FTO inhibitors

Abstract

The invention provides compounds that inhibit FTO (fat mass and obesity), including pharmaceutically acceptable salts, hydrides and stereoisomers thereof. The compounds are employed in pharmaceutical compositions, and methods of making and use, including treating a person in need thereof, particularly obesity, with an effective amount of the compound or composition, and detecting a resultant improvement in the person's health or condition.

Claims

1. A pharmaceutical composition comprising an FTO inhibitor selected from a compound formula I, a stereoisomer thereof, a hydrate thereof, and a pharmaceutically-acceptable salt thereof, and a pharmaceutically-acceptable excipient, formulated in a unit dosage form, and suitable for administration to a person in need thereof, the inhibitor of structure: ##STR00197## wherein: (a) R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or an optionally substituted, optionally hetero-, optionally cyclic C1-C18 hydrocarbyl; and R4 is optionally substituted, optionally hetero-, optionally cyclic C1-C18 hydrocarbyl; (b) R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or C1-C4 alkyl; R4 is CONHR5; and R5 is optionally substituted, optionally hetero-, optionally cyclic C1-C18 hydrocarbyl; (c) R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or C1-C4 alkyl; R4 is COR5; and R5 is optionally substituted, heterocyclic C3-C18 hydrocarbyl comprising an n-membered ring wherein n=3-18, including 1 to n1 heteroatoms independently selected from N, O, S and P; or (d) R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or C1-C4 alkyl; and R4 is optionally substituted, heterocyclic C3-C18 hydrocarbyl comprising an n-membered ring wherein n=3-18, including 1 to n1 heteroatoms independently selected from N, O, S and P.

2. The composition of claim 1 wherein: R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or an optionally substituted, optionally hetero-, optionally cyclic C1-C18 hydrocarbyl; and R4 is optionally substituted, optionally hetero-; optionally cyclic C1-C18 hydrocarbyl.

3. The composition of claim 1 wherein: R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or C1-C4 alkyl; R4 is CONHR5; and R5 is optionally substituted, optionally hetero-, optionally cyclic C1-C18 hydrocarbyl.

4. The composition of claim 1 wherein: R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or C1-C4 alkyl; R4 is COR5; and R5 is optionally substituted, heterocyclic C3-C18 hydrocarbyl comprising an n-membered ring wherein n=3-18, including 1 to n1 heteroatoms independently selected from N, O, S and P.

5. The composition of claim 1 wherein: R1 and R2 are independently H or Me; R3 is OH or NHR, wherein R is H or C1-C4 alkyl; and R4 is optionally substituted, heterocyclic C3-C18 hydrocarbyl comprising an n-membered ring wherein n=3-18, including 1 to n1 heteroatoms independently selected from N, O, S and P.

6. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00198## ##STR00199## ##STR00200## ##STR00201##

7. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00202## ##STR00203## ##STR00204## ##STR00205##

8. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00206## ##STR00207## ##STR00208##

9. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00209## ##STR00210##

10. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00211## ##STR00212## ##STR00213## ##STR00214##

11. The composition of claim 1 wherein: R3 is OH.

12. The composition of claim 2 wherein: R3 is OH.

13. The composition of claim 3 wherein: R3 is OH.

14. The composition of claim 4 wherein: R3 is OH.

15. The composition of claim 5 wherein: R3 is OH.

16. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00215##

17. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00216##

18. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00217## ##STR00218##

19. The composition of claim 1, wherein the FTO inhibitor is a compound of formula: ##STR00219##

20. A method comprising administering to a person in need thereof a composition of claim 1 to inhibit FTO, inhibit weight gain, promote weight loss, reduce serum LDL, cholesterol, LDL-c, or triglycerides, or treat Obesity or an obesity related disease or Alzheimer's disease.

Description

EXAMPLES: COMPOUND PREPARATION

(1) Compound 347:

(2) 347 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(3) ##STR00156##

Step 1: Synthesis of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (2)

(4) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (1.0 g, 4.4 mmol) in toluene (11 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (10 mL).

(5) Under a nitrogen atmosphere, 60% NaH (0.35 g, 8.8 mmol) was added to solution of 2-cyano-N,N-diethylacetamide (0.56 g, 4.0 mmol) in anhydrous THF (15 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoyl chloride was added over 10 min and stirred for an additional 1 h at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound as an orange solid (705 mg, 99%). MS [MH].sup.+ calcd for C.sub.16H.sub.19N.sub.3O.sub.6 350.1, found 350.1.

Step 2: Synthesis of 2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (3)

(6) A solution of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (500 mg, 1.43 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (5 mL, 5 mmol) at 15 C. under a nitrogen atmosphere. The resulting red suspension was stirred for 1 h at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried over Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as a bright yellow solid (80 mg, 17%). .sup.1H NMR (400 MHz, CDCl.sub.3) 10.92 (s, 1H), 8.28 (d, J=2.0 Hz, 1H), 7.74 (d, J=1.9 Hz, 1H), 7.26 (s, 3H), 5.93 (s, 1H), 3.66 (d, J=6.0 Hz, 3H), 1.33 (t, J=7.0 Hz, 6H). MS [MH].sup.+ calcd for C.sub.14H.sub.15N.sub.3O.sub.6 322.0, found 322.0.

(7) Compound 315:

(8) 315 was prepared in one synthetic step from 3,4-dihydroxy-5-nitrobenzaldehyde, according to the following procedure:

(9) ##STR00157##

Step 1: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyridin-2-yl)acrylonitrile (2)

(10) A solution of 2-(pyridin-2-yl)acetonitrile (142 mg, 1.2 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (182 mg, 1 mmol) and NH.sub.4OAc (462 mg, 6 mmol) in MeOH (10 mL) was heated to reflux for overnight. LCMS showed no 3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixture was cooled to room temperature. The solid was filtered and washed by MeOH and H.sub.2O. The solid was re-dissolved in MeOH (5 mL). 5 mL of 1N aqueous HCl was added to adjust pH 3-4. The desired product was obtained by filter as a bright solid (56 mg, 20%). .sup.1H NMR (400 MHz, DMSO) 8.57 (m, 1H), 8.12 (s, 1H), 7.92 (d, J=2.2 Hz, 1H), 7.84 (td, J=7.8, 1.8 Hz, 1H), 7.70 (d, J=8.1 Hz, 1H), 7.56 (d, J=2.4 Hz, 1H), 7.31-7.25 (m, 1H). MS [MH].sup.+ calcd for C.sub.14H.sub.9N.sub.3O.sub.4 284.0, found 284.0.

(11) Compound 361:

(12) 361 was prepared in four synthetic steps from malonamide, according to the following procedure:

(13) ##STR00158##

Step 1: Synthesis of 2-(4,6-dihydroxypyrimidin-2-yl)acetamide (2)

(14) To a solution of NaOEt (21% in EtOH, 167 mL, 450 mmol) in EtOH (170 mL) was added malonamide (22.9 g, 224 mmol). After being refluxed for 2 hours, half of EtOH was removed under reduced pressure and the precipitated solid was filtered and dried under high vacuum for overnight. The dried solid sodium salt (24 g) was dissolved in ice-cold H.sub.2O (70 mL) and brought to pH 2-3 using 3N. HCl (50 mL), recrystallization from water gave 2-(4,6-dihydroxypyrimidin-2-yl)acetamide as a pale yellow solid (6.28 g, 33%).

Step 2: Synthesis of 2-(4,6-dichloropyrimidin-2-yl)acetonitrile (3)

(15) To a solution of 2-(4,6-dihydroxypyrimidin-2-yl)acetamide (6.28 g, 37.1 mmol) in POCl.sub.3 (19 mL, 204 mmol) was placed in a flask which was then attached to a reflux condenser. Through the condenser was added N,N-dimethylaniline (10 mL, 79 mmol). The mixture was warmed cautiously in an oil bath which is quickly removed when the reaction began. After the initial vigorous reaction had subsided, the reaction was refluxed for ten minutes longer. The hot material was poured over 100 g ice and the resulting suspension was extracted (DCM). The combined organic layers were dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The product was purified by column chromatography (SiO.sub.2, PE/EA=4/1) to provide the desired product as a yellow solid (5.1 g, 27.1 mmol). MS [MH].sup.+ calcd for C.sub.6H.sub.3Cl.sub.2N.sub.3 189.0, found 189.0.

