Selective BACE1 inhibitors

Abstract

The present invention provides a compound of Formula I: ##STR00001##
or a pharmaceutically acceptable salt thereof.

Claims

1. A compound of the formula: ##STR00046## or a pharmaceutically acceptable salt thereof.

2. The compound or salt thereof according to claim 1 wherein the compound is: ##STR00047##

3. The compound or salt thereof according to claim 2 wherein the compound is: ##STR00048##

4. The salt according to claim 3 which is: ##STR00049##

5. The salt according to claim 3 which is: ##STR00050##

6. The salt according to claim 3 which is: ##STR00051##

7. The compound or salt thereof according to claim 3 wherein the compound is N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide.

8. The compound according to claim 3 which is N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide.

9. The salt according to claim 4 which is N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide 4-methylbenzenesulfonate.

10. The salt according to claim 4 which is N-[3-[(4aS,5S,7aS)-2-Amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide 4-methylbenzenesulfonate hemihydrate.

11. The salt according to claim 10 which is characterized by a substantial peak in the X-ray diffraction spectrum, at diffraction angle 2-theta of 6.8 in combination with one or more of the peaks selected from the group consisting of 19.7, 14.9, and 10.3; with a tolerance for the diffraction angles of 0.2 degrees.

12. The salt according to claim 6 which is N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide methanesulfonate.

13. A method of treating Alzheimer's disease in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof.

14. A method of treating the progression of mild cognitive impairment to Alzheimer's disease in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of claim 1, or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition, comprising a compound or a pharmaceutically acceptable salt thereof according to claim 1 with one or more pharmaceutically acceptable carriers, diluents, or excipients.

16. A process for preparing a pharmaceutical composition, comprising admixing a compound or a pharmaceutically acceptable salt thereof according to claim 1 with one or more pharmaceutically acceptable carriers, diluents, or excipients.

17. A method of treating Alzheimer's disease in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of claim 3, or a pharmaceutically acceptable salt thereof.

18. A method of treating the progression of mild cognitive impairment to Alzheimer's disease in a patient, comprising administering to a patient in need of such treatment an effective amount of a compound of claim 3, or a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition, comprising a compound or a pharmaceutically acceptable salt thereof according to claim 3 with one or more pharmaceutically acceptable carriers, diluents, or excipients.

20. A process for preparing a pharmaceutical composition, comprising admixing a compound or a pharmaceutically acceptable salt thereof according to claim 3 with one or more pharmaceutically acceptable carriers, diluents, or excipients.

Description

(1) The following preparations and examples further illustrate the invention.

Preparation 1

(2S)-1-Trityloxybut-3-en-2-ol

(2) ##STR00014##

(3) Scheme 1, step A: Stir trimethylsulfonium iodide (193.5 g, 948.2 mmol) in THF (1264 mL) at ambient temperature for 75 minutes. Cool mixture to 50 C. and add n-butyllithium (2.5 mol/L in hexanes, 379 mL, 948.2 mmol) via cannula, over a period of 30 minutes. Allow the reaction to gradually warm to 30 C. and stir for 60 minutes. Add (2S)-2-trityloxymethyl oxirane (100 g, 316.1 mmol) portion wise, keeping the temperature below 10 C. After the complete addition, allow the reaction mixture to warm to room temperature and stir for 2 hours. Pour the reaction into saturated ammonium chloride, separate the phases, and extract the aqueous phase with ethyl acetate. Combine the organic layers and dry over magnesium sulfate. Filter and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with methyl t-butyl ether:hexanes (10-15% gradient), to give the title compound (56.22 g, 54%). ES/MS m/z 353 (M+Na).

Alternate Preparation 1

(2S)-1-Trityloxybut-3-en-2-ol

(4) Scheme 1a, step A starting material: Add triphenylmethyl chloride (287 g, 947.1 mmol), DMAP (7.71 g, 63.1 mmol) and triethylamine (140 g, 1383.5 mmol) to a solution of (2S)-but-2-ene-1,2-diol (prepared as in JACS, 1999, 121, 8649) (64.5 g, 631 mmol) in dichloromethane (850 mL). Stir for 24 hours at 24 C. Add 1 N aqueous citric acid (425 mL). Separate the layers and concentrate the organic extract under reduced pressure to dryness. Add methanol (900 mL) and cool to 5 C. for 1 hour. Collect the solids by filtration and wash with 5 C. methanol (50 mL). Discard the solids and concentrate the mother liquor under reduced pressure to dryness. Add toluene (800 mL) and concentrate to a mass of 268 g to obtain the title compound (129 g, 67%) in a 48 wt % solution of toluene.

Preparation 2

1-Morpholino-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone

(5) ##STR00015##

(6) Scheme 1a, step A: Add tetrabutyl ammonium hydrogen sulfate (83.2 g, 245.0 mmol) and 4-(2-chloroacetyl)morpholine (638.50 g, 3902.7 mmol) to a solution of 1-trityloxybut-3-en-2-ol (832.4, 2519 mmol) in toluene (5800 mL) that is between 0 and 5 C. Add sodium hydroxide (1008.0 g, 25202 mmol) in water (1041 mL). Stir for 19 hours between 0 and 5 C. Add water (2500 mL) and toluene (2500 mL). Separate the layers and wash the organic extract with water (23500 mL). Concentrate the organic extract under reduced pressure to dryness. Add toluene (2500 mL) to the residue and then add n-heptane (7500 mL) slowly. Stir for 16 hours. Collect the resulting solids by filtration and wash with n-heptane (1200 mL). Dry the solid under vacuum to obtain the title compound (1075.7 g, 98%).

Preparation 3

1-(5-Bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone

(7) ##STR00016##

(8) Scheme 1a, step B: Add a 1.3 M solution of isopropyl magnesium chloride lithium chloride complex (3079 mL, 2000 mmol) in THF to a solution of 4-bromo-1-fluoro-2-iodobenze (673.2 g, 2237.5 mmol) in toluene (2500 mL) at a rate to maintain the reaction temperature below 5 C. Stir for 1 hour. Add the resulting Grignard solution (5150 mL) to a solution of 1-morpholino-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone (500 g, 1093 mmol) in toluene (5000 mL) at a rate to maintain the reaction temperature below 5 C. Stir for 3 hours maintaining the temperature below 5 C. Add additional prepared Grignard solution (429 mL) and stir for 1 hour. Add a 1 N aqueous citric acid solution (5000 mL) at a rate to maintain the temperature below 5 C. Separate the layers and wash the organic extract with water (5000 mL). Concentrate the solution under reduced pressure to dryness. Add methanol (2000 mL) to the residue and concentrate to give the title compound as a residue (793 g, 73.4% potency, 83%).

Preparation 4

1-(5-Bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone oxime

(9) ##STR00017##

(10) Scheme 1a, step C: Add hydroxylamine hydrochloride (98.3 g) to 1-(5-bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone (450 g, 707 mmol) and sodium acetate (174 g) in methanol (3800 mL). Heat the solution to 50 C. for 2 hours. Cool to 24 C. and concentrate. Add water (1000 mL) and toluene (1500 mL) to the residue. Separate the layers and extract the aqueous phase with toluene (500 mL). Combine the organic extract and wash with water (2400 mL). Concentrate the solution under reduced pressure to give the title compound as a residue (567 g, 61.4% potency, 88%).

Preparation 5

tert-Butyl 2-[(1S)-1-(trityloxymethyl)allyloxy]acetate

(11) ##STR00018##

(12) Scheme 1, step B: Add (2S)-1-trityloxybut-3-en-2-ol (74.67 g, 226.0 mmol) to a solution of tetra-N-butylammonium sulfate (13.26 g, 22.6 mmol) in toluene (376 mL). Add sodium hydroxide (50% mass) in water (119 mL) followed by tert-butyl-2-bromoacetate (110.20 g, 565.0 mmol). Stir reaction mixture for 18 hours at ambient temperature. Pour into water, separate the phases, and extract the aqueous phase with ethyl acetate. Combine the organic layers and dry over magnesium sulfate. Filter the mixture and concentrate under reduced pressure to give the title compound (77.86 g, 77%). ES/MS m/z 467 (M+Na).

