DECARBOXYLASE INHIBITORS FOR TREATING PARKINSON'S DISEASE

Abstract

Provided are inhibitors of pathogenic, bacterial metabolite production and conjugates of the inhibitors. Also provided are pharmaceutical compositions containing the inhibitors or conjugates and methods of using the same.

Claims

1. A compound chosen from the following compounds ##STR00069## ##STR00070## ##STR00071## ##STR00072## and pharmaceutically acceptable salts thereof.

2. A compound chosen from the following compounds ##STR00073## ##STR00074## and pharmaceutically acceptable salts thereof.

3. A compound of formula (I): ##STR00075## or a pharmaceutically acceptable salt thereof, wherein n is 0 or 1; R.sup.1 is H or —OR.sup.A, wherein R.sup.A is H, —C(O)C.sub.1-6 alkyl, or an acylated sugar; R.sup.2 is H, halogen, amino, C.sub.1-6 alkyl, or —OR.sup.A, wherein R.sup.A is H or an acylated sugar; R.sup.3 is H, a halogen, —OH, or C.sub.1-6 alkyl optionally substituted with one or more halogens; R.sup.4 is H, —NH.sub.2, —C(O)OCH.sub.3, or an acylated sugar; R.sup.5 is H, —C(O)OH, —C(O)OC.sub.1-6 alkyl, —C(O)Oglycoside, —C(O)NHOH, or —C(O)O(acylated sugar); and R.sup.6 is H, halogen, or optionally substituted C.sub.1-6 alkyl; provided that at least one R.sup.A is present; or provided that R.sup.3 and/or R.sup.6 comprise a halogen.

4. The compound of formula (I) according to claim 3, or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (I-a): ##STR00076##

5. The compound of formula (I) according to any one of claims 3 and 4, or a pharmaceutically acceptable salt thereof, wherein n is 0 or 1; R.sup.1 is H, —C(O)C.sub.1-6 alkyl, or —OR.sup.A, wherein R.sup.A is H or an acylated sugar; R.sup.2 is H, or —OR.sup.A, wherein R.sup.A is H or an acylated sugar; R.sup.3 is H, or a halogen; R.sup.4 is H, —NH.sub.2, or an acylated sugar; R.sup.5 is —C(O)OH, —C(O)OC.sub.1-6 alkyl, —C(O)Oglycoside, or —C(O)O(acylated sugar); and R.sup.6 is H or optionally substituted C.sub.1-6 alkyl; provided that at least one R.sup.A is present; or provided that R.sup.3 and/or R.sup.6 comprise a halogen.

6. The compound of formula (I) according to any one of claims 3 to 5, or a pharmaceutically acceptable salt thereof, wherein R.sup.1 is —OR.sup.A.

7. The compound of formula (I) according to any one of claims 3 to 6, or a pharmaceutically acceptable salt thereof, wherein R.sup.2 is H or —OR.sup.A.

8. The compound of formula (I) according to any one of claims 3 to 7, wherein each R.sup.A is H.

9. The compound of formula (I) according to any one of claims 3 and 4, or a pharmaceutically acceptable salt thereof, wherein R.sup.2 is a halogen.

10. The compound of formula (I) according to any one of claims 3 to 9, or a pharmaceutically acceptable salt thereof, wherein R.sup.3 is fluoro or chloro.

11. The compound of formula (I) according to any one of claims 3 to 9, or a pharmaceutically acceptable salt thereof, wherein R.sup.3 is H.

12. The compound of formula (I) according to any one of claims 3 to 11, or a pharmaceutically acceptable salt thereof, wherein R.sup.4 is H.

13. The compound of formula (I) according to any one of claims 3 to 11, or a pharmaceutically acceptable salt thereof, wherein R.sup.4 is —NH.sub.2.

14. The compound of formula (I) according to any one of claims 3 to 13, or a pharmaceutically acceptable salt thereof, wherein R.sup.5 is —C(O)OH.

15. The compound of formula (I) according to any one of claims 3 to 13, or a pharmaceutically acceptable salt thereof, wherein R.sup.5 is —C(O)Oacylated sugar.

16. The compound of formula (I) according to any one of claims 3, 4, and 6 to 13, or a pharmaceutically acceptable salt thereof, wherein R.sup.5 is H.

17. The compound of formula (I) according to any one of claims 3 to 16, or a pharmaceutically acceptable salt thereof, wherein R.sup.6 is H.

18. The compound of formula (I) according to any one of claims 3 to 16, or a pharmaceutically acceptable salt thereof, wherein R.sup.6 is a C.sub.1-6 alkyl.

19. The compound of formula (I) according to any one of claims 3 to 16, or a pharmaceutically acceptable salt thereof, wherein R.sup.6 is a C.sub.1-6 alkyl substituted with one, two, or three halogens.

20. The compound of formula (I) according to any one of claims 3 to 16, or a pharmaceutically acceptable salt thereof, wherein R.sup.6 is a C.sub.1-6 alkyl substituted with one, two, or three fluorine atoms.

21. The compound of formula (I) according to any one of claims 3 to 20, or a pharmaceutically acceptable salt thereof, wherein n is 0.

22. The compound of formula (I) according to any one of claims 3 to 20, or a pharmaceutically acceptable salt thereof, wherein n is 1.

23. The compound of formula (I) according to any one of claims 3 and 4, or a pharmaceutically acceptable salt thereof, wherein n is 0; R.sup.1 is —OH; R.sup.2 is halogen; R.sup.3 is H, a halogen, or —OH, C.sub.1-6 alkyl optionally substituted with one or more halogens; R.sup.4 is H, —NH.sub.2, or an acylated sugar; R.sup.5 is H, —C(O)OH, —C(O)OC.sub.1-6 alkyl, —C(O)Oglycoside, —C(O)NHOH, or —C(O)O(acylated sugar); and R.sup.6 is H or optionally substituted C.sub.1-6 alkyl.

24. The compound of formula (I) according to anyone of claims 3, 4, and 23, or a pharmaceutically acceptable salt thereof, wherein n is 0; R.sup.1 is —OH; R.sup.2 is halogen; R.sup.3 is H; R.sup.4 is H; R.sup.5 is —C(O)OH; and R.sup.6 is optionally substituted alkyl.

25. A pharmaceutical composition comprising at least one pharmaceutically acceptable excipient and at least one entity chosen from the compounds according to any one of claims 1 to 24 and pharmaceutically acceptable salts thereof.

26. A method of treating Parkinson's disease comprising administering to a subject in need thereof a therapeutically effective amount of at least one entity chosen from the compounds according to any one of claims 1 to 24 and pharmaceutically acceptable salts thereof or a pharmaceutical composition according to claim 25.

27. A method of inhibiting a decarboxylase-mediated conversion of L-DOPA to L-dopamine comprising contacting the decarboxylase with at least one entity chosen from the compounds according to any one of claims 1 to 24 and pharmaceutically acceptable salts thereof or a pharmaceutical composition according to claim 25.

28. The method of claim 27, wherein the decarboxylase is tyrosine decarboxylase.

Description

EXAMPLES

Example 1: Preparation of Exemplary Compounds

[0229] ##STR00044##

Compound A: (S)-2-(4-acetoxybenzyl)-2-amino-3-fluoropropanoic Acid

[0230] (2S)-2-amino-3-fluoro-2-[(4-hydroxyphenyl)methyl]propanoic acid (1 equiv), is treated with Na.sub.2CO.sub.3, acetic anhydride to afford the title compound (S)-2-(4-acetoxybenzyl)-2-amino-3-fluoropropanoic acid.

##STR00045##

Compound B: (S)-2-amino-3-fluoro-2-(4-(propionyloxy)benzyl)propanoic Acid

[0231] (2S)-2-amino-3-fluoro-2-[(4-hydroxyphenyl)methyl]propanoic acid (1 equiv), is treated with Na.sub.2CO.sub.3, propionic anhydride to afford the title compound (S)-2-amino-3-fluoro-2-(4-(propionyloxy)benzyl)propanoic acid.

##STR00046##

Compound C: (S)-2-amino-2-(4-(butyryloxy)benzyl)-3-fluoropropanoic Acid

[0232] (2S)-2-amino-3-fluoro-2-[(4-hydroxyphenyl)methyl]propanoic acid (1 equiv), is treated with Na.sub.2CO.sub.3, butyric anhydride to afford the title compound (S)-2-amino-2-(4-(butyryloxy)benzyl)-3-fluoropropanoic acid.

##STR00047##

Compound D: (S)-3-(4-acetoxyphenyl)-2-amino-2-methylpropanoic Acid

[0233] ((2R)-2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid (1 equiv), is treated with Na.sub.2CO.sub.3, acetic anhydride to afford the title compound (S)-3-(4-acetoxyphenyl)-2-amino-2-methylpropanoic acid.

##STR00048##

Compound E: (S)-2-amino-2-methyl-3-(4-(propionyloxy)phenyl)propanoic Acid

[0234] (((2R)-2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid (1 equiv), is treated with Na.sub.2CO.sub.3, propionic anhydride to afford the title compound (S)-2-amino-2-methyl-3-(4-(propionyloxy)phenyl)propanoic acid

##STR00049##

Compound F: (2S,3R,4S,5R)-2-(((S)-2-amino-2-(4-(butyryloxy)benzyl)-3-fluoropropanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tributyrate

[0235] (2S)-2-amino-3-fluoro-2-[(4-hydroxyphenyl)methyl]propanoic acid (1 equiv), is treated with 1 eq of Na.sub.2CO.sub.3 and butyric anhydride and the corresponding butyric acid will be DCC coupled to (2S,3S,4R,5S)-2-hydroxytetrahydro-2H-pyran-3,4,5-triyl tributyrate (which can be synthesized from (2S,3S,4R,5S)-tetrahydro-2H-pyran-2,3,4,5-tetraol) to afford the title compound (2S,3R,4S,5R)-2-(((S)-2-amino-2-(4-(butyryloxy)benzyl)-3-fluoropropanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tributyrate.

