Histone deacetylase inhibitors and compositions and methods of use thereof

Abstract

Provided are certain histone deacetylase (HDAC) inhibitors of Formula I, compositions thereof, and methods of their use. ##STR00001##

Claims

1. A method of treating a condition or disorder selected from a neurodegenerative condition, amyotrophic lateral sclerosis (ALS), a cardiovascular condition, and cancer in a subject in need of such a treatment, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of Formula I: ##STR00075## or a pharmaceutically acceptable salt thereof, wherein: R.sub.1 and R.sub.2 are independently optionally substituted aryl or optionally substituted heteroaryl; R.sub.3 is C(O)NH(OH) or N(OH)C(O)R.sub.4; R.sub.3a is halo; and R.sub.4 is hydrogen or lower alkyl; and wherein: the neurodegenerative condition is selected from Alzheimer's disease, Parkinson's disease, neuronal intranuclear inclusion disease (NIID), Dentatorubral pallidoluysian atrophy (DRPLA), Friedreich's ataxia, Rubenstein-Taubi Syndrome, Huntington's disease, spinocerebellar ataxia 1 (SCA 1), spinocerebellar ataxia 7 (SCA 7), seizures, striatonigral degeneration, progressive supranuclear palsy, torsion dystonia, spasmodic torticollis, dyskinesis, familial tremor, Gilles de la Tourette syndrome, diffuse Lewy body disease, progressive supranuclear palsy, Pick's disease, primary lateral sclerosis, progressive neural muscular atrophy, spinal muscular atrophy, hypertrophic interstitial polyneuropathy, retinitis pigmentosa, hereditary optic atrophy, hereditary spastic paraplegia, Shy-Drager syndrome, Kennedy's disease, protein-aggregation-related neurodegeneration, Machado-Joseph's disease, spongiform encephalopathy, prion-related disease, multiple sclerosis (MS), progressive supranuclear palsy (Steel-Richardson-Olszewski disease), Hallervorden-Spatz disease, progressive familial myoclonic epilepsy, cerebellar degeneration, motor neuron disease, Werdnig-Hoffman disease, Wohlfart-Kugelberg-Welander disease, Charcot-Marie-Tooth disease, Dejerine-Sottas disease, Leber's disease, progressive systemic sclerosis, dermatomyositis, and mixed connective tissue disease; the cardiovascular condition is selected from cardiomyopathy, cardiac hypertrophy, myocardial ischemia, heart failure, cardiac restenosis, and arteriosclerosis; and the cancer is selected from lymphoma, pancreatic cancer, colorectal cancer, hepatocellular carcinoma, Waldenstrom macroglobulinemia, hormone refractory cancer of the prostate, leukemia, breast cancer, lung cancer, ovarian cancer, prostate cancer, head and neck cancer, renal cancer, gastric cancer, brain cancer, B-cell lymphoma, peripheral T-cell lymphoma, and cutaneous T-cell lymphoma.

2. The method of claim 1, wherein the neurodegenerative condition is Huntington's disease.

3. The method of claim 1, wherein the compound of Formula I is chosen from compounds of Formula II: ##STR00076##

4. The method of claim 1, wherein the compound of Formula I is chosen from compounds of Formula III: ##STR00077##

5. The method of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sub.3a is fluoro or chloro.

6. The method of claim 5, or a pharmaceutically acceptable salt thereof, wherein R.sub.3a is fluoro.

7. The method of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sub.3 is C(O)NH(OH).

8. The method of claim 1, or a pharmaceutically acceptable salt thereof, wherein R.sub.3 is N(OH)C(O)R.sub.4 wherein R.sub.4 is hydrogen.

9. The method of claim 1, wherein R.sub.3 is N(OH)C(O)R.sub.4 wherein R.sub.4 is methyl.

10. The method of claim 1, wherein R.sub.2 is aryl or heteroaryl, each of which is optionally substituted with one or two groups independently chosen from lower alkyl, halo, hydroxyl, and lower alkoxy.

11. The method of claim 10, wherein R.sub.2 is aryl optionally substituted with one or two groups independently chosen from lower alkyl, halo, hydroxyl, and lower alkoxy.

12. The method of claim 11, wherein R.sub.2 is phenyl, 2-methylphenyl, or 3-fluoro-2-methylphenyl.

13. The method of claim 12, wherein R.sub.2 is phenyl.

14. The method of claim 10, wherein R.sub.2 is heteroaryl optionally substituted with one or two groups independently chosen from lower alkyl, halo, hydroxyl, and lower alkoxy.

15. The method of claim 14, R.sub.2 is pyridin-3-yl or 6-oxo-1,6-dihydropyridin-2-yl, each of which is optionally substituted with one or two groups independently chosen from lower alkyl, halo, hydroxyl, and lower alkoxy.

16. The method of claim 15, or a pharmaceutically acceptable salt thereof, wherein R.sub.2 is 2-methylpyridin-3-yl or 1-methyl-6-oxo-1,6-dihydropyridin-2-yl.

17. The method of claim 1, wherein R.sub.1 is aryl or heteroaryl, each of which is optionally substituted with 1, 2, or 3 groups independently chosen from halo, cyclopropyl, trifluoromethyl, lower alkyl optionally substituted with 1, 2 or 3 groups independently chosen from halo, lower alkoxy, and hydroxyl, phenyl optionally substituted with 1 or 2 groups independently chosen from cyclopropyl, halo, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, and lower alkyl, heteroaryl optionally substituted with 1 or 2 groups independently chosen from cyclopropyl, halo, difluoromethyl, trifluoromethyl, trifluoromethoxy, difluoromethoxy, and lower alkyl, and -L-(CR.sub.5R.sub.6).sub.nN(R.sub.7)R.sub.8 where L is chosen from C(O)NR.sub.9 and NR.sub.10, n is 1 or 2, each occurrence of R.sub.5 and R.sub.6 is independently selected from hydrogen and lower alkyl, R.sub.7 is hydrogen or lower alkyl, and R.sub.8 is hydrogen or lower alkyl or R.sub.7 and R.sub.8, taken together with the nitrogen to which they are bound, form an optionally substituted 4- to 8-membered heterocycloalkyl ring, R.sub.9 is hydrogen, and R.sub.10 is selected from hydrogen and lower alkyl.

18. The method of claim 17, wherein R.sub.1 is 1,2,3,4-tetrahydroquinolin-6-yl, 1H-pyrazol-4-yl, 2,3-dihydrobenzo[b][1,4]dioxin-6-yl, benzo[d]oxazol-6-yl, benzo[d]thiazol-6-yl, chroman-6-yl, phenyl, pyridazin-4-yl, pyridin-3-yl, pyridin-4-yl, or thiazol-5-yl, each of which is optionally substituted with 1, 2, or 3 groups independently chosen from halo, cyclopropyl, lower alkyl optionally substituted with 1, 2, or 3 groups independently chosen from halo, lower alkoxy, and hydroxyl, phenyl optionally substituted with halo, oxazol-5-yl optionally substituted with cyclopropyl, pyrimidin-4-yl optionally substituted with 1 or 2 groups independently chosen from halo, difluoromethoxy, difluoromethyl, trifluoromethoxy, trifluoromethyl and lower alkyl, pyrimidin-2-yl optionally substituted with 1 or 2 groups independently chosen from halo, difluoromethoxy, difluoromethyl, trifluoromethoxy, trifluoromethyl and lower alkyl, pyrazin-2-yl optionally substituted with 1 or 2 groups independently chosen from halo, difluoromethoxy, difluoromethyl, trifluoromethoxy, trifluoromethyl or lower alkyl, pyridin-2-yl optionally substituted with 1 or 2 groups independently chosen from halo, difluoromethoxy, difluoromethyl, trifluoromethoxy, trifluoromethyl or lower alkyl, and -L-(CR.sub.5R.sub.6).sub.nN(R.sub.7)R.sub.8 where L is chosen from C(O)NR.sub.9 and NR.sub.10, n is 1 or 2, each occurrence of R.sub.5 and R.sub.6 is independently selected from hydrogen and lower alkyl, R.sub.7 is hydrogen or lower alkyl, and R.sub.8 is hydrogen or lower alkyl or R.sub.7 and R.sub.8, taken together with the nitrogen to which they are bound, form an optionally substituted 4- to 8-membered heterocycloalkyl ring, R.sub.9 is hydrogen, and R.sub.10 is selected from hydrogen and lower alkyl.

19. The method of claim 17, wherein R.sub.1 is 1,2,3,4-tetrahydroquinolin-6-yl, 1H-pyrazol-4-yl, 2,3-dihydrobenzo[b][1,4]dioxin-6-yl, benzo[d]oxazol-6-yl, benzo[d]thiazol-6-yl, chroman-6-yl, phenyl, pyridazin-4-yl, pyridin-3-yl, pyridin-4-yl, or thiazol-5-yl, each of which is optionally substituted with 1, 2, or 3 groups independently chosen from 2-(trifluoromethyl)pyrimidin-4-yl, 2-cyclopropyloxazol-5-yl, 2-hydroxypropan-2-yl, 4-(trifluoromethyl)pyrimidin-2-yl, 4-fluorophenyl, 5-(trifluoromethyl)pyridin-2-yl, 5-fluoropyrimidin-2-yl, 5-chloropyrimidin-2-yl, 5-methylpyrimidin-2-yl, 5-(difluoromethoxy)pyrimidin-2-yl, 5-(difluoromethyl)pyrimidin-2-yl, 2-methylpyrimidin-5-yl, 5-fluoropyridin-2-yl, 5-(trifluoromethyl)pyrazin-2-yl, bromo, chloro, cyclopropyl, fluoro, and oxazol-5-yl.

