-2-(8-Chloro-3-methyl-3,4-dihydro-2H-1-benzopyran-6-carbonyl)cyclo-propane-1-carboxylic acid
[0325] .sub.H NMR (500 MHz, Chloroform-d) 7.88 (d, J=2.1 Hz, 1H), 7.66-7.63 (m, 1H), 4.40 (ddd, J=10.7, 3.4, 2.0 Hz, 1H), 3.91-3.82 (m, 1H), 3.13 (ddd, J=9.4, 5.8, 3.8 Hz, 1H), 2.92 (dd, J=16.3, 3.9 Hz, 1H), 2.52 (dd, J=16.3, 9.8 Hz, 1H), 2.37 (dddd, J=7.0, 5.4, 3.8, 1.2 Hz, 1H), 2.26-2.14 (m, 1H), 1.66 (ddd, J=8.7, 5.8, 4.0 Hz, 1H), 1.62 (ddd, J=9.1, 5.8, 3.5 Hz, 1H), 1.09 (d, J=6.7 Hz, 3H). Tr=3.09 min m/z (ES.sup.+) (M+H.sup.+) 295, 297.
TABLE-US-00007 TABLE 7 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 8
Scheme for Method 8
[0326] ##STR00024##
Step 1, Method 8: 5-Bromo-3-chloro-2-hydroxybenzaldehyde
[0327] Powdered sodium hydroxide (23.14 g, 578 mmol) was added to a solution of 4-bromo-2-chlorophenol (20 g, 96 mmol) and water (1.8 mL, 96 mmol) in chloroform (200 mL) and the reaction mixture was stirred at reflux for 7.5 hours. Powdered sodium hydroxide (7.71 g, 193 mmol) was added and the reaction mixture was refluxed for an additional 19 hours, allowed to cool to room temperature, diluted with water (250 mL), acidified to pH=1 with 2N HCl and extracted with EtOAc (3500 mL). The combined organic extracts were filtered to remove a small amount of insoluble off-white solid, washed with brine (2500 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a dark brown oil (18.42 g). Purification by column chromatography (Biotage, 2-20% EtOAc in heptane, Rf=0.35 in 10% EtOAc in heptane) afforded the title compound as a pale yellow solid (3.42 g, 14% yield), which was used without further purification. .sub.H NMR (500 MHz, DMSO-d.sub.6) 11.21 (s, 1H), 10.12 (s, 1H), 8.00 (d, J=2.5 Hz, 1H), 7.84 (d, J=2.5 Hz, 1H). Tr=1.94 min, no mass ion.
Step 2, Method 8:5-Bromo-3-chlorobenzene-1,2-diol
[0328] A solution of hydrogen peroxide (30 wt % in water, 1.6 ml, 15.7 mmol) in water (20 mL) was added dropwise over 10 minutes to a solution of 5-bromo-3-chloro-2-hydroxybenzaldehyde (90%, 3.42 g, 13.1 mmol) and sodium hydroxide (0.63 g, 15.7 mmol) in water (30 mL). The reaction mixture was then stirred at room temperature for 20 minutes. A saturated sodium sulfite solution (20 mL) was added. The mixture was stirred for 10 minutes, acidified to pH=2 with 1N HCl and extracted with DCM (350 mL). The combined organic extracts were washed with brine (50 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a thick orange oil (3.07 g). Purification by column chromatography (Biotage, 7-60% EtOAc in heptane, Rf=0.30 in 30% EtOAc in heptane) afforded the title compound as a beige solid (2.01 g, 95% purity, 65% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.04 (d, J=2.2 Hz, 1H), 7.01 (d, J=2.2 Hz, 1H), 5.56 (s, 1H), 5.50 (s, 1H). Tr=1.63 min m/z (ES.sup.) (MH.sup.) 221, 223, 225.