##STR00050##

Compound G: 4-((S)-2-amino-2-(fluoromethyl)-3-oxo-3-(((2S,3R,4S,5R)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)propyl)phenyl Butyrate

[0236] (2S)-2-amino-3-fluoro-2-[(4-hydroxyphenyl)methyl]propanoic acid (1 equiv) is treated with 1 equivalent of Na.sub.2CO.sub.3 and butyric anhydride, and the corresponding butyric acid will be DCC coupled to (2S,3S,4R,5S)-2-hydroxytetrahydro-2H-pyran-3,4,5-triyl acetate (which can be synthesized from (2S,3S,4R,5S)-tetrahydro-2H-pyran-2,3,4,5-tetraol). This material is then treated with dilute lithium hydroxide in water to afford the title compound.

##STR00051##

Compound H: (2S,3R,4S,5R)-2-(((S)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl Tributyrate

[0237] (2S)-2-amino-3-fluoro-2-[(4-hydroxyphenyl)methyl]propanoic acid (1 equiv), is treated with 1 eq of BnBr, K.sub.2CO.sub.3 in THF and the corresponding benzyl acid will be DCC coupled to (2S,3S,4R,5S)-2-hydroxytetrahydro-2H-pyran-3,4,5-triyl tributyrate (which can be synthesized from (2S,3S,4R,5S)-tetrahydro-2H-pyran-2,3,4,5-tetraol) and hydrogenated with Pd(OH).sub.2/H.sub.2, to afford the title compound hydrogenated with Pd(OH).sub.2/H.sub.2 to afford the title compound (2S,3R,4S,5R)-2-(((S)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tributyrate.

##STR00052##

Compound I: (2S,3R,4S,5R)-2-(((S)-2-amino-3-(4-(butyryloxy)phenyl)-2-methylpropanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl Tributyrate

[0238] ((2R)-2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid (1 equiv), is treated with 1 eq of is treated with 1 eq of Na.sub.2CO.sub.3 and butyric anhydride and the corresponding carboxylic acid will be DCC coupled to (2S,3S,4R,5S)-2-hydroxytetrahydro-2H-pyran-3,4,5-triyl tributyrate (which can be synthesized from (2S,3S,4R,5S)-tetrahydro-2H-pyran-2,3,4,5-tetraol) to afford the title compound (2S,3R,4S,5R)-2-(((S)-2-amino-3-(4-(butyryloxy)phenyl)-2-methylpropanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tributyrate.

##STR00053##

Compound J: (2S,3R,4S,5R)-2-(((S)-2-amino-3-(4-hydroxyphenyl)-2-methylpropanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl Tributyrate

[0239] ((2R)-2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid (1 equiv), is treated with 1 eq of BnBr, K.sub.2CO.sub.3 in THF and the corresponding benzyl acid will be DCC coupled to (2S,3S,4R,5S)-2-hydroxytetrahydro-2H-pyran-3,4,5-triyl tributyrate (which can be synthesized from (2S,3S,4R,5S)-tetrahydro-2H-pyran-2,3,4,5-tetraol) and hydrogenated with Pd(OH).sub.2/H.sub.2, to afford the title compound (2S,3R,4S,5R)-2-(((S)-2-amino-3-(4-hydroxyphenyl)-2-methylpropanoyl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tributyrate.

##STR00054##

Compound K: 2-amino-3,3-difluoro-2-(4-hydroxybenzyl)propanoic Acid

Step 1:

[0240] To a solution of LDA (2 M, 60.18 mL, 2 eq, THF) in THF (50 mL) was added 2-(4-methoxyphenyl)acetic acid (10 g, 60.18 mmol, 1 eq) in THF (50 mL) at −70° C. and the mixture was stirred at 0° C. for 3 h. Then the mixture was cooled to −70° C. and ethyl 2,2-difluoroacetate (8.21 g, 66.20 mmol, 1.1 eq) in THF (50 mL) was added to the mixture at −70° C. and stirred at −70° C. for 2 h. The reaction mixture was quenched by addition 1N HCl 150 mL at 0° C., and then extracted with EtOAc 300 mL (100 mL*3). The combined organic layers were washed with sat. NaHCO.sub.3150 mL (50 mL*3) and brine 100 mL (50 mL*2), dried over Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/0 to 0/1) to give 1,1-difluoro-3-(4-methoxyphenyl)propan-2-one (2.3 g, 11.49 mmol, 19.09% yield) as yellow liquid.

Step 2:

[0241] A mixture of 1,1-difluoro-3-(4-methoxyphenyl)propan-2-one (2.3 g, 11.49 mmol, 1 eq) and (NH.sub.4).sub.2CO.sub.3 (5.19 g, 54.00 mmol, 5.77 mL, 4.7 eq) in EtOH (12 mL) and H.sub.2O (8 mL) was stirred at 55° C., degassed and purged with N.sub.2 3 times, and then NaCN (608.12 mg, 12.41 mmol, 1.08 eq) was added to the mixture and stirred at 55° C. for 21 hr under N.sub.2 atmosphere. Then the mixture was stirred at 90° C. for 0.5 hr. The reaction mixture was diluted with H.sub.2O 20 mL and extracted with EtOAc 120 mL (20 mL*6). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/0 to 0/1) to give 5-(difluoromethyl)-5-[(4-methoxyphenyl)methyl]imidazolidine-2,4-dione (1.9 g, 5.98 mmol, 52.02% yield, 85% purity) as a yellow solid.

Step 3:

[0242] A mixture of 5-(difluoromethyl)-5-[(4-methoxyphenyl)methyl]imidazolidine-2,4-dione (1.8 g, 6.66 mmol, 1 eq) in aq. HBr (18 mL, 48%) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 110° C. for 5 hr under N.sub.2 atmosphere. The reaction mixture was washed with EtOAc 30 mL (10 mL*3). The aqueous phase was concentrated under reduced pressure to give a residue. The residue was used sat. NaHCO.sub.3 to adjust pH to 7˜8, then 6 M HCl was added to the mixture and the pH was adjusted to 3˜4. The residue was purified by prep-HPLC (column: Welch Xtimate C18 150*25 mm*5 um; mobile phase: [water (0.04% HCl)-ACN]; B %: 1%-5%, 10 min) to give 2-amino-3,3-difluoro-2-[(4-hydroxyphenyl)methyl]propanoic acid (56 mg, 202.95 umol, 3.05% yield, 97% purity, HCl) as a white solid. LCMS m/z=232.1. .sup.1H NMR (400 MHz, DMSO-d6) δ 9.26 (s, 1H), 7.37 (br s, 1H), 7.06 (d, J=9.6 Hz, H), 6.63 (d, J=9.0 Hz, 2H), 6.12 (t, J=32.8 Hz, 1H), 3.03 (d, J=13.6 Hz, 1H), 2.67 (d, J=13.6 Hz, 1H).

##STR00055##

Compound L: 2-amino-2-(3-chloro-4-hydroxybenzyl)-3-fluoropropanoic Acid

Step 1:

[0243] To a solution of 4-(bromomethyl)-2-chloro-1-methoxy-benzene (3 g, 12.74 mmol, 1 eq) and 2-(benzhydrylideneamino)acetonitrile (1.84 g, 8.34 mmol, 6.55e-1 eq) in DCM (30 mL) was added benzyl(trimethyl)ammonium chloride (189.24 mg, 1.02 mmol, 176.86 uL, 0.08 eq), then aq. NaOH (10 M, 1.91 mL, 1.5 eq) was added dropwise at 0° C. The mixture was warmed to 25° C. and stirred for 12 hr. TLC indicated Reactant was consumed completely and two new spots formed. The reaction mixture was concentrated under reduced pressure to remove DCM (30 mL). The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to 10/1). Compound 2-(benzhydrylideneamino)-3-(3-chloro-4-methoxy-phenyl)propanenitrile (2.4 g, 6.40 mmol, 50.26% yield) was obtained as a yellow oil.

Step 2:

[0244] To a solution of 2-(benzhydrylideneamino)-3-(3-chloro-4-methoxy-phenyl)propanenitrile (2.4 g, 6.40 mmol, 1 eq) in THF (25 mL) was added fluoro(iodo)methane (5.12 g, 32.01 mmol, 5 eq) and KOtBu (3.59 g, 32.01 mmol, 5 eq). The mixture was stirred at 25° C. for 1 hr. LC-MS showed 2-(benzhydrylideneamino)-3-(3-chloro-4-methoxy-phenyl)propanenitrile was consumed completely and the desired MS was detected. The reaction mixture was filtered and filtrate concentrated under reduced pressure to give a residue. The residue was purified by prep-TLC (SiO.sub.2, Petroleum Ether: Ethyl Acetate=3:1). Compound 2-(benzhydrylideneamino)-2-[(3-chloro-4-methoxy-phenyl) methyl]-3-fluoro-propanenitrile (800 mg, 1.97 mmol, 30.71% yield) was obtained as yellow oil.