Preparation 6

(1E)-2-[(1S)-1-(Trityloxymethyl)allyloxy]acetaldehyde oxime

(13) ##STR00019##

(14) Scheme 1, step C: Cool a solution of tert-butyl 2-[(1S)-1-(trityloxymethyl)allyloxy]acetate (77.66 g, 174.7 mmol) in dichloromethane (582.2 mL) to 78 C. Add a solution of diisobutylaluminum hydride in hexanes (1 mol/L, 174.7 mL) dropwise over a period of 35 minutes and maintain the temperature below 70 C. Stir at 78 C. for 5 hours. Add hydrochloric acid in water (2 mol/L, 192.1 mL) to the reaction mixture dropwise, keeping the temperature below 60 C. Allow the reaction to gradually warm to ambient temperature and stir for 60 minutes. Separate the organic extract and wash with saturated sodium bicarbonate. Dry the solution over magnesium sulfate, filter, and concentrate under reduced pressure to give a residue. Dissolve the residue in dichloromethane. Add sodium acetate (28.66 g, 349.3 mmol), followed by hydroxylamine hydrochloride (18.21 g, 262.0 mmol). Stir at ambient temperature for 18 hours. Pour into water, separate the phases, and extract the aqueous phase with dichloromethane. Combine the organic layers and dry over magnesium sulfate. Filter the mixture and concentrate under reduced pressure to give the title compound (68.38 g, 101%). ES/MS m/z 386 (MH).

Preparation 7

(3aR,4S)-4-(Trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole

(15) ##STR00020##

(16) Scheme 1, step D: Cool a solution of (1E)-2-[(1S)-1-(trityloxymethyl)allyloxy]acetaldehyde oxime (55.57 g, 143.4 mmol) in tert-butyl methyl ether (717 mL) to 5 C. Add sodium hypochlorite (5% in water, 591 mL, 430.2 mmol) dropwise, keeping the temperature below 10 C. Stir at 10 C. for 30 minutes. Allow the reaction to warm to 15 C. Stir at 15 C. for 18 hours. Dilute the reaction mixture with ethyl acetate and wash with saturated sodium bicarbonate. Separated the phases, wash the organic phase with a 5% sodium hydrogen sulphite solution and brine. Dry the solution over magnesium sulfate, filter, and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with 50% methyl tert-butyl ether/dichloromethane:hexanes (20-27% gradient), to give the title compound (35.84 g, 65%). ES/MS m/z 408 (M+Na).

Preparation 8

(3aR,4S,6aR)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole

(17) ##STR00021##

(18) Scheme 1, step E: Cool a solution of 4-bromo-1-fluoro-2-iodo-benzene (86.94 g, 288.9 mmol) in THF (144.5 mL) and toluene (1445 mL) to 78 C. Add n-butyllithium (2.5 M in hexanes, 120 mL, 288.9 mmol) dropwise, keeping the temperature below 70 C. Stir for 30 minutes at 78 C. Add boron trifluoride diethyl etherate (36.5 mL, 288.9 mmol) dropwise, keeping temperature below 70 C. Stir the solution for 30 minutes at 78 C. Add a solution of (3aR,4S)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole (55.69 g, 144.5 mmol) in THF (482 mL) dropwise to the reaction, over a period of 30 minutes, keeping temperature below 65 C. Stir at 78 C. for 90 minutes. Rapidly add saturated ammonium chloride, keeping temperature below 60 C. Pour into brine, and extract the aqueous phase with ethyl acetate. Combine the organic extract and dry over magnesium sulfate. Filter and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with a gradient of 100% hexanes to 30% hexanes/70% diethyl ether, to give the title compound (36.52 g, 45%). ES/MS m/e (.sup.79Br/.sup.81Br) 560/562 [M+H].

Alternate Preparation 8

(19) Scheme 1a, step D: Heat a solution of 1-(5-bromo-2-fluoro-phenyl)-2-[(1S)-1-(trityloxymethyl)allyloxy]ethanone oxime (458 g, 502 mmol) and hydroquinone (56.3 g 511 mmol) in toluene (4000 mL) to reflux under nitrogen for 27 hours. Cool the solution to 24 C. and add aqueous sodium carbonate (800 mL). Separate the layers and extract the aqueous phase with toluene (300 mL). Combine the organic extract and wash with water (2500 mL). Concentrate the solution under reduced pressure to give a residue. Add isopropyl alcohol (1500 mL) and heat to reflux. Cool to 24 C. and collect the solids by filtration. Dry the solid under vacuum to obtain the title compound (212 g, 75%).

Preparation 9

1-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone

(20) ##STR00022##

(21) Scheme 1a, step E: Add acetyl chloride (35.56 g, 503.9 mmol) to a solution of (3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole (235.3 g, 420 mmol), DMAP (5.13 g, 42.0 mmol), and pyridine (66.45 g, 840.1 mmol) in dichloromethane (720 mL) under nitrogen, maintaining internal temperature below 5 C. Stir for 1 hour and then add water (300 mL) and 1 M sulfuric acid (300 mL). Stir the mixture for 10 minutes and allow the layers to separate. Collect the organic extract and wash with saturated sodium carbonate (500 mL) and water (500 mL). Dry the solution over magnesium sulfate. Filter and concentrate under reduced pressure to give 1-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone (235 g, 93%) as a grey solid.

Preparation 10

1-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluorophenyl)-4-(hydroxymethyl)tetrahydro-1H,3H-furo[3,4-c][1,2]oxazol-1-yl]ethanone

(22) ##STR00023##

(23) Scheme 2, step A: In a 20 L jacketed reactor add acetyl chloride (290 mL, 4075 mmol) to a solution of (3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole (1996 g, 3384 mmol), DMAP (56.0 g, 458 mmol), pyridine (500 mL, 6180 mmol) in dichloromethane (10 L) under nitrogen maintaining internal temperature below 10 C. After complete addition (1 hour) warm to 20 C. and stir overnight. If reaction is incomplete, add acetyl chloride, DMAP, pyridine, and dichloromethane until complete reaction is observed. Cool the reaction mixture to 0 C. and slowly add water (5 L), stir the reaction mixture at 10 C. for 30 minutes and allow the layers to separate. Collect the organic extract and wash the aqueous with dichloromethane (1 L). Wash the combined organic extracts with 1 N aqueous hydrochloric acid (24 L), extract the aqueous with dichloromethane (21 L). Wash the combined organic extracts with water (4 L) and remove the solvent under reduced pressure give total volume of approximately 5 L. Add 90% formic acid (1800 mL) and stand at ambient temperature for 3 days. Warm to 40 C. for 2 hours then remove the solvent under reduced pressure. Dilute the residue with methanol (4 L) and slowly add saturated aqueous sodium carbonate (3 L). Add solid sodium carbonate (375 g) to adjust the pH to 8-9. Stir at 45 C. for 1 hour then cool to ambient temperature. Remove the solids by filtration, washing with methanol (4500 mL) then treat with 2 N aqueous sodium hydroxide (100 mL) and stand at ambient temperature for 1 hour. Remove the solids by filtration, washing with methanol (2100 mL). Evaporate the solvent under reduced pressure and partition the residue between ethyl acetate (5 L) and water (2 L). Extract the aqueous with ethyl acetate (2 L) and wash the combined organic extracts with brine (21 L). Remove the solvent under reduced pressure, add methyl tert-butyl ether (2.5 L) and evaporate to dryness. Add methyl tert-butyl ether (4 L) and stir at 65 C. for 1 hour cool to ambient temperature and collect the solids by filtration, washing with methyl tert-butyl ether (3500 mL). Dry under vacuum to a beige solid. Heat this solid in toluene (7.5 L) to 110 C. until fully dissolved, cool to 18 C. over 1 hour, and stir at this temperature for 1 hour. Warm to 40 C. and when precipitate forms, cool to 18 C. once more. Stir for 45 minutes then collect solids by filtration, washing with toluene (2500 mL). Dry the solid under vacuum to obtain the title compound (443.1 g, 36%, 95% purity by LCMS). Evaporate the filtrate under vacuum to give a residue. Purify the residue by silica gel flash chromatography, eluting with 20% to 100% ethyl acetate in isohexane. Slurry the product containing fractions in methyl tert-butyl ether (2 L) at 60 C. for 30 minutes, cool to ambient temperature, and collect the solids by filtration, washing with methyl tert-butyl ether (2200 mL). Dry the solids under vacuum to give the title compound as a beige crystalline solid (304 g, 24%, 88% purity by LCMS). Evaporate the filtrate under vacuum to a residue. Purify the residue by silica gel flash chromatography, eluting with 20% to 100% ethyl acetate in isohexane to give the title compound (57.8 g, 5%, 88% purity by LCMS). ES/MS: m/z (.sup.79Br/.sup.81Br) 360.0/362.0 [M+H].