Step 3, Method 8:7-Bromo-5-chloro-2,3-dihydro-1,4-benzodioxine
[0329] Cesium carbonate (5.57 g, 17.1 mmol) was added to a solution of 5-bromo-3-chlorobenzene-1,2-diol (95%, 2.01 g, 8.6 mmol) and 1,2-dibromoethane (0.81 ml, 9.4 mmol) in anhydrous DMF (20 mL). The reaction mixture was stirred at 80 C. under a nitrogen atmosphere for 4 hours. The reaction mixture was allowed to cool to room temperature and partitioned between water (20 mL) and EtOAc (20 mL). The aqueous layer was separated and extracted further with EtOAc (220 mL). the combined organic extracts were washed with water (220 mL), brine (20 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a brown oil (1.71 g). Purification by column chromatography (Biotage, 1-10% EtOAc in heptane, Rf=0.26 in 10% EtOAc in heptane) afforded the title compound as a white solid (1.38 g, 63% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.08 (d, J=2.3 Hz, 1H), 6.95 (d, J=2.3 Hz, 1H), 4.36-4.33 (m, 2H), 4.28-4.25 (m, 2H). Tr=2.11 min, no mass ion.
Step 4, Method 8: (8-Chloro-2,3-dihydro-1,4-benzodioxin-6-yl)trimethylstannane
[0330] n-Butyllithium (3.7 mL of a 1.6M solution in hexanes, 5.9 mmol) was added dropwise over 10 minutes to a cold (78 C.) solution of 7-Bromo-5-chloro-2,3-dihydro-1,4-benzodioxine (97%, 1.38 g, 15.4 mmol) in anhydrous THF (20 mL) under a nitrogen atmosphere. The reaction mixture was stirred for 45 minutes and chloro(trimethyl)stannane (1M solution in THF, 5.9 mL, 5.9 mmol) was added dropwise over 10 minutes. After 20 minutes, the reaction mixture was allowed to warm to room temperature slowly and stirred for 21 hours. The reaction mixture was poured into brine (50 mL). The aqueous layer was separated and extracted further with EtOAc (250 mL). The combined organic extracts were washed with brine (50 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a yellow oil (1.85 g). Purification by column chromatography (Biotage, 1-10% EtOAc in heptane, Rf=0.33 in 5% EtOAc in heptane) afforded the title compound as a pale yellow oil (1.40 g, 54% yield), which was used without further purification. Tr=2.70 min, no mass ion.
Step 5, Method 8: Methyl (1S,2S)-2-(8-chloro-2,3-dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylate
[0331] A solution of (8-chloro-2,3-dihydro-1,4-benzodioxin-6-yl)trimethylstannane (69%, 1.4 g, 2.9 mmol) and methyl (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (716 mg, 4.4 mmol) in anhydrous toluene (20 mL) was degassed by bubbling nitrogen for 30 minutes. PdCl.sub.2(PPh.sub.3).sub.2 (102 mg, 0.14 mmol) was added and the reaction mixture was stirred at 110 C. for 3 hours. The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure to leave a thick yellow oil (1.46 g). Purification by column chromatography (Biotage, 7-40% EtOAc in heptane, Rf=0.25 in 30% EtOAc in heptane) afforded the title compound as an off-white solid (390 mg, 100% purity, 45% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.66 (d, J=2.1 Hz, 1H), 7.48 (d, J=2.1 Hz, 1H), 4.46-4.40 (m, 2H), 4.34-4.29 (m, 2H), 3.73 (s, 3H), 3.06 (ddd, J=8.7, 5.8, 3.8 Hz, 1H), 2.36 (ddd, J=8.7, 5.9, 3.8 Hz, 1H), 1.59 (ddt, J=12.0, 6.0, 3.5 Hz, 2H). Tr=1.95 min m/z (ES.sup.+) (M+H.sup.+) 297, 299.
Step 6, Method 8: (1S,2S)-2-(8-Chloro-2,3-dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylic acid
[0332] 2M aqueous sodium hydroxide (1.3 mL, 2.6 mmol) was added to a solution of methyl (1S,2S)-2-(8-chloro-2,3-dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylate (390 mg, 1.3 mmol) in dioxane (5 mL) and the reaction mixture was stirred at room temperature for 4 hours. 2N aqueous HCl (1.3 mL, 2.6 mmol) was added and the reaction mixture was concentrated under reduced pressure to leave a pale yellow solid. Purification by acidic preparative HPLC afforded the title compound as a white solid (58 mg, 15% yield).