20. The method of claim 19, wherein R.sub.1 is (1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazol-4-yl, 2-(2-(trifluoromethyl)pyrimidin-4-yl)thiazol-5-yl, 2-(2-hydroxypropan-2-yl)benzo[d]oxazol-6-yl, 2-(2-hydroxypropan-2-yl)benzo[d]thiazol-6-yl, 2-(2-hydroxypropan-2-yl)pyridin-4-yl, 2-(2-hydroxypropan-2-yl)thiazol-5-yl, 2-(4-fluorophenyl)thiazol-5-yl, 2-(5-fluoropyrimidin-2-yl)thiazol-5-yl, 2-cyclopropyl-5-fluoropyridin-4-yl, 2-cyclopropylbenzo[d]oxazol-6-yl, 2-cyclopropylpyridin-4-yl, 2-cyclopropylthiazol-5-yl, 3-(2-cyclopropyloxazol-5-yl)phenyl, 3-(5-fluoropyrimidin-2-yl)phenyl, 4-(2-cyclopropyloxazol-5-yl)phenyl, 4-(4-(trifluoromethyl)pyrimidin-2-yl)phenyl, 4-(5-fluoropyrimidin-2-yl)phenyl, 4-(5-methylpyrimidin-2-yl)phenyl, 4-(oxazol-5-yl)phenyl, 4-bromophenyl, 6-cyclopropylpyridazin-4-yl, 8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl, 8-chloro-4,4-difluoro-1,2,3,4-tetrahydroquinolin-6-yl, or 8-chloro-4,4-difluorochroman-6-yl.

21. A method of treating a condition or disorder selected from a neurodegenerative condition, amyotrophic lateral sclerosis (ALS), a cardiovascular condition, and cancer in a subject in need of such a treatment, wherein the method comprises administering to the subject a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, chosen from: (1S,2S,3S)-1-fluoro-N-hydroxy-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(3-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-2-(3-(5-fluoropyrimidin-2-yl)phenyl)-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-chloro-N-hydroxy-2-(4-(oxazol-5-yl)phenyl)-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-(4-(oxazol-5-yl)phenyl)-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-phenyl-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)phenyl)cyclopropanecarboxamide, (1S,2S,3S)-2-(4-bromophenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-chloro-N-hydroxy-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-2-(4-(5-fluoropyrimidin-2-yl)phenyl)-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(4-(5-(difluoromethoxy)pyrimidin-2-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-phenyl-3-(4-(5-(trifluoromethyl)pyrimidin-2-yl)phenyl)cyclopropanecarboxamide, (1S,2S,3S)-2-(4-(5-(difluoromethyl)pyrimidin-2-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(4-(5-chloropyrimidin-2-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-(4-(2-methylpyrimidin-5-yl)phenyl)-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-phenyl-3-(4-(5-(trifluoromethyl)pyrazin-2-yl)phenyl)cyclopropanecarboxamide, (1S,2S,3S)-2-(6-((2-(diethylamino)ethyl)amino)pyridin-3-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, N((S)-1-(dipropylamino)propan-2-yl)-4-((1S,2S,3S)-2-fluoro-2-(hydroxycarbamoyl)-3-phenylcyclopropyl)benzamide, 4-((1S,2S,3S)-2-fluoro-2-(hydroxycarbamoyl)-3-phenylcyclopropyl)-N-(2-(pyrrolidin-1-yl)ethyl)benzamide, (1R,2S,3S)-1-fluoro-N-hydroxy-2-(2-(2-hydroxypropan-2-yl)pyridin-4-yl)-3-o-tolylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-(2-(2-hydroxypropan-2-yl)pyridin-4-yl)-3-phenylcyclopropanecarboxamide, (1R,2S,3S)-2-(2-cyclopropylpyridin-4-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(2-cyclopropyl-5-fluoropyridin-4-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(2-cyclopropylpyridin-4-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(6-cyclopropylpyridazin-4-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(6-cyclopropylpyridazin-4-yl)-1-fluoro-N-hydroxy-3-o-tolylcyclopropanecarboxamide, (1S,2S,3S)-2-(6-cyclopropylpyridazin-4-yl)-1-fluoro-3-(3-fluoro-2-methylphenyl)-N-hydroxycyclopropanecarboxamide, (1S,2S,3S)-2-(8-chloro-4,4-difluorochroman-6-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1R,2S,3S)-2-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(8-chloro-4,4-difluoro-1,2,3,4-tetrahydroquinolin-6-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-2-(2-cyclopropylbenzo[d]oxazol-6-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-(2-(2-hydroxypropan-2-yl)benzo[d]oxazol-6-yl)-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-N-hydroxy-2-(2-(2-hydroxypropan-2-yl)benzo[d]thiazol-6-yl)-3-phenylcyclopropanecarboxamide, (1S,2S,3S)-1-fluoro-2-(3-fluoro-2-methylphenyl)-N-hydroxy-3-(1-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazol-4-yl)cyclopropanecarboxamide, (1S,2R,3S)-2-(2-cyclopropylthiazol-5-yl)-1-fluoro-3-(3-fluoro-2-methylphenyl)-N-hydroxycyclopropanecarboxamide, (1S,2S,3R)-1-fluoro-2-(3-fluoro-2-methylphenyl)-3-(2-(5-fluoropyrimidin-2-yl)thiazol-5-yl)-N-hydroxycyclopropanecarboxamide, (1S,2R,3S)-1-fluoro-2-(2-(4-fluorophenyl)thiazol-5-yl)-N-hydroxy-3-(2-methylpyridin-3-yl)cyclopropanecarboxamide, (1S,2S,3R)-1-fluoro-2-(3-fluoro-2-methylphenyl)-3-(2-(4-fluorophenyl)thiazol-5-yl)-N-hydroxycyclopropanecarboxamide, (1S,2R,3S)-1-fluoro-2-(2-(4-fluorophenyl)thiazol-5-yl)-N-hydroxy-3-phenylcyclopropanecarboxamide, (1S,2R,3S)-1-fluoro-2-(2-(5-fluoropyridin-2-yl)thiazol-5-yl)-N-hydroxy-3-phenylcyclopropanecarboxamide, and (1S,2R,3S)-1-fluoro-2-(2-(5-fluoropyridin-2-yl)thiazol-5-yl)-N-hydroxy-3-o-tolylcyclopropanecarboxamide; and wherein: the neurodegenerative condition is selected from Alzheimer's disease, Parkinson's disease, neuronal intranuclear inclusion disease (NIID), Dentatorubral pallidoluysian atrophy (DRPLA), Friedreich's ataxia, Rubenstein-Taubi Syndrome, Huntington's disease, spinocerebellar ataxia 1 (SCA 1), spinocerebellar ataxia 7 (SCA 7), seizures, striatonigral degeneration, progressive supranuclear palsy, torsion dystonia, spasmodic torticollis, dyskinesis, familial tremor, Gilles de la Tourette syndrome, diffuse Lewy body disease, progressive supranuclear palsy, Pick's disease, primary lateral sclerosis, progressive neural muscular atrophy, spinal muscular atrophy, hypertrophic interstitial polyneuropathy, retinitis pigmentosa, hereditary optic atrophy, hereditary spastic paraplegia, Shy-Drager syndrome, Kennedy's disease, protein-aggregation-related neurodegeneration, Machado-Joseph's disease, spongiform encephalopathy, prion-related disease, multiple sclerosis (MS), progressive supranuclear palsy (Steel-Richardson-Olszewski disease), Hallervorden-Spatz disease, progressive familial myoclonic epilepsy, cerebellar degeneration, motor neuron disease, Werdnig-Hoffman disease, Wohlfart-Kugelberg-Welander disease, Charcot-Marie-Tooth disease, Dejerine-Sottas disease, Leber's disease, progressive systemic sclerosis, dermatomyositis, and mixed connective tissue disease; the cardiovascular condition is selected from cardiomyopathy, cardiac hypertrophy, myocardial ischemia, heart failure, cardiac restenosis, and arteriosclerosis; and the cancer is selected from lymphoma, pancreatic cancer, colorectal cancer, hepatocellular carcinoma, Waldenstrom macroglobulinemia, hormone refractory cancer of the prostate, leukemia, breast cancer, lung cancer, ovarian cancer, prostate cancer, head and neck cancer, renal cancer, gastric cancer, brain cancer, B-cell lymphoma, peripheral T-cell lymphoma, and cutaneous T-cell lymphoma.

22. The method of claim 21, wherein the neurodegenerative condition is Huntington's disease.

Description

EXAMPLES

(1) The compounds, or pharmaceutically acceptable salts thereof, compositions, and methods described herein are further illustrated by the following non-limiting examples.

(2) As used herein, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.

Abbreviations

(3) BOP: Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate DABCO: 1,4-Diazabicyclo[2.2.2]octane DCM: Dichloromethane DMF: Dimethylformamide DMSO: Dimethylsulfoxide ES+: Electrospray Positive Ionisation ES: Electrospray Negative Ionisation Et.sub.3N: Triethylamine Et.sub.2O: Diethyl ether EtOAc: Ethyl acetate h: Hour HPLC: High Performance Liquid Chromatography i-hex: iso-Hexane IPA: Isopropanol LCMS: Liquid Chromatography Mass Spectrometry LDA: Lithium diisopropylamide LiHMDS: Lithium bis(trimethylsilyl)amide M: Mass MeCN: Acetonitrile MeOH: Methanol NFSI: N-Fluorobenzenesulfonimide Pd(dppf)Cl.sub.2: [1,1-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(PPh.sub.3).sub.4: Tetrakis(triphenylphosphine)palladium(O) P(o-tol).sub.3: Tri(ortho-Tolyl)phosphine RT: Retention time r.t.: Room temperature THF: Tetrahydrofuran
Analytical Conditions

(4) Compounds were named with the aid of the Cambridgesoft Chemistry Cartridge (v. 9.0.0.182) software.

(5) All reactions involving air- or moisture-sensitive reagents were performed under a nitrogen atmosphere using dried solvents and glassware.

(6) Racemic mixtures of the cyclopropyl core are denoted using asterisks e.g. (1R*,2R*,3R*). Chirally pure compounds are denoted without asterisks e.g. (1R,2R,3R).