Alternate Preparation 10

(24) Scheme 2, step A: Add 1-[(3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone (69 g, 114.5 mmol) to a 15 C. solution of p-toluenesulfonic acid monohydrate (2.2 g, 11.45 mmol), dichloromethane (280 mL) and methanol (700 mL). Stir for 18 hours and then remove the solvent under reduced pressure. Dilute the residue with dichloromethane (350 mL) and add 1 M aqueous sodium carbonate (140 mL) and water (140 mL). Separate the layers and evaporate the organic layer under reduced pressure. Add toluene (350 mL) to the residue and heat to reflux for 1 hour. Cool to 10-15 C. at a rate of 10 C./hour.

(25) Collect the solids by filtration and wash with toluene (70 mL). Dry the solid under vacuum to obtain the title compound (30 g, 65%) as a grey solid.

Preparation 11

(3aR,4S,6aS)-1-Acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole-4-carboxylic acid

(26) ##STR00024##

(27) Scheme 2, step B: Add water (2 L) to a suspension of 1-[(3aR,4S,6aS)-6a-(5-bromo-2-fluorophenyl)-4-(hydroxymethyl)tetrahydro-1H,3H-furo[3,4-c][1,2]oxazol-1-yl]ethanone (804.9 g, 2177 mmol), (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (40.0 g, 251 mmol) in acetonitrile (4.5 L) in a 20 L jacketed reactor and cool to an internal temperature of 5 C. Add (diacetoxyiodo)benzene (1693 g, 4993.43 mmol) portionwise over 30 minutes. Control the exotherm using reactor cooling and then hold at 20 C. until LCMS shows complete reaction. Slowly add a suspension of sodium bisulfite (70 g, 672.68 mmol) in water (300 mL) at ambient temperature, maintaining the internal temperature below 25 C. Stir for 30 minutes and then cool to 5 C. Add water (2 L), then slowly add 47 wt % aqueous sodium hydroxide (780 mL) over a period of 1 hour maintaining the internal temperature below 10 C. Add ethyl acetate (2 L) and isohexane (5 L), stir vigorously and separate the layers. Extract the biphasic organic layers with water (1 L) and wash the combined aqueous with methyl tert-butyl ether (2.5 L). Cool the aqueous extracts to 5 C. and slowly add 37% hydrochloric acid (1.4 L) over 30 minutes maintaining the internal temperature around 5 C. Add ethyl acetate (5 L), separate the layers and wash the organic with brine (31 L). Extract the combined aqueous extracts with ethyl acetate (2.5 L), wash the combined organics with brine (1 L), then dry with sodium sulfate, and filter. Dilute the organics with heptane (2.5 L) and evaporate to dryness under reduced pressure. Add methyl tert-butyl ether (1.5 L) and heptane (1.5 L) and evaporate to dryness. Add heptane (2.5 L) and evaporate to dryness twice. Add heptane (500 mL) and methyl tert-butyl ether (500 mL) and stir at 40 C. for 30 minutes then collect the precipitate by filtration, washing with heptane/methyl tert-butyl ether (1:1, 1 L) then methyl tert-butyl ether (3300 mL) and air dry to give the title compound as a beige crystalline solid (779 g, 91%). ES/MS: m/z (.sup.79Br/.sup.81Br) 374.0/376.0 [M+H]. [].sub.D.sup.20=19.0 (C=1.004, chloroform).

Alternate Preparation 11

(28) Scheme 2, step B: Add water (150 mL) and acetonitrile (150 mL) to 1-[(4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(hydroxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-1-yl]ethanone (30 g, 73.3 mmol), TEMPO (1.14 g, 7.30 mmol) and (diacetoxyiodo) benzene (51.9 g, 161 mmol). Cool to 15 C. and stir for 2 hours. Slowly add sodium thiosulfate (21 g) and potassium carbonate (22 g) in water (150 mL) at ambient temperature. Stir for 1 hour and then add methyl tert-butyl ether (150 mL). Separate the layers and adjust the pH of the aqueous layer to 2-3 with concentrated sulfuric acid. Add ethyl acetate (150 mL) and separate the layers. Evaporate the organic layer to dryness under reduced pressure. Add n-heptane (90 mL) and heat to reflux for 1 hour. Cool to 15 C. and then collect the precipitate by filtration, washing with n-heptane (90 mL). Dry under vacuum to give the title compound as a white solid (27 g, 98%).

Preparation 12

(3aR,4S,6aS)-1-Acetyl-6a-(5-bromo-2-fluorophenyl)-N-methoxy-N-methyltetrahydro-1H,3H-furo[3,4-c][1,2]oxazole-4-carboxamide

(29) ##STR00025##

(30) Scheme 2, step C: In a 10 L jacketed reactor, cool a solution of (3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole-4-carboxylic acid (771 g, 2019 mmol) in dichloromethane (7.0 L) to 0 C. under nitrogen and add CDI (400 g, 2421 mmol) portionwise over 40 minutes. Cool the reactor jacket to 20 C. and stir for 1 hour and then add N,O-dimethylhydroxylamine hydrochloride (260.0 g, 2612 mmol) portionwise over about 30 minutes. Stir at 20 C. for 1 hour, at 0 C. for 2 hours, and at 10 C. for 7 hours. Add CDI (175 g, 1058 mmol) and stir at 10 C. overnight. Add further CDI (180 g, 1088 mmol) at 10 C. and stirred for 1 hour then add N,O-dimethylhydroxylamine hydrochloride (140 g, 1407 mmol) and continue stirring at 10 C. If the reaction is incomplete, further charges of CDI followed by N,O-dimethylhydroxylamine hydrochloride can be made until complete reaction is observed. Cool the reaction mixture to 5 C. and wash with 1 N aqueous hydrochloric acid (5 L) then 2 N aqueous hydrochloric acid (5 L). Extract the combined aqueous solution with dichloromethane (1 L), combine the organic extract and wash with water (2.5 L), 1 N aqueous sodium hydroxide (2.5 L), and water (2.5 L), dry over magnesium sulfate, filter, and evaporate under reduced pressure to give a residue. Add methyl tert-butyl ether (3 L) and evaporate under reduced pressure. Add further methyl tert-butyl ether (2 L) and stir at 50 C. for 1 hour, cool to 25 C. and stir for 30 minutes. Collect the resulting solids by filtration, wash with methyl tert-butyl ether (2500 mL) and dry under vacuum to give the title compound (760 g, 88%) as a white solid. ES/MS: m/z (.sup.79Br/.sup.81Br) 417.0/419.0 [M+H].

Alternate Preparation 12

(31) Scheme 2, step C: Cool a solution of (3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole-4-carboxylic acid (27 g, 70.7 mmol) in N,N-dimethylformamide (135 mL) to 0 C. under nitrogen and add CDI (14.9 g, 91.9 mmol). Stir for 1 hour and then add N,O-dimethylhydroxylamine hydrochloride (9.0 g, 92 mmol) and triethylamine (14.3 g, 141 mmol). Stir at 15 C. for 16 hours. Cool the reaction mixture to 0 C. and add 0.5 M aqueous sulfuric acid (675 mL). Stir for 1 hour. Collect the resulting solids by filtration. Slurry the solids in methyl tert-butyl ether (90 mL) for 1 hour. Collect the solids by filtration, wash with methyl tert-butyl ether (30 mL). Dry under vacuum to give the title compound (23 g, 78%) as a solid.

Preparation 13

1-[(3aR,4S,6aS)-1-Acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone

(32) ##STR00026##

(33) Scheme 2, step D: In a 20 L jacketed reactor, cool a solution of (3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluorophenyl)-N-methoxy-N-methyltetrahydro-1H,3H-furo[3,4-c][1,2]oxazole-4-carboxamide (654.0 g, 1536 mmol) in THF (10 L) to 60 C. and add a 3.2 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (660 mL, 2110 mmol) dropwise, while maintaining the internal temperature below 40 C. Stir the reaction mixture at 40 C. for 30 minutes then cool to 50 C. and add a solution of 1 N aqueous hydrochloric acid (2 L) in THF (2 L) maintaining the internal temperature below 38 C. Increase the temperature to 10 C. and add ethyl acetate (5 L) and water (1 L), stir and allow internal temperature to reach 5 C. and separate the layers. Extract the aqueous layer with ethyl acetate (1 L) and combine the organic extracts. Wash the organic extracts with water (2 L) and extract the aqueous layer with ethyl acetate (1 L). Combine the organic extract and wash with brine (32 L) then dry over magnesium sulfate, filter, and evaporate under reduced pressure to a residue. Add cyclohexane (2.5 L), stir at 60 C. for 1 hour then at 20 C. for 30 minutes, and collect the solid by filtration, washing with cyclohexane (500 mL). Dry the solid under vacuum to obtain the title compound as a white solid (565 g, 99%). ES/MS: m/z (.sup.79Br/.sup.81Br) 372.0/374.0 [M+H], [].sub.D.sup.20=58.0 (C=1.000, chloroform).