Example 1, Method 8: (1S,2S)-2-(8-Chloro-2,3-dihydro-1,4-benzodioxine-6-carbonyl)cyclopropane-1-carboxylic acid
[0333] .sub.H NMR (500 MHz, Chloroform-d) 7.66 (d, J=2.1 Hz, 1H), 7.48 (d, J=2.1 Hz, 1H), 4.47-4.40 (m, 2H), 4.34-4.28 (m, 2H), 3.11 (ddd, J=9.4, 5.8, 3.8 Hz, 1H), 2.36 (ddd, J=8.8, 5.8, 3.8 Hz, 1H), 1.64 (dddd, J=18.2, 9.1, 5.8, 3.6 Hz, 2H). Tr=2.55 min m/z (ES.sup.+) (M+H.sup.+) 283, 285.
TABLE-US-00008 TABLE 8 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 9
Scheme for Method 9
[0334] ##STR00026##
Step 1, Method 9: 6-Bromo-4-chloro-2H-1,3-benzodioxole
[0335] Cesium carbonate (5.18 g, 15.9 mmol) was added to a solution of 5-bromo-3-chlorobenzene-1,2-diol (96%, 1.85 g, 7.95 mmol) and dibromomethane (0.6 mL, 8.7 mmol) in anhydrous DMF (20 mL). The reaction mixture was stirred at 80 C. under a nitrogen atmosphere for 19 hours. The reaction mixture was partitioned between water (20 mL) and EtOAc (20 mL). The aqueous layer was separated and extracted further with EtOAc (220 mL). The combined organic extracts were washed with water (220 mL), brine (20 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a sticky brown solid (1.83 g). Purification by column chromatography (Biotage, 1-10% EtOAc in heptane, Rf=0.40 in 5% EtOAc in heptane) afforded the title compound as a white solid (1.57 g, 83% yield). .sub.H NMR (500 MHz, Chloroform-d) 7.00 (d, J=1.8 Hz, 1H), 6.87 (d, J=1.8 Hz, 1H), 6.05 (s, 2H). Tr=2.12 min, no mass ion.
Step 2, Method 9: (7-Chloro-2H-1,3-benzodioxol-5-yl)trimethylstannane
[0336] n-Butyllithium (4.5 mL of a 1.6M solution in hexanes, 7.3 mmol) was added dropwise over 15 minutes to a cold (78 C.) solution of 6-bromo-4-chloro-2H-1,3-benzodioxole (99%, 1.57 g, 6.6 mmol) in anhydrous THF (20 mL) under a nitrogen atmosphere. The reaction mixture was stirred for 45 minutes and chloro(trimethyl)stannane (1M solution in THF, 7.3 mL, 7.3 mmol) was added dropwise over 15 minutes. After 20 minutes, the reaction mixture was allowed to warm to room temperature and stirred for 21 hours. The reaction mixture was poured into brine (50 mL). The aqueous layer was separated and extracted further with EtOAc (250 mL). The combined organic extracts were washed with brine (50 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a yellow oil (1.98 g). Purification by column chromatography (Biotage, 1-10% EtOAc in heptane, Rf=0.50 in 5% EtOAc in heptane) afforded the title compound as a colourless oil (944 mg, 33% yield), which was used without further purification in next step. Tr=2.49 min, no mass ion.
Step 3, Method 9: Methyl (1S,2S)-2-(7-chloro-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylate
[0337] A solution of (7-chloro-2H-1,3-benzodioxol-5-yl)trimethylstannane (73%, 944 mg, 2.2 mmol) and methyl (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (534 mg, 3.3 mmol) in anhydrous toluene (20 mL) was degassed by bubbling nitrogen for 30 minutes. PdCl.sub.2(PPh.sub.3).sub.2 (76 mg, 0.11 mmol) was added and the reaction mixture was stirred at 110 C. for 3 hours. The reaction mixture was allowed to cool to room temperature and concentrated under reduced pressure to leave a thick yellow oil (1.21 g). Purification by column chromatography (Biotage, 7-60% EtOAc in heptane, Rf=0.22 in 20% EtOAc in heptane) afforded the title compound as a white solid (309 mg, 90% purity, 46% yield), which was used in next step without further purification. .sub.H NMR (500 MHz, Chloroform-d) 7.63 (d, J=1.5 Hz, 1H), 7.36 (d, J=1.5 Hz, 1H), 6.14 (s, 2H), 3.74 (s, 3H), 3.04 (ddd, J=8.7, 5.8, 3.8 Hz, 1H), 2.37 (ddd, J=8.7, 6.0, 3.8 Hz, 1H), 1.64-1.56 (m, 2H). Tr=1.95 min m/z (ES.sup.+) (M+H.sup.+) 285, 287.