Step 3:

[0245] The mixture of 2-(benzhydrylideneamino)-2-[(3-chloro-4-methoxy-phenyl)methyl]-3-fluoro-propanenitrile (800 mg, 1.97 mmol, 1 eq) in aq. HBr (331.43 mg, 1.97 mmol, 222.44 uL, 48% purity, 1 eq) was stirred at 110° C. for 12 hr. LC-MS showed 2-(benzhydrylideneamino)-2-[(3-chloro-4-methoxy-phenyl)methyl]-3-fluoro-propanenitrile was consumed completely. The residue was diluted with H.sub.2O (10 mL) and extracted with EtOAc 15 mL (5 mL*3). The H.sub.2O phase was freeze-dried. The residue was purified by prep-HPLC (column: Phenomenex luna C.sub.18 250*50 mm*10 um; mobile phase: [water (0.1% TFA)-ACN]; B %: 1%-20%, 10 min) to give the crude product. The crude product in H.sub.2O (3 mL) was adjusted pH to 7˜8 with sat. NaHCO.sub.3 aq. then adjusted the pH to 3˜4 with 6M HCl. The aqueous phase was purified by purified by prep-HPLC (column: Phenomenex luna C.sub.18 250*50 mm*10 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 1%-20%, 10 min). Compound 2-amino-2-[(3-chloro-4-hydroxy-phenyl)methyl]-3-fluoro-propanoic acid (150 mg, 527.96 mol, 54.48% yield, HCl) was obtained as a white solid. LCMS m/z=248.0. .sup.1H NMR (400 MHz, DMSO-d6) δ 10.29 (br s, 1H), 7.21 (d, J=2.0 Hz, 1H), 7.00 (dd, J=8.4, 2.0 Hz, 1H), 6.92 (d, J=8.4 Hz, 1H), 4.80 (dd, J=46.8 9.8 Hz, 1H), 4.70 (dd, J=46.8, 9.8 Hz, 1H), 2.99 (d, J=14.4 Hz, 1H), 2.94 (d, J=14.4 Hz, 1H).

##STR00056##

Compound M: 2-amino-3-fluoro-2-(3-fluoro-4-hydroxybenzyl)propanoic Acid

Step 1:

[0246] To a solution of 4-(bromomethyl)-2-fluoro-1-methoxy-benzene (3 g, 13.70 mmol, 1.51 eq) and 2-(benzhydrylideneamino)acetonitrile (2 g, 9.08 mmol, 1 eq) in DCM (30 mL) was added benzyl(trimethyl)ammonium chloride (134.89 mg, 726.39 umol, 126.06 uL, 0.08 eq), then aq. NaOH (10 M, 1.36 mL, 1.5 eq) was added dropwise at 0° C. The mixture was warmed to 50° C. and stirred for 12 hr. TLC indicated 4-(bromomethyl)-2-fluoro-1-methoxy-benzene was not consumed completely and two new spots formed. The reaction mixture was concentrated under reduced pressure to remove DCM (30 mL). The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to 5/1). Compound 2-(benzhydrylideneamino)-3-(3-fluoro-4-methoxy-phenyl)propanenitrile (2.6 g, 7.25 mmol, 79.89% yield) was obtained as yellow oil.

Step 2:

[0247] To a solution of 2-(benzhydrylideneamino)-3-(3-fluoro-4-methoxy-phenyl)propanenitrile (2.6 g, 7.25 mmol, 1 eq) and fluoro(iodo)methane (5.80 g, 36.27 mmol, 5 eq) in THF (30 mL) was added KOtBu (4.07 g, 36.27 mmol, 5 eq, solid). The mixture was stirred at 25° C. for 1.5 hr. LC-MS showed 2-(benzhydrylideneamino)-3-(3-fluoro-4-methoxy-phenyl)propanenitrile was consumed completely and desired MS was detected. The reaction mixture was filtered and filtrate concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=15/1 to 5/1). Compound 2-(benzhydrylideneamino)-3-fluoro-2-[(3-fluoro-4-methoxy-phenyl)methyl]propanenitrile (1 g, 2.56 mmol, 35.31% yield) was obtained as a yellow oil.

Step 3:

[0248] The mixture of 2-(benzhydrylideneamino)-3-fluoro-2-[(3-fluoro-4-methoxy-phenyl) methyl]propanenitrile (600 mg, 1.54 mmol, 1 eq) in aq. HBr (4.47 g, 26.52 mmol, 3 mL, 48% purity, 17.26 eq) was stirred at 110° C. for 12 hr. LC-MS showed 2-(benzhydrylideneamino)-3-fluoro-2-[(3-fluoro-4-methoxy-phenyl)methyl]propanenitrile was consumed completely and one main peak with desired MS was detected. The reaction mixture was concentrated under reduced pressure to remove HBr (3 mL). Then adjust pH to 7-8 by saturated NaHCO.sub.3 aqueous and then adjust pH to 7-8 with 6N HCl. The aqueous phase was purified by prep-HPLC (column: Phenomenex luna C18 250*50 mm*10 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 1%-10%, 10 min). Compound 2-amino-3-fluoro-2-[(3-fluoro-4-hydroxy-phenyl)methyl]propanoic acid (77 mg, 277.61 umol, 18.06% yield, 96.5% purity, HCl) was obtained as a white solid. LCMS m/z=232.0. .sup.1H NMR (400 MHz, DMSO-d6) δ 10.01 (br s, 1H), 8.85 (br s, 3H), 7.04 (d, 12.4 Hz, 1H), 6.95 (t, J=8.4 Hz, 1H), 6.86 (d, J=8.4 Hz), 4.89 (dd, J=46.6, 10 Hz, 1H), 4.71 (dd, J=46.6, 10 Hz, 1H, 3.12-3.04 (m, 2H).

##STR00057##

Compound N: 2-amino-3-fluoro-2-(4-hydroxy-3-methylbenzyl)propanoic Acid

Step 1:

[0249] To a solution of 4-(chloromethyl)-1-methoxy-2-methyl-benzene (3 g, 17.58 mmol, 1 eq) in acetone (30 mL) was added NaI (5.27 g, 35.16 mmol, 2 eq) at 25° C. Then the mixture was stirred at 25° C. for 10 hr. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was diluted with H.sub.2O 10 mL and extracted with EtOAc 30 mL (10 mL*3). The combined organic layers were washed with brine 20 mL (10 mL*2) and aq. sodium thiosulfate 20 mL (10 mL*2), dried over Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/0 to 1/1) to give 4-(iodomethyl)-1-methoxy-2-methyl-benzene (4 g, 15.26 mmol, 86.81% yield) as a yellow liquid.

Step 2:

[0250] To a solution of 4-(iodomethyl)-1-methoxy-2-methyl-benzene (4 g, 15.26 mmol, 1.2 eq), 2-(benzhydrylideneamino)acetonitrile (2.80 g, 12.72 mmol, 1 eq) and N,N,N-trimethyl-1-phenylmethanaminium chloride (236.17 mg, 1.27 mmol, 220.72 uL, 0.1 eq) in DCM (40 mL) was added aq. NaOH (10 M, 2.29 mL, 1.8 eq) at 0° C. The mixture was stirred at 25° C. for 10 hr and stirred at 50° C. for 24 hr. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was diluted with H.sub.2O 15 mL and extracted with EtOAc 60 mL (20 mL*3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Agela DuraShell C18 250*80 mm*10 um; mobile phase: [water (0.04% NH.sub.3H.sub.2O+10 mM NH.sub.4HCO.sub.3)-ACN]; B %: 55%-85%, 20 min) to give 2-(benzhydrylideneamino)-3-(4-methoxy-3-methyl-phenyl)propanenitrile (1.9 g, 5.36 mmol, 42.15% yield) as yellow oil.

Step 3:

[0251] To a solution of 2-(benzhydrylideneamino)-3-(4-methoxy-3-methyl-phenyl)propanenitrile (0.5 g, 1.41 mmol, 1 eq) in THF (10 mL) was added t-BuOK (791.45 mg, 7.05 mmol, 5 eq) and fluoro(iodo)methane (2.26 g, 14.11 mmol, 10 eq). Then the mixture was stirred at 25° C. for 1 hr. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=1/0 to 5/1) to give 2-(benzhydrylideneamino)-2-(fluoromethyl)-3-(4-methoxy-3-methyl-phenyl)propanenitrile (0.45 g, 1.16 mmol, 82.54% yield) as yellow oil.

Step 4:

[0252] A mixture of 2-(benzhydrylideneamino)-2-(fluoromethyl)-3-(4-methoxy-3-methyl-phenyl) propanenitrile (0.44 g, 1.14 mmol, 1 eq) in aq. HBr (8 mL, 48%) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 110° C. for 10 hr under N.sub.2 atmosphere. The reaction mixture was washed with EtOAc 30 mL (10 mL*3). The aqueous phase was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Luna Omega 5 u Polar C18 100 Å; mobile phase: [water (0.04% HCl)-ACN]; B %: 1%-10%, 7 min) to give the product. The product in H.sub.2O (2 mL) was adjusted pH to 7˜8 with sat. NaHCO.sub.3 aq. then adjusted the pH to 3˜4 with 6M HCl. The aqueous phase was purified by prep-HPLC (column: Luna Omega 5 u Polar C.sub.18 100 Å; mobile phase: [water (0.04% HCl)-ACN]; B %: 1%-15%, 7 min) to give 2-amino-2-(fluoromethyl)-3-(4-hydroxy-3-methyl-phenyl)propanoic acid (54 mg, 204.78 umol, 20.87% yield, 100% purity, HCl) as a white solid. LCMS m/z=228.1. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 6.96 (s, 1H), 6.89 (d, J=8.0 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 4.89 (dd, J=47.0, 10.0 Hz, 1H), 4.65 (dd, J=47.0, 10.0 Hz, 1H), 3.17 (d, J=7.2 Hz, 1H), (2.96, J=14.2 Hz, 1H), 2.17 (s, 3H).