Alternate Preparation 13

(34) Scheme 2, step D: Cool a solution of (3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluorophenyl)-N-methoxy-N-methyltetrahydro-1H,3H-furo[3,4-c][1,2]oxazole-4-carboxamide (4.0 g, 9.59 mmol) in THF (60 mL) to 5 C. and add a 3.0 M solution of methylmagnesium bromide in 2-methyltetrahydrofuran (5.0 mL, 15 mmol) dropwise, while maintaining the internal temperature between 5 and 0 C. Stir the reaction mixture between 5 and 0 C. for 60 minutes then add a solution of saturated ammonium chloride (20 mL). Add methyl tert-butyl ether (40 mL), allow the internal temperature to reach 5 C. and separate the layers. Evaporate the organic layer under reduced pressure to a residue. Add n-heptane (50 mL), stir, and collect the solid by filtration. Dry the solid under vacuum to obtain the title compound as a solid (3.0 g, 77%).

Preparation 14

1-[(3aR,4S,6aS)-6a-(5-Bromo-2-fluorophenyl)-4-(1,1-difluoroethyl)tetrahydro-1H,3H-furo[3,4-c][1,2]oxazol-1-yl]ethanone

(35) ##STR00027##

(36) Scheme 2, step E: Add 1-[(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone (5.08 g, 13.6 mmol) in a single portion to a stirred suspension of difluoro(morpholino)sulfonium tetrafluoroborate (10.02 g, 39.18 mmol) in anhydrous dichloromethane (100 mL) at 0-5 C. Stir the mixture for 10 minutes and add triethylamine trihydrofluoride (4.5 mL, 27 mmol) dropwise over 10 minutes. Stir the reaction mixture in the ice-bath for 8 hours then warm to ambient temperature and stir overnight. Add saturated aqueous sodium carbonate (100 mL) and stir for 1 hour. Separate the layers and extract the aqueous with dichloromethane (250 mL). Combine the organic extracts and wash with saturated aqueous sodium bicarbonate (100 mL), 2 N aqueous hydrochloric acid (2100 mL), and brine (100 mL). Evaporate to dryness to a light brown solid and dissolve in methyl tert-butyl ether (300 mL) at 60 C. Filter the hot solution and evaporate the filtrate to give a brown solid (5.3 g, 81%, 82% purity by LCMS) that is used without further purification. ES/MS: m/z (.sup.79Br/.sup.81Br) 393.8/395.8 [M+H].

Alternate Preparation 14

(37) Scheme 2, step E: Add XtalFluor-M (1.21 kg, 4.73 mol) in portions to a stirred solution of 1-[(3aR,4S,6aS)-1-acetyl-6a-(5-bromo-2-fluoro-phenyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazol-4-yl]ethanone (565 g, 1.51 mol) in anhydrous dichloromethane (5 L) at 14 C. Stir the mixture for 10 minutes and add triethylamine trihydrofluoride (550 g, 3.34 mol) dropwise over 20 minutes. Stir the reaction mixture at 10 C. for approximately 10 hours then warm to ambient temperature and stir overnight. Add 50% aqueous sodium hydroxide (750 mL) slowly, maintaining the internal temperature below 10 C., then add water (1.5 L) and saturated aqueous sodium hydrogen carbonate (1 L) and stir for 30 minutes. Separate the layers and extract the aqueous with dichloromethane (1 L). Combine the organic extracts and wash with brine (3 L), 2 N aqueous hydrochloric acid (5 L), and brine (3 L). Evaporate to give a residue and purify by silica gel chromatography eluting with 50-100% dichloromethane in iso-hexane then 10% methyl tert-butyl ether in dichloromethane to give the title compound as a white powder (467 g, 73%, 94% purity by LCMS). ES/MS: m/z (.sup.79Br/.sup.81Br) 393.8/395.8 [M+H].

Preparation 15

(3 aR,4S,6aS)-6a-(5-Bromo-2-fluoro-phenyl)-4-(1,1-difluoroethyl)-3,3a,4,6-tetrahydro-1H-furo[3,4-c]isoxazole

(38) ##STR00028##

(39) Scheme 2, step F: Add 37 wt % aqueous hydrochloric acid (1.3 L, 16 mol) to a solution of 1-[(3aR,4S,6aS)-6a-(5-bromo-2-fluorophenyl)-4-(1,1-difluoroethyl)tetrahydro-1H,3H-furo[3,4-c][1,2]oxazol-1-yl]ethanone (570 g, 1.45 mol) in 1,4-dioxane (5 L) in a 10 L jacketed reactor and stir at 100 C. for approximately 3 hours or until LCMS shows complete reaction. Cool the reaction mixture to 10 C., dilute with water (1 L) and add a mixture 50 wt % aqueous sodium hydroxide solution (800 mL) and water (1 L) slowly, maintaining the internal temperature below 20 C. Add ethyl acetate (2.5 L) and stir vigorously, before separating the layers and washing the organic phase with brine (2 L), further brine (1 L), and water (1 L). Dry over magnesium sulfate, filter and concentrate to dryness under reduced pressure to give a residue. Add cyclohexane (2.5 L) and evaporate to dryness then repeat to obtain the title compound as a brown oil (527 g, 89%, 86% purity by LCMS). ES/MS: m/z (.sup.79Br/.sup.81Br) 351.8/353.8 [M+H].

Preparation 16

[(2S,3R,4S)-4-Amino-4-(5-bromo-2-fluorophenyl)-2-(1,1-difluoroethyl)tetrahydrofuran-3-yl]methanol

(40) ##STR00029##

(41) Scheme 2, step G: Add zinc powder (6.0 g, 92 mmol) to a solution of (3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(1,1-difluoroethyl)-3,3a,4,6-tetrahydro-1H-furo[3,4-c]isoxazole (5.06 g, 13.4 mmol) in acetic acid (100 mL) at ambient temperature and stir overnight. Dilute the mixture with ethyl acetate (200 mL) and water (300 mL) and stir vigorously while adding sodium carbonate (97 g, 915 mmol). Separate the layers and wash the organic layer with brine (2200 mL), dry over magnesium sulfate, filter, and concentrate to give a residue. Purify the residue by silica gel chromatography eluting with 0% to 100% methyl tert-butyl ether in isohexane to give the title compound as a waxy solid (4.67 g, 89%, 90% purity by LCMS). ES/MS: m/z (.sup.79Br/.sup.81Br) 354.0/356.0 [M+H].

Alternate Preparation 16

(42) Scheme 2, step G: Add zinc powder (200 g, 3.06 mol) portionwise to a solution of (3aR,4S,6aS)-6a-(5-bromo-2-fluoro-phenyl)-4-(1,1-difluoroethyl)-3,3a,4,6-tetrahydro-1H-furo[3,4-c]isoxazole (304 g, 75% purity, 647 mmol) in acetic acid (2 L) and water (2 L) at 20 C. then warm to 40 C. and stir overnight. Dilute the mixture with water (2 L) and stir vigorously while adding sodium carbonate (4 kg, 43.4 mol) then adjust to pH 8-9 with further sodium carbonate. Add ethyl acetate (5 L) and water (2.5 L), stir for 30 minutes and filter through diatomaceous earth washing with 2:1 acetonitrile/water. Separate the layers, extract the aqueous with ethyl acetate (22.5 L) and wash the combined organic extracts with brine (22.5 L), dry over magnesium sulfate, filter, and concentrate to give a residue. Purify the residue by SFC, column: Chiralpak AD-H (5), 50250 mm; eluent: 12% ethanol (0.2% diethylmethylamine in CO.sub.2; flow rate: 340 g/minute at UV 220 nm to give the title compound as a white solid (197.7 g, 84%). [].sub.D.sup.20=6.93 (C=0.678, chloroform). ES/MS: m/z (.sup.79Br/.sup.81Br) 354.0/356.0 [M+H].

Preparation 17

[(2S,3R,4S)-4-Amino-4-(5-bromo-2-fluoro-phenyl)-2-(trityloxymethyl)tetrahydrofuran-3-yl]methanol

(43) ##STR00030##

(44) Scheme 1, step F: Add (3aR,4S,6aR)-6a-(5-bromo-2-fluoro-phenyl)-4-(trityloxymethyl)-3,3a,4,6-tetrahydrofuro[3,4-c]isoxazole (31.30 g, 55.9 mmol) to acetic acid (186 mL) to give a suspension. Add zinc (25.6 g, 391 mmol) and stir the reaction mixture vigorously for 18 hours. Dilute the mixture with toluene and filter through diatomaceous earth. Concentrate the filtrate under reduced pressure. Solubilize the residue with ethyl acetate, wash with brine, and saturated sodium bicarbonate. Separate the phases, dry over magnesium sulfate, filter, and concentrate under reduced pressure to give the title compound (31.35 g, 99%). ES/MS m/e (.sup.79Br/.sup.81Br) 562/564 [M+H].