Step 4, Method 9: (1S,2S)-2-(7-Chloro-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylic acid
[0338] 2M aqueous sodium hydroxide (0.98 mL, 1.97 mmol) was added to a solution of methyl (1S,2S)-2-(7-chloro-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylate (90%, 309 mg, 0.98 mmol) in dioxane (5 mL) and the reaction mixture was stirred at room temperature for 4.5 hours. 2N aqueous HCl (0.98 mL, 1.97 mmol) was added and the reaction mixture was concentrated under reduced pressure to leave a pale yellow oil. Purification by acidic preparative HPLC afforded the title compound as a white solid (48 mg, 18% yield).
Example 1, Method 9: (1S,2S)-2-(7-Chloro-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylic acid
[0339] .sub.H NMR (500 MHz, Chloroform-d) 7.63 (d, J=1.5 Hz, 1H), 7.37 (d, J=1.5 Hz, 1H), 6.15 (s, 2H), 3.09 (ddd, J=9.4, 5.8, 3.8 Hz, 1H), 2.38 (ddd, J=8.8, 5.8, 3.8 Hz, 1H), 1.65 (dddd, J=17.0, 9.2, 5.8, 3.6 Hz, 2H). Tr=2.57 min m/z (ES.sup.+) (M+H.sup.+) 269, 271.
TABLE-US-00009 TABLE 9 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Method 10
Scheme for Method 10
[0340] ##STR00028##
Step 1, Method 10: 6-Bromo-4-chloro-2-methyl-2H-1,3-benzodioxole
[0341] Cesium carbonate (5.18 g, 15.9 mmol) was added to a solution of 5-bromo-3-chlorobenzene-1,2-diol (96%, 1.85 g, 7.95 mmol) and 1,1-dibromoethane (0.76 mL, 8.74 mmol) in anhydrous DMF (20 mL) and the reaction mixture was stirred at 80 C. under a nitrogen atmosphere for 21.5 hours. 1,1-Dibromethane (1.4 mL, 15.9 mmol) was added. The reaction mixture was stirred at 80 C. for an additional 23 hours, allowed to cool to room temperature and partitioned between water (50 mL) and EtOAc (50 mL). The aqueous layer was separated and extracted further with EtOAc (250 mL). The combined organic extracts were washed with water (250 mL), brine (50 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a dark purple oil (1.84 g). Purification by column chromatography (Biotage, heptane, Rf=0.34 in heptane) afforded the title compound as a colourless oil (1.18 g, 58% yield). .sub.H NMR (500 MHz, Chloroform-d) 6.97 (d, J=1.8 Hz, 1H), 6.81 (d, J=1.8 Hz, 1H), 6.35 (q, J=5.0 Hz, 1H), 1.72 (d, J=5.0 Hz, 3H). Tr=2.23 min, no mass ion.
Step 2, Method 10: (7-Chloro-2-methyl-2H-1,3-benzodioxol-5-yl)trimethylstannane
[0342] n-Butyllithium (3.2 mL of a 1.6M solution in hexanes, 5.0 mmol) was added dropwise over 15 minutes to a cold (78 C.) solution of 6-bromo-4-chloro-2-methyl-2H-1,3-benzodioxole (97%, 1.18 g, 4.59 mmol) in anhydrous THF (20 mL) under a nitrogen atmosphere. The reaction mixture was stirred for 45 minutes and chloro(trimethyl)stannane (1M solution in THF, 5.0 mL, 5.0 mmol) was added dropwise over 15 minutes. After 20 minutes, the reaction mixture was allowed to warm slowly to room temperature and stirred for 22 hours. The reaction mixture was poured in brine (50 mL). the aqueous layer was separated and extracted further with EtOAc (250 mL). The combined organic extracts were washed with brine (50 mL), dried (MgSO4), filtered and concentrated under reduced pressure to leave a yellow oil (m=1.51 g). Purification by column chromatography (Biotage, 0-5% EtOAc in heptane, Rf=0.23 in heptane) afforded the title compound as a colourless oil (805 mg, 44% yield), which was used in the next step without further purification. .sub.H NMR (500 MHz, Chloroform-d) 6.86 (s, 1H), 6.77 (s, 1H), 6.31-6.28 (m, 1H), 1.72 (d, J=5.0 Hz, 3H), 0.28 (s, 9H). Tr=2.59 min, no mass ion.