##STR00058##

Compound M: 2-amino-2-(4-hydroxybenzyl)butanoic Acid

Step 1:

[0253] To a mixture of tert-butyl 2-amino-3-(4-hydroxyphenyl)propanoate (30 g, 126.43 mmol, 1 eq) and diphenylmethanone (23.04 g, 126.43 mmol, 1 eq) in Toluene (300 mL) was added TsOH (2.18 g, 12.64 mmol, 0.1 eq). The mixture was stirred at 120° C. for 48 h and remove water by Dean-Stark trap. TLC (PE:EtOAc=5:1) indicated a little starting materials remained, and one major new spot was detected. The reaction mixture was concentrated to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to 5/1). Compound tert-butyl 2-(benzhydrylideneamino)-3-(4-hydroxyphenyl) propanoate (8.5 g, 21.17 mmol, 16.75% yield) was obtained as a yellow oil.

Step 2:

[0254] To the solution of tert-butyl (2S)-2-(benzhydrylideneamino)-3-(4-hydroxyphenyl)propanoate (12.5 g, 31.13 mmol, 1 eq) in THF (125 mL) was added NaH (1.62 g, 40.47 mmol, 60% purity, 1.3 eq) at 0° C. The mixture was stirred at 0° C. for 0.5 h. Then MOMCl (3.26 g, 40.47 mmol, 3.07 mL, 1.3 eq) was added drop-wise to the mixture at 0° C. The mixture was allowed to warm to 25° C. and stirred at 25° C. for 2.5 h. TLC (PE:EtOAc=5:1) indicated the starting material was consumed completely and one new spot formed. The mixture was poured into sat. NaHCO.sub.3 (100 mL) at 0-5° C. The aqueous phase was extracted with EtOAc (100 mL*3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuum. Compound tert-butyl (2S)-2-(benzhydrylideneamino)-3-[4-(methoxymethoxy)phenyl]propanoate (10 g, 22.44 mmol, 72.09% yield) was obtained as a yellow oil.

Step 3:

[0255] To a solution of tert-butyl (25)-2-(benzhydrylideneamino)-3-[4-(methoxymethoxy)phenyl] propanoate (2.00 g, 4.49 mmol, 1 eq) in THF (40 mL) and HMPA (7.45 g, 41.57 mmol, 7.30 mL, 9.26 eq) was added dropwise LDA (2 M, 15.71 mL, 7 eq) at −70° C. under N.sub.2. The mixture was stirred at −70° C. for 0.5 h. Then CH3CH2I (7.00 g, 44.89 mmol, 3.59 mL, 10 eq) was drop-wise to the above mixture at −70° C. The reaction mixture was allowed to warm to 25° C. and stirred at 25° C. for 1.5 h. TLC (PE:EtOAc=5:1) indicated the starting material was consumed completely and one new spot formed. The reaction was clean according to TLC. The reaction mixture was quenched by sat. NaHCO.sub.3100 mL at 0° C. The organic phase was separated, washed with EtOAc (50 mL*3), dried over NaSO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to 8/1). Compound tert-butyl 2-(benzhydrylideneamino)-2-[[4-(methoxymethoxy) phenyl] methyl] butanoate (1 g, 2.11 mmol, 47.04% yield) was obtained as a yellow oil.

Step 4:

[0256] To a solution of tert-butyl 2-(benzhydrylideneamino)-2-[[4-(methoxymethoxy)phenyl] methyl] butanoate (1.03 g, 2.17 mmol, 1 eq) in THF (30 mL) was added aq. citric acid (21.81 g, 5.68 mmol, 21.83 mL, 5% purity, 2.61 eq) and the reaction was stirred at 25° C. for 6 h. LC-MS (ET28600-23-P1A, RT=2.371 min) showed the starting material was consumed completely. The mixture was diluted with EtOAc (30 mL) and the mixture was extracted with EtOAc (60 mL*3). The combined organic phases were dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by prep-HPLC (HPLC: ET28600-23-P1A, RT=2.522 min, 89.8% purity; Kromasil C18 (250*50 mm*10 um); mobile phase: [water (10 mM NH.sub.4HCO.sub.3)-ACN]; B %: 30%-60%, 10 min) to give desired compound. Compound tert-butyl 2-amino-2-[[4-(methoxymethoxy)phenyl]methyl]butanoate (0.25 g, 808.02 umol, 37.15% yield) was obtained as a yellow oil. The product was detected by .sup.1H NMR (ET28600-23-P1A, MeOD).

Step 5:

[0257] To the tert-butyl 2-amino-2-[[4-(methoxymethoxy)phenyl]methyl]butanoate (0.25 g, 808.02 umol, 1 eq) in dioxane (3 mL) was added aq. HCl (2.5 M, 6.46 mL, 20 eq). The mixture was stirred at 60° C. for 3 h. LC-MS (ET28600-33-P1B, product: M+1=210, RT=0.877 min) showed the starting material was consumed completely. The reaction mixture on notebook page ET28600-24-P1 was combined to ET28600-33-P1 for work up. The reaction mixture was partitioned between water (20 mL) and EtOAc (30 mL). The organic phase was separated, washed with sat. NaHCO.sub.3 (5 mL*3), dried over though Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (HPLC: ET28600-33-P1A, RT=1.772 min; column: Waters Atlantis T3 150*30 mm*5 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 1%-30%, 12 min) to give desired compound as a white solid. Total 106 mg of 2-amino-2-[(4-hydroxyphenyl) methyl] butanoic acid (HCl salt) was obtained (ET28600-33-P1&ET28600-24-P1, combined together) as a white solid. LCMS m/z=210.1. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.08 (d, J=8.4 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 3.22 (d, J=14.4 Hz, 1H) 2.99 (d, J=14.4 Hz, 1H), 2.12-2.07 (m, 1H), 1.90-1.84 (m, 1H), 1.04 (t, J=7.2 Hz, 1H).

##STR00059##

Compound P: 4-(2-aminoethyl)-2-methylphenol

Step 1:

[0258] To a solution of 4-hydroxy-3-methyl-benzaldehyde (1 g, 7.34 mmol, 1 eq) in CH.sub.3NO.sub.2 (10 mL) was added NH.sub.4OAc (113.23 mg, 1.47 mmol, 0.2 eq). The mixture was stirred at 110° C. for 2 hr. LC-MS showed 4-hydroxy-3-methyl-benzaldehyde was consumed completely and one main peak with desired m/z was detected. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=80/1 to 0/1). Compound 2-methyl-4-[(E)-2-nitrovinyl]phenol (700 mg, 3.91 mmol, 53.19% yield) was obtained as yellow solid.

Step 2:

[0259] To a mixture of LiAlH.sub.4 (105.92 mg, 2.79 mmol, 10 eq) in THF (10 mL) was added 2-methyl-4-[(E)-2-nitrovinyl]phenol (50 mg, 279.06 umol, 1 eq) in THF (5 mL) at 0° C. under N.sub.2. The mixture was stirred at 0° C. for 2 hr, and then the mixture was stirred at 70° C. for 12 hr. LCMS showed 2-methyl-4-[(E)-2-nitrovinyl]phenol was consumed completely. The suspension was cooled to 0° C. and the excess of LiAlH.sub.4 was quenched with 6 M aqueous sodium hydroxide (1 mL). The precipitate was filtered off and the filter cake was washed with EtOAc (5 mL). The combined organic layers were washed with brine and dried Na.sub.2SO.sub.4. The residue was purified by prep-HPLC (column: Nano-micro Kromasil C18 80*25 mm 3 um; mobile phase: [water (0.04% HCl)-ACN]; B %: 5%-25%, 7 min). Compound 4-(2-aminoethyl)-2-methyl-phenol (3 mg, 15.83 umol, 2.39% yield, 99% purity, HCl) was obtained as a white solid. LCMS m/z=152.0. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 6.97 (s, 1H), 6.90 (d, J=8.2 Hz, 1H), 6.71 (d, J=8.2 Hz, 1H), 3.10 (t, J=7.6 Hz, 2H), 2.81 (t, J=7.6 Hz, 2H), 2.18 (s, 3H).

##STR00060##

Compound Q: 2-amino-3-(3,4-dihydroxyphenyl)-2-methylpropanoic Acid

Step 1:

[0260] To a mixture of tert-butyl 2-(benzhydrylideneamino)acetate (2 g, 6.77 mmol, 1 eq) in DMF (20 mL) was added NaH (324.98 mg, 8.13 mmol, 60% purity, 1.2 eq) in one portion at 0° C. The mixture was stirred at 0° C. for 0.5 hr. Then to the mixture was added 4-(bromomethyl)-1,2-dimethoxy-benzene (1.88 g, 8.13 mmol, 1.2 eq). The mixture was stirred at 25° C. for 2 hr. LC-MS showed Reactant was consumed completely and one main peak with desired m/z (M+1=446.2, RT=2.434 min) was detected. The mixture was poured to sat. NaHCO.sub.3 (40 mL) at 0-5° C. The mixture was extracted with ethyl acetate (20 mL*3). The combined organic phase was washed with brine (15 mL*4), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO2, Petroleum ether/Ethyl acetate=20/1 to 3/1). Compound tert-butyl 2-(benzhydrylideneamino)-3-(3,4-dimethoxyphenyl)propanoate (1.9 g, 4.26 mmol, 62.98% yield) was obtained as a yellow oil.