Preparation 18

N-[[(3S,4R,5S)-3-(5-Bromo-2-fluoro-phenyl)-4-(hydroxymethyl)-5-(trityloxymethyl)tetrahydrofuran-3-yl]carbamothioyl]benzamide

(45) ##STR00031##

(46) Scheme 1, step G: Dissolve [(2S,3R,4S)-4-amino-4-(5-bromo-2-fluoro-phenyl)-2-(trityloxymethyl) tetrahydrofuran-3-yl]methanol (31.35 g, 55.73 mmol) in dichloromethane (557 mL) and cool to 5 C. Add benzoyl isothiocyanate (9.74 mL, 72.45 mmol). After addition is complete, allow the reaction mixture to warm to room temperature and stir for 2 hours. Pour into saturated sodium bicarbonate, separate the phases, and extract the aqueous phase with dichloromethane. Combine the organic extract and dry over magnesium sulfate. Filter the solution and concentrate under reduced pressure to give the title compound (42.95 g, 106%). ES/MS m/e (.sup.79Br/.sup.81Br) 747/749 [M+Na].

Preparation 19

N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

(47) ##STR00032##

(48) Scheme 2, step H: Add benzoyl isothiocyanate (1.80 mL, 13.3 mmol) to a solution of [(2S,3R,4S)-4-amino-4-(5-bromo-2-fluorophenyl)-2-(1,1-difluoroethyl)tetrahydrofuran-3-yl]methanol (4.67 g, 11.9 mmol) in dichloromethane (20 mL) at ambient temperature for 1 hour until LCMS shows reaction is complete. Evaporate the reaction mixture to a residue under vacuum. Add cyclohexane (50 mL), warm to 60 C. and add methyl tert-butyl ether until precipitate is fully dissolved (100 mL). Filter the hot solution, cool to room temperature and slowly evaporate under reduced pressure until formation of a white precipitate. Remove the solvent under reduced pressure and dissolve the residue in anhydrous dichloromethane (30 mL), add pyridine (2.4 mL, 30 mmol), and cool the solution to 25 C. Add trifluoromethanesulfonic anhydride (2.2 mL 13 mmol) dropwise over 30 minutes and allow to warm 0 C. over 1 hour. Wash the reaction mixture with water (25 mL), 2 N aqueous hydrochloric acid (25 mL), water (25 mL), aqueous saturated sodium bicarbonate (25 mL), and water (25 mL), dry over magnesium sulfate, filter, and concentrated to dryness. Purify the residue by silica gel chromatography eluting with 5% methyl tert-butyl ether in dichloromethane to give the title compound as a light yellow foam (5.0 g, 76%, 90% purity by LCMS). ES/MS: m/z (.sup.79Br/.sup.81Br) 499.0/501.0 [M+H].

Alternate Preparation 19

(49) Scheme 2, step H: Add benzoyl isothiocyanate (98 mL, 724.9 mmol) to a solution of [(2S,3R,4S)-4-amino-4-(5-bromo-2-fluorophenyl)-2-(1,1-difluoroethyl)tetrahydrofuran-3-yl]methanol (197.6 g, 546.7 mmol) in dichloromethane (1.2 L) at 30 C. for 1 hour. Add CDI (101 g, 610.4 mmol) and stir at ambient temperature for 3 hours. Further charges of CDI can be made to ensure complete consumption of the thiourea intermediate. Heat to 90 C. for 42 hours and cool the solution to ambient temperature. Dilute the reaction mixture with ethyl acetate (2 L) and add 2 N aqueous hydrochloric acid (2 L), stir, add brine (1 L) and separate the layers. Wash the organic layer with 2 N aqueous hydrochloric acid (0.5 L), brine (21 L) and aqueous saturated sodium bicarbonate (1 L). Dry over magnesium sulfate, filter, and concentrate to give a residue. Purify the residue by silica gel chromatography eluting with 0-100% ethyl acetate in iso-hexane to give the title compound as a light yellow solid (234 g, 83%). ES/MS: m/z (.sup.79Br/.sup.81Br) 499.0/501.0 [M+H].

Preparation 20

N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(trityloxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

(50) ##STR00033##

(51) Scheme 1, step H: Dissolve N-[[(3S,4R,5S)-3-(5-bromo-2-fluoro-phenyl)-4-(hydroxymethyl)-5-(trityloxymethyl)tetrahydrofuran-3-yl]carbamothioyl]benzamide (42.95 g, 59.18 mmol) in dichloromethane (591 mL) and cool to 20 C. Add pyridine (12.0 mL, 148.0 mmol), followed by trifluoromethanesulfonic anhydride (10.97 mL, 65.10 mmol). Monitor the addition keeping the temperature below 20 C. Stir the reaction mixture at 20 C. for 30 minutes. Allow the reaction mixture to warm to room temperature. Pour into saturated ammonium chloride, separate the phases, and extract the aqueous phase with dichloromethane. Combine the organic extract and dry over magnesium sulfate. Filter the solution and concentrate under reduced pressure to give the title compound (45.24 g, 108%). ES/MS m/e (.sup.79Br/.sup.81Br) 707/709 [M+H].

Preparation 21

N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(hydroxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

(52) ##STR00034##

(53) Scheme 1, step I: Dissolve N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-(trityloxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide (45.24 g, 63.93 mmol) in formic acid (160 mL) and stir at ambient temperature for 1 hour. Add water (29 mL) over a period of 5 minutes. Stir for 50 minutes. Concentrate the mixture under reduced pressure to a residue. Dissolve the residue in methanol (639 mL), add triethylamine (26.7 mL, 191.8 mmol), and stir overnight at ambient temperature. Pour into brine, separate the phases, and extract the aqueous phase with chloroform. Combine the organic extract and dry over magnesium sulfate. Filter and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with acetone:hexanes (25-38% gradient), to give the title compound (16.04 g, 54%). ES/MS m/e (79Br/81Br) 465/467 [M+H].

Preparation 22

(4aS,5S,7aS)-2-Benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxylic acid

(54) ##STR00035##

(55) Scheme 1, step J: Add N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-(hydroxymethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide (16.04 g, 34.47 mmol) to DMSO (172 mL). Add 2-iodoxybenzoic acid (35.56 g, 120.70 mmol) and stir at ambient temperature for 3 hours. Dilute the reaction mixture with chloroform (300 mL) and pour into saturated ammonium chloride (400 mL). Separate the organic phase and dry over magnesium sulfate. Filter the solution and concentrate under reduced pressure to give a residue. Dissolve the residue in ethyl acetate (400 mL) and wash with saturated ammonium chloride (2250 mL). Separate the organic phase, dry over magnesium sulfate, filter, and concentrate under reduced pressure to give a residue. Dissolve the residue in a dichloromethane:methanol mixture and add diethyl ether until a solid precipitates. Collect the solid by filtration and dry under reduced pressure to give the title compound (5.78 g, 35%). ES/MS m/e (.sup.79Br/.sup.81Br) 479/481 [M+H].

Preparation 23

(4aS,5 S,7aS)-2-Benzamido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxamide

(56) ##STR00036##

(57) Scheme 1, step K: Dissolve (4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxylic acid (5.78 g, 12.1 mmol) in dichloromethane (201 mL) and N,O-dimethylhydroxylamine hydrochloride (1.76 g, 18.1 mmol). Add triethylamine (5.29 mL, 36.2 mmol) followed by HATU (7.02 g, 18.1 mmol). Stir at ambient temperature for 3 days. Pour into saturated ammonium chloride, separate the phases, and extract the aqueous phase with ethyl acetate. Combine the organic extracts and dry over magnesium sulfate. Filter and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with ethyl acetate:dichloromethane (0-50% gradient) to give the title compound (4.15 g, 66%). ES/MS m/e (.sup.79Br/.sup.81Br) 522/524 [M+H].