Step 3: Synthesis of 2-(pyrimidin-2-yl)acetonitrile (4)

(16) To a solution of 2-(4,6-dichloropyrimidin-2-yl)acetonitrile (2.2 g, 11.7 mmol) and triethylamine (3.0 mL, 20.8 mmol) in ethyl acetate/MeOH (1/1, 40 mL) was added 10% Pd/C (400 mg) and the solution was vigorously stirred for 2.5 hours under H.sub.2 atmosphere (1 atm). The reaction was filtered through celite and washed the celite with MeOH. The combined filtrates were concentrated under reduced pressure and purified by flash chromatography (SiO.sub.2, PE/EA=1/1) to give the 2-(pyrimidin-2-yl)acetonitrile as a pale red liquid (618 mg, 54%). MS [MH].sup.+ calcd for C.sub.6H.sub.5N.sub.3 120.1, found 120.1.

Step 4: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyrimidin-2-yl)acrylonitrile (6)

(17) A solution of 2-(pyrimidin-2-yl)acetonitrile (120 mg, 1 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (182 mg, 1 mmol) and NH.sub.4OAc (462 mg, 6 mmol) in MeOH (10 mL) was heated to reflux for 5 hours. LCMS showed no starting materials left. The solid was filtered and washed by MeOH and H.sub.2O, then dissolved in MeOH (5 mL). 5 mL of 1N.HCl was added to adjust pH 34, the solid was filtered and dried in vacuo to give the desired product as a bright yellow solid (250 mg, 88%). .sup.1H NMR (400 MHz, DMSO) 8.78 (d, J=4.8 Hz, 2H), 8.35 (s, 1H), 7.96 (d, J=2.5 Hz, 1H), 7.59 (d, J=2.5 Hz, 1H), 7.32 (t, J=4.8 Hz, 1H). MS [MH].sup.+ calcd for C.sub.13H.sub.8N.sub.4O.sub.4 285.0, found 285.0.

(18) Compound 395:

(19) 395 was prepared in four synthetic steps from 4-methylpyrimidine, according to the following procedure:

(20) ##STR00159##

Step 1: Synthesis of 4-(chloromethyl)pyrimidine (2)

(21) 4-methylpyrimidine (53.1 mmol, 5 g) was dissolved in CHCl.sub.3 (100 mL), the mixture was heated to 75 C., then 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione (26.6 mmol, 6.2 g) was added slowly in two portions. The mixture was stirred at 75 C. overnight. After the completion of the reaction, it was filtered and concentrated in vacco. The residue was purified by column chromatograph (silica gel, PE/EA=30/1 to 10/1) to obtain the desired product (1.64 g, 24%) as a yellow oil. .sup.1H-NMR (400 MHz, CDCl.sub.3) (ppm) 9.16 (s, 1H), 8.77 (d, J=5.2 Hz, 1H), 7.54 (d, J=5.1 Hz, 1H), 4.60 (s, 2H); MS [MH]+ calcd for C.sub.5H.sub.6ClN.sub.2 129.0, found 129.1;

Step 2: Synthesis of 2-(pyrimidin-4-yl)acetonitrile (3)

(22) Anhydrous potassium carbonate (7.78 mmol, 1.08 g), sodium iodide (3.89 mmol, 583 mg) and trimethylsilanecarbonitrile (5.83 mmol, 579 mg) were dissolved in acetonitrile (12 mL), the mixture was heated to 50 C. Finally 4-(chloromethyl)-pyrimidine (3.89 mmol, 500 mg) was dropped into the reaction mixture. The mixture was stirred 50 C. for 2 hours. Then it was concentrated in vacco and the residue was purified by column chromatograph (silica gel, PE/EA=1/1) to obtain the desired product (120 mg, 26%) as a black oil. .sup.1H-NMR (400 MHz, CDCl.sub.3) (ppm) 9.21 (s, 1H), 8.81 (d, J=5.2 Hz, 1H), 7.52 (d, J=5.0 Hz, 1H), 3.94 (s, 2H); MS [MH]+ calcd for C.sub.6H.sub.6N.sub.3 120.1, found 120.2.

Step 3: Synthesis of 3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyrimidin-4-yl)acrylonitrile (4)

(23) A mixture of 2-(pyrimidin-4-yl)acetonitrile (0.95 mmol, 113 mg), 3,4-dihydroxy-5-nitrobenzaldehyde (0.79 mmol, 145 mg) and ammonium acetate (4.75 mmol, 366 mg) in methanol (8 mL) was stirred at 80 C. for 4 hours, then it was filtered and washed with methanol and water to obtain the desired product (200 mg, 89%). .sup.1H-NMR (400 MHz, DMSO) (ppm) 9.08 (s, 1H), 8.72 (d, J=5.5 Hz, 1H), 8.31 (s, 1H), 8.02 (s, 1H), 7.74 (d, J=5.5 Hz, 1H), 7.59 (s, 1H), 7.08 (s, 2H); MS [MH].sup. calcd for C.sub.13H.sub.7N.sub.4O.sub.4 283.1, found 283.0;

(24) Compound 505:

(25) 505 was prepared in three synthetic steps from 3-chloropyridazine, according to the following procedure:

(26) ##STR00160##

Step 1: Synthesis of tert-butyl 2-cyano-2-(pyridazin-3-yl)acetate (2)

(27) To a solution of 3-chloropyridazine (0.5 g, 4.38 mmol) in NMP (2.5 mL) was added potassium carbonate (1.8 g, 13.15 mmol). Then Cert-Butyl 2-cyanoacetate (0.88 mL, 6.14 mmol) was added. The yellow suspension was warmed up to 80 C. and stirred 3 hours at 80 C. The brown suspension was cooled down to room temperature. Then it was added to water (10 mL). The brown solution was acidified with HCl (gas evolution, strong foaming) There was a precipitation. The suspension was filtrated and the filter cake was washed with water. The filter cake was dissolved in ethyl acetate, dried with Na.sub.2SO.sub.4, filtrated and the organic phase evaporated to yield 600 mg of desired product as a yellow oil. .sup.1H-NMR (400 MHz, CDCl.sub.3) (ppm): 14.3 (bs, 1H), 7.68 (dd, 1H), 7.35 (d, 1H), 1.55 (s, 9H). MS [MH].sup.+ calcd for C.sub.10H.sub.11N.sub.3O.sub.2 206.1, found 206.1;

Step 2: Synthesis of 2-(pyridazin-3-yl)acetonitrile (3)

(28) The product prepared above was combined with TsOH (142 mg) in toluene (50 mL). After being stirred at refluxing for 12 hours, the reaction was cooled to 25 C., diluted with sat. NaHCO.sub.3 and extracted (10 percent MeOH/CH.sub.2Cl.sub.23). The organic layers were washed with brine, dried with Na2SO.sub.4, filtered and concentrated under reduced pressure. Purification of the crude material by flash chromatography (silica gel, 40-45 percent EtOAc/Hexanes) gave the desired product (87 mg, 17% for two steps) as light yellow oil. MS [MH].sup.+ calcd for C.sub.6H.sub.5N.sub.3 120.0, found 120.0.

Step 3: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(pyridazin-3-yl)acrylonitrile (4)

(29) A mixture of 2-(pyrazin-2-yl)acetonitrile (87 mg, 0.73 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (0.79 mmol, 145 mg) and ammonium acetate (366 mg, 4.75 mmol) in methanol (8 mL) was stirred at 80 C. for 4 hours. Then it was filtered and washed with methanol and water, and dried in vacuo to obtain the desired product (155 mg, 75%) as a yellow solid. .sup.1H NMR (400 MHz, DMSO) 10.92 (s, 2H), 9.25 (d, J=4.8 Hz, 1H), 8.43 (s, 1H), 8.20 (d, J=8.7 Hz, 1H), 8.08 (d, J=2.0 Hz, 1H), 7.95 (d, J=2.0 Hz, 1H), 7.85 (dd, J=8.7, 4.9 Hz, 1H). MS [MH].sup. calcd for C.sub.6H.sub.5N.sub.3 283.1, found 283.0.