(7) TABLE-US-00002 Analytical Condition Method Description 10cm_ESI_Formic_MeCN, 1 Solvents: Acetonitrile (far UV grade) with 0.1% 10cm_ESCI_Formic_MeCN (v/v) formic acid. Water (high purity via Pure Lab Option unit) with 0.1% formic acid Column: Phenomenex Luna 5 m C18 (2), 100 4.6 mm (Plus guard cartridge) Flow Rate: 2 mL/min gradient: A: Water/formic acid B: MeCN/formic acid Time A % B % 0.00 95 5 3.50 5 95 5.50 5 95 5.60 95 5 6.50 95 5 Typical Injections 2-7 L (concentration~0.2-1.0 mg/mL) 15cm_Bicarb_GeminiNX_ 2 Solvents: 100% Acetonitrile (Far UV grade) HPLC_MeCN Water (High purity via PureLab Ultra unit) with 10 mM Ammonium Bicarbonate Column: Phenomenex, Gemini NX, 3 m C18, 150 4.6 mm. Flow Rate: 1 mL/min gradient: A: 10 mM Ammonium Bicarbonate in water B: 100% MeCN Time A % B % 0.00 95.5 4.5 3.00 95.5 4.4 9.00 0 100 13.6 0 100 13.7 95.5 4.5 15 95.5 4.5 Typical Injections 2-7 L (concentration~0.2-1 mg/mL) 15cm_Formic_Ascentis_ 3 Solvents: Acetonitrile (Far UV grade) with 0.1% HPLC_MeCN (V/V) formic acid Water (High purity via PureLab Ultra unit) with 0.1% formic acid Column: Supelco, Ascentis Express C18 or Hichrom Halo C18, 2.7 m C18, 150 4.6 mm. Flow Rate: 1 mL/min gradient: A: Water/formic B: MeCN/formic Time A % B % 0.00 96 4 3.00 96 4 9.00 0 100 13.6 0 100 13.7 96 4 15 96 4 Typical Injections 2-7 L (concentration~0.2-1 mg/mL) 10cm_ESCI_bicarb_MeCN 4 Solvents: Acetonitrile (Far UV grade) Water (High purity via Pure Lab Option unit) with 10 mM ammonium bicarbonate (ammonium hydrogen carbonate) Column: Waters Xterra MS 5 m C18, 100 4.6 mm. (Plus guard cartridge) Flow Rate: 2 mL/min gradient: A: Water/Bicarb B: MeCN Time A % B % 0.00 95 5 0.50 95 5 4.00 5 95 5.50 5 95 5.60 95 5 6.50 95 5 Typical Injections 2-7 L (concentration~0.2-1 mg/mL) 10cm_Formic_ACE- 5 Solvents: Acetonitrile (Far UV grade) with 0.1% AR_HPLC_CH3CN (V/V) formic acid Water (High purity via PureLab Ultra unit) with 0.1% formic acid Column: Hichrom ACE 3 C18-AR mixed mode column 100 4.6 mm Flow Rate: 1 mL/min gradient: A: Water/formic B: MeCN/formic Time A % B % 0.00 98 2 3.00 98 2 12.00 0 100 15.4 0 100 15.5 98 2 17 98 2 Typical Injections 2-10 L 10cm_Formic_ACE- 6 Solvents: Methanol (AR grade) with 0.1% AR_HPLC_CH3OH_Slow (V/V) formic acid Water (High purity via PureLab Ultra unit) with 0.1% formic acid Column: Hichrom ACE 3 C18-AR mixed mode column 100 4.6 mm Flow Rate: 1 mL/min gradient: A: Water/formic B: MeOH/formic Time A % B % 0.00 98 2 3.00 98 2 12.00 0 100 15.4 0 100 15.5 98 2 17 98 2 Typical Injections 2-10 L
Synthetic Section
General Methods
Method A (Cyclopropanation Reaction)

(8) A mixture of sulfonium salt (8.92 mmol), the enoate (5.96 mmol) and 12-crown-4 (8.92 mmol) in DCM (20 mL) was cooled to 20 C. LiHMDS (8.92 mL) was then added dropwise. After complete addition, the mixture was warmed to r.t, stirred for 2 h and quenched with H.sub.2O (30 mL). The biphasic mixture was separated and the organic layers were washed with brine (230 mL), separated, dried (MgSO.sub.4), filtered and concentrated.

(9) Method B (Hydroxamic Acid Formation)

(10) To a stirred solution of ester (0.30 mmol) in THF/MeOH (1:1, 3 mL) was added hydroxylamine (0.2 mL, 50% aqueous solution, 3.00 mmol) and potassium hydroxide (33 mg, 0.60 mmol). The mixture was stirred at r.t. for 2 h, neutralized with 1 M HCl.sub.(aq) and extracted with DCM. The combined organic layers were washed with brine (10 mL), passed through a phase separator and concentrated.

(11) Method C (Heck Reaction)

(12) A stirred mixture of aryl bromide (10.0 mmol), ethyl acrylate (15.0 mmol), palladium acetate (1.00 mmol), P(o-tol).sub.3 (2.00 mmol) and Et.sub.3N (20.0 mmol) in DMF (50 mL) was degassed with nitrogen for 15 min and heated to 80 C. for 3-18 h. The reaction mixture was cooled and diluted with water (100 mL) and extracted into DCM (350 mL). The combined organics were washed with water (5100 mL) and brine (100 mL). The organic layers were passed through a phase separator and concentrated.

(13) Method D (Wittig Reaction)

(14) To a stirred solution of triethyl phosphonoacetate (24.4 mmol) in THF (30 mL) at 0 C. was added sodium hydride (24.4 mmol) portionwise. The mixture was stirred for 1 h before addition of aldehyde (12.2 mmol). The reaction mixture was allowed to warm to r.t. and stirred for 17 h, before quenching with water (50 mL) and extracting into EtOAc (250 mL). The organic layers were combined and washed with water (250 mL), dried (MgSO.sub.4), filtered and concentrated.

(15) Method E (Heck Reaction-2)

(16) A stirred mixture of aryl bromide (10.0 mmol), ethyl acrylate (15.0 mmol), palladium acetate (1.00 mmol), P(o-tol).sub.3 (2.00 mmol) and Et.sub.3N (20.0 mmol) in MeCN (50 mL) was degassed with nitrogen for 15 min. and heated to 80 C. for 3-18 h. The reaction mixture was cooled and the MeCN removed in vacuo. The residue was partitioned between DCM and H.sub.2O and the organic layers were passed through a phase separator and concentrated.

Example 1

(17) ##STR00048##

(1R*,2R*,3R*)-Methyl-2-(4-bromophenyl)-3-phenylcyclopropanecarboxylate

(18) To a stirred solution of benzyl bromide (27 mL, 227 mmol) in acetone at r.t. was added tetrahydrothiophene (10.0 mL, 114 mmol). The solution was stirred for 16 h and the resulting precipitate filtered and washed with acetone (350 mL) and dried under air, to give 1-benzyltetrahydrothiophenium bromide as a white solid (51.9 g, 88%).

(19) A mixture of 1-benzyltetrahydrothiophenium bromide (3.39 g, 13.1 mmol) and (E)-methyl 3-(4-bromophenyl)acrylate (2.10 g, 8.71 mmol) in DCM (50 mL) was cooled to 78 C. and slowly treated with LiHMDS (13.1 mL, 1 M solution in THF) (via syringe pump, 1 mL/h). After complete addition, the mixture was warmed to r.t., stirred for 16 h and was quenched with H.sub.2O (50 mL). The biphasic mixture was separated and the organic layer washed with brine (250 mL), dried (MgSO.sub.4) and concentrated. Purification by flash silica column chromatography (gradient elution i-hex to 5% EtOAc in i-hex) gave the title compound as a colourless oil (600 mg, 20%). LCMS (ES+) 332, 334 (M+H).sup.+.

(1S*,2S*,3S*)-Methyl 2-(4-bromophenyl)-1-fluoro-3-phenylcyclopropanecarboxylate

(20) To a solution of (1R*,2R*,3R*)-methyl-2-(4-bromophenyl)-3-phenylcyclopropanecarboxylate (662 mg, 2 mmol) and LiCl (500 mg, 12 mmol) in dry THF (50 mL) stirred at 78 C. for 20 min, was added LDA (2N, 3.3 mL, 6.6 mmol) and the reaction mixture was stirred at 78 C. for 30 min. Then a solution of NFSI (2.08 g, 6.6 mmol) in dry THF (20 mL) was added slowly and the reaction mixture was stirred for 2 h. The reaction was quenched with sat. NH.sub.4Cl (20 mL) and extracted with DCM (50 mL). The organic phase was passed through a phase separator and concentrated to afford a crude compound that was purified by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex). The target compound was isolated (230 mg) as an enriched mixture (3:1) of the desired diastereoisomer. LCMS (ES+) 350 (M+H).sup.+.

(1S*,2S*,3S*)-(Bromophenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide

(21) Following method B from (1S*,2S*,3S*)-methyl-2-(4-bromophenyl)-1-fluoro-3-phenylcyclopropanecarboxylate (100 mg, 0.29 mmol). Purification by flash silica column chromatography (gradient elution DCM to 5% MeOH in DCM) gave the racemic compound (22 mg, 22%). LCMS (ES) 348, 350 (MH).sup., RT 3.88 min. (Analytical method 1); .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.17 (1H, s), 8.98 (1H, s), 7.63-7.60 (2H, m), 7.47-7.40 (2H, m), 7.38-7.25 (5H, m), 3.58 (2H, dd, J=19.1, 9.3 Hz), 3.54 (1H, s)

Example 2

(22) ##STR00049##

(1S*,2S*,3S*)-1-Fluoro-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxylic acid

(23) A solution of (1S*,2S*,3S*)-methyl 2-(4-bromophenyl)-1-fluoro-3-phenylcyclopropanecarboxylate (226 mg, 0.65 mmol), bis-pinacolato diboron (165 mg, 0.65 mmol), Pd(dppf).sub.2Cl.sub.2 (48 mg, 0.06 mmol), KOAc (279 mg, 2.8 mmol) in dioxane (7 mL) was stirred at 90 C. for 17 h. Water was added and the mixture was extracted with DCM and passed through a phase separator. The organic phase was concentrated and the crude used in the next step.

(24) A solution of (1S*,2S*,3S*)-methyl-1-fluoro-2-phenyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclopropanecarboxylate (0.65 mmol), Pd(PPh.sub.3).sub.4 (78 mg, 0.067 mmol), 5-methylpyrimidine bromide (137 mg, 0.79 mmol), 2N Na.sub.2CO.sub.3 (2.15 mL) in dioxane (5 mL) was stirred at 90 C. for 17 h. The reaction mixture was diluted with water and extracted into DCM. The organic phase was passed through a phase separator and concentrated. Purification by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex) afforded the acid compound (145 mg, 63%). LCMS (ES+) 349 (M+H).sup.+.