Preparation 24

N-[(4aS,5S,7aS)-5-Acetyl-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

(58) ##STR00037##

(59) Scheme 1, step L: Add dropwise to a 78 C. solution of (4aS,5S,7aS)-2-benzamido-7a-(5-bromo-2-fluoro-phenyl)-N-methoxy-N-methyl-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazine-5-carboxamide (1.51 g, 2.89 mmol) in THF (57.8 mL) methylmagnesium bromide (3.0 mol/L in diethyl ether, 4.8 mL, 14.5 mmol). Stir the reaction at 78 C. for 5 minutes and allow to gradually warm to ambient temperature. Stir for 30 minutes. Quench the reaction with methanol (4 mL), dilute with saturated ammonium chloride, and extract with ethyl acetate. Combine the organic extract and dry over sodium sulfate. Filter and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with ethyl acetate:hexanes (0-100% gradient) to give the title compound (1.28 g, 93%). ES/MS m/e (.sup.79Br/.sup.81Br) 477/479 [M+H].

Preparation 25

N-[(4aS,5S,7aS)-7a-(5-Bromo-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

(60) ##STR00038##

(61) Scheme 1, step M: Add together dichloromethane (34 mL), bis(2-methoxyethyl)aminosulfur trifluoride (1.52 mL, 6.88 mmol), and boron trifluoride diethyl etherate (0.89 mL, 6.88 mmol). Stir at ambient temperature for 2 hours. Add N-[(4aS,5S,7aS)-5-acetyl-7a-(5-bromo-2-fluoro-phenyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide (0.821 g, 1.72 mmol) in one portion, followed by triethylamine trihydrofluoride (1.13 mL, 6.88 mmol). Stir at ambient temperature for 18 hours. Pour into saturated ammonium chloride, separate the phases, and extract the aqueous phase with ethyl acetate. Combine the organic extract and dry over magnesium sulfate. Filter and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with dichloromethane:hexanes (80-100% gradient), to give the title compound (0.552 g, 64%). ES/MS m/e (.sup.79Br/.sup.81Br) 499/501 [M+H].

Preparation 26

N-[(5S,7aS)-5-(1,1-Difluoroethyl)-7a-{2-fluoro-5-[(trifluoroacetyl)amino]phenyl}-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide

(62) ##STR00039##

(63) Scheme 5, step A: Dissolve N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluorophenyl)-5-(1,1-difluoroethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide (234 g, 454.6 mmol) in 1,4-dioxane (2 L) and add 4 molecular sieves (37 g), 2,2,2-trifluoroacetamide (91 g, 780.9 mmol), finely ground potassium carbonate (114 g, 824.9 mmol), sodium iodide (117 g, 780.6 mmol), copper (I) iodide (17.5 g, 91.9 mmol) and racemic trans-N,N-dimethyl-1,2-cyclohexane diamine (20 g, 140.6 mmol) under a stream of nitrogen. Purge the vessel with 3 vacuum nitrogen switches and heat to 123 C. for 18 hours. Cool to ambient temperature and filter the solution through diatomaceous earth, and wash with ethyl acetate. Add saturated aqueous ammonium chloride (2 L) and vigorously stir for 45 minutes. Separate the layers and wash the organic layer with saturated aqueous ammonium chloride (31 L), brine (300 mL), dry over magnesium sulfate, filter, and evaporate to give a residue. Purify the residue by silica gel chromatography eluting with 0-100% ethyl acetate in iso-hexane to give the title compound as a light yellow solid (297.9 g, 95%, 81% purity). ES/MS: m/z 532.0 [M+H].

Preparation 27

N-[(4aS,5S,7aS)-7a-(5-Amino-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide

(64) ##STR00040##

(65) Scheme 1, step N: Combine N-[(4aS,5S,7aS)-7a-(5-bromo-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide (0.372 g, 0.74 mmol) and (1R,2R)N,N-dimethyl-1,2-cyclohexanediamine (0.037 mL, 0.22 mmol) in ethanol (30 ml). Add sodium azide (0.194 g, 2.98 mmol), followed by sodium ascorbate (0.66 M solution, 0.50 ml, 0.33 mmol). Purge the top of the flask with nitrogen and add cupric sulfate (0.33 M solution, 0.68 ml, 0.22 mmol). Heat the reaction mixture to 80 C. and stir for 5 hours. Cool the reaction and add cold water. Extract the mixture with ethyl acetate. Combine the organic extract and dry over sodium sulfate. Filter and concentrate under reduced pressure to give a residue. Combine the residue with palladium (10 mass % on carbon, 0.35 g, 0.16 mmol) in ethanol (50 ml) and THF (10 ml). Purge the mixture with nitrogen and with hydrogen. Stir at ambient temperature under 50 psi of hydrogen for 1 hour. Filter off the catalyst and wash with ethyl acetate. Concentrate the solution under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with ethyl acetate:dichloromethane (0-20% gradient), to give the title compound (0.2184 g, 67%). ES/MS m/z 436 (M+H).

Alternate Preparation 27

(66) Scheme 5, step B: Add 7 N ammonia in methanol (600 mL, 4.2 mol) to a stirred suspension of N-[(5S,7aS)-5-(1,1-difluoroethyl)-7a-{2-fluoro-5-[(trifluoroacetyl)amino]phenyl}-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]benzamide (250 g, 80% purity, 376.3 mmol) in methanol (200 mL) at room temperature and stir at ambient temperature for 18 hours. Evaporate to dryness to give the title compound as a brown gum (190 g, 375.2 mmol, 86% purity). ES/MS: m/z 436.0 [M+H].

Preparation 28

(4aS,5S,7aS)-7a-(5-Amino-2-fluorophenyl)-5-(1,1-difluoroethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

(67) ##STR00041##

(68) Scheme 4, step A: Dissolve N-[(4aS,5S,7aS)-7a-(5-amino-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide (216.4 g, 88% purity, 435.9 mmol) in pyridine (400 mL), ethanol (100 mL) and THF (300 mL). Add O-methylhydroxylamine hydrochloride (190 g, 2275.0 mmol) and stir at ambient temperature for 18 hours. Dilute with 2-methyltetrahydrofuran (1 L) and wash with water (2300 mL). Isolate the organic layer and add 35% aqueous ammonium hydroxide (100 mL) to the aqueous. Extract with 2-methyltetrahydrofuran (300 mL) then saturate with sodium chloride and extract with 2-methyltetrahydrofuran (2300 mL). Combine the organic extracts, wash with brine (300 mL), and evaporate to a residue. Dissolve in methanol (200 mL), add 7 N ammonia in methanol (100 mL, 700 mmol) and stir at room temperature for 18 hours. Further ammonia can be added if any trifluoracetamide impurity remains. Remove the solvent under reduced pressure and dissolve the residue in aqueous 2 N aqueous hydrochloric acid (1.5 L). Extract with dichloromethane (6500 mL), combine the organic layers and remove the solvent under reduced pressure to a total volume of about 1 L. Wash with 2 N aqueous hydrochloric acid (300 mL) and combine all aqueous washings. Add 2-methyltetrahydrofuran (1 L) and stir vigorously while adjusting the pH to basic with sodium bicarbonate until no gas evolution is observed. Separate the layers and extract the aqueous with 2-methyltetrahydrofuran (2500 mL). Dry the combined organic extracts with magnesium sulfate, filter, and evaporate to give a brown solid. Purify the residue by silica gel chromatography eluting with 0-100% dichloromethane in THF. Evaporate the product containing fractions with ethyl acetate/heptane to give the title compound as a fine beige powder (106 g, 70%, 95% purity). ES/MS: m/z 332.0 [M+H], [].sub.D.sup.20=+42.11 (C=0.532, chloroform).

Preparation 29

N-[3-[(4aS,5S,7aS)-2-Benzamido-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide

(69) ##STR00042##

(70) Scheme 3, Step A: Add N,N-diisopropylethylamine (0.032 mL, 0.1837 mmol) to a mixture of N-[(4as,5s,7as)-7a-(5-amino-2-fluoro-phenyl)-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-2-yl]benzamide (0.040 g, 0.09185 mmol), 5-cyanopyridine-2-carboxylic acid (0.0203 g, 0.1378 mmol) and 1-hydroxy-7-azabenzotriazole (0.0191 g, 0.1378 mmol) in dichloromethane (2 ml) and dimethylformamide (0.5 mL). Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.026 g, 0.1378 mmol) in one portion. Stir the reaction mixture at ambient temperature for 18 hours. Dilute with ethyl acetate, and wash with water and brine. Extract with ethyl acetate. Combine the organic extracts and dry over sodium sulfate. Filter and concentrate under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with methyl-tert-butyl ether:dichloromethane (0-10% gradient), to give the title compound (0.0465 g, 90%). ES/MS m/z 566 (M+1).