(30) Compound 331:

(31) 331 was prepared in one synthetic step from 3,4-dihydroxy-5-nitrobenzaldehyde, according to the following procedure:

(32) ##STR00161##

Step 1: Synthesis of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-(thiazol-2-yl)acrylamide (2)

(33) A solution of 2-cyano-N-(thiazol-2-yl)acetamide (184 mg, 1.1 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (200 mg, 1.1 mmol) and NH.sub.4OAc (462 mg, 6 mmol) in MeOH (10 mL) was heated to reflux for overnight. LCMS showed no 3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixture was cooled to room temperature. The solid was filtered and washed by MeOH and H.sub.2O. The solid was re-dissolved in MeOH (5 mL). 5 mL of 1N aqueous HCl was added to adjust pH 3-4. The desired product was obtained by filter as a bright solid (90 mg, 25%). .sup.1H NMR (400 MHz, DMSO) 8.17 (s, 1H), 7.95 (s, 1H), 7.50 (s, 2H), 7.20 (s, 1H). MS [MH].sup.+ calcd for C.sub.13H.sub.8N.sub.4O.sub.5S 333.0, found 333.0.

(34) Compound 394:

(35) 394 was prepared in four synthetic steps from benzene-1,2-diamine, according to the following procedure:

(36) ##STR00162##

Step 1: Synthesis of 1-(1H-benzo[d]imidazol-2-yl)ethanone (2)

(37) A mixture of 2-oxosuccinic acid (4.9 g, 37 mmol), benzene-1,2-diamine (4 g, 37 mmol) and 4N hydrochloride solution (9 mL) in MeOH (30 mL) was refluxed for 7 hours. After the completion of the reaction, it was concentrated in vacuo to remove the solvent. The residue was dissolved in ethyl acetate, washed with aq. sodium bicarbonate and brine. The organic layers were combined and concentrated under reduced pressure. The residue was purified by column chromatograph (silica gel, PE/EA=5/1) to obtain the desired product (4 g, 67%). MS [MH].sup.+ calcd for C.sub.9H.sub.9N.sub.2O 161.06, found 161.1.

Step 2: Synthesis of 1-(1H-benzo[d]imidazol-2-yl)-2-bromoethanone (3)

(38) 1-(1H-benzo[d]imidazol-2-yl)ethanone (4 g, 25 mmol) was dissolved in tetrachloromethane (50 mL). 1-Bromopyrrolidine-2,5-dione (5.3 g, 30 mmol) and 2,2-(diazene-1,2-diyl)bis(2-methylpropane-nitrile) (411 mg, 2.5 mmol) were added. The mixture was stirred at 100 C. for 2 hours, then it was concentrated in vacuo and re-dissolved in ethyl acetate. The organic layer was washed with water and concentrated to obtain the crude product (2 g, 33%), which was used in the next step without further purification. MS [MH].sup.+ calcd for C.sub.9H.sub.8BrN.sub.2O 238.97, found 239.0.

Step 3: Synthesis of 3-(1H-benzo[d]imidazol-2-yl)-3-oxopropanenitrile (4)

(39) A mixture of 1-(1H-benzo[d]imidazol-2-yl)-2-bromoethanone (2 g, 8.4 mmol), trimethylsilane-carbonitrile (1.66 g, 16.7 mmol), TBAF (2.2 g, 8.4 mmol) in dichloromethane (15 mL) was stirred at room temperature for 24 hours. Then it was concentrated in vacuo and re-dissolved in ethyl acetate. The organic layer was washed with water and concentrated to obtain the crude product (400 mg, 26%), which was used in the next step without further purification. MS [MH].sup.+ calcd for C.sub.10H.sub.7N.sub.3O 186.06, found 186.1.

Step 4: Synthesis of 2-(1H-benzo[d]imidazole-2-carbonyl)-3-(3,4-dihydroxy-5-nitrophenyl)acrylo-nitrile (5)

(40) A solution of 3,4-dihydroxy-5-nitrobenzaldehyde (107 mg, 0.59 mmol) and 3-(1H-benzo[d]imida-zole-2-yl)-3-oxopropanenitrile (130 mg, 0.7 mmol) and NH.sub.4OAc (273 mg, 3.54 mmol) in methanol (10 mL) was heated to reflux for overnight. LCMS showed the desired product was formed, the reaction mixture was cooled to room temperature and concentrated in vacuo to remove the solvent. The desired product was obtained by Prep-HPLC (50 mg, 24%). .sup.1H-NMR (400 MHz, DMSO-d.sup.6) (ppm) 12.63 (s, 1H), 8.95 (s, 1H), 7.85 (s, 1H), 7.76 (d, J=8.2 Hz, 1H), 7.65 (s, 1H), 7.50 (t, J=7.5 Hz, 1H), 7.38-7.26 (m, 2H), 7.12 (s, 2H). MS [MH].sup.+ calcd for C.sub.17H.sub.11N.sub.4O.sub.5 351.07, found 351.0.

(41) Compound 382:

(42) 382 was prepared in two synthetic steps from ethyl 2-cyanoacetate, according to the following procedure:

(43) ##STR00163##

Step 1: Synthesis of 3-morpholino-3-oxopropanenitrile (2)

(44) A mixture of sodium ethoxide (0.1 mmol) in ethanol (3 mL), ethyl cyanoacetate (1.13 g, 10 mmol) and morpholine (0.85 g, 10 mmol) was stirred at room temperature for 24 hours. The precipitate was collected by filtration, washed with diethylether and recrystallised in ethanol to provide a white solid of 3-morpholino-3-oxopropanenitrile (0.56 g, 35%).

Step 2: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrile (3)

(45) A solution of 3-morpholino-3-oxopropanenitrile (300 mg, 2.0 mmol), 3,4-dihydroxy-5-nitro-benzaldehyde (188 mg, 1.1 mmol) and NH.sub.4OAc (462 mg, 6 mmol) in MeOH (10 mL) was heated to reflux for 5 hours. LCMS showed no 3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixture was cooled to room temperature, concentrated in vacuo to dryness. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) afforded the desired product as a yellow solid (60 mg, 19%). .sup.1H NMR (400 MHz, DMSO) 10.87 (s, 2H), 7.94 (d, J=2.1 Hz, 1H), 7.77 (d, J=2.1 Hz, 1H), 7.68 (s, 1H), 3.56-3.66 (m, 8H). MS [MH].sup.+ calcd for C.sub.14H.sub.13N.sub.3O.sub.6 320.1, found 320.0.

(46) Compound 351:

(47) 351 was prepared in three synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(48) ##STR00164##

Step 1: Synthesis of 3,4-dimethoxy-5-nitrobenzoyl chloride (2)

(49) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. for 15 hours. The solvent was removed under reduced pressure. The resulting yellowish solid (500 mg, 92%) was used in the next step without further workup.

Step 2: Synthesis of 3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(pyridin-2-yl)propanenitrile (3)

(50) Under a nitrogen atmosphere, NaH (60% w/w, 176 mg, 4.4 mmol) was added to solution of 2-(pyridin-2-yl)acetonitrile (236 mg, 2.0 mmol) in anhydrous THF (10 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride (500 mg, 2.2 mmol) in THF (5 mL) was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature. The mixture was extracted with ethyl acetate (25 mL2). The combined organic layers were dried with anhydrous sodium sulfate and concentrated in vacuo to give the desired product (425 mg, 64%). MS [MH].sup.+ calcd for C16H14N3O5 328.09, found 328.1.

Step 3: Synthesis of 3-(3,4-dihydroxy-5-nitrophenyl)-3-oxo-2-(pyridin-2-yl)propanenitrile (4)

(51) A solution of 3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(pyridin-2-yl)propanenitrile (425 mg, 1.3 mmol) in dichloromethane (5 mL) was added 1.0 M solution of BBr.sub.3 in dichloromethane (10 mL, 10 mmol) at 15 C. under a nitrogen atmosphere. The resulting suspension was stirred for 1 hours at 15 C. and allowed to warm to room temperature for overnight. The reaction was quenched slowly by the addition of water (4 mL) and stirred for another 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried over with anhydrous sodium sulfate. The solvent was removed under reduced pressure to give the crude product. Further purification was conducted by Prep-HPLC to obtain the desired product (65 mg, 17%). .sup.1H-NMR (400 MHz, DMSO-d.sup.6) (ppm) 16.11 (s, 1H), 10.65 (s, 2H), 8.38 (t, J=5.7 Hz, 1H), 8.28-7.96 (m, 1H), 7.84 (d, J=2.0 Hz, 1H), 7.55 (d, J=2.1 Hz, 1H), 7.47 (d, J=8.7 Hz, 1H), 7.26 (t, J=6.6 Hz, 1H). MS [MH].sup.+ calcd for C.sub.14H.sub.10N.sub.3O.sub.5 300.05, found 300.0.