(1S,2S,3S)-1-Fluoro-N-hydroxy-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxamide

(25) A solution of the (1S*,2S*,3S*)-1-fluoro-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxylic acid (144 mg, 0.41 mmol), BOP (185 mg, 0.41 mmol), NH.sub.2OH.HCl (42 mg, 0.62 mmol), Et.sub.3N (175 L, 1.24 mmol) in pyridine (2.5 mL) was stirred at r.t. for 2 h. The solvent was evaporated and water was added. The mixture was extracted into DCM, passed through a phase separator and concentrated. Preparative achiral and chiral purification afforded the title compound (8.2 mg) (Chiralpak IC 30/70 [EtOH/MeOH (0.1% formic acid)]/heptane 1.0 mL/min, RT 16.3 min). LCMS (ES+) 364 (M+H).sup.+ RT 3.69 min (Analytical method 1); .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.12 (1H, s), 8.93 (1H, s), 8.74 (2H, s), 8.35 (2H, d, J=8.2 Hz), 7.54 (2H, d, J=8.2 Hz), 7.38-7.26 (4H, m), 7.26-7.18 (1H, m), 3.62-3.52 (2H, m), 2.31 (3H, s).

(1R,2R,3R)-1-Fluoro-N-hydroxy-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxamide

(26) (Chiralpak IC 30/70 [EtOH/MeOH (0.1% formic acid)]/heptane 1.0 mL/min, RT 13.6 min).

Example 3

(27) ##STR00050##

(1S*,2S*,3S*)-Methyl-2-(4-bromophenyl)-1-chloro-3-phenylcyclopropanecarboxylate

(28) To a solution of (1R*,2R*,3R*)-methyl-2-(4-bromophenyl)-3-phenylcyclopropanecarboxylate (1.72 g, 4 mmol) in dry THF (70 mL) stirred at 78 C. for 20 min, was added LDA (2 N, 7.5 mL, 15 mmol) and the reaction mixture was stirred at 78 C. for 30 min. Then CCl.sub.4 (1.2 mL, 12 mmol) was added slowly and the reaction mixture was stirred for 2 h. The reaction was quenched with sat. NH.sub.4Cl (40 mL) and extracted with DCM (100 mL). The organic phase was passed through a phase separator and concentrated to afford a crude compound that was purified by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex). The target compound was isolated (711 mg, 49%) as an enriched mixture (5:1) of the desired diastereoisomer. LCMS (ES+) 367 (M+H).sup.+.

(1S*,2S*,3S*)-Methyl-1-chloro-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxylate

(29) A solution of (1S*,2S*,3S*)-methyl-2-(4-bromophenyl)-1-chloro-3-phenylcyclopropanecarboxylate (710 mg, 1.9 mmol), bis-pinacolato diboron (476 mg, 1.9 mmol), pd(dppf).sub.2O.sub.2 (140 mg, 0.2 mmol), KOAc (816 mg, 8.3 mmol) in dioxane (15 mL) was stirred at 80 C. for 17 h. Water was added and the mixture was extracted with DCM, passed through a phase separator. The organic phase was concentrated and the crude used in the next step.

(30) A solution of (1S*,2S*,3S*)-methyl-1-chloro-2-phenyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclopropanecarboxylate (1.8 mmol), Pd(dppf).sub.2Cl.sub.2 (73 mg, 0.1 mmol), 5-methylpyrimidine bromide (280 mg, 1.8 mmol), and CsF (820 mg, 6.6 mmol) in dioxane (10 mL) was stirred at 90 C. for 17 h. The reaction mixture was diluted with water and extracted into DCM. The organic phase was passed through a phase separator and concentrated. Purification by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex) followed by crystallization in EtOAc-i-hex (3:1) afforded the target compound (285 mg, 10%). LCMS (ES+) 379 (M+H).sup.+.

(1S,2S,3S)-1-Chloro-N-hydroxy-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxamide

(31) Following method B from (1S*,2S*,3S*)-methyl-1-chloro-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxylate (280 mg, 0.74 mmol). Purification by flash silica column chromatography (gradient elution DCM to 5% MeOH in DCM) gave the racemic compound (45 mg, 16%). Preparative chiral purification gave the title compound (Chiralpak IC 30/70 [IPA/MeOH (50/50/0.1% formic acid)]/heptane 1.0 mL/min, RT 12.1 min). LCMS (ES+) 379 (M+H).sup.+, RT 3.68 min. (Analytical method 1); .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.00 (1H, s), 8.89 (1H, s), 8.68 (2H, s), 8.29 (2H, d, J=8.2 Hz), 7.49 (2H, d, J=8.2 Hz), 7.30-7.16 (5H, m), 3.70 (1H, d, J=8.9 Hz), 3.39 (1H, d, J=8.9 Hz), 2.25 (3H, s).

(1R,2R,3R)-1-Chloro-N-hydroxy-2-(4-(5-methylpyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxamide

(32) (Chiralpak IC 30/70 [IPA/MeOH (50/50/0.1% formic acid)]/heptane 1.0 mL/min, RT 9.5 min).

Example 4

(33) ##STR00051##

(1R*,2R*,3R*)-Ethyl-2-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-phenylcyclopropanecarboxylate

(34) (E)-Ethyl-3-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylate was prepared following method D from 8-chloro-2,3-dihydrobenzo[b][1,4]dioxine-6-carbaldehyde (700 mg, 3.53 mmol). The resulting yellow oil was used without further purification. LCMS (ES+) 269, 271 (M+H).sup.+.

(35) Following method A from (E)-ethyl-3-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylate (946 mg, 3.52 mmol) and 1-benzyltetrahydrothiophenium bromide (1.37 mg, 5.28 mmol). Purification by flash silica column chromatography (gradient elution i-hex to 10% EtOAc in i-hex) gave the title compound as a colourless oil (484 mg, 38%, 4:1 trans:cis). LCMS (ES+) 359, 361 (M+H).sup.+.

(1R*,2S*,3*S)-Ethyl-2-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-fluoro-3-phenylcyclopropanecarboxylate

(36) To a solution of (1R*,2R*,3R*)-ethyl-2-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-3-phenylcyclopropanecarboxylate, (360 mg, 1.0 mmol), LiCl (252 mg, 6.0 mmol) at 78 C., was added LDA (2N, 1.66 mL, 3.3 mmol). After stirring for 45 min, NFSI (1.04 g, 3.3 mmol) in dry THF (35 mL) was added and the reaction mixture was stirred for 1 h. The reaction was quenched with sat. NH.sub.4Cl (50 mL) and extracted with DCM (150 mL). The organic phase was passed through a phase separator and concentrated to afford a crude compound that was purified by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex). The target compound was isolated (280 mg, 75%) as a mixture of diastereoisomer. LCMS (ES+) 377 (M+H).sup.+.

(1R*,2S*,3S*)-2-(8-Chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide

(37) Following method B from (1R*,2S*,3S*)-ethyl-2-(8-chloro-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-fluoro-3-phenylcyclopropanecarboxylate (280 mg, 0.75 mmol). Purification by preparative achiral HPLC gave the title compound (108 mg, 40%). LCMS (ES+) 365 (M+H).sup.+, RT 3.77 min (Analytical method 1); .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.15 (1H, s), 9.02 (1H, s), 7.44-7.31 (5H, m), 7.01 (1H, d, J=2.0 Hz), 6.88 (1H, d, J=2.0 Hz), 4.39-4.30 (4H, m), 3.53-3.42 (2H, m).

Example 5

(38) ##STR00052##

(1S*,2S*,3S*)-Methyl 2-(4-(5-(difluoromethoxy)pyrimidin-2-yl)phenyl)-1-fluoro-3-phenylcyclopropanecarboxylate

(39) A solution of (1S*,2S*,3S*)-methyl 2-(4-bromophenyl)-1-fluoro-3-phenylcyclopropanecarboxylate (337 mg, 0.96 mmol), bis-pinacolato diboron (295 mg, 1.2 mmol), Pd(dppf).sub.2Cl.sub.2 (78 mg, 0.09 mmol), KOAc (114 mg, 1.2 mmol) in dioxane (10 mL) was stirred at 90 C. for 17 h. Water was added and the mixture was extracted with DCM and passed through a phase separator. The organic phase was concentrated and the crude used in the next step.

(40) A solution of (1S*,2S*,3S*)-methyl-1-fluoro-2-phenyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclopropanecarboxylate (0.8 mmol), Pd(dppf).sub.2Cl.sub.2 (33 mg, 0.04 mmol), 2-chloro-5-(difluoromethoxy)pyrimidine (222 mg, 1.2 mmol), and Cs.sub.2CO.sub.3 (390 mg, 1.2 mmol) in dioxane (5 mL) and water (1 mL), was stirred at 90 C. for 17 h. The reaction mixture was diluted with water and extracted into DCM. The organic phase was passed through a phase separator and concentrated. Purification by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex) afforded the title compound (161 mg, 49%). LCMS (ES+) 415 (M+H).sup.+.

(1S,2S,3S)-2-(4-(5-(Difluoromethoxy)pyrimidin-2-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide

(41) Following method B from (1S*,2S*,3S*)-methyl 2-(4-(5-(difluoromethoxy)pyrimidin-2-yl)phenyl)-1-fluoro-3-phenylcyclopropanecarboxylate (156 mg, 0.38 mmol). Purification by achiral HPLC chromatography followed by chiral HPLC separation afforded the title compound (16.3 mg) (Chiralpak IA 50/50 [IPA/MeOH (0.1% formic acid)]/heptane 1.0 mL/min, RT 13.5 min). .sup.1H NMR (ppm)(DMSO-d.sub.6): .sup.1H NMR (400 MHz, DMSO) 11.10 (1H, s), 8.90 (1H, s), 8.81 (2H, s), 8.28 (2H, d, J=8.6 Hz), 7.52 (2H, d, J=8.3 Hz), 7.34 (1H, t, J=73.0 Hz), 7.30-7.21 (4H, m), 7.19-7.14 (1H, m), 3.55 (1H, dd, J=9.6, 21.4 Hz), 3.51 (1H, s); LCMS (ES+) 416 (M+H).sup.+, RT 3.82 min (Analytical method 1).

(1R,2R,3R)-2-(4-(5-(Difluoromethoxy)pyrimidin-2-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide

(42) (Chiralpak IA 50/50 [IPA/MeOH (0.1% formic acid)]/heptane 1.0 mL/min, RT 7.8 min); LCMS (ES+) 416 (M+H).sup.+, RT 3.83 min (Analytical method 1).