EXAMPLE 1

N-[3-[(4aS,5S,7aS)-2-Amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide

(71) ##STR00043##

(72) Scheme 3, Step B; Heat at 50 C. for 18 hours a mixture of N-[3-[(4aS,5S,7aS)-2-benzamido-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide (0.0465 g, 0.0822 mmol), O-methylhydroxylamine hydrochloride (0.0687 g, 0.8220 mmol) and pyridine (0.066 ml, 0.8220 mmol) in THF (1.5 mL) and ethanol (1.5 mL). Concentrate the mixture under reduced pressure to give a residue. Purify the residue by silica gel chromatography, eluting with 7 N NH.sub.3 in methanol:dichloromethane (0-2% gradient), to give the title compound (0.026 g, 68%). ES/MS m/z 462 (M+1).

EXAMPLE 1A

N-[3-[(4aS,5S,7aS)-2-Amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide 4-methylbenzenesulfonate hemihydrate (1:1:0.5)

(73) ##STR00044##

(74) Add N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide (150 mg, 0.33 mmol) and THF (2 mL) together and stir at room temperature to dissolve. Add p-toluenesulfonic acid hydrate (0.095 g, 0.5 mmol) and heat the solution to 50 C. Add water in 200 microliter aliquots and observe precipitation after about 2 mL total addition. Stir at 50 C. for several hours to give a thick suspension. Add additional THF (1 mL) to improve mixing. Cool to room temperature over a few hours and filter by vacuum filtration. Wash with minimal THF. Allow to air dry overnight to give the title compound.

Alternate Preparation Example 1a

N-[3-[(4aS,5S,7aS)-2-Amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide 4-methylbenzenesulfonate hemihydrate (1:1:0.5)

(75) Add N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide (1.5 g, 3.3 mmol) and THF (12 mL) together and stir at room temperature to dissolve. Heat to 60 C. and add p-toluenesulfonic acid hydrate (0.75 g, 3.96 mmol) and water (5 mL). A white precipitate forms after 5 minutes of stirring. Stir at 60 C. for several hours to give a thick suspension. Cool to room temperature over a few hours and filter by vacuum filtration. Allow to air dry overnight to give the title compound.

(76) X-Ray Powder Diffraction (XRD)

(77) The XRD patterns of crystalline solids are obtained on a Bruker D4 Endeavor X-ray powder diffractometer, equipped with a CuKa source =1.54060 ) and a Vantec detector, operating at 35 kV and 50 mA. The sample is scanned between 4 and 40 in 20, with a step size of 0.009 in 2 and a scan rate of 0.5 seconds/step, and with 0.6 mm divergence, 5.28 fixed anti-scatter, and 9.5 mm detector slits. The dry powder is packed on a quartz sample holder and a smooth surface is obtained using a glass slide. The crystal form diffraction patterns are collected at ambient temperature and relative humidity. It is well known in the crystallography art that, for any given crystal form, the relative intensities of the diffraction peaks may vary due to preferred orientation resulting from factors such as crystal morphology and habit. Where the effects of preferred orientation are present, peak intensities are altered, but the characteristic peak positions of the polymorph are unchanged. See, e.g., The United States Pharmacopeia #23, National Formulary #18, pages 1843-1844, 1995. Furthermore, it is also well known in the crystallography art that for any given crystal form the angular peak positions may vary slightly. For example, peak positions can shift due to a variation in the temperature or humidity at which a sample is analyzed, sample displacement, or the presence or absence of an internal standard. In the present case, a peak position variability of 0.2 in 2 will take into account these potential variations without hindering the unequivocal identification of the indicated crystal form. Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks (in units of 2), typically the more prominent peaks. The crystal form diffraction patterns, collected at ambient temperature and relative humidity, are adjusted based on NIST 675 standard peaks at 8.853 and 26.774 2-theta.

(78) A prepared sample of crystalline N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide 4-methylbenzenesulfonate hemihydrate (1:1:0.5) is characterized by an XRD pattern using CuKa radiation as having diffraction peaks (2-theta values) as described in Table 1, and in particular having peaks at 6.80 in combination with one or more of the peaks selected from the group consisting of 19.7, 14.9, and 10.3; with a tolerance for the diffraction angles of 0.2 degrees.

(79) TABLE-US-00001 TABLE 1 X-ray powder diffraction peaks of the crystalline Example 1a Example 1a Peak Positions Angle (2-Theta) +/ Relative Intensity Peak 0.2 (% of most intense peak) 1 5.9 12.5% 2 6.8 100.0% 3 10.3 17.2% 4 14.9 18.7% 5 18.8 3.7% 6 19.7 46.5% 7 21.0 14.3% 8 24.5 4.8% 9 28.6 8.1% 10 39.7 5.1%

EXAMPLE 1B

N-[3-[(4aS,5S,7aS)-2-Amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide methanesulfonate

(80) ##STR00045##

(81) Add N-[3-[(4aS,5S,7aS)-2-amino-5-(1,1-difluoroethyl)-4,4a,5,7-tetrahydrofuro[3,4-d][1,3]thiazin-7a-yl]-4-fluoro-phenyl]-5-cyano-pyridine-2-carboxamide (150 mg, 0.33 mmol) and THF (2 mL) together and stir at room temperature to dissolve. Add methanesulfonic acid (0.095 g, 0.5 mmol) and heat the solution to 50 C. Add water in 200 microliter aliquots up to 2 mL total addition. Stir at 25 C. and precipitation is not observed. Concentrate under nitrogen to volume and a precipitate is observed. Heat the suspension to 60 C. and a clear solution is observed after about 10 minutes. Heat at 60 C. for 1 hour. Cool to room temperature to give a white suspension and stir the mixture for several hours. Isolate the solid by vacuum filtration and wash with a minimal amount of water. Allow to air dry overnight to give the title compound as a crystalline solid.

(82) In Vitro Assay Procedures:

(83) To assess selectivity of BACE1 over BACE2, the test compound is evaluated in FRET and immunoassay detection base enzymatic assays using specific substrates for BACE1 and BACE2 as described below. For in vitro enzymatic and cellular assays, the test compound is prepared in DMSO to make up a 10 mM stock solution. The stock solution is serially diluted in DMSO to obtain a ten-point dilution curve with final compound concentrations ranging from 10 M to 0.05 nM in a 96-well round-bottom plate before conducting the in vitro enzymatic and whole cell assays.

(84) In Vitro Protease Inhibition Assays:

Expression of huBACE1:Fc and huBACE2:Fc

(85) Human BACE1 (accession number: AF190725) and human BACE2 (accession number: AF 204944) are cloned from total brain cDNA by RT-PCR. The nucleotide sequences corresponding to amino acid sequences #1 to 460 are inserted into the cDNA encoding human IgG.sub.1 (Fc) polypeptide (Vassar et al., Science, 286, 735-742 (1999)). This fusion protein of BACE1(1-460) or BACE2(1-460) and human Fc, named huBACE1:Fc and huBACE2:Fc respectively, are constructed in the pJB02 vector. Human BACE1(1-460):Fc (huBACE1:Fc) and human BACE2(1-460):Fc (huBACE2:Fc) are transiently expressed in HEK293 cells. cDNA (250 g) of each construct are mixed with Fugene 6 and added to 1 liter HEK293 cells. Four days after the transfection, conditioned media are harvested for purification. huBACE1:Fc and huBACE2:Fc are purified by Protein A chromatography as described below. The enzymes are stored at 80 C. in small aliquots. (See Yang, et. al., J. Neurochemistry, 91(6) 1249-59 (2004).

Purification of huBACE1:Fc and huBACE2:Fc

(86) Conditioned media of HEK293 cells transiently transfected with huBACE1:Fc or huBACE2:Fc cDNA are collected. Cell debris is removed by filtering the conditioned media through 0.22 m sterile filter. 5 ml Protein A-agarose (bed volume) is added to 4 liter conditioned media. This mixture is gently stirred overnight at 4 C. The Protein A-agarose resin is collected and packed into a low-pressure chromatography column. The column is washed with 20 bed volumes of PBS at a flow rate 20 ml per hour. Bound huBACE1:Fc or huBACE2:Fc protein is eluted with 50 mM acetic acid, pH 3.6, at flow rate 20 ml per hour. 1 ml fractions of eluent are neutralized immediately with ammonium acetate (0.5 ml 200 mM), pH 6.5. The purity of final product is assessed by electrophoresis in 4-20% Tris-Glycine SDS-PAGE. The enzyme is stored at 80 C. in small aliquots.