Step 2:

[0261] To the mixture LDA (2 M, 3.93 mL, 7 eq) in THF (6 mL) was added the solution tert-butyl 2-(benzhydrylideneamino)-3-(3,4-dimethoxyphenyl)propanoate (0.5 g, 1.12 mmol, 1 eq) in HMPA (1.86 g, 10.39 mmol, 1.83 mL, 9.26 eq) and THF (3 mL) at −70° C. under N.sub.2. The mixture was stirred at −70° C. for 0.5 hr. Then to the mixture was added Mel (1.59 g, 11.22 mmol, 698.62 uL, 10 eq) drop-wise at −70° C. The mixture was allowed to warm to 25° C. and stirred at 25° C. for 1 hr. LC-MS indicated Reactant was consumed completely. The mixture was poured into sat. NaHCO.sub.3 (15 mL) and extracted with ethyl acetate (15 mL*3), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=20/1 to 8/1). Compound tert-butyl 2-(benzhydrylideneamino)-3-(3,4-dimethoxyphenyl)-2-methyl-propanoate (0.32 g, 696.30 umol, 62.05% yield) was obtained as a yellow oil.

Step 3:

[0262] The mixture of tert-butyl 2-(benzhydrylideneamino)-3-(3,4-dimethoxyphenyl)-2-methyl-propanoate (0.27 g, 587.50 umol, 1 eq) in aq. HBr (8.32 g, 41.12 mmol, 5.58 mL, 40% purity, 70 eq) was stirred at 100° C. for 4 hr. TLC (Petroleum ether: Ethyl acetate=10:1) indicated rt-butyl 2-(benzhydrylideneamino)-3-(3,4-dimethoxyphenyl)-2-methyl-propanoate was consumed completely. The reaction mixture was extracted with EtOAc (15 mL*3). The aqueous layer was concentrated under reduced pressure to remove the organic. The crude was purified by prep-HPLC (column: Nano-micro Kromasil C18 80*25 mm 3 um; mobile phase: [water (0.04% HCl)-ACN]; B %: 1%-8%, 7 min). The crude product was further purified by prep-HPLC (column: Welch Xtimate C18 150*25 mm*5 um; mobile phase: [water (0.04% HCl)-ACN]; B %: 1%-10%, 10 min). 2-amino-3-(3,4-dihydroxyphenyl)-2-methyl-propanoic acid (15 mg, 70.31 umol, 12% yield, 99% purity) was obtained as white solid as HCl salt. LCMS m/z=212.1. .sup.1H NMR (400 MHz, CD.sub.3OD) δ 6.76 (d, J=8.4 Hz, 1H), 6.71 (s, 1H), 6.59 (d, J=8.4 Hz, 1H), 3.19 (d, J=14.2 Hz, 1H), 2.93 (d, J=14.2 Hz, 1H), 1.61 (s, 3H).

##STR00061##

Compound R: 2-amino-3-fluoro-2-(3-hydroxybenzyl)propanoic Acid

Step 1:

[0263] To the solution of 2-amino-3-(3-hydroxyphenyl)propanoic acid (10 g, 55.19 mmol, 1 eq) in tert-butyl acetate (86.60 g, 745.54 mmol, 100.00 mL, 13.51 eq) was added perchloric acid (12.67 g, 88.31 mmol, 7.63 mL, 70% purity, 1.6 eq) drop-wise at 0° C. The mixture was stirred at 25° C. for 10 hr. TLC (Dichloromethane:Methanol=10:1, R.sub.f=0.30) showed ˜20% of R2-amino-3-(3-hydroxyphenyl)propanoic acid remained. One new spot was shown on TLC. Added EtOAc (50 mL) to the mixture, then washed the mixture with H.sub.2O (50 mL). Then the organic phase was extracted with 1N HCl (10 mL). The combined aqueous phase was adjusted to pH=9 by 10% K.sub.2CO.sub.3 solution. Then the aqueous phase was extracted with DCM (30 mL*3). The combined organic phase was washed with brine (20 mL), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. Tert-butyl 2-amino-3-(3-hydroxyphenyl)propanoate (4.35 g, 18.33 mmol, 33.21% yield) was obtained as off-white solid.

Step 2:

[0264] To the solution of tert-butyl 2-amino-3-(3-hydroxyphenyl)propanoate (4.35 g, 18.33 mmol, 1 eq) in toluene (90 mL) was added 4 A molecular sieve (4.35 g) and TsOH (157.84 mg, 916.58 umol, 0.05 eq). The mixture was stirred at 25° C. for 30 min under N.sub.2. To the mixture was added diphenylmethanone (3.67 g, 20.16 mmol, 1.1 eq). The mixture was stirred at 110° C. for 9.5 hr. TLC (Petroleum ether: Ethyl acetate=5:1, R.sub.f=0.50) indicated tert-butyl 2-amino-3-(3-hydroxyphenyl)propanoate was consumed completely. The reaction mixture was cooled to 25° C. Then the mixture was filtered. The filter cake was washed with EtOAc (50 mL*2). The combined organic phase was concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 100 g SepaFlash® Silica Flash Column, Eluent of 0˜15% Ethyl acetate/Petroleum ethergradient @ 80 mL/min). Tert-butyl 2-(benzhydrylideneamino)-3-(3-hydroxyphenyl)propanoate (2.4 g, 5.98 mmol, 32.61% yield) was obtained as yellow solid.

Step 3:

[0265] To the mixture of tert-butyl 2-(benzhydrylideneamino)-3-(3-hydroxyphenyl)propanoate (2.4 g, 5.98 mmol, 1 eq) in DMF (25 mL) was added NaH (286.90 mg, 7.17 mmol, 60% purity, 1.2 eq) at 0° C. The mixture was stirred at 15° C. for 30 min. To the mixture was added MOMCl (673.79 mg, 8.37 mmol, 635.65 uL, 1.4 eq) drop-wise at 0° C. The mixture was stirred at 25° C. for 2 hr. TLC (Petroleum ether: Ethyl acetate=5:1, R.sub.f=0.65) indicated tert-butyl 2-(benzhydrylideneamino)-3-(3-hydroxyphenyl)propanoate was consumed completely. The mixture was added slowly to saturated aq. NaHCO.sub.3 (75 mL). The aqueous phase was extracted with MTBE (30 mL*3). The combined organic phase was washed with brine (15 mL*3), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. tert-butyl 2-(benzhydrylideneamino)-3-[3-(methoxymethoxy)phenyl]propanoate (2.28 g, 5.12 mmol, 85.61% yield) was obtained as yellow oil.

Step 4:

[0266] To the solution of THF (30 mL) was added LDA (2 M, 17.91 mL, 7 eq) under N.sub.2. Then Cool to −70° C. To the mixture was added tert-butyl 2-(benzhydrylideneamino)-3-[3-(methoxymethoxy) phenyl]propanoate (2.28 g, 5.12 mmol, 1 eq) in HMPA (8.49 g, 47.39 mmol, 8.33 mL, 9.26 eq) and THF (20 mL) drop-wise at −70° C. The mixture was stirred at −70° C. for 0.5 hr. Then fluoro(iodo)methane (8.18 g, 51.17 mmol, 10 eq) was added drop-wise at −70° C. The mixture was stirred at 25° C. for 1 hr. TLC (Petroleum ether: Ethyl acetate=10:1, R.sub.f=0.66) showed the starting material was consumed completely. The mixture was poured into aq. NaHCO.sub.3 (50 mL) slowly at 0-5° C. The mixture was extracted with ethyl acetate (10 mL*3). The combined organic phase was washed with brine (5 mL*2), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by flash silica gel chromatography (ISCO®; 10 g SepaFlash® Silica Flash Column, Eluent of 0˜5% Ethyl acetate/Petroleum ethergradient @ 40 m L/m in). tert-butyl 2-(benzhydrylideneamino)-2-(fluoromethyl)-3-[3-(methoxymethoxy)phenyl]propanoate (1.38 g, 2.89 mmol, 56.47% yield) was obtained as yellow oil.

Step 5:

[0267] To the solution of tert-butyl 2-(benzhydrylideneamino)-2-(fluoromethyl)-3-[3-(methoxymethoxy) phenyl]propanoate (1.38 g, 2.89 mmol, 1 eq) in THF (35 mL) was added citric acid (28.98 g, 7.54 mmol, 29.01 mL, 5% purity, 2.61 eq). The mixture was stirred at 25° C. for 6 hr. TLC (Petroleum ether: Ethyl acetate=10:1, R.sub.f=0.13) indicated tert-butyl 2-(benzhydrylideneamino)-2-(fluoromethyl)-3-[3-(methoxymethoxy)phenyl]propanoate was consumed completely. One new spot with large polarity was detected. The mixture was concentrated in reduced pressure to remove THF. The aqueous phase was extracted with ethyl acetate (10 m L*3). The combined organic phase was washed with brine (10 mL), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by column chromatography (SiO.sub.2, Petroleum ether/Ethyl acetate=10/1 to 0/1). tert-butyl 2-amino-2-(fluoromethyl)-3-[3-(methoxymethoxy)phenyl]propanoate (460 mg, 1.35 mmol, 46.74% yield, 92% purity) was obtained as white solid.

Step 6:

[0268] To the mixture of tert-butyl 2-amino-2-(fluoromethyl)-3-[3-(methoxymethoxy)phenyl]propanoate (240 mg, 765.88 umol, 1 eq) in THF (20 mL) was added NaHCO.sub.3 (64.34 mg, 765.88 umol, 29.79 uL, 1 eq) in H.sub.2O (10 mL). The mixture was cooled to 0° C. To the mixture was added CbzCl (156.78 mg, 919.06 umol, 130.65 uL, 1.2 eq) slowly at 0° C. The mixture was stirred at 25° C. for 2 hr. TLC (Petroleum ether: Ethyl acetate=5:1, Rf=0.60) indicated Reactant was consumed completely. Combine two same scale batches together for work-up and purification. The mixture was extracted with ethyl acetate (10 mL*4). The combined organic phase was washed with brine (10 mL), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The residue was purified by prep-TLC (SiO.sub.2, Petroleum ether: Ethyl acetate=5:1). tert-butyl 2-(benzyloxycarbonylamino)-2-(fluoromethyl)-3-[3-(methoxymethoxy)phenyl]propanoate (400 mg, 893.86 umol, 58.36% yield) was obtained as light yellow oil.