Example 6

(43) ##STR00053##

(E)-Ethyl-3-(2-cyclopropylpyridin-4-yl)acrylate

(44) To a solution of 4-bromo-2-cyclopropylpyridine (1.7 g, 8.6 mmol), ethyl acrylate (1.2 mL, 11.2 mmol) and DABCO (1.92 g, 17.2 mmol) in DMF (25 mL) was added potassium carbonate (2.37 g, 17.2 mmol) and palladium acetate (192 mg, 0.86 mmol) and the mixture was stirred under N.sub.2, at 125 C. for 17 h. The reaction mixture was diluted with water and extracted into EtOAc. The organic phase was washed with brine, dried over Mg.sub.2SO.sub.4, filtered and concentrated to give a yellow oil. Purification by flash silica column chromatography (gradient elution 5% EtOAc in i-hex to 15% EtOAc in i-hex) gave the title compound as a yellow oil (1.35 g 72%). LCMS (ES+) 218 (M+H).sup.+.

(1R*,2R*,3R*)-Ethyl 2-(2-cyclopropylpyridin-4-yl)-3-phenylcyclopropanecarboxylate

(45) Following method A from (E)-ethyl-3-(2-cyclopropylpyridin-4-yl)acrylate (1.3 g, 6 mmol) and 1-benzyltetrahydrothiophenium triflate (2.5 g, 7.8 mmol). Purification by flash silica column chromatography (gradient elution 30% EtOAc in i-hex to 50% EtOAc in i-hex) gave the title compound as a colourless oil (1.45 mg, 79%, 2:1 trans:cis). LCMS (ES+) 308 (M+H).sup.+.

(1R*,2S*,3S*)-Ethyl-2-(2-cyclopropylpyridin-4-yl)-1-fluoro-3-phenylcyclopropanecarboxylate

(46) To a solution of (1R*,2R*,3R*)-ethyl-2-(2-cyclopropylpyridin-4-yl)-3-phenylcyclopropanecarboxylate (750 mg, 2.4 mmol) and LiCl (1 g, 12.4 mmol) in dry THF (60 mL) stirred at 78 C. for 20 min, was added LDA (2 N, 4.1 mL, 8.2 mmol) and the reaction mixture was stirred at 78 C. for 45 min. Then a solution of NFSI (2.6 g, 8.2 mmol) in dry THF (10 mL) was added slowly and the reaction mixture was stirred for 45 min. The reaction was quenched with sat. NH.sub.4Cl (20 mL) and extracted with DCM (50 mL). The organic phase was passed through a phase separator and concentrated. Purification by flash silica column chromatography (gradient elution i-hex 10% to 20% EtOAc in i-hex), and achiral preparative HPLC purification (130 mg) gave the title compound as a mixture of diastereoisomers (4:1, (1R*,2S*,3S*):(1S*,2S*,3S*)). LCMS (ES+) 326 (M+H).sup.+.

(1R,2S,3S)-2-(2-Cyclopropylpyridin-4-yl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide

(47) Following method B from (1R*,2S*,3S*)-ethyl-2-(2-cyclopropylpyridin-4-yl)-1-fluoro-3-phenylcyclopropanecarboxylate (130 mg, 0.4 mmol). Purification by preparative achiral HPLC gave the title compound (10 mg). LCMS (ES+) 313 (M+H).sup.+, RT 2.32 min (Analytical method 1); .sup.1H NMR (ppm)(DMSO-d.sub.6).sup.1H NMR (400 MHz, DMSO) 11.19 (1H, s), 9.02 (1H, s), 8.29 (1H, d, J=5.0 Hz), 7.44-7.35 (4H, m), 7.34-7.30 (1H, m), 7.26 (1H, s), 7.08 (1H, dd, J=1.4, 5.1 Hz), 3.62-3.48 (2H, m), 2.10-2.03 (1H, m), 0.95-0.90 (4H, m).

Example 7

(48) ##STR00054## ##STR00055##

(E)-Ethyl 3-(4-(2-cyclopropyloxazol-5-yl)phenyl)acrylate

(49) Following method E from 5-(4-bromophenyl)-2-cyclopropyloxazole (5.29 g, 19.7 mmol). The crude product was purified by flash silica column chromatography (gradient elution i-hex to 70% EtOAc in i-hex) affording the target compound as an oil (5.13 g). LCMS (ES+) 284 (M+H).sup.+.

(1R*,2R*,3R*)-Ethyl 2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-3-phenylcyclopropanecarboxylate

(50) Following method A using (E)-ethyl 3-(4-(2-cyclopropyloxazol-5-yl)phenyl)acrylate (1.5 g, 5.29 mmol). Purification by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex) and afforded the title compound (1.93 g, 43%, trans:cis 85:15). LCMS (ES+) 374 (M+H).sup.+.

(1S*,2S*,3S*)-Ethyl 2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-3-phenylcyclopropanecarboxylate

(51) To a solution of (1R*,2R*,3R*)-ethyl-2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-3-phenylcyclopropanecarboxylate (1.93 mg, 15.5 mmol) in dry THF (70 mL) stirred at 78 C. for 20 min, was added LDA (2N, 7.75 mL, 15.5 mmol) and the reaction mixture was stirred at 78 C. for 30 min. Then a solution of NFSI (4.89 g, 15.5 mmol) in dry THF (25 mL) was added slowly and the reaction mixture was stirred for 20 h. The reaction was quenched with sat. NH.sub.4Cl (200 mL) and extracted with DCM (100 mL). The organic phase was passed through a phase separator and concentrated. Purification by flash silica column chromatography (gradient elution i-hex to 30% EtOAc in i-hex). The target compound was isolated (780 mg) as a mixture of diastereoisomers (1:1, (1S*,2S*,3S*):(1R*,2S*,3S*)). LCMS (ES+) 392 (M+H).sup.+.

(1S*,2S*,3S*)-2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide and (1R*,2S*,3S*)-2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide

(52) Following method B using (1S*,2S*,3S*)-ethyl 2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-3-phenylcyclopropanecarboxylate (108 mg, 0.27 mmol). Purification by achiral HPLC chromatography afforded the two diastereoisomers. (1S*,2S*,3S*)-2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropane carboxamide: LCMS (ES+) 379 (M+H).sup.+ RT=3.67 min. .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.15 (1H, s), 8.91 (1H, s), 7.71-7.61 (2H, m), 7.49 (3H, d, J=9.07 Hz), 7.41-7.18 (5H, m), 3.59-3.46 (2H, m), 2.21-2.13 (1H, m), 1.11-0.98 (4H, m). (1R*,2S*,3S*)-2-(4-(2-cyclopropyloxazol-5-yl)phenyl)-1-fluoro-N-hydroxy-3-phenylcyclopropanecarboxamide: LCMS (ES+) 379 (M+H).sup.+ RT=3.75 min. .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.14 (1H, s), 8.97 (1H, s), 7.58 (2H, d, J=8.1 Hz), 7.48-7.33 (7H, m), 7.34-7.27 (1H, m), 3.61-3.51 (2H, m), 2.20-2.12 (1H, m), 1.11-0.98 (4H, m).

Example 8

(53) ##STR00056##

(E)-Ethyl-3-(2-bromothiazol-5-yl)acrylate

(54) Following method D from 2-bromothiazole-5-carbaldehyde (10 g, 52.1 mmol). Purification by flash silica column chromatography (gradient elution i-hex to 20% EtOAc in i-hex) gave the title compound as a colourless oil (12.3 g, 90%). LCMS (ES+) 262, 264 (M+H).sup.+.

(1R*,2R*,3S*)-Ethyl-2-(2-bromothiazol-5-yl)-3-phenylcyclopropanecarboxylate

(55) Following method A from (E)-ethyl-3-(2-bromothiazol-5-yl)acrylate (540 mg, 2.06 mmol) and 1-tetrahydro-1H-thiophenium triflate (1.01 g, 3.09 mmol). Purification by flash silica column chromatography (gradient elution i-hex to 10% EtOAc in i-hex) gave the title compound (600 mg, 83%, 1:1 trans:cis). LCMS (ES+) 352, 354 (M+H).sup.+.

(1R*,2R*,3S*)-Ethyl-2-(2-(4-fluorophenyl)thiazol-5-yl)-3-phenylcyclopropanecarboxylate

(56) A suspension of (1R*,2R*,3S*)-ethyl-2-(2-bromothiazol-5-yl)-3-phenylcyclopropanecarboxylate (1.0 g, 2.8 mmol), p-fluoro phenyl boronic acid (441 mg, 3.15 mmol), Pd(PPh.sub.3).sub.4 (181 mg, 0.15 mmol), 2N Na.sub.2CO.sub.3 (4 mL) in dioxane (20 mL) was stirred at 90 C. overnight. Water was added and the reaction mixture was extracted in DCM, the organic phase was passed through a phase separator cartridge, concentrated and purified by silica gel column chromatography to afford the target compound (815 mg, cis:trans 1:1). LCMS (ES+) 368 (M+H).sup.+.

(1S*,2R*,3S*)-Ethyl-1-fluoro-2-(2-(4-fluorophenyl)thiazol-5-yl)-3-phenylcyclopropanecarboxylate

(57) To a solution of (1R*,2R*,3S*)-ethyl-2-(2-(4-fluorophenyl)thiazol-5-yl)-3-phenylcyclopropanecarboxylate (813 mg, 2.0 mmol) and 12-crown-4 (1.3 mL, 6.6 mmol) in dry THF (30 mL) stirred at 78 C. for 20 min, was added LDA (2N, 3.3 mL, 6.6 mmol) and the reaction mixture was stirred at 78 C. for 30 min. Then a solution of NFSI (2.08 g, 6.6 mmol) in dry THF (20 mL) was added slowly and the reaction mixture was stirred for 2 h. The reaction was quenched with sat NH.sub.4Cl (20 mL) and extracted with DCM (50 mL). The organic phase was passed through a phase separator and concentrated to afford a crude that was purified by flash silica column chromatography (gradient elution i-hex to 40% EtOAc in i-hex). The target compound was isolated as a mixture of diastereoisomers (513 mg, 1:1, (1S*,2R*,3S*):(1R*,2R*,3S*)). (LCMS (ES+) 386 (M+H)+.