BACE1 FRET Assay

(87) Serial dilutions of the test compound are prepared as described above. The compound is further diluted 20 in KH.sub.2PO.sub.4 buffer. 10 L of each dilution is added to each well on row A to H of a corresponding low protein binding black plate containing the reaction mixture (25 L of 50 mM KH.sub.2PO.sub.4, pH 4.6, 1 mM TRITON X-100, 1 mg/mL BSA, and 15 M of FRET substrate based upon the sequence of APP) (See Yang, et. al., J. Neurochemistry, 91(6) 1249-59 (2004)). The content is mixed well on a plate shaker for 10 minutes. 15 L of 200 pM human BACE1(1-460):Fc (See Vasser, et al., Science, 286, 735-741 (1999)) in the KH.sub.2PO.sub.4 buffer is added to the plate containing substrate and the test compound to initiate the reaction. The RFU of the mixture at time 0 is recorded at excitation wavelength 355 nm and emission wavelength 460 nm, after brief mixing on a plate shaker. The reaction plate is covered with aluminum foil and kept in a dark humidified oven at room temperature for 16 to 24 hours. The RFU at the end of incubation is recorded with the same excitation and emission settings used at time 0. The difference of the RFU at time 0 and the end of incubation is representative of the activity of BACE1 under the compound treatment. RFU differences are plotted versus inhibitor concentration and a curve is fitted with a four-parameter logistic equation to obtain the IC.sub.50 value. (May, et al., Journal of Neuroscience, 31, 16507-16516 (2011)).

(88) The compound of Example 1 is tested essentially as described above and exhibits an IC.sub.50 for BACE1 of 0.509 nM0.104, n=4 (Meanstandard deviation of the mean). This data demonstrates that the compound of Example 1 inhibits purified recombinant BACE1 enzyme activity in vitro.

BACE2 MBP-C125Swe Assay

(89) 10 point serial dilutions of test compounds are prepared in the appropriate range. Compounds are further diluted 6 in ammonium acetate assay buffer (50 mmol ammonium acetate, pH 4.6, 1 mM Triton X-100, 1 mg/mL BSA). 10 L of each dilution is added to each well on row A to H of a corresponding low protein binding plate to which 10 L of an affinity purified Escherichia coli derived substrate (MBPC125swe, 1 g/mL) for BACE2 activity are pre-added. The content is mixed well on a plate shaker for 10 minutes. 10 L of 200 picomolar human BACE2 (1-460):Fc in the same reaction buffer described above is added to the plate containing substrate and test compounds to initiate the reaction. After 4 hours, the reaction is stopped by adding stop buffer (40 L). The amount of product is measured by ELISA using MBP-C26swe standard. Anti-MBP antibody is immobilized on the surface of a high binding polystyrene plate and blocked using a casein/PBS blocking buffer. Sample or standard (40 L) is added to the ELISA plate and incubated at 4 C. overnight. The plates are then washed and 40 L of the cleavage specific detection antibody (GN405) is added and allowed to sit for one hour at room temperature. Unbound GN405 is then removed by washing and 40 L of goat anti-rabbit-HRP conjugate (Southern Biotech, 4010-05) is added to the plate and allowed to sit for 1 hour at room temperature. The plate is again washed and TMB substrate (40 L) is added. The corresponding amount of product released is a measure of BACE2 activity in the solution at any tested concentration of inhibitor. The 10-point inhibition curve is plotted and fitted with the four-parameter logistic equation to obtain the EC.sub.50 and IC.sub.50 values. (See: Sinha, et al., Nature, 402, 537-540 (2000)).

(90) The compound of Example 1 is tested essentially as described above and exhibits a BACE2 IC.sub.50 of 17.6 nM7.4, n=6 (Meanstandard deviation of the mean). The ratio of BACE1 (FRET IC.sub.50 enzyme assay) to BACE2 (MBP-C125Swe cell assay) is approximately 35-fold, indicating functional selectivity for inhibiting the BACE1 enzyme. The data set forth above demonstrates that the compound of Example 1 is selective for BACE1 over BACE2.

SH-SY5YAPP695Wt Whole Cell Assay

(91) The routine whole cell assay for the measurement of inhibition of BACE1 activity utilizes the human neuroblastoma cell line SH-SY5Y (ATCC Accession No. CRL2266) stably expressing a human APP695Wt cDNA. Cells are routinely used up to passage number 6 and then discarded.

(92) SH-SY5YAPP695Wt cells are plated in 96 well tissue culture plates at 5.010.sup.4 cells/well in 200 L culture media (50% MEM/EBSS and Ham's F12, lx each sodium pyruvate, non-essential amino acids and NaHCO.sub.3 containing 10% FBS). The following day, media is removed from the cells, fresh media added then incubated at 37 C. for 24 hours in the presence/absence of test compound at the desired concentration range.

(93) At the end of the incubation, conditioned media are analyzed for evidence of beta-secretase activity by analysis of Abeta peptides 1-40 and 1-42 by specific sandwich ELISAs. To measure these specific isoforms of Abeta, monoclonal 2G3 is used as a capture antibody for Abeta 1-40 and monoclonal 21F12 as a capture antibody for Abeta 1-42. Both Abeta 1-40 and Abeta 1-42 ELISAs use biotinylated 3D6 as the reporting antibody (for description of antibodies, see Johnson-Wood, et al., Proc. Natl. Acad. Sci. USA 94, 1550-1555 (1997)). The concentration of Abeta released in the conditioned media following the compound treatment corresponds to the activity of BACE1 under such conditions. The 10-point inhibition curve is plotted and fitted with the four-parameter logistic equation to obtain the IC.sub.50 values for the Abeta-lowering effect.

(94) The compound of Example 1 is tested essentially as described above and exhibits an IC.sub.50 of 0.157 nM0.048, n=4 for SH-SYSYAPP695Wt A-beta (1-40) ELISA and an IC.sub.50 of 0.177 nM0.050, n=4 for SH-SY5YAPP695Wt A-beta (1-42) ELISA (Meanstandard deviation of the mean). The data set forth above demonstrates that the compound of Example 1 inhibits BACE1 in the whole cell assay.

In Vivo Inhibition of Beta-Secretase

(95) Several animal models, including mouse, guinea pig, dog, and monkey, may be used to screen for inhibition of beta-secretase activity in vivo following compound treatment. Animals used in this invention can be wild type, transgenic, or gene knockout animals. For example, the PDAPP mouse model, prepared as described in Games et al., Nature 373, 523-527 (1995), and other non-transgenic or gene knockout animals are useful to analyze in vivo inhibition of Abeta and sAPPbeta production in the presence of inhibitory compounds. Generally, 2 month old PDAPP mice, gene knockout mice or non-transgenic animals are administered compound formulated in vehicles, such as corn oil, beta-cyclodextran, phosphate buffers, PHARMASOLVE, or other suitable vehicles via oral, subcutaneous, intra-venous, feeding, or other route of administration. 1 to 24 hours following the administration of compound, animals are sacrificed, and brains are removed for analysis of Abeta 1-x. Abeta 1-x as used herein refers to the sum of Abeta species that begin with residue 1 and end with a C-terminus greater than residue 28. This detects the majority of Abeta species and is often called total Abeta. Total Abeta peptides (Abeta 1-x) levels are measured by a sandwich ELISA, using monoclonal 266 as a capture antibody and biotinylated 3D6 as reporting antibody. (See May, et al., Journal of Neuroscience, 31, 16507-16516 (2011)).

(96) For acute studies, compound or appropriate vehicle is administered and animals are sacrificed at about 3 hours after dosing. Brain tissue, is obtained from selected animals and analyzed for the presence of Abeta 1-x. After chronic dosing brain tissues of older APP transgenic animals may also be analyzed for the amount of beta-amyloid plaques following compound treatment.

(97) Animals (PDAPP or other APP transgenic or non-transgenic mice) administered an inhibitory compound may demonstrate the reduction of Abeta in brain tissues, as compared with vehicle-treated controls or time zero controls. For example, a 0.1, 0.3, and 1 mg/kg oral dose of Example 1, to young female PDAPP mice reduced Abeta 1-x peptide levels in brain hippocampus by 32%, 40%, and 55% (all values p<0.01), respectively. In brain cortical tissue, doses of 0.1, 0.3, and 1 mg/kg of Example 1 reduced Abeta 1-x levels by 38%, 50%, and 67% (all values p<0.01) compared to vehicle-treated mice three hours after dosing.

(98) Given the activity of the compound of Example 1 against the BACE1 enzyme in vitro, these Abeta-lowering effects are consistent with BACE1 inhibition in vivo, and further demonstrate CNS penetration of the compound of Example 1.

(99) These studies show that compounds of the present invention inhibit BACE1 and are, therefore, useful in reducing Abeta levels.