(52) Compound 371:

(53) 371 was prepared in two synthetic steps from 2-cyanoacetyl chloride, according to the following procedure:

(54) ##STR00165##

Step 1: Synthesis of 3-oxo-3-(piperidin-1-yl)propanenitrile (2)

(55) A mixture of piperidine (5 mL, 50.6 mmol), in DCM (25 mL) was added 2-cyanoacetyl chloride (5 mL) at 0 C., then warmed to room temperature overnight. The reaction mixture was quenched by H.sub.2O, and concentrated in vacuo to dryness, the residue was purified by column chromatography (SiO.sub.2, PE/EA=1/1) to give the 3-oxo-3-(piperidin-1-yl)propanenitrile as a yellow oil (500 mg, 7%). MS [MH].sup.+ calcd for C.sub.8H.sub.12N.sub.2O 153.1, found 153.1.

Step 2: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrile (3)

(56) A solution of 3-oxo-3-(piperidin-1-yl)propanenitrile (300 mg, 2 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (273 mg, 1.5 mmol) and NH.sub.4OAc (924 mg, 12 mmol) in MeOH (15 mL) was heated to reflux for 3 hours. The solid was filtered and washed by MeOH and H.sub.2O to give the crude product. The solid dissolved in MeOH (5 mL) was added 1N.HCl (0.5 mL), the color was changed and the solid was formed, the solid was filtered and washed by H.sub.2O, dried in vacuo to give the (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrile as a bright yellow solid (60 mg, 13%). .sup.1H NMR (400 MHz, DMSO) 10.86 (s, 2H), 7.92 (d, J=2.1 Hz, 1H), 7.75 (d, J=2.0 Hz, 1H), 7.63 (s, 1H), 3.56-3.45 (m, 4H), 1.55-1.62 (m, 6H). MS [MH].sup.+ calcd for C.sub.15H.sub.15N.sub.3O.sub.5 318.3, found 318.0.

(57) Compound 518:

(58) 518 was prepared in two synthetic steps from 2-(pyridin-3-yl)acetonitrile, according to the following procedure:

(59) ##STR00166##

Step 1: Synthesis of 3-(cyanomethyl)pyridine 1-oxide (2)

(60) A solution of 2-(pyridin-3-yl)acetonitrile (625 mg, 5.3 mmol) and m-CPBA (1.36 g, 7.95 mmol) in CHCl.sub.3 (20 mL) was stirred at room temperature for overnight. The reaction mixture was quenched by sat.NaHCO.sub.3 and extracted by DCM and MeOH (DCM/MeOH=10/1). The combined organic layers were dried over anhydrous Na.sub.2SO.sub.4, filtered and dried in vacuo to give the crude product as a white solid (780 mg, >100%), which was used to the next step without further purification. MS [MH].sup.+ calcd for C.sub.7H.sub.6N.sub.2O 135.0 found 135.0.

Step 2: Synthesis of (E)-3-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyridine 1-oxide (3)

(61) A solution of 3-(cyanomethyl)pyridine 1-oxide (400 mg, 3.0 mmol), 3,4-dihydroxy-5-nitro-benzaldehyde (270 mg, 1.5 mmol) and NH.sub.4OAc (693 mg, 9 mmol) in MeOH (10 mL) was heated to reflux for overnight. LCMS showed no 3,4-dihydroxy-5-nitrobenzaldehyde left. The reaction mixture was cooled to room temperature. The solid was filtered and washed by MeOH and H.sub.2O. The solid was re-dissolved in MeOH (5 mL). 5 mL of 1N aqueous HCl was added to adjust pH 3-4. The desired product was obtained by filter as a bright solid (110 mg, 18%). .sup.1H NMR (301 MHz, DMSO) 13.71 (s, 1H), 10.89 (s, 2H), 8.65 (s, 1H), 8.26 (d, J=6.2 Hz, 1H), 8.14 (s, 1H), 7.99 (d, J=2.0 Hz, 1H), 7.84 (d, J=2.0 Hz, 1H), 7.67-7.50 (m, 2H). MS [MH].sup.+ calcd for C.sub.14H.sub.9N.sub.3O.sub.5 300.0 found 300.0.

(62) Compound 523:

(63) 523 was prepared in three synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(64) ##STR00167##

Step 1: Synthesis of 3,4-dimethoxy-5-nitrobenzoyl chloride (2)

(65) Under a nitrogen atmosphere, SOCl.sub.2 (0.76 mL, 10.56 mmol) and anhydrous DMF (0.02 mL, 0.44 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (1 g, 4.4 mmol) in toluene (20 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The solvent was removed under reduced pressure. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride (1 g, 93%) was used in the next step without further workup.

Step 2: Synthesis of 3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrazin-2-yl)acrylonitrile (3)

(66) Under a nitrogen atmosphere, 60% NaH (336 mg, 8.4 mmol) was added to solution of 2-(pyrazin-2-yl)acetonitrile (500 mg, 4.2 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoyl chloride (500 mg, 4.07 mmol) was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (8 mL) and stirred for 10 min at room temperature, then extracted with ethyl acetate (30 mL3), the organic layer was dried with anhydrous sodium sulfate and concentrated in vacuo to obtain the desired product (440 mg, 33%). MS [MH].sup. calcd for C.sub.15H.sub.11N.sub.4O.sub.5 327.08, found 327.1.

Step 3: Synthesis of 3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(pyrazin-2-yl)acrylonitrile (4)

(67) To a solution of 3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrazin-2-yl)acrylonitrile (200 mg, 0.61 mmol) in anhydrous dichloromethane (5 mL) was added 1.0 M solution of BBr.sub.3 in dichloromethane (3 mL, 3 mmol) at 15 C. under a nitrogen atmosphere. The resulting suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature for overnight. The reaction was quenched by the addition of water (2 mL) and stirred for another 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to give the crude product. Further purification was conducted by Prep-HPLC to obtain the desired product (35 mg, 19%). .sup.1H-NMR (400 MHz, DMSO-d.sup.6) (ppm) 15.85 (s, 1H), 10.75 (s, 2H), 8.88 (s, 1H), 8.34 (d, J=3.7 Hz, 1H), 8.22 (d, J=2.5 Hz, 1H), 7.88 (d, J=2.1 Hz, 1H), 7.56 (d, J=2.1 Hz, 1H). MS [MH].sup. calcd for C.sub.13H.sub.7N.sub.4O.sub.5 299.05, found 299.0.

(68) Compound 525:

(69) 525 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(70) ##STR00168##

Step 1: Synthesis of (E)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrimidin-4-yl)acrylonitrile (2)

(71) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(72) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to solution of 2-(pyrimidin-4-yl)acetonitrile (0.44 g, 2.0 mmol) in anhydrous THF (10 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoyl chloride was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound as an orange solid (550 mg, 85%). MS [MH].sup.+ calcd for C.sub.15H.sub.12N.sub.4O.sub.5 329.0 found 329.0.

Step 2: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(pyrimidin-4-yl)acrylonitrile (3)

(73) A solution of (E)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(pyrimidin-4-yl)acrylonitrile (250 mg, 0.76 mmol) in DCM (5 mL) was added BBr.sub.3 (0.5 mL, 5 mmol) at 5 C. under a nitrogen atmosphere. The resulting red suspension was stirred for 1 h at 5 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried over Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as a bright yellow solid (46 mg, 19%). .sup.1H NMR (400 MHz, DMSO) 15.35 (s, 1H), 10.67 (s, 2H), 8.95-7.16 (m, 5H). MS [MH].sup.+ calcd for C.sub.15H.sub.14N.sub.2O.sub.5 337.0 found 301.0 (free).

(74) Compound 503:

(75) 503 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(76) ##STR00169##

It Step 1: Synthesis of 3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(thiazol-2-yl)propanenitrile (2)

(77) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(78) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 2-(thiazol-2-yl) acetonitrile (0.24 g, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound as an orange solid (240 mg, 39%). MS [MH].sup.+ calcd for C.sub.14H.sub.11N.sub.3O.sub.5S 334.3, found 334.3.