(1S,2R,3S)-1-Fluoro-2-(2-(4-fluorophenyl)thiazol-5-yl)-N-hydroxy-3-phenylcyclopropanecarboxamide

(58) Following method B using (1S*,2R*,3S*)-ethyl 1-fluoro-2-(2-(4-fluorophenyl)thiazol-5-yl)-3-phenylcyclopropanecarboxylate (510 mg, 1.3 mmol). Purification by achiral HPLC chromatography followed by chiral HPLC separation afforded the title compound. (Chiralpak IC 20/80 IPA/MeOH (50/50/0.1% formic acid/heptane, 1.0 mL/min, RT=13.8 min) LCMS (ES+) 373 (M+H).sup.+, RT=3.83 min (Analytical method 1). .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.31 (1H, s), 9.04 (1H, s), 8.06-7.99 (3H, m), 7.43-7.26 (7H, m), 3.78 (1H, dd, J=9.2, 3.9 Hz), 3.48 (1H, dd, J=23.3, 9.2 Hz).

Example 9

(59) ##STR00057##

(1S*,2S*,3S*)-Methyl-1-fluoro-2-(4-(5-fluoropyrimidin-2-yl)phenyl)-3-phenylcyclopropanecarboxylate

(60) A solution of (1S*,2S*,3S*)-methyl-2-(4-bromophenyl)-1-fluoro-3-phenylcyclopropanecarboxylate (3.88 g, 12.2 mmol), bis-pinacolato diboron (3.40 g, 13.4 mmol), Pd(dppf).sub.2Cl.sub.2 (995 mg, 1.22 mmol), KOAc (5.97 g, 61.0 mmol) in dioxane (100 mL) was stirred at 90 C. for 17 h. Water was added and the mixture was extracted with DCM and passed through a phase separator. The organic phase was concentrated and the crude used in the next step.

(61) A solution of (1S*,2S*,3S*)-methyl-1-fluoro-2-phenyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclopropanecarboxylate (4.83 g, 12.2 mmol), Pd(dppf).sub.2Cl.sub.2 (995 mg, 1.22 mmol), 2-chloro-5-fluoropyrimidine (1.76 g, 14.6 mmol), and CsF (6.12 g, 40.3 mmol) in dioxane (100 mL) was stirred at 100 C. for 17 h. The reaction mixture was diluted with water and extracted into DCM. The organic phase was passed through a phase separator and concentrated. Purification by flash silica column chromatography (gradient elution i-hex to 30% EtOAc in i-hex) afforded the target compound (3.78 g, 85%). LCMS (ES+) 367 (M+H).sup.+.

(1S,2S,3S)-1-Fluoro-2-(4-(5-fluoropyrimidin-2-yl)phenyl)-N-hydroxy-3-phenylcyclopropanecarboxamide

(62) Following method B from (1S*,2S*,3S*)-methyl-1-fluoro-2-(4-(5-fluoropyrimidin-2-yl)phenyl)-3-phenyl cyclopropanecarboxylate (130 mg, 0.36 mmol). The target compound was obtained after flash silica column chromatography (gradient elution DCM to 5% MeOH in DCM) followed by chiral HPLC purification (37.3 mg). (Chiralpak IC EtOH (0.1% formic acid/heptane 1.0 mL/min, RT 7.0 min). LCMS (ES+) 368 (M+H)+, RT 3.73 min (Analytical method 1); .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.07 (1H, s), 8.91 (2H, s), 8.87 (1H, s), 8.26 (2H, d, J=8.2 Hz), 7.51 (2H, d, J=8.19 Hz), 7.31-7.20 (4H, m), 7.16 (1H, t, J=7.0 Hz), 3.58-3.48 (2H, m).

(1R,2R,3R)-1-Fluoro-2-(4-(5-fluoropyrimidin-2-yl)phenyl)-N-hydroxy-3-phenylcyclopropanecarboxamide

(63) (Chiralpak IC EtOH (0.1% formic acid)/heptane 1.0 mL/min, RT 5.5 min). LCMS (ES+) 368 (M+H)+, RT 3.73 min (Analytical method 1).

Example 10

(64) ##STR00058##

(1S*,2S*,3S*)-Methyl-1-fluoro-2-phenyl-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)phenyl)cyclopropanecarboxylate

(65) To a stirred solution of (1S*,2S*,3S*)-methyl-2-(4-bromophenyl)-1-fluoro-3-phenylcyclopropane carboxylate (470 mg, 1.47 mmol) in dioxane (10 mL) was added bis-pinacolato diboron (412 mg, 1.62 mmol), Pd(dppf)Cl.sub.2 (120 mg, 0.15 mmol) and potassium acetate (720 mg, 7.35 mmol). The mixture was degassed with nitrogen and heated to 100 C. for 2 h. The reaction mixture was diluted with H.sub.2O (20 mL) and extracted into DCM (220 mL). The organic phase was concentrated and the crude used in the next step.

(66) A solution of (1S*,2S*,3S*)-methyl-1-fluoro-2-phenyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)cyclopropanecarboxylate (538 mg, 1.47 mmol), Pd(dppf).sub.2Cl.sub.2 (169 mg, 0.15 mmol), 2-bromo-4-(trifluoromethyl)pyrimidine (367 mg, 1.62 mmol), and 2 M Na.sub.2CO.sub.3 (aq) (2.2 mL, 4.41 mmol) in dioxane (15 mL) was stirred at 100 C. for 17 h. The reaction mixture was diluted with water and extracted into DCM. The organic phase was passed through a phase separator and concentrated. Purification by flash silica column chromatography (gradient elution i-hex to 30% EtOAc in i-hex) afforded the target compound (230 mg, 41%). LCMS (ES+) 417 (M+H).sup.+.

(1S,2S,3S)-1-Fluoro-N-hydroxy-2-phenyl-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)phenyl)cyclopropane carboxamide

(67) Following method B from (1S*,2S*,3S*)-methyl-1-fluoro-2-phenyl-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)phenyl)cyclopropanecarboxylate (230 mg, 0.55 mmol) The target compound was obtained after flash silica column chromatography (gradient elution DCM to 3% MeOH in DCM) followed by chiral HPLC purification (46 mg). (Chiralpak IC IPA/MeOH (50/50/0.1% formic acid/heptane 1.0 mL/min, RT 15.3 min). LCMS (ES+) 418 (M+H)+, RT 4.14 min (Analytical method 1); .sup.1H NMR (ppm)(DMSO-d.sub.6): 11.21 (1H, s), 9.33 (1H, d, J=5.0 Hz), 9.01 (1H, s), 8.47 (2H, d, J=8.2 Hz), 8.00 (1H, d, J=5.0 Hz), 7.70 (2H, d, J=8.2 Hz), 7.45-7.35 (4H, m), 7.33-7.27 (1H, m), 3.69-3.62 (2H, m).

(1R,2R,3R)-1-Fluoro-N-hydroxy-2-phenyl-3-(4-(4-(trifluoromethyl)pyrimidin-2-yl)phenyl)cyclopropane carboxamide

(68) (Chiralpak IC IPA/MeOH (50/50/0.1% formic acid)/heptane 1.0 mL/min, RT 11.4 min). LCMS (ES+) 418 (M+H)+, RT 4.14 min (Analytical method 1).

Example 11

(69) Analysis of Inhibition of HDAC4 with Class IIa Histone Deacetylase (HDAC) Inhibitors.

(70) The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the Histone Deacetylase 4 (HDAC4) catalytic domain enzymatic activity using the Class IIa selective substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC4. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.

(71) Serially Dilute HDAC Inhibitor Compounds.

(72) Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% dimethyl sulfoxide (DMSO). Stocks of 60 l aliquots of the 10 mM compound in DMSO are prepared and stored at 20 C. From one stock aliquot of each compound to be tested and the reference compound, a 16-point serial dilution is prepared according to Table 1 using a 125 l 16-channel Matrix multi-channel pipette (Matrix Technologies Ltd).

(73) TABLE-US-00003 TABLE 1 Serial Dilution of Compounds Concen- Diluted tration Dilution Solutions Well (M) ratio Volumes Concentration 1 A 10000 60 l 10 mM Test compound/reference control Concentration 2 B 5000 1:2 30 l A + 30 l DMSO Concentration 3 C 2500 1:2 30 l B + 30 l DMSO Concentration 4 D 1000 1:2.5 30 l C + 45 l DMSO Concentration 5 E 500 1:2 30 l D + 30 l DMSO Concentration 6 F 250 1:2 30 l E + 30 l DMSO Concentration 7 G 125 1:2 30 l F + 30 l DMSO Concentration 8 H 62.5 1:2 30 l G + 30 l DMSO Concentration 9 I 31.25 1:2 30 l H + 30 l DMSO Concentration 10 J 15.63 1:2 30 l I + 30 l DMSO Concentration 11 K 7.81 1:2 30 l J + 30 l DMSO Concentration 12 L 3.91 1:2 30 l K + 30 l DMSO Concentration 13 M 1.95 1:2 30 l L + 30 l DMSO Concentration 14 N 0.98 1:2 30 l M + 30 l DMSO Concentration 15 O 0.49 1:2 30 l N + 30 l DMSO Concentration 16 P 0.24 1:2 30 l O + 30 l DMSO

(74) 2 l (200) of each diluted solution and each control (full activity: 100% DMSO alone or full inhibition 1 mM) is stamped into V-bottomed polypropylene 384-well compound plates using either the Bravo (384-well head from Agilent) or 12.5 l 16-channel Matrix multi-channel pipette (Matrix Technologies Ltd). Each well with the 200 compound solution is diluted 1:20 by the addition of 38 l assay buffer+DMSO (10.5% DMSO, 45 mM Tris-HCl, 123 mM NaCl, 2.4 mM KCl, and 0.9 mM MgCl.sub.2 at pH 8.0 and equilibrated to room temperature).

(75) Prepare HDAC4 Catalytic Domain Enzyme (0.86 g/ml).

(76) The HDAC4 catalytic domain enzyme is human catalytic domain HDAC4 protein (amino acids 648-1057, but with a replacement of amino acids 730-744 with 4 amino acid GSGS linker) made from VOID 3428 and provided by Emerald Biostructures at 1.2 mg/ml. A working solution of enzyme is prepared from a 1.2 mg/ml stock aliquot of HDAC4 catalytic domain (thawed on ice) diluted to 0.86 g/ml with assay buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl.sub.2 at pH 8 and equilibrated to room temperature) just prior to the addition of the enzyme to the assay.