Step 7:

[0269] To the mixture of tert-butyl 2-(benzyloxycarbonylamino)-2-(fluoromethyl)-3-[3-(methoxymethoxy)phenyl]propanoate (300 mg, 670.40 umol, 1 eq) in THF (10 mL) was added aq. HCl (2.5 M, 5.36 mL, 20 eq). The mixture was stirred at 60° C. for 3 hr. LCMS showed tert-butyl 2-(benzyloxycarbonylamino)-2-(fluoromethyl)-3-[3-(methoxymethoxy) phenyl]propanoate was consumed completely. The mixture was concentrated in reduced pressure to remove THF. The aqueous phase was extracted with ethyl acetate (5 mL*3). The combined organic phase was washed with brine (3 mL), dried with anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuum. The product was purified by prep-HPLC (column: Welch Xtimate C18 150*30 mm*5 um; mobile phase: [water (10 mM NH4HCO.sub.3)-ACN]; B %: 45%-75%, 3 min). tert-butyl 2-(benzyloxycarbonylamino)-2-(fluoromethyl)-3-(3-hydroxyphenyl)propanoate (157 mg, 389.15 umol, 58.05% yield) was obtained as colorless oil.

Step 8:

[0270] To the mixture of tert-butyl 2-(benzyloxycarbonylamino)-2-(fluoromethyl)-3-(3-hydroxyphenyl) propanoate (100 mg, 247.87 umol, 1 eq) in ACN (20 mL) was added TMSI (148.79 mg, 743.60 umol, 101.22 uL, 3 eq). The mixture was stirred at 25° C. for 2 hr. TLC (Petroleum ether: Ethyl acetate=5:1, R.sub.f=0.02) indicated the starting material was consumed completely. The mixture was concentrated in reduced pressure. The crude was purified by prep-HPLC (column: Welch Xtimate C18 150*25 mm*5 um; mobile phase: [water (0.04% HCl)-ACN]; B %: 1%-3%, 10 min). 2-amino-2-(fluoromethyl)-3-(3-hydroxyphenyl)propanoic acid (27 mg, 96.36 umol, 38.87% yield, 89.1% purity, HCl) was obtained as yellow solid. LCMS m/z=214.1 .sup.1H NMR (400 MHz, CD.sub.3OD) δ 7.19 (app t, J=8.2 Hz, 1H), 6.78 (d, J=8.2 Hz, 1H), 6.71 (d, J=8.2 Hz, 1H), 6.69 (s 1H), 4.96 (dd, J=46, 10.4 Hz, 1H), 4.69 (dd, J=46, 10.4 Hz, 1H), 3.29 (d, J=14.4 Hz, 1H), 3.05 (d, J=14.4 Hz, 1H).

##STR00062##

Compounds S and T: (S)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoic Acid and (R)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoic Acid

Step 1:

[0271] Two reactions were carried out in parallel and combined together for work-up. To a solution of tert-butyl L-tyrosinate (60 g, 253 mmol, 1.00 eq) in dry DCM (300 mL) were added TsOH (6.53 g, 37.9 mmol, 0.15 eq) and MgSO.sub.4 (60.9 g, 505 mmol, 2.00 eq) in one portion. After the addition, the suspension was stirred at 25° C. for 0.5 hr. Benzaldehyde (29.5 g, 278 mmol, 28.1 mL, 1.10 eq) was added to the solution in one portion. After addition, the suspension was stirred at 25° C. for 12 hrs. HNMR (ET27430-13-P1A1) showed the starting material was consumed completely. Two reactions were combined together for work-up. The suspension was filtered, and the filter cake was washed by DCM (200 mL×2). The filtrate was washed by cold aqueous solution of NaHCO.sub.3(Sat. 800 mL). The organic phase was dried over Na.sub.2SO.sub.4, and concentrated under vacuum to give a solid. The solid was dried in the air for 12 hrs to oxidize PhCHO into PhCOOH. The solid was triturated with MTBE/Petroleum ether (2/1) at 25° C. for 30 mins. The suspension was filtered, and the filter cake was dissolved in DCM (300 mL). The organic layer was washed with cold aqueous solution of NaHCO.sub.3(Sat. 200 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to give tert-butyl (S,E)-2-(benzylideneamino)-3-(4-hydroxyphenyl)propanoate (110 g, 338 mmol, 66.9% yield) as an off-white solid.

Step 2:

[0272] Four reactions were carried out in parallel and combined together for work-up. To a solution of tert-butyl (S,E)-2-(benzylideneamino)-3-(4-hydroxyphenyl)propanoate (20 g, 61.5 mmol, 1.00 eq) in DMF (200 mL) was added NaH (2.70 g, 67.6 mmol, 60% purity, 1.10 eq) at 0° C. in portions. After the addition, the resulting suspension was stirred at 0° C. for 1.5 hrs. MOMCl (4.95 g, 61.5 mmol, 4.67 mL, 1.00 eq) was added to the suspension in one portion, and the suspension was stirred at 0° C. for 1 hr. LCMS (ET27430-22-P1A2) showed the starting material was consumed completely and one main peak with desired mass was detected. Four reactions were combined together for work-up. The suspension was slowly poured into cold saturated aqueous solution of NaHCO.sub.3 (1200 mL) and extracted with MTBE (300 mL×4). The combined organic layer was washed with cold brine (600 mL×2), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give tert-butyl (S,E)-2-(benzylideneamino)-3-(4-(methoxymethoxy)phenyl)propanoate (90.3 g, crude) as a yellow oil which was used to next step directly.

Step 3:

[0273] Four reactions were carried out in parallel and combined together for work-up. LDA (2 M, 29.8 mL, 2.20 eq) was added to THF (50 mL) at −70° C. drop-wise. After the addition, HMPA (12.1 g, 67.7 mmol, 11.9 mL, 2.50 eq) was added to the solution in one portion, and followed by a solution of tert-butyl (S,E)-2-(benzylideneamino)-3-(4-(methoxymethoxy)phenyl)propanoate (10.0 g, 27.1 mmol, 1.00 eq) in THF (20 mL) drop-wise at −70° C. The solution was stirred at −70° C. for 1 hr. Fluoroiodomethane (10.8 g, 67.7 mmol, 2.50 eq) was added to the solution drop-wise at −70° C. After the addition, the solution was stirred at −70° C. for 1 hr. LCMS (ET27430-25-P1A2) showed the starting material was consumed completely and one main peak with desired mass was detected. Four reactions were combined together for work-up. The solution was slowly poured into cold aqueous solution of NaHCO.sub.3(Sat. 600 mL) and extracted with MTBE (300 mL×4). The combined organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated under vacuum to give tert-butyl (E)-2-(benzylideneamino)-3-fluoro-2-(4-(methoxymethoxy)benzyl)propanoate (50 g, crude) as a brown oil which was used to next step directly.

Step 4:

[0274] To a solution of tert-butyl (E)-2-(benzylideneamino)-3-fluoro-2-(4-(methoxymethoxy)benzyl)propanoate (60 g, 149 mmol, 1.00 eq) in THF (150 mL) was added citric acid monohydrate (1.08 kg, 257 mmol, 1200 mL, 5% purity, 1.72 eq). The solution was stirred at 20° C. for 5 hrs. TLC (Petroleum ether: Ethyl acetate=10:1, R.sub.f of material=0.5) showed the starting material was consumed completely, and one major new spot with higher polarity was detected. LCMS (ET27430-27-P1A) showed the reaction was completed. The solution was extracted with MTBE: Petroleum ether=1:1 (500 mL×2). The organic layer was washed by water (500 mL), and the organic layer was discarded. The combined aqueous layer was poured into aqueous solution of NaHCO.sub.3(Sat. 600 mL). The aqueous phase was extracted with MTBE (600 mL×3). The combined organic layers were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO.sub.2, NH.sub.3. H.sub.2O/Petroleum ether/Dichloromethane=0/4/1, 0/2/1, 0.005/2/1, 0.005/1/1, 0.005/0/1) to give tert-butyl 2-amino-3-fluoro-2-(4-(methoxymethoxy)benzyl)propanoate (18 g, 57.44 mmol, 38.43% yield) as a brown oil. The racemic mixture (18 g, 57.44 mmol) was purified by SFC (column: DAICEL CHIRALPAK AD-H (250 mm*30 mm, 5 um); mobile phase: [0.1% NH3.H2O ETON]; B %: 15%-15%, 2.3 min). (S)tert-butyl 2-amino-3-fluoro-2-(4-(methoxymethoxy)benzyl)propanoate (7.4 g, 23.6 mmol, 92.5% yield) was obtained as a brown oil and (R)-tert-butyl 2-amino-3-fluoro-2-(4-(methoxymethoxy)benzyl)propanoate (7.1 g, 22.7 mmol, 88.8% yield) was obtained as a brown oil.