Step 2: Synthesis of 3-(3,4-dihydroxy-5-nitrophenyl)-3-oxo-2-(thiazol-2-yl)propanenitrile (3)

(79) A solution of 3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-2-(thiazol-2-yl)propanenitrile (224 mg, 0.67 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (3 mL, 3 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried over Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as a bright yellow solid (24 mg, 12%). .sup.1H NMR (400 MHz, DMSO) 7.88 (d, J=2.0 Hz, 1H), 7.60 (d, J=4.0 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.31 (d, J=4.0 Hz, 1H). MS [MH].sup. calcd for C.sub.12H.sub.7N.sub.3O.sub.5S 304.0, found 304.0.

(80) Compound 374:

(81) 374 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(82) ##STR00170##

Step 1: Synthesis of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-N-(thiazol-2-yl)propanamide (2)

(83) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(84) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to solution of 2-cyano-N-(thiazol-2-yl)acetamide (0.35 g, 2.0 mmol) in anhydrous THF (10 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoyl chloride was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound as an orange solid (510 mg, 67%). MS [MH].sup.+ calcd for C.sub.15H.sub.12N.sub.4O.sub.6S 377.0, found 377.0.

Step 2: Synthesis of 2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-3-oxo-N-(thiazol-2-yl)propanamide (3)

(85) A solution of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-oxo-N-(thiazol-2-yl)propanamide (400 mg, 1.1 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (5 mL, 5 mmol) at 15 C. under a nitrogen atmosphere. The resulting red suspension was stirred for 1 h at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried over Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as a bright yellow solid (100 mg, 28%). .sup.1H NMR (400 MHz, DMSO) 7.88 (d, J=2.0 Hz, 1H), 7.60 (d, J=4.0 Hz, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.31 (d, J=4.0 Hz, 1H). MS [MH].sup.+ calcd for C.sub.13H.sub.8N.sub.4O.sub.6S 349.0, found 349.0.

(86) Compound 655:

(87) 655 was prepared in four synthetic steps from methyl 5-chloropyrazine-2-carboxylate, according to the following procedure:

(88) ##STR00171##

Step 1: Synthesis of methyl 5-(2-(tert-butoxy)-1-cyano-2-oxoethyl)pyrazine-2-carboxylate (2)

(89) A solution of tert-butyl 2-cyanoacetate (2.1 g, 15 mmol) and t-BuOK (1.6 g, 15 mmol) in dry THF (50 mL) stirred at rt for 30 min, then methyl 5-chloropyrazine-2-carboxylate (1.7 g, 10.0 mmol) was added, the reaction mixture was heated to reflux overnight, After the reaction was completed, cooled it to rt and quenched by H.sub.2O (100 mL), the solid was filtered and dried in vacuo to afford the desired product as yellow solid (1.7 g, 61%). MS [M+H].sup.+ calcd for C.sub.13H.sub.15N.sub.3O.sub.4 278.1, found 278.1.

Step 2: Synthesis of methyl 5-(cyanomethyl)pyrazine-2-carboxylate (3)

(90) A solution of 5-(2-(tert-butoxy)-1-cyano-2-oxoethyl)pyrazine-2-carboxylate (1.7 g, 6.14 mmol) and p-TsOH (314 mg, 1.84 mmol) was heated to reflux for 3 h, then TLC showed no starting materials left. The reaction mixture was quenched by H.sub.2O (5 mL), extracted by EA, washed with sat. NaHCO.sub.3, the organic layer was dried with Na.sub.2SO.sub.4, filtered and dried in vacuo to afford the crude product, further purification by column chromatography (SiO.sub.2, 100 g, 200-300 m, eluted by PE/EA=5/1) to afford the desired product methyl 5-(cyanomethyl)pyrazine-2-carboxylate (800 mg, 74%) as yellow solid. MS [M+H].sup.+ calcd for C.sub.8H.sub.7N.sub.3O.sub.2 178.1 found 178.1.

Step 3: Synthesis of methyl (Z)-methyl 5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate (4)

(91) A solution of methyl 5-(cyanomethyl)pyrazine-2-carboxylate (170 mg, 1.0 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and NH.sub.4OAc (554 mg, 7.2 mmol) in MeOH (15 mL) was heated to reflux for 3 hours, then cooled it to room temperature. The solid was filtered and washed by H.sub.2O (15 mL), The solid was re-dissolved in MeOH (5 mL). 5 mL of 1N aqueous HCl was added to till pH=3-4. The desired product was obtained by filter and dried in vacuo as bright solid (210 mg, 61%). MS [M+H].sup.+ calcd for C.sub.15H.sub.10N.sub.4O.sub.6 343.0, found 343.0.

Step 4: Synthesis of (Z)-5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylic acid (5)

(92) A solution of (Z)-methyl 5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate (150 mg, 0.44 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (2 mL, 2 mmol) at 15 C. under a nitrogen atmosphere. The suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated in vacuo to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as bright yellow solid (55 mg, 38%). .sup.1H NMR (300 MHz, DMSO) 9.27 (s, 1H), 9.22 (s,1H), 8.57 (s, 1H), 8.12 (s, 1H), 7.98 (s, 1H), 2.52 (s, 19H), 0.02 (s, 1H). MS [MH].sup.+ calcd for C.sub.14H.sub.8N.sub.4O.sub.6 329.0, found 329.0.

(93) Compound 656:

(94) 656 was prepared in two synthetic steps from 5-chloro-1,2,4-thiadiazole, according to the following procedure:

(95) ##STR00172##

Step 1: Synthesis of 2-(1,2,4-thiadiazol-5-yl)acetonitrile (2)

(96) A solution of dry MeCN (226 mg, 11 mmol) in dry THF (25 ml) was added LiHMDS (5.5 mmol, 5.5 mL) at 0 C., then the mixture was stirred at 0 C. for 30 min, 5-chloro-1,2,4-thiadiazole (691 mg, 5.5 mmol) in dry THF (5 mL) was added to the mixture at 0 C., then stirred at rt overnight. The reaction mixture was quenched by H.sub.2O (1 mL), and extracted by EA (30 mL3), dried with Na.sub.2SO.sub.4, filtered and dried in vacuo to afford the crude product. Further purification by column chromatography (SiO.sub.2, 100 g, 200-300 m, eluted by PE/EA=5:1) gave the desired product (410 mg, 60%) as yellow solid. MS [M+H].sup.+ calcd for C.sub.4H.sub.3N.sub.3S 126.0, found 126.0.

Step 2: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(1,2,4-thiadiazol-5-yl)acrylonitrile (3)

(97) A solution of 2-(1,2,4-thiadiazol-5-yl)acetonitrile (150 mg, 1.2 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (182 mg, 1 mmol) and NH.sub.4OAc (462 mg, 6 mmol) in MeOH (10 mL) was heated to reflux for 5 hours. LC-MS showed no starting materials left. The solid was filtered and washed by MeO and H.sub.2O, then dissolved in MeOH (5 mL). 5 ml of 1N.HCl was added till pH=3-4. The solid was filtered and dried in vacuo to give the desired product as bright yellow solid (200 mg, 69%). .sup.1H NMR (400 MHz, DMSO) 10.96 (s, 1H), 8.97 (s, 1H), 8.45 (s, 1H), 8.14 (d, J=2.1 Hz, 1H), 7.95 (d, J=2.2 Hz, 1H). MS [M+H].sup.+ calcd for C.sub.11H.sub.6N.sub.4O.sub.4S 291.0, found 291.0.

(98) Compound 660:

(99) 660 was prepared in two synthetic steps from (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid, according to the following procedure:

(100) ##STR00173##

Step 1: Synthesis of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acryloyl chloride (2)

(101) A solution of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid (25 mg, 0.1 mmol) in DCM (25 ml) was added a drop of DMF and oxalyl dichloride (25 mg,0.2 mmol). The reaction mixture was heated till the solid dissolved, cooled to rt, concentrated in vacuo to dryness. It was used in the next step without purification (27 mg, 100%).