Step 3, Method 10: Methyl (1S,2S)-2-(7-chloro-2-methyl-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylate
[0343] A solution of (7-chloro-2-methyl-2H-1,3-benzodioxol-5-yl)trimethylstannane (84%, 805 mg, 2.03 mmol) and (1S,2S)-2-(carbonochloridoyl)cyclopropane-1-carboxylate (522 mg, 3.21 mmol) in anhydrous toluene (10 mL) was degassed for 20 minutes by bubbling nitrogen. PdCl.sub.2(PPh.sub.3).sub.2 (71 mg, 0.1 mmol) was added. The reaction mixture was stirred at 110 C. under an atmosphere of nitrogen for 3 hours, allowed to cool to room temperature and concentrated under reduced pressure to leave a sticky yellow solid (1.09 g). Purification by column chromatography (Biotage, 5-40% EtOAc in heptane, Rf=0.31 in 20% EtOAc in heptane) afforded the title compound as a colourless oil (401 mg, 59% yield), which was used in the next step without further purification. .sub.H NMR (500 MHz, Chloroform-d) 7.61 (d, J=1.6 Hz, 1H), 7.30 (d, J=1.5 Hz, 1H), 6.44 (qd, J=5.0, 1.5 Hz, 1H), 3.74 (s, 3H), 3.03 (ddd, J=9.3, 5.8, 3.8 Hz, 1H), 2.40-2.31 (m, 1H), 1.76 (dd, J=5.0, 0.8 Hz, 3H), 1.61-1.55 (m, 2H). Tr=2.05 min m/z (ES.sup.+) (M+H.sup.+) 297, 299.
Step 4, Method 10: (1S,2S)-2-(7-Chloro-2-methyl-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylic acid
[0344] 2M Sodium hydroxide (1.2 mL, 2.4 mmol) was added to a solution of methyl (1S,2S)-2-(7-chloro-2-methyl-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylate (89%, 401 mg, 1.2 mmol) in dioxane (5 mL). The reaction mixture was stirred at room temperature for 3.5 hours. 2M Sodium hydroxide (0.6 mL, 1.2 mmol), The reaction mixture was stirred at room temperature for an additional 16 hours. 2N HCl (1.8 mL, 3.6 mmol) was added and the reaction mixture was concentrated under reduced pressure to leave a pale yellow solid Purification by acidic preparative HPLC afforded the title compound as a white solid (m=160 mg, 97% purity, 46% yield).
Example 1, Method 10: (1S,2S)-2-(7-Chloro-2-methyl-2H-1,3-benzodioxole-5-carbonyl)cyclopropane-1-carboxylic acid
[0345] .sub.H NMR (500 MHz, Chloroform-d) 7.61 (d, J=1.5 Hz, 1H), 7.31 (d, J=1.4 Hz, 1H), 6.45 (qd, J=4.9, 1.4 Hz, 1H), 3.09 (ddd, J=9.2, 5.8, 3.9 Hz, 1H), 2.40-2.34 (m, 1H), 1.76 (dd, J=5.0, 0.9 Hz, 3H), 1.65 (dddt, J=20.5, 9.1, 5.7, 2.8 Hz, 2H). Tr=2.84 min m/z (ES.sup.+) (M+H.sup.+) 283, 285.
TABLE-US-00010 TABLE 10 Molecular LCMS % Inhibition Structure IUPAC Name Weight Data at 30 M
Prophetic Examples
[0346] The following examples may be prepared using the methods described above
TABLE-US-00011 Structure IUPAC Name
Biology Example 1
[0347] A generalized procedure for monitoring L-Kynurenine (KYN) hydroxylation to form product 3-Hydroxy-Kynurenine (3OH-KYN) by LC/MS is described below. Product is quantified by multiple reaction monitoring using MS.