(77) Prepare 5 (50 M) Boc-Lys(Tfa)-AMC Substrate.

(78) 5 (50 M) substrate is prepared just prior to the addition to the assay. A 1 mM substrate stock is made by diluting a 100 mM Boc-Lys(Tfa)-AMC in DMSO solution 1:100 by adding it drop-wise to assay buffer (equilibrated to room temperature) while vortexing at slow speed to prevent precipitation. The 5 substrate is prepared by diluting the 1 mM substrate solution 1:20 by adding it drop-wise to assay buffer (equilibrated to room temperature) while vortexing at slow speed to prevent precipitation.

(79) Prepare 3 (30 M) Developer/Stop Solution.

(80) 3 (30 M) Developer/Stop Solution is prepared just prior to addition to the plate by diluting a stock solution of 10 mM reference compound 1:333 in 25 mg/ml trypsin (PAA Laboratories Ltd.) equilibrated to room temperature.

(81) Assay.

(82) 5 l of each solution of 1:20 diluted compound from above is transferred to a clear bottomed, black, 384-well assay plate using the Bravo or the Janus (384-well MDT head from Perkin Elmer). Using a 16-channel Matrix multi-channel pipette, 35 l of the working solution of HDAC4 catalytic domain enzyme (0.86 g/ml in assay buffer) is transferred to the assay plate. The assay is then started by adding 10 l of 5 (50 M) substrate to the assay plates using either the Bravo, Janus or 16-channel Matrix multi-channel pipette. The assay plate is then shaken for two minutes on an orbital shaker at 900 rpm (rotations per minute). Next the plate is incubated for 15 minutes at 37 C. The reaction is stopped by adding 25 l of 3 (30 M) developer/stop solution to the assay plates using either the Bravo, Janus or a 16-channel Matrix multi-channel pipette. Assay plates are then shaken for 5 minutes on an orbital shaker at 1200 rpm. Next, the assay plates are incubated at 37 C. for 1 hour in a tissue culture incubator. Finally, the fluorescence is measured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmer EnVision in top read mode.

Example 12: Analysis of Inhibition of HDAC5 with Class IIa Histone Deacetylase (HDAC) Inhibitors

(83) The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the Histone Deacetylase 5 (HDAC5) enzymatic activity using the Class IIa selective substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC5. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.

(84) Serially Dilute HDAC Inhibitor Compounds.

(85) Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% DMSO. Stocks of 60 l aliquots of the 10 mM compound in DMSO are prepared and stored at 20 C. From one stock aliquot of each compound to be tested and the reference compound, a 16-point serial dilution is prepared according to Table 1 using a 125 l 16-channel Matrix multi-channel pipette.

(86) 2 l (200) of each diluted solution and each control (full activity: 100% DMSO alone or full inhibition 1 mM) is stamped into V-bottom polypropylene 384-well compound plates using either Bravo, Janus, or a 12.5 l 16-channel Matrix multi-channel pipette. Each well with the 2 l of the 200 stamped compound solution is diluted 1:20 by the addition of 38 l assay buffer+DMSO (10.5% DMSO, 45 mM Tris-HCl, 123 mM NaCl, 2.4 mM KCl, and 0.9 mM MgCl.sub.2 at pH 8.0 and equilibrated to 37 C.).

(87) Prepare HDAC5 Catalytic Domain Enzyme (0.57 g/ml).

(88) The HDAC5 catalytic domain enzyme is human HDAC5 catalytic domain (Gen Bank Accession No. NM_001015053), amino acids 657-1123 with a C-terminal His tag and can be obtained from BPS BioScience. The protein is 51 kDa and is expressed in a baculovirus expression system. A working solution of enzyme is prepared from a 1.65 mg/ml stock aliquot of HDAC5 catalytic domain (thawed on ice) diluted to 0.57 g/ml with assay buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl.sub.2 at pH 8 and equilibrated to 37 C.) just prior to the addition of the enzyme to the assay.

(89) Prepare 5 (40 M) Boc-Lys(Tfa)-AMC Substrate.

(90) 5 (40 M) substrate is prepared just prior to the addition to the assay. The 5 substrate is prepared by diluting the 100 mM Boc-Lys(Tfa)-AMC in DMSO solution 1:2500 by adding it drop-wise to assay buffer (equilibrated to 37 C.) while vortexing at slow speed to prevent precipitation.

(91) Prepare 3 (30 M) Developer/Stop Solution.

(92) 3 (30 M) Developer/Stop Solution is prepared just prior to addition to the plate by diluting a stock solution of 10 mM reference compound 1:333 in 25 mg/ml trypsin equilibrated to 37 C.

(93) Assay.

(94) 5 l of each solution of the 1:20 diluted inhibitor compounds and controls from above is transferred to a clear bottomed, black, 384-well assay plate using the Bravo or Janus. Using a 16-channel Matrix multi-channel pipette, 35 l of the working solution of the HDAC5 catalytic domain enzyme (0.57 g/ml in assay buffer) is transferred to the assay plate. The assay is then started by adding 10 l of 5 (40 M) substrate to the assay plates using either the Bravo, Janus or 16-channel Matrix multi-channel pipette. The assay plate is then shaken for one minute on an orbital shaker at 900 rpm. Next, the plates are incubated for 15 minutes at 37 C. The reaction is stopped by adding 25 l of 3 (30 M) developer/stop solution to the assay plates using either the Bravo, Janus or a 16-channel Matrix multi-channel pipette. Assay plates are then shaken for 2 minutes on an orbital shaker at 900 rpm. Next, the assay plates are incubated at 37 C. for 1 hour in a tissue culture incubator followed by shaking for 1 minute at the maximum rpm on an orbital shaker before reading on the EnVision. Finally, the fluorescence is measured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmer EnVision in top read mode.

Example 13: Analysis of Inhibition of HDAC7 with Class IIa Histone Deacetylase (HDAC) Inhibitors

(95) The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the Histone Deacetylase 7 (HDAC7) enzymatic activity using the Class IIa selective substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC7. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.

(96) Serially Dilute HDAC Inhibitor Compounds.

(97) Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% DMSO. Stocks of 60 l aliquots of the 10 mM compound in DMSO are prepared and stored at 20 C. From one stock aliquot of each compound to be tested and the reference compound, a 16-point serial dilution is prepared according to Table 1 using a 125 l 16-channel Matrix multi-channel pipette.

(98) 2 l (200) of each diluted solution and each control (full activity: 100% DMSO alone or full inhibition 1 mM) is stamped into V-bottom polypropylene 384-well compound plates using either the Bravo, Janus, or a 12.5 l 16-channel Matrix multi-channel pipette. Each well with the 200 compound solution is diluted 1:20 by the addition of 38 l assay buffer+DMSO (10.5% DMSO, 45 mM Tris-HCl, 123 mM NaCl, 2.4 mM KCl, and 0.9 mM MgCl.sub.2 at pH 8.0 and equilibrated to 37 C.).

(99) Prepare HDAC7 Enzyme (71 ng/ml).

(100) The HDAC7 enzyme is human HDAC7 (GenBank Accession No. AY302468) amino acids 518-end with a N-terminal Glutathione S-transferase (GST) tag and can be obtained from BPS BioScience. The protein is 78 kDa and is expressed in a baculovirus expression system. A working solution of enzyme is prepared from a 0.5 mg/ml stock aliquot of HDAC7 (thawed on ice) diluted to 71 ng/ml with assay buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl.sub.2 at pH 8 and equilibrated to 37 C.) just prior to the addition of enzyme to the assay.

(101) Prepare 5 (50 M) Boc-Lys(Tfa)-AMC Substrate.

(102) 5 (50 M) substrate is prepared just prior to the addition to the assay. The 5 substrate is prepared by diluting a 100 mM Boc-Lys(Tfa)-AMC in DMSO solution 1:2000 by adding it drop-wise to assay buffer (equilibrated to 37 C.) while vortexing at slow speed to prevent precipitation.

(103) Prepare 3 (30 M) Developer/Stop Solution.

(104) 3 (30 M) Developer/Stop Solution is prepared just prior to addition to the plate by diluting a stock solution of 10 mM reference compound 1:333 in 25 mg/ml trypsin equilibrated to 37 C.

(105) Assay.

(106) 5 l of each solution of 1:20 diluted compound from above is transferred to a clear bottomed, black, 384-well assay plate using the Bravo or Janus. Using a 16-channel Matrix multi-channel pipette, 35 l of the working solution of the HDAC7 enzyme (71 ng/ml in assay buffer) is transferred to the assay plate. The assay is then started by adding 10 l of 5 (50 M) substrate to the assay plate using either the Bravo, Janus or 16-channel Matrix multi-channel pipette. The assay plate is then shaken for one minute on an orbital shaker at 900 rpm. Next, the plate is incubated for 15 minutes at 37 C. The reaction is then stopped by adding 25 l of 3 (30 M) developer/stop solution to the assay plates using either the Bravo, Janus or a 16-channel Matrix multi-channel pipette. The assay plate is then shaken for 2 minutes on an orbital shaker at 900 rpm. Next, the assay plate is incubated at 37 C. for 1 hour in a tissue culture incubator followed by shaking for 1 minute at maximum rpm on an orbital shaker. Finally, the fluorescence is measured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmer EnVision in top read mode.

Example 14: Analysis of Inhibition of HDAC9 with Class IIa Histone Deacetylase (HDAC) Inhibitors

(107) The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the Histone Deacetylase 9 (HDAC9) enzymatic activity using the Class IIa selective substrate, Boc-Lys(Tfa)-AMC. The substrate is deacetylated to Boc-Lys-AMC by HDAC9. Cleavage by trypsin results in the release of the fluorophore AMC from the deacetylated substrate. The fluorescence of the sample is directly related to the histone deacetylase activity in the sample.

(108) Serially Dilute HDAC Inhibitor Compounds.

(109) Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% DMSO. Stocks of 60 l aliquots of the 10 mM compound in DMSO are prepared and stored at 20 C. From one stock aliquot of each compound to be tested and the reference compound, a 16-point serial dilution is prepared according to Table 1 using a 125 l 16-channel Matrix multi-channel pipette.