Step 2: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(1,3-oxazinane-3-carbonyl)acrylonitrile (3)

(102) A solution of 1,3-oxazinane (9 mg, 0.12 mmol), Et.sub.3N (24 mg, 0.24 mmol) in DCM (3 mL) was added acyl chloride dissolved in DCM (3 mL) dropwise at 0 C., when the addition was completed, the reaction mixture was slowly warmed to rt overnight. The reaction mixture was quenched by H.sub.2O, separated the organic layer, and dried with Na.sub.2SO.sub.4, concentrated in vacuo to afford the crude product, further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) afford the desired product as yellow solid (5 mg, 16%). 1H NMR (400 MHz, DMSO) 10.87 (s, 2H), 7.95 (d, J=2.0 Hz, 1H), 7.76 (d, J=2.1 Hz, 1H), 7.69 (s, 1H), 5.01 (s, 2H), 3.88-3.84 (m, 2H), 3.74 (s, 2H), 1.72-1.66 (m, 2H). MS [M+H]+ calcd for C.sub.14H.sub.14N.sub.3O.sub.6 320.1 found. 319.9

(103) Compound 661:

(104) 661 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(105) ##STR00174##

Step 1: Synthesis of (Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl) acrylonitrile (2)

(106) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(107) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 3-oxo-3-(piperidin-1-yl)propanenitrile (0.30 g, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound 3 as an orange solid (260 mg, 36%). MS [MH].sup.+ calcd for C.sub.17H.sub.20N.sub.3O.sub.6 362.1, found 362.1.

Step 2: Synthesis of (Z)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile (3)

(108) A solution of (Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile (180 mg, 0.5 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (3 mL, 3 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as yellow solid (63 mg, 38%). 1H NMR (400 MHz, DMSO) 10.96 (s, 1H), 7.87 (d, J=2.0 Hz, 1H), 7.49 (s, 1H), 3.65 (s, 4H), 1.60 (s, 6H). MS [MH]-calcd for C.sub.15H.sub.16N.sub.3O.sub.6 334.1, found 333.9

(109) Compound 666:

(110) 666 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(111) ##STR00175##

Step 1: Synthesis of 2-(cyanomethyl)thiazole-4-carboxylic acid (3)

(112) Under a nitrogen atmosphere, 3-bromo-2-oxopropanoic acid in dry THF (5 mL) was added to a suspension of 2-cyanoethanethioamide (1.2 g, 12 mmol) in THF (20 ml) at 0 C. The mixture was heated at 70 C. and stirred for 3 hours. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by column chromatography (SiO.sub.2, 100 g, 200-300 m, eluted by PE/EA=1/1) afforded the desired product as white solid (350 mg, 18%).

Step 2: Synthesis of (E)-2-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)vinyl)thiazole-4-carboxylic acid (4)

(113) A solution of 2-(cyanomethyl)thiazole-4-carboxylic acid (168 mg, 1.0 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and NH.sub.4OAc (554 mg, 7.2 mmol) in MeOH (15 mL) was heated to reflux for 3 hours, then cooled to room temperature. The solid was filtered and washed by H.sub.2O (15 mL), The solid was re-dissolved in MeOH (5 mL). 5 mL of 1N aqueous HCl was added to adjust pH 3-4. The desired product was obtained by filter and dried in vacuo as bright solid (150 mg, 45%). .sup.1H NMR (400 MHz, DMSO) 7.98 (s, 1H), 7.91 (s, 1H), 7.84 (s, 1H), 7.15-7.75 (m, 4H). MS [MH].sup.+ calcd for C.sub.13H.sub.7N.sub.3O.sub.6S 334.0, found 334.0.

(114) Compound 668:

(115) 668 was prepared in three synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(116) ##STR00176##

Step 1: Synthesis of 2-cyano-N-(1,2,4-thiadiazol-5-yl)acetamide (2)

(117) Under a nitrogen atmosphere, NaH (200 mg, 5 mmol, 60%) was added to a suspension of 1,2,4-thiadiazol-5-amine (500 mg, 5 mmol) in THF (25 mL) at 0 C. The resulting suspension was stirred at 0 C. for 15 min and the solution of 2-cyanoacetyl chloride (500 mg, 5 mmol) in THF was added over 10 min and stirred for an additional 1 h at RT, then quenched by the addition of 1N.HCl solution and stirred for 10 min at room temperature. Extracted it by ethyl acetate (25 mL2), and the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the crude product. Washed it by PE/EA=1:1 (5 mL) to afford the purity product (260 mg, 30%). MS [MH].sup.+ calcd for C.sub.5H.sub.4N.sub.4OS 169.0, found 169.0.

Step 2: Synthesis of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(1,2,4-thiadiazol-5-yl)acrylamide (3)

(118) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. When the reaction was complete, the organic solvent was eliminated by distillation under reduced pressure. Additional toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL)

(119) Under a nitrogen atmosphere, 60% NaH (80 mg, 2.0 mmol) was added to solution of 2-cyano-N-(1,2,4-thiadiazol-5-yl)acetamide (250 mg, 1.5 mmol) in anhydrous THF (10 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the THF solution of 3,4-dimethoxy-5-nitrobenzoyl chloride was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C.; quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature. Extracted by ethyl acetate (25 mL2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound as orange solid (210 mg, 37%). MS [M+H].sup.+ calcd for C.sub.14H.sub.11N.sub.5O.sub.6S 378.0, found 378.0.

Step 3: Synthesis of 2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-N-(1,2,4-thiadiazol-5-yl)acrylamide (4)

(120) A solution of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(1,2,4-thiadiazol-5-yl)acrylamide (150 mg, 0.40 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (2 mL, 2 mmol) at 15 C. under a nitrogen atmosphere. The suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as bright yellow solid (50 mg, 36%).'H NMR (400 MHz, DMSO) 13.80 (s, 1H), 8.33 (s, 1H), 7.72 (d, J=1.8 Hz, 1H), 7.47 (d, J=1.8 Hz, 1H). MS [MH].sup.+ calcd for C.sub.12H.sub.7N.sub.5O.sub.6S 350.0, found 350.0.

(121) Compound 673:

(122) 673 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(123) ##STR00177##

Step 1: Synthesis of (Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(pyrimidin-4-ylmethyl)acrylamide (2)

(124) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(125) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 2-cyano-N-(pyrimidin-4-ylmethyl)acetamide (0.35 g, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound 3 as an orange solid (169 mg, 22%). MS [M+H].sup.+ calcd for C.sub.17H.sub.16N.sub.5O.sub.6 386.1, found 386.0.

Step 2: Synthesis of (Z)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-N-(pyrimidin-4-ylmethyl)acrylamide (3)

(126) A solution of (Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-(pyrimidin-4-ylmethyl)acrylamide (115 mg, 0.3 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (3 mL, 3 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as yellow solid (8 mg, 7%). 1H NMR (400 MHz, DMSO) 14.18 (s, 1H), 10.74 (s, 2H), 9.12 (s, 1H), 8.75 (d, J=5.2 Hz, 1H), 7.88 (s, 1H), 7.55 (d, J=2.1 Hz, 1H), 7.45 (d, J=4.7 Hz, 1H), 4.52 (s, 2H). MS [M+H].sup.+ calcd for C.sub.15H.sub.12N.sub.5O.sub.6 356.1, found 356.0

(127) Compound 675:

(128) 675 was prepared by four synthetic steps from methyl 3-hydroxyisoxazole-5-carboxylate according to the following procedure:

(129) ##STR00178##

Step 1: Synthesis of methyl 3-methoxyisoxazole-5-carboxylate (2)

(130) A mixture of methyl 3-hydroxyisoxazole-5-carboxylate (1.0 g, 7.0 mmol) and K.sub.2CO.sub.3 (1.9 g, 14 mmol) in dry DMF (15 mL) was added Me.sub.2SO.sub.4 (1.0 g, 8.4 mmol) at 0 C. The reaction solution was stirred at 0 C. continuously, and monitored by TLC until all the starting material was consumed completely; 50 mL of water was added. The residue was extracted by EA for two times (25 mL2), and the organic layer was washed with brine (25 mL3), and dried over anhydrous Na.sub.2SO.sub.4. The organic layer was concentrated in vacuo to afford the desired product (850 mg, 77%) without further purification. MS [M+H].sup.+ calcd for C.sub.6H.sub.7NO.sub.4 158.0, found 158.0.