Step 5:

[0275] To a solution of (S)tert-butyl 2-amino-3-fluoro-2-(4-(methoxymethoxy)benzyl)propanoate (6.52 g, 20.8 mmol, 1.00 eq) in THF (30 mL) was added aqueous solution of HCl (2.5 M, 83.2 mL, 10.0 eq). The solution was stirred at 60° C. for 3 hrs. LCMS (ET27430-31-P1A2) showed the reaction was completed. The reaction solution was lyophilized to give a crude product. The crude product was purified by pre-HPLC (column: Phenomenex luna C18 250*80 mm*10 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 0%-9%, 20 min) to give (S)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoic acid (2.3 g, 10.8 mmol, 51.9% yield) as a white solid. LCMS m/z=214. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.68 (br s, 1H), 7.01 (d, J=8.4 Hz, 1H), 6.72 (d, J=8.4 Hz, 1H), 4.84 (dd, J=45.2, 10.0 Hz, 1H), 4.63 (d, J=45.2, 10.0 Hz, 1H), 3.04 (d, J=14.0 Hz, 1H), 2.96 (d, J=14.0 Hz, 1H).

[0276] To a solution of (R)-tert-butyl 2-amino-3-fluoro-2-(4-(methoxymethoxy)benzyl)-propanoate (6.77 g, 21.6 mmol, 1.00 eq) in THF (30 mL) was added aqueous solution of HCl (2.5 M, 86.4 mL, 10.0 eq). The solution was stirred at 60° C. for 3 hrs. LCMS showed the reaction was completed. The reaction solution was lyophilized to give a crude product. The crude product was purified by pre-HPLC (column: Phenomenex luna C18 250*80 mm*10 um; mobile phase: [water (0.05% HCl)-ACN]; B %: 0%-9%, 20 min) to give (R)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoic acid (2.3 g, 10.8 mmol, 49.9% yield) as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 8.68 (br s, 1H), 7.01 (d, J=8.4 Hz, 1H), 6.72 (d, J=8.4 Hz, 1H), 4.84 (dd, J=45.2, 10.0 Hz, 1H), 4.63 (d, J=45.2, 10.0 Hz, 1H), 3.04 (d, J=14.0 Hz, 1H), 2.96 (d, J=14.0 Hz, 1H).

##STR00063##

Compound U: 2-amino-2-(fluoromethyl)-4-(4-methoxyphenyl)butanoic Acid

[0277] To a solution of 3-(4-methoxyphenyl)propanal in 250-mL round-bottomed flask charged with NH.sub.4Cl (1.2 eq), ammonia (3 eq), ethanol (0.2 M), and water (0.2M). Dissolve the mixture into a clear solution. Add NaCN (1.5 eq) to the mixture. Seal the flask quickly with a rubber stopper. Stir the mixture for 3 days. Extract the mixture with CH.sub.2Cl.sub.2 (100 mL). Wash the combined organic layer with water to remove the remaining NaCN. Dry the mixture with anhydrous sodium sulfate. Concentrate the mixture under reduced pressure to afford the product. The residue was purified by column chromatography to give 2-amino-4-(4-methoxyphenyl)butanenitrile in 70-80%. Yield. A mixture of the 2-amino-4-(4-methoxyphenyl)butanenitrile, Et.sub.3N and benzophenone (1:1.3:8 molar ratio, respectively) and DMF (7 mL/g ketone) was loaded in a round-bottomed, two-necked flask fitted with a refluxing condenser. A toluene (1 M) solution of TiCl.sub.4, (0.9 molar with respect to the substrate) was carefully added dropwise to the solution After the addition was completed, the mixture was refluxed (35-40° C.) for 1 h and then allowed to stand 6 h at room temperature. The suspension was concentrated and extracted by diethyl ether and purified by column chromatography to give 2-((diphenylmethylene)amino)-4-(4-methoxyphenyl)butanenitrile in 23-30%. Yield. 2-amino-4-(4-methoxyphenyl)butanenitrile was treated with 11 eq of HBr (48 wt. % in H.sub.2O) was added and the solution was heated to 60° C. for five days. Progress of the reaction was monitored by LCMS. The crude compound was purified by reverse phase column chromatography to give 2-amino-2-(fluoromethyl)-4-(4-hydroxyphenyl)butanoic acid 2-amino-2-(fluoromethyl)-4-(4-hydroxyphenyl)butanoic acid in 20% yield. 1H NMR (500 MHz, Deuterium Oxide) δ 7.34-7.26 (m, 2H), 7.02-6.96 (m, 2H), 5.11-4.76 (m, 2H), 2.89-2.78 (m, 1H), 2.70 (td, J=12.9, 5.0 Hz, 1H), 2.34-2.12 (m, 1H). LCMS: (M+1) 228.3, (M−1) 226.2.

##STR00064##

Compound V: 3-(4-hydroxyphenyl)-2-((methoxycarbonyl)amino)-2-methylpropanoic Acid

[0278] Added methyl chloroformate (1 eq) to a solution of 2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid and NaHCO.sub.3 (20 eq) in a mixture of H.sub.2O/THF (2 M). Stir the mixture at room temperature overnight. Dilute the mixture with H.sub.2O. Wash the mixture with Et.sub.2O. Acidify the aqueous layer to pH ˜2-3. Evaporate to dryness and purified by reverse phase column chromatography to give 3-(4-hydroxyphenyl)-2-((methoxycarbonyl)amino)-2-methylpropanoic acid with 70% yield. .sup.1H NMR (400 MHz, Methanol-d.sub.4) δ 7.02-6.89 (m, 2H), 6.77-6.59 (m, 2H), 3.64 (s, 3H), 3.21-2.96 (m, 2H), 1.41 (s, 3H). LCMS: (M+1): 254.2, (M−1): 252.2.

##STR00065##

Compound W: 3-fluoro-2-(4-hydroxybenzyl)-2-((methoxycarbonyl)amino)-propanoic Acid

[0279] Added methyl chloroformate (1 eq) to a solution of (S)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoic acid and NaHCO.sub.3 (20 eq) in a mixture of H.sub.2O/THF (2 M). Stir the mixture at room temperature overnight. Dilute the mixture with H.sub.2O. Wash the mixture with Et.sub.2O. Acidify the aqueous layer to pH˜2-3. Evaporate to dryness and purified by reverse phase column chromatography to give 3-fluoro-2-(4-hydroxybenzyl)-2-((methoxycarbonyl)amino)propanoic acid with 73% yield. .sup.1H NMR (400 MHz, Methanol-d4) δ 6.95 (d, J=8.5 Hz, 2H), 6.79-6.59 (m, 2H), 4.70 (dt, J=47.2, 8.8 Hz, 2H), 3.66 (s, 3H), 3.15-2.93 (m, 2H). LCMS: (M+1): 272.2, (M−1): 270.1.

##STR00066##

Compound X: 2-acetamido-3-(4-hydroxyphenyl)-2-methylpropanoic Acid

[0280] To a solution of 2-amino-3-(4-hydroxyphenyl)-2-methylpropanoic acid and Hunig's base (5 eq) in THF (0.2 M) was added acetic anhydride (1.5 eq). Stir the mixture at room temperature overnight. Dilute the mixture with H.sub.2O. Wash the mixture with Et.sub.2O. Acidify the aqueous layer to pH˜2-3. Evaporate to dryness and purified by reverse phase column chromatography to give 2-acetamido-3-(4-hydroxyphenyl)-2-methylpropanoic acid with 75% yield. 1H NMR (400 MHz, Methanol-d.sub.4) δ 6.93 (d, J=8.4 Hz, 2H), 6.68 (d, J=8.5 Hz, 2H), 3.26 (d, J=13.6 Hz, 2H), 3.03 (d, J=13.6 Hz, 1H), 1.92 (s, 3H), 1.37 (s, 3H). LCMS: (M+1): 238.2, (M−1): 236.2.

##STR00067##

Compound Y: 3-fluoro-2-(4-hydroxybenzyl)-2-((methoxycarbonyl)amino)propanoic Acid

[0281] To a solution of (S)-2-amino-3-fluoro-2-(4-hydroxybenzyl)propanoic acid and Hunig's base (5 eq) in THF (0.2 M) was added acetic anhydride (1.5 eq). Stir the mixture at room temperature overnight. Dilute the mixture with H.sub.2O. Wash the mixture with Et.sub.2O. Acidify the aqueous layer to pH˜2-3. Evaporate to dryness and purified by reverse phase column chromatography to give 2-acetamido-3-fluoro-2-(4-hydroxybenzyl)propanoic acid with 66% yield. .sup.1H NMR (400 MHz, Methanol-d4) δ 7.06-6.90 (m, 2H), 6.75-6.62 (m, 2H), 4.79-4.70 (m, 1H), 4.68-4.57 (m, 2H), 3.08 (s, 2H), 1.98 (s, 3H). LCMS: (M+Na): 278.2, (M−1): 254.2.