Key Reagents:
[0348] Compound: Stock concentrations: 10 mM in 100% DMSO [0349] Cell line: CHO GST HIS KMO cell line, 1E4 cells/well/100 l in 96 well cell plate [0350] Substrate: L-Kynurenine (Sigma: Cat #K3750, stock concentration: 10 mM in 100 mM potassium phosphate buffer, pH 7.4)
Assay Conditions:
[0351] Medium: OptiMem (Reduced Serum Medium 1, +L-Glutamine+HEPES-Phenol Red; GIBCO: Cat #11058) [0352] Assay Volume: 200 l [0353] Plate Format: 96 well plate, transparent (Corning) [0354] Read-Out: product (3OH-KYN) quantification using product specific MRM [0355] Reader: LC/MS/MS
Assay Protocol:
[0356] prepare serial dilution (factor 3) of compound in 100% DMSO (top concentration=6.67 mM, 100% DMSO) [0357] [8 points: 6.67 mM; 2.22 mM; 0.74 mM; 0.247 mM; 0.082 mM; 0.027 mM; 0.009 mM; 0.003 mM] [0358] prepare 300-fold concentrated solution of each compound concentration (top concentration 22.22 M, 0.3% DMSO) in OptiMem medium [0359] [22.2 M; 7.41 M; 2.47 M; 0.82 M; 0.27 M; 0.09 M; 0.03 M; 0.01 M] [0360] prepare substrate (10 mM) at concentration of 1.1 mM in medium [0361] medium of cell plate is drawed off [0362] cells are washed with OptiMem (100 l/well) and drawed off again [0363] assay mix: 90 l OptiMem/well+90 l compound/well of each concentration [0364] [final compound top concentration: 10 M; 0.15% DMSO] [0365] [final compound bottom concentration: 0.004 M; 0.15% DMSO] [0366] pre-incubation: 30 min at 37 C. [0367] add 20 l/well of the 1.1 mM substrate solution (final assay concentration: 100 M) [0368] positive control: 200 l OptiMem [0369] negative control: 180 l OptiMem+20 l 1.1 mM substrate [0370] incubate 24 h at 37 C. [0371] transfer 100 l of each well in a transparent 96 well plate (Corning) [0372] add 100 l/well 10% trichloro acetic acid (TCA) in water [0373] centrifugate plate for 3 min at 4000 rpm [0374] detect product by LC/MS (injection of 50 l/well; 2.5 fold overfill of the 20 l sample loop)
Data Analysis:
[0375] IC.sub.50's are calculated using automated fitting algorithm (A+ Analysis).
Biology Example 2
[0376] A method of monitoring L-Kynurenine (KYN) hydroxylation to form product 3-Hydroxy-Kynurenine (3OH-KYN) by LC/MS is described below. Product is quantified by multiple reaction monitoring.
Key Reagents:
[0377] Compound: Stock concentrations: 10 mM in 100% DMSO [0378] Enzyme: KMO enzyme prepared at Evotec via mitochondria isolation from CHO-GST HIS KMO cells [0379] Substrate: L-Kynurenine (Sigma: Cat #K3750) [0380] [stock concentration: 10 mM in 100 mM potassium phosphate buffer, pH 7.4]
Assay Conditions:
[0381] Buffer: 100 mM potassium phosphate, pH 7.4, 200 M NADPH, 0.4 U/ml G6P-DH (Glucose 6-phosphate dehydrogenase), 3 mM G6P (D-Glucose 6-phosphate) [0382] Assay Volume: 40 l [0383] Plate Format: 384 well plate, transparent (Matrix) [0384] Read-Out: product (3OH-KYN) quantification using product specific MRM [0385] Reader: LC/MS/MS
Assay Protocol:
[0386] prepare serial dilution (factor 3) of compound in 100% DMSO (top concentration=10 mM, 100% DMSO) [0387] [8 points: 10 mM; 3.33 mM; 1.11 mM; 0.37 mM; 0.12 mM; 0.04 mM; 0.0137 mM; 0.0045 mM, 0.0015 mM] [0388] prepare 3.33-fold concentrated solution of each compound concentration (top concentration 300 M, 3% DMSO) in assay buffer [0389] [concentrations: 300 M; 100 M; 33.3 M; 11.1 M; 3.70 M; 1.23 M; 0.41 M; 0.137 M] [0390] prepare substrate (10 mM) at concentration of 1 mM in assay buffer [0391] assay mix: 4 l compound/well of each concentration+24 l assay buffer/well+8 l KMO human enzyme+4 l 1 mM substrate (final concentration=100 M) [0392] [final compound top concentration: 30 M; 0.3% DMSO] [0393] [final compound bottom concentration: 0.0137 M; 0.3% DMSO] [0394] positive control: 4 l 50 M FCE28833 in assay buffer [0.5% DMSO] (final assay concentration=5 M)+24 l assay buffer/well+8 l KMO human enzyme+4 l 1 mM substrate (final concentration=100 M) [0395] negative control: 28 l assay buffer/well+8 l KMO human enzyme+4 l 1 mM substrate (final concentration=100 M) [0396] incubate 400 min at RT [0397] add 40 l/well 10% trichloro acetic acid in water to stop the assay and precipitate protein [0398] centrifuge plate for 3 min at 4000 rpm [0399] product detection by LC/MS (injection of 50 l/well; 2.5 fold overfill of the 20 l sample loop)
Data Analysis:
[0400] IC.sub.50's are calculated using automated fitting algorithm (A+ Analysis).