Step 2: Synthesis of3-(3-methoxyisoxazol-5-yl)-3-oxopropanenitrile (3)

(131) A mixture of MeCN (553 mg, 13.5 mmol) and t-BuOK (1.5 g, 13.5 mmol) in dry THF (25 mL) and toluene (15 mL) was added methyl 3-methoxyisoxazole-5-carboxylate (850 mg, 5.4 mmol) at rt. The reaction solution was stirred at 80 C. continuously for 24 h, and monitored by TLC until all the starting material was consumed completely. 50 mL of water was added. The aqueous phase was extracted by EA for two times (25 mL2), and the organic layer was combined and washed with brine (25 mL3), and dried over anhydrous Na.sub.2SO.sub.4. The organic layer was concentrated in vacuo to afford the crude product which was purified by silica chromatograph (100 g, 200-300 m, eluted by PE/EA=3/1) to afford the desired product as bright yellow solid (650 mg, 72%). MS [M+H].sup.+ calcd for C.sub.7H.sub.6N.sub.2O.sub.3167.0, found 167.0.

Step 3: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(3-methoxyisoxazole-5-carbonyl)acrylonitrile (5)

(132) A mixture of 3-(3-methoxyisoxazol-5-yl)-3-oxopropanenitrile (200 mg, 1.2 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and NH.sub.4OAc (554 mg, 7.2 mmol) in MeOH (15 mL) was refluxed for 3 hours, then cooled to room temperature. The reaction mixture was filtered and the solid was collected and washed by H.sub.2O (15 mL). The solid was re-dissolved in MeOH (5 mL) and the PH was adjusted to 3-5 by adding 5 mL of 1N aqueous HCl. The desired product was obtained by filter and dried in vacuo as bright yellow solid (250 mg, 76%). MS [MH].sup.+ calcd for C.sub.14H.sub.9N.sub.3O.sub.7 332.0, found 332.0.

Step 4: Synthesis of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(3-hydroxyisoxazole-5-carbonyl)acrylonitrile (6)

(133) A solution of (E)-3-(3,4-dihydroxy-5-nitrophenyl)-2-(3-methoxyisoxazole-5-carbonyl) acrylonitrile (150 mg, 0.45 mmol) in AcOH (5 mL) was added 0.5 mL 33% HBr (in AcOH) at rt. The reaction solution was stirred at 80 C. continuously for 3 h and monitored by LC-MS until all the starting material was consumed completely. The reaction mixture was concentrated in vacuo to afford the crude product which was purified by by Prep-HPLC (0.5% TFA, MeCN/H.sub.2O) to afford the desired product as yellow solid (22 mg, 15%). MS [MH].sup.+ calcd for C.sub.13H.sub.7N.sub.3O.sub.7 318.0, found 318.0. .sup.1H NMR (400 MHz, DMSO) 11.96 (s, 2H), 8.32 (s, 1H), 8.18 (d, J=2.1 Hz, 1H), 7.88 (d, J=2.2 Hz, 1H), 6.93 (s, 1H)

(134) Compound 687:

(135) 687 was prepared in two synthetic steps from 2-cyanoacetate according, according to the following procedure:

(136) ##STR00179##

Step 1: Synthesis of 3-(azetidin-1-yl)-3-oxopropanenitrile (2)

(137) A mixture of ethyl 2-cyanoacetate (565 mg, 5.0 mmol) and azetidine (285 mg, 5.0 mmol) was stirred at rt overnight. The reaction solution was purified by HPLC to afford the desire product. MS [MH+H].sup.+ calcd for C.sub.6H.sub.9N.sub.2O.sub.1 125.1, found 125.1

Step 2: Synthesis of 3-(azetidin-1-yl)-3-oxopropanenitrile (3)

(138) A mixture of 3-(azetidin-1-yl)-3-oxopropanenitrile (124 mg, 1.0 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1.0 mmol) and CH.sub.3COONH.sub.4 (770 mg, 10.0 mmol) in MeOH (15 mL) was stirred at 80 C. overnight and then cooled to rt. The reaction mixture was filtered to collect the solid. The solid was re-dissolved in MeOH and the PH was adjusted to 3-5 by adding 1N HCl. The desire product was collected by filtered and dried in vavuo. (116 mg, 40%). MS [M+H].sup.+ calcd for C.sub.13H.sub.12N.sub.3O.sub.5 290.2, found 289.9. .sup.1H NMR (400 MHz, DMSO) 10.93 (s, 2H), 8.01 (d, J=2.1 Hz, 1H), 7.97 (s, 1H), 7.81 (d, J=2.1 Hz, 1H), 4.48 (t, J=7.2 Hz, 2H), 4.03 (t, J=7.3 Hz, 2H), 2.32-2.22 (m, 2H).

(139) Compound 688:

(140) 688 was prepared in two synthetic steps from 2-cyanoacetate according to the following procedure:

(141) ##STR00180##

Step 1: Synthesis of 2-cyano-N-(2-hydroxyethyl)acetamide (2)

(142) A mixture of ethyl 2-cyanoacetate (113 mg, 1.0 mmol) and 2-aminoethanol (305 mg, 5.0 mmol) was stirred at rt overnight. The reaction solution was purified by HPLC to afford the desire product. MS [M+H].sup.+ calcd for C.sub.5H.sub.9N.sub.2O.sub.2 129.1, found 129.1

Step 2: Synthesis of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-(2-hydroxyethyl)acrylamide (3)

(143) A mixture of 2-cyano-N-(2-hydroxyethyl)acetamide (65 mg, 0.5 mmol), 3,4-dihydroxy-5-nitrobenzaldehyde (92 mg, 0.5 mmol) in MeOH (15 mL) and CH.sub.3COONH.sub.4 (385 mg, 5.0 mmol) was stirred at 80 C. overnight and then cooled to rt. The reaction mixture was filtered to collect the solid. The solid was re-dissolved in MeOH and the PH was adjusted to 3-5 by adding 1N HCl. The desire product was collected by filtered and dried in vavuo. (80 mg, 54%). MS [M+H].sup.+ calcd for C.sub.13H.sub.12N.sub.3O.sub.6 294.2, found 293.8. .sup.1H NMR (400 MHz, DMSO) 10.92 (s, 2H), 8.30 (t, J=5.5 Hz, 1H), 8.06 (s, 1H), 7.95 (d, J=2.1 Hz, 1H), 7.78 (d, J=2.2 Hz, 1H), 3.49 (t, J=6.1 Hz, 2H), 3.28 (q, J=5.9 Hz, 2H), 3.17 (s, 1H).

(144) Compound 691:

(145) 691 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(146) ##STR00181##

Step 1: Synthesis of (Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide (3)

(147) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(148) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 2-cyano-N-ethylacetamide (0.23 g, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 h at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound 3 as an yellow solid (288 mg, 39%).

Step 2: Synthesis of (Z)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-ethyl-3-hydroxyacrylamide (4)

(149) A solution of (Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide (167 mg, 0.5 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (3 mL, 3 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product as yellow solid (48 mg, 33%). 1H NMR (400 MHz, DMSO) 14.18 (s, 1H), 10.74 (s, 2H), 9.12 (s, 1H), 8.75 (d, J=5.2 Hz, 1H), 7.88 (s, 1H), 7.55 (d, J=2.1 Hz, 1H), 7.45 (d, J=4.7 Hz, 1H), 4.52 (s, 2H). MS [M+H].sup.+ calcd for C.sub.12H.sub.12N.sub.3O.sub.6 294.2, found 293.8

(150) Compound 692:

(151) 692 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(152) ##STR00182##

Step 1: Synthesis of (Z)-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylamide (3)

(153) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(154) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 2-cyano-N-cyclopropylacetamide (250 mg, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 h at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the compound 3 as an yellow solid (242 mg, 36%).

Step 2: Synthesis of (Z)-2-cyano-N-cyclopropyl-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxyacrylamide (4)

(155) A solution of (Z)-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylamide (166 mg, 0.5 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (3 mL, 3 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product as yellow solid (25 mg, 26%). 1H NMR (400 MHz, DMSO) 10.81 (s, 1H), 8.82 (s, 1H), 7.88 (d, J=1.9 Hz, 1H), 7.54 (d, J=2.1 Hz, 1H), 2.83-2.77 (m, 1H), 0.74-0.64 (m, 4H). MS [MH].sup. calcd for C.sub.13H.sub.10N.sub.3O.sub.6 304.2, found 303.9.