(110) 2 l (200) of each diluted solution and each control (full activity: 100% DMSO alone or full inhibition 1 mM) is stamped into V-bottom polypropylene 384-well compound plates using either the Bravo, Janus, or 12.5 l 16-channel Matrix multi-channel pipette. Each well with the stamped 200 compound solution is diluted 1:20 by the addition of 38 l assay buffer+DMSO (10.5% DMSO, 45 mM Tris-HCl, 123 mM NaCl, 2.4 mM KCl, and 0.9 mM MgCl.sub.2 at pH 8.0 and equilibrated to 37 C.).

(111) Prepare HDAC9 Enzyme (0.57 g/ml).

(112) The HDAC9 enzyme is human HDAC9 (GenBank Accession No. NM_178423) amino acids 604-1066 with a C-terminal His tag and can be obtained from BPS BioScience. The protein is 50.7 kDa and is expressed in a baculovirus expression system. A working solution of enzyme is prepared from a 0.5 mg/ml stock aliquot of HDAC9 (thawed on ice) diluted to 0.57 g/ml with assay buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl.sub.2 at pH 8 and equilibrated to 37 C.) just prior to the addition of enzyme to the assay.

(113) Prepare 5 (125 M) Boc-Lys(Tfa)-AMC Substrate.

(114) 5 (125 M) substrate is prepared just prior to the addition to the assay. The 5 substrate is prepared by diluting a 100 mM Boc-Lys(Tfa)-AMC in DMSO solution 1:800 by adding it drop-wise to assay buffer (equilibrated to 37 C.) while vortexing at slow speed to prevent precipitation.

(115) Prepare 3 (30 M) Developer/Stop Solution.

(116) 3 (30 M) Developer/Stop Solution is prepared just prior to addition to the plate by diluting a stock solution of 10 mM reference compound 1:333 in 25 mg/ml trypsin equilibrated to 37 C.

(117) Assay.

(118) 5 l of each solution of 1:20 diluted compound from above is transferred to a clear bottomed, black, 384-well assay plate using the Bravo or Janus. Using a 16-channel Matrix multi-channel pipette, 35 l of the working solution of the HDAC9 enzyme (0.57 g/ml in assay buffer) is transferred to the assay plate. The assay is then started by adding 10 l of 5 (125 M) substrate to the assay plate using either the Bravo, Janus or 16-channel Matrix multi-channel pipette. The assay plate is then shaken for one minute on an orbital shaker at 900 rpm. Next, the plate is incubated for 15 minutes at 37 C. The reaction is stopped by adding 25 l of 3 developer/stop solution to the assay plates using either the Bravo, Janus or a 16-channel Matrix multi-channel pipette. The assay plate is then shaken for 2 minutes on an orbital shaker at 900 rpm. Next, the assay plate is incubated at 37 C. for 1 hour in a tissue culture incubator followed by shaking for 1 minute at maximum rpm on an orbital shaker before reading on the enVision. Finally, the fluorescence is measured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmer EnVision in top read mode.

Example 15: Analysis of Inhibition of Cellular HDAC Activity with Class IIa Histone Deacetylase (HDAC) Inhibitors

(119) The potency of Class IIa Histone Deacetylase (HDAC) inhibitors is quantified by measuring the cellular histone deacetylase enzymatic activity using the Class IIa selective substrate, Boc-Lys(Tfa)-AMC. After penetration in Jurkat E6-1 cells, the substrate is deacetylated to Boc-Lys-AMC. After cell lysis and cleavage by trypsin, the fluorophore AMC is released from the deacetylated substrate only. The fluoresence of the sample is directly related to the histone deacetylase activity in the sample.

(120) Jurkat E6.1 Cell Culture and Plating.

(121) Jurkat E6.1 cells are cultured according to standard cell culture protocols in Jurkat E6.1 Growth Media (RPMI without phenol red, 10% FBS, 10 mM HEPES, and 1 mM Sodium Pyruvate). Jurkat E6.1 cells are counted using a Coulter Counter and resuspended in Jurkat E6.1 growth media at a concentration of 75,000 cells/35 l. 35 l or 75,000 cells is seeded into Greiner microtitre assay plates. The plates are then incubated at 37 C. and 5% CO.sub.2 while other assay components are being prepared.

(122) Serially Dilute HDAC Inhibitor Compounds.

(123) Serial dilutions of the HDAC inhibitors and control reference compound (1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone) are made by first resuspending the lyophilized compound to a final concentration of 10 mM in 100% DMSO. Stocks of 70 l aliquots of the 10 mM compound in DMSO are prepared and stored at 20 C. From one stock aliquot of each compound to be tested and the reference compound, a 16-point serial dilution is prepared according to Table 1 using a 125 l 16-channel Matrix multi-channel pipette.

(124) 2 l (200) of each diluted solution and each control (full activity: 100% DMSO alone or full inhibition 1 mM) is stamped into V-bottom polypropylene 384-well compound plates using either the Bravo, Janus, or 12.5 l 16-channel Matrix multi-channel pipette. Each well with the 200 compound solution is diluted 1:20 by the addition of 38 l Jurkat assay buffer+DMSO (9.5% DMSO, RPMI without phenol red, 0.09% FBS, 9 mM Hepes, and 0.9 mM Sodium Pyruvate equilibrated to room temperature)

(125) Prepare 5 (500 M) Boc-Lys(Tfa)-AMC Substrate.

(126) 5 (500 M) substrate is prepared just prior to the addition to the assay. The 5 substrate is prepared by diluting a 100 mM Boc-Lys(Tfa)-AMC in DMSO solution 1:200 by adding it drop-wise to Jurkat assay medium (RPMI without phenol red, 0.1% FBS, 10 mM Hepes, and 1 mM Sodium Pyruvate equilibrated to 37 C.) while vortexing at slow speed to prevent precipitation.

(127) Prepare 3 Lysis Buffer.

(128) 10 ml of 3 lysis buffer is prepared with 8.8 ml of 3 stock lysis buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl.sub.2, 1% Nonidet P40 Substitute equilibrated to room temperature) and 1.2 ml of 3 mg/ml Trypsin equilibrated to room temperature.

(129) Assay.

(130) 5 l of each solution of 1:20 diluted compound from above is transferred to the Greiner microtitre assay plates with 75,000 cells/well using the Bravo. Cells are then incubated for 2 hours at 37 C. and 5% CO.sub.2. The assay is then started by adding 10 l of 5 (500 M) substrate to the assay plate using either the Bravo or 16-channel Matrix multi-channel pipette. The cells are then incubated for 3 hours at 37 C. and 5% CO.sub.2. Next, 25 l of 3 lysis buffer is added to each well using either the 125 l 16 channel pipette or the Bravo. The assay plate is then incubated overnight (15-16 hours) at 37 C. and 5% CO.sub.2. The following day, the plates are shaken on an orbital shaker for 1 minute at 900 rpm. Finally the top read fluorescence (Excitation: 355 nm, Emission: 460 nm) is measured using PerkinElmer EnVision.

Example 16

(131) Using the synthetic methods similar to those described above and the assay protocols described above, the following compounds were synthesized and tested.

(132) TABLE-US-00004 Biochemical Cellular HDAC-4 IC.sub.50 IC.sub.50 Chemical Name Structure (M) (M) (1S*,2S*,3S*)-2-(4-Bromophenyl)-1- fluoro-N-hydroxy-3- phenylcyclopropanecarboxamide 0.06 0.87 (1R*,2S*,3S*)-2-(8-Chloro-2,3- dihydrobenzo[b][1,4]dioxin-6-yl)-1-fluoro- N-hydroxy-3- phenylcyclopropanecarboxamide 0 6.47 40 (1S,2S,3S)-1-Fluoro-N-hydroxy-2-(4-(5- methylpyrimidin-2-yl)phenyl)-3- phenylcyclopropanecarboxamide 0.02 0.12 (1S,2S,3S)-1-Chloro-N-hydroxy-2-(4-(5- methylpyrimidin-2-yl)phenyl)-3- phenylcyclopropanecarboxamide 0.11 1.1 (1R,2R,3R)-1-Chloro-N-hydroxy-2-(4-(5- methylpyrimidin-2-yl)phenyl)-3- phenylcyclopropanecarboxamide 1.58 15.3 (1R,2R,3R)-1-Fluoro-N-hydroxy-2-(4-(5- methylpyrimidin-2-yl)phenyl)-3- phenylcyclopropanecarboxamide 1.43 8.9 (1S,2S,3S)-2-(4-(5- (Difluoromethoxy)pyrimidin-2-yl)phenyl)-1- fluoro-N-hydroxy-3- phenylcyclopropanecarboxamide 0.04 0.22 (1R,2R,3R)-2-(4-(5- (Difluoromethoxy)pyrimidin-2-yl)phenyl)-1- fluoro-N-hydroxy-3- phenylcyclopropanecarboxamide 26 >50 (1R*,2S*,3S*)-2-(4-(2-Cyclopropyloxazol- 5-yl)phenyl)-1-fluoro-N-hydroxy-3- phenylcyclopropanecarboxamide 4.65 28 (1S*,2S*,3S*)-2-(4-(2-Cyclopropyloxazol- 5-yl)phenyl)-1-fluoro-N-hydroxy-3- phenylcyclopropanecarboxamide 0.02 0.31 (1S,2R,3S)-1-Fluoro-2-(2-(4- fluorophenyl)thiazol-5-yl)-N-hydroxy-3- phenylcyclopropanecarboxamide 0.06 0.69 (1S,2S,3S)-1-Fluoro-N-hydroxy-2-phenyl- 2-(4-(4-(trifluoromethyl)pyrimidin-2- yl)phenyl)cyclopropanecarboxamide 0 0.04 0.26 (1R,2R,3R)-1-Fluoro-N-hydroxy-2-phenyl- 3-(4-(4-(trifluoromethyl)pyrimidin-2- yl)phenyl)cyclopropanecarboxamide 6.87 >50 (1S,2S,3S)-1-Fluoro-2-(4-(5- fluoropyrimidin-2-yl)phenyl)-N-hydroxy-3- phenylcyclopropanecarboxamide 0.01 0.12 (1R,2R,3R)-1-Fluoro-2-(4-(5- fluoropyrimidin-2-yl)phenyl)-N-hydroxy-3- phenylcyclopropanecarboxamide 0.41 4.5 (1R*,2S*,3S*)-2-(2-Cyclopropylpyridin-4- yl)-1-fluoro-N-hydroxy-3- phenylcyclopropanecarboxamide >50 >50

(133) 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.