Biology Example 3
[0401] A method of monitoring L-Kynurenine (KYN) hydroxylation to form 3-Hydroxy-Kynurenine (3OH-KYN) by LC/MS is described. Product is quantified by multiple reaction monitoring (MRM method).
Key Reagents:
[0402] Compound: Stock concentrations: 10 mM in 100% DMSO [0403] Enzyme: KMO enzyme prepared at Evotec from mouse liver (4-6 weeks old) via mitochondria isolation as described in the literature [0404] Substrate: L-Kynurenine (Sigma: Cat #K3750, stock concentration: 10 mM in 100 mM potassium phosphate buffer, pH 7.4)
Assay Conditions:
[0405] Buffer: 100 mM potassium phosphate, pH 7.4, 200 M NADPH, 0.4 U/ml G6P-DH (Glucose 6-phosphate Dehydrogenase), 3 mM G6P (D-Glucose 6-phosphate) [0406] Assay Volume: 40 l [0407] Plate Format: 384 well plate, transparent (Matrix) [0408] Read-Out: product (3OH-KYN) quantification using product specific MRM [0409] Reader: LC/MS/MS
Assay Protocol:
[0410] prepare serial dilution (factor 3) of compound in 100% DMSO (top concentration=10 mM, 100% DMSO) [0411] [8 points: 10 mM; 3.33 mM; 1.11 mM; 0.37 mM; 0.12 mM; 0.04 mM; 0.0137 mM; 0.0045 mM, 0.0015 mM] [0412] prepare 3.33-fold concentrated solution of each compound concentration (top concentration 300 M, 3% DMSO) in assay buffer [0413] [concentrations: 300 M; 100 M; 33.3 M; 11.1 M; 3.70 M; 1.23 M; 0.41 M; 0.137 M] [0414] prepare substrate (10 mM) at concentration of 1 mM in assay buffer [0415] assay mix: 4 l compound/well of each concentration+24 l assay buffer/well+8 l KMO mouse enzyme+4 l 1 mM substrate (final concentration=100 M) [0416] [final compound top concentration: 30 M; 0.3% DMSO] [0417] [final compound bottom concentration: 0.0137 M; 0.3% DMSO] [0418] positive control: 4 l 50 M FCE28833 in assay buffer, 0.5% DMSO [final assay concentration=5 M]+24 l assay buffer/well+8 l KMO mouse enzyme+4 l mM substrate [final concentration=100 M] [0419] negative control: 28 l assay buffer/well+8 l KMO mouse enzyme+4 l 1 mM substrate [0420] [final concentration=100 M] [0421] incubate 40 min at RT [0422] add 40 l/well 10% trichloro acetic acid in water to stop the assay and precipitate protein [0423] centrifuge plate for 3 min at 4000 rpm [0424] product detection by LC/MS (injection of 20 l/well, 2 fold overfill of the 10 l sample loop)
Data Analysis:
[0425] IC.sub.50's are calculated using automated fitting algorithm (A+ Analysis).
Biology Example 4
[0426] Using the assay protocols substantially similar to those of Biology Example 3, the following compounds were tested.
TABLE-US-00012 Table of compounds with inhibition information % Inhibition Structure IUPAC Name at 30 M
[0427] While some embodiments have been shown and described, various modifications and substitutions may be made thereto without departing from the spirit and scope of the invention. For example, for claim construction purposes, it is not intended that the claims set forth hereinafter be construed in any way narrower than the literal language thereof, and it is thus not intended that exemplary embodiments from the specification be read into the claims. Accordingly, it is to be understood that the present invention has been described by way of illustration and not limitations on the scope of the claims.