##STR00068##

Compound Z: 2-amino-2-benzyl-3-fluoropropanoic Acid

[0282] Dissolve the 2-fluoroacetonitrile (1 eq) in anhydrous Toluene (1 M). Cool the solution to 0° C. Add slowly phenylmagnesium chloride (2 M, 1 eq) to the reaction mixture under an argon atmosphere. Stir the reaction for 2 hours. Quench by adding water (5 mL) and 1M HCl (5 mL). Add 50 mL of ethyl acetate to this solution, wash the organic layer with water (2×20 ml), brine (2×20 ml). Dry over anhydrous sodium sulfate. Isolate the product by filtration and remove the solvent. Purified by column chromatography and carried to the next step directly. Ketone was transferred to a round-bottomed flask and charged with NH.sub.4Cl (1.2 eq), ammonia (3 eq), ethanol (0.2 M), and water (0.2 M). Dissolve the mixture into a clear solution. Add NaCN (1.5 eq) to the mixture. Seal the flask quickly with a rubber stopper. Stir the mixture for 3 days. Extract the mixture with CH.sub.2Cl.sub.2 (100 mL). Wash the combined organic layer with water to remove the remaining NaCN. Dry the mixture with anhydrous sodium sulfate. Concentrate the mixture under reduced pressure to afford the corresponding amino nitrile. The residue was purified by column chromatography and carried to the next step directly. The resulting nitrile is heated under reflux with a conc HCl in dioxane (0.4 M) such as dilute hydrochloric acid. A carboxylic acid is formed was evaporate to dryness and purified by reverse phase column chromatography to give 2-amino-2-benzyl-3-fluoropropanoic acid with 59% yield. 1H NMR (400 MHz, DMSO-d6) δ 6.68-6.41 (m, 5H), 4.21 (d, J=10.3 Hz, 1H), 3.94 (dd, J=47.2, 10.3 Hz, 1H), 2.58-2.44 (m, 2H), 2.36 (d, J=14.2 Hz, 2H). LCMS: (M+): 198.2.

Example 2: Inhibition of Tyrosine Decarboxylase In Vitro

[0283] Tyrosine decarboxylase (tdc) was obtained by following a previously published literature procedure (Science, 14 Jun. 2019: Vol. 364, Issue 6445, eaau6323). Tdc (220 nM final concentration) was thawed on ice and then mixed with pyridoxal-5-phosphate (2.2 mM final concentration) in 200 mM sodium acetate buffer, pH 5.5 optionally containing 1 mM TCEP. To this mixture was added inhibitor at a final concentration of 1000, 333, 111, 37, 12, 4.1, 1.4, or 0 μM (final volume: 100 μL; inhibitor was 100-fold concentrated in a solution of DMSO, H.sub.2O, or DMSO:H.sub.2O (1/1 v/v)). The protein-inhibitor mixture was incubated at room temperature for 60 min. 6 μL of this mixture was then withdrawn from each solution and mixed with 54 μL of 10 mM levodopa in 200 mM sodium acetate buffer pH 5.5. The final concentration of the reaction was 22 nM tdc, 220 μM pyridoxal-5-phosphate, 9 mM levodopa in 200 mM sodium acetate buffer pH 5.5 with 0-100 μM inhibitor. The reaction was proceeded for 5 min at room temperature before quenching by addition of 540 μL acetonitrile containing 0.1 (v/v) formic acid supplemented with 200 nM tolbutamide as an internal standard. The reactions were centrifuged (3,000 g, 10 min), and then 100 μL of each supernatant was transferred to a fresh plate. 100 μL of acetonitrile containing 0.1% (v/v) formic acid supplemented with 200 nM tolbutamide was added. An external standard curve containing 0-150 μM dopamine was prepared in the exact same manner.

[0284] Dopamine formed in each reaction was quantified by using an Agilent 6470 triple quadrupole mass spectrometer equipped with an Acquity UPLC. Mobile phase A consisted of H.sub.2O containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. Mobile phase B consisted of acetonitrile containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. 5 μL of each sample was injected onto a BEH Amide column (Waters Corporation, 2.1×50 mm, 1.7 pm). The gradient was set to: 100% mobile phase B at 0 min, decreasing linearly to 65% mobile phase B by 1.5 min, held constant at 65% mobile phase B until 2.5 min, ramped back up to 100% mobile phase B by 2.6 min, and held constant at 100% mobile phase B until 4.2 min. The flow rate was 0.6 mL/min. The dopamine was detected by using the mass spectrometer in multiple reaction monitoring (MRM) mode, quantifying the transition 154.1 to 137.0 m/z in positive mode. The fragmentor setting was 74, the collision energy was 9, and the cell accelerator voltage was 4, and the dwell time was 20. Tolbutamide was monitored using MRM and quantifying the transition of 271.1 to 91.0 m/z in positive mode. The fragmentor setting was 88, the collision energy was 37, and the cell accelerator voltage was 4, and the dwell time was 20.

[0285] The amount of dopamine was quantified by normalizing the area to the area of tolbutamide internal standard within each sample. This relative response was then compared to that of the standard curve to obtain the dopamine formed within each sample. The concentration of dopamine formed as a function of the inhibitor concentration at the preincubation stage was plotted in GraphPad Prism 8, and the IC.sub.50 was calculated using the non-linear fit for the standard IC.sub.50 curve equation “[inhibitor] vs response (three parameters).”

TABLE-US-00001 TABLE 1 IC.sub.50 @ 60 min Compound (μM) U >1000 K Minor inhibition M 3.2 L 2.5 N 2.6 V >1000 W >1000 Y >1000 Q >1000 P >1000 X >1000 O >1000 R Minor inhibition Z >1000 T 82.3 S 4

Example 3: Inhibition of E. faecalis Decarboxylation Activity In Vitro

[0286] A vial of 200 μL of Enterococcus faecalis v583 was removed from the −80° C. freezer and thawed in an anaerobic chamber containing an atmosphere of either 95/5 N.sub.2/H.sub.2 (v/v) or 90/5/5 N.sub.2/H.sub.2/CO.sub.2 (v/v). 200 μL was inoculated into 10 mL of sterile, anaerobic BHI broth, pH 5 (adjusted with NaOH). The culture was grown overnight at 37° C. under anaerobic conditions.

[0287] After overnight incubation, 40 μL of the saturated starter culture was mixed with 744 μL of sterile, anaerobic BHI broth, pH 5 that had been supplemented with 1.5 mM levodopa. To this was added 16 μL of a 50-fold concentrated stock solution of inhibitor that had been dissolved in either DMSO, H.sub.2O, or DMSO:H.sub.2O (1:1 v/v). The final concentration of the inhibitor in each condition was 0, 0.001, 0.01, 0.1, 1, 10 or 100 μM. The contents of each incubation were mixed, and then 100 μL was transferred into a fresh 96-well plate. A standard curve of levodopa (0-1.5 mM) in BHI broth, pH 5.5 was likewise prepared on a 100 μL scale and aliquoted into the plate. The plate was sealed and incubated for 24 h at 37° C. under an atmosphere of either 95/5 N.sub.2/H.sub.2 (v/v) or 90/5/5 N.sub.2/H.sub.2/CO.sub.2 (v/v) in an anaerobic chamber.

[0288] After 24 h incubation, the seal was removed, and the contents of each plate was mixed with 400 μL acetonitrile containing 0.1% (v/v) formic acid and 200 nM tolbutamide as an internal standard. The samples were mixed and then centrifuged (4,000 g, 10 min). 200 μL of each supernatant was transferred to a separate plate.

[0289] The samples were analyzed by using an Agilent 6470 triple quadrupole mass spectrometer equipped with an Acquity UPLC. Mobile phase A consisted of H.sub.2O containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. Mobile phase B consisted of acetonitrile containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. 5 μL of each sample was injected onto a BEH Amide column (Waters Corporation, 2.1×50 mm, 1.7 pm). The gradient was set to: 100% mobile phase B at 0 min, decreasing linearly to 65% mobile phase B by 1.5 min, held constant at 65% mobile phase B until 2.5 min, ramped back up to 100% mobile phase B by 2.6 min, and held constant at 100% mobile phase B until 4.2 min. The flow rate was 0.6 mL/min. The levodopa was detected by using the mass spectrometer in multiple reaction monitoring (MRM) mode, quantifying the transition 198.1 to 151.9 m/z in positive mode. The fragmentor setting was 78, the collision energy was 13, and the cell accelerator voltage was 4, and the dwell time was 20. Tolbutamide was monitored using MRM and quantifying the transition of 271.1 to 91.0 m/z in positive mode. The fragmentor setting was 88, the collision energy was 37, and the cell accelerator voltage was 4, and the dwell time was 20.

[0290] The amount of levodopa was quantified by comparing normalizing the area to the area of tolbutamide internal standard within each sample. This relative response was then compared to that of the standard curve to obtain the residual levodopa within each sample. The concentration of levodopa remaining as a function of inhibitor concentration was then plotted in GraphPad Prism 8, and the IC.sub.50 was calculated using the non-linear fit for the standard IC.sub.50 curve equation “[inhibitor] vs response (three parameters).”

TABLE-US-00002 TABLE 2 IC.sub.50 @ 60 min Compound (μM) V >1000 W >1000 Y >1000 Q >1000 P >1000 X >1000 O >1000 R >1000 Z >1000 T 18.5 S 2

Example 4: Inhibition of Dopamine Production in Fecal Matter

[0291] This example is representative of the methods described in Example 8. Fecal samples may be assayed for the presence of the tydc gene by attempting to amplify the gene with primers specific for it by qPCR. Samples that give a signal below the detection limit may be used in subsequent steps.

[0292] Following steps 1-2 as detailed in Example 6, E. faecalis v583 may be grown.

[0293] E. faecalis v583 may be added to the samples obtained in step 1 at a dilution level calculated to represent 0, 0.1, 1, 2, 5, or 10% of all organisms present. The substrate (d4-L-DOPA, 1 mM final concentration) may be added to the mixture. An inhibitor of TyDC may also be added at this time at a final concentration of 10 μM. Optionally, the IC.sub.50 of an inhibitor may instead be determined by adding an inhibitor across a range of appropriate concentrations, for example, 0, 0.001, 0.01, 0.1, 1, and 10 μM.

[0294] After incubation for a designated period of time and at a certain temperature (for example, 8 h at 37° C.), samples will be rendered compatible with LCMS analysis and the amount of product will be determined using LCMS analysis as in Example 4.

Other Embodiments

[0295] Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure.

[0296] Other embodiments are in the claims.