(156) Compound 697:

(157) 697 was prepared in two synthetic steps from methyl 5-(cyanomethyl)pyrazine-2-carboxylate, according to the following procedure:

(158) ##STR00183##

Step 1: Synthesis of (E)-methyl 5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate (3)

(159) Under a nitrogen atmosphere, 60% NaH (0.072 g, 1.8 mmol) was added to the solution of methyl 5-(cyanomethyl)pyrazine-2-carboxylate (0.15 g, 0.9 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound (E)-methyl 5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate as an orange solid(125 mg, 30%). MS [M+H].sup.+ calcd for C.sub.17H.sub.14N.sub.4O.sub.7 387.0, found 387.0.

Step 2: Synthesis of (E)-5-(1-cyano-2-(3,4-dihydroxy-5-nitrophenyl)-2-hydroxyvinyl) pyrazine-2-carboxylic acid (4)

(160) A solution of (E)-methyl 5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl) pyrazine-2-carboxylate (125 mg, 0.3 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (3 mL, 3 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as yellow solid (15 mg, 15%). 1H NMR (400 MHz, DMSO) 10.57 (s, 1H), 9.17 (s, 1H),8.68 (s, 1H), 7.81 (s, 1H), 7.51 (s, 1H). MS [M+H].sup.+ calcd for C.sub.14H.sub.8N.sub.4O.sub.7 345.0, found 345.0

(161) Compound 701:

(162) 701 was prepared in two synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(163) ##STR00184##

Step 1: Synthesis of (Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile (3)

(164) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(165) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 2-cyano-N-cyclopropylacetamide (250 mg, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 h at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the compound 3 (146 mg, 22%).

Step 2: Synthesis of (Z)-2-(azetidine-1-carbonyl)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxyacrylonitrile (4)

(166) A solution of (Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile (146 mg, 0.4 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (2 mL, 2 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na2SO4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product (18 mg, 15%). 11H NMR (400 MHz, DMSO) 10.93 (s, 2H), 7.93 (d, J=2.1 Hz, 1H), 7.55 (d, J=2.1 Hz, 1H), 4.36 (s, 4H), 2.35-2.27 (m, 2H). MS [M+H]+ calcd for C13H12N306 306.2, found 305.9

(167) Compound 711:

(168) 711 was prepared in two synthetic steps from 3-oxo-3-(thiazol-4-yl)propanenitrile, according to the following procedure:

(169) ##STR00185##

Step 1: Synthesis of (Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile (2)

(170) Under a nitrogen atmosphere, 60% NaH (0.072 g, 1.8 mmol) was added to the solution of 3-oxo-3-(thiazol-4-yl)propanenitrile (0.41 g, 2.7 mmol) in anhydrous THF (15 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride (661 mg, 2.7 mmol) in THF was added over 5 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound (E)-methyl 5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate as an orange solid (600 mg, 62%). MS [M+H]+ calcd for C.sub.15H.sub.11N.sub.3O.sub.6S 362.0, found 362.0.

Step 2: Synthesis of (Z)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile (4)

(171) A solution of (Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile (70 mg, 0.2 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (1 mL, 1 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of 0.5 N.NH.sub.4OH (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as yellow solid (11 mg, 17%). 1H NMR (400 MHz, DMSO) 10.66 (s, 1H), 8.61 (s,1H), 7.97 (s, 1H), 7.65 (s, 1H). MS [M+H].sup.+ calcd for C.sub.13H.sub.7N.sub.3O.sub.6S 334.0, found 334.0.

(172) Compound 709:

(173) 709 was prepared in two synthetic steps from (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid, according to the following procedure:

(174) ##STR00186##

Step 1: Synthesis of (E)-methyl 2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)acetate (2)

(175) A solution of (E)-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylic acid (1) (250 mg, 1 mmol) and methyl 2-aminoacetate (89 mg, 1 mmol) in THF (15 mL) was added HBTU (600 mg, 1.5 mmol) and DIPEA (388 mg, 3 mmol) at rt. The resulting suspension was stirred for 5 hours at 60 C. and allowed to cool to room temperature overnight. The reaction was added H.sub.2O and extracted with ethyl acetate (30 mL*3). The organic layers were combined to afford the crude product which was used in the next step without further purification (120 mg, 37.4%).

Step 2: Synthesis of (E)-2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)acetic acid (3)

(176) A solution of (E)-methyl 2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)acetate (2) (120 mg, 0.37 mmol) in THF/H.sub.2O (5 mL/5 mL) was added LiOH (24 mg, 0.55 mmol) at 0 C. The resulting suspension was stirred for 1 hour at 0 C. and was quenched by adding aqueous HCl. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product (7 mg, 6.2%). 1H NMR (400 MHz, DMSO) 8.65 (t, J=5.8 Hz, 1H), 8.10 (s, 1H), 7.98 (d, J=2.1 Hz, 1H), 7.79 (d, J=2.1 Hz, 1H), 3.88 (d, J=5.8 Hz, 2H). MS [M+H].sup.+ calcd for C.sub.12H.sub.10N.sub.3O.sub.7 308.2, found 308.0.

(177) Compound 693:

(178) 693 was prepared in three synthetic steps from ethyl 2-cyanoacetate, according to the following procedure:

(179) ##STR00187##

Step 1: Synthesis of (S)-methyl 2-(2-cyanoacetamido)-3-hydroxypropanoate (2)

(180) A mixture of ethyl 2-cyanoacetate (1130 mg, 10.0 mmol) and (S)-methyl 2-amino-3-hydroxypropanoate (1190 mg, 10.0 mmol) was stirred at r.t. overnight. The reaction solution was added 10 mL MeOH and filtered to collect the solid as crude product, which was used in next step without further purification. MS [M+H].sup. calcd for C.sub.7H.sub.11N.sub.2O.sub.4 187.2, found 187.2.

Step 2: Synthesis of (S,E)-methyl 2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate (3)

(181) A solution of (S)-methyl 2-(2-cyanoacetamido)-3-hydroxypropanoate (930 mg, 5 mmol) (2) and 3,4-dihydroxy-5-nitrobenzaldehyde (915 mg, 5 mmol) in MeOH (25 mL) was added CH.sub.3COONH.sub.4 (3850 mg, 50 mmol) at rt. The reaction solution was stirred at 60 C. continuously for 5h and monitored by TLC until all the starting material was consumed completely. Then the reaction mixture was cooled to rt and the solvent was eliminated under reduced pressure, then the Sat. aq. NaCl (100 mL) was added. The aqueous solution was extracted by EA for three times (50 mL*3). The organic layer was concentrated in vacuo to afford crude product which was purified by silica chromatograph chromatography to afford the desired product (705 mg, 40%). MS [M+H].sup.+ calcd for C.sub.14H.sub.14N.sub.3O.sub.8 352.3, found 352.3.

Step 3: Synthesis of (S,E)-2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoic acid (4)

(182) A solution of (S,E)-methyl 2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate (3) (705 mg, 2 mmol) in THF/H.sub.2O (10 mL/10 mL) was added LiOH (126 mg, 3 mmol) at 0 C. The resulting suspension was stirred for 1 hour at 0 C. and was quenched by adding aqueous HCl. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product (220 mg, 32.6%). 1H NMR (400 MHz, DMSO) 12.89 (s, 1H), 10.94 (s, 2H), 8.22 (d, J=7.6 Hz, 1H), 8.15 (s, 1H), 7.97 (d, J=2.1 Hz, 1H), 7.82 (d, J=2.1 Hz, 1H), 5.05 (s, 1H), 4.41 (m, 1H), 3.79 (m, 2H). MS [M+H].sup.+ calcd for C.sub.13H.sub.12N.sub.3O.sub.8 338.2, found 337.8

(183) Compound 702:

(184) 702 was prepared in three synthetic steps from ethyl 2-cyanoacetate, according to the following procedure:

(185) ##STR00188##

Step 1: Synthesis of (S)-methyl 1-(2-cyanoacetyl)pyrrolidine-2-carboxylate (2)

(186) A mixture of ethyl 2-cyanoacetate (113 mg, 1.0 mmol) and (S)-methyl pyrrolidine-2-carboxylate (129 mg, 1.0 mmol) was stirred at r.t. overnight. The reaction solution was added 10 mL MeOH and filtered to collect the solid as crude product, which was used in next step without further purification. MS [M+H].sup.+ calcd for C.sub.9H.sub.13N.sub.2O.sub.3197.2, found 197.2.

Step 2: Synthesis of (S,E)-methyl 2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate (3)

(187) A solution of (S)-methyl 1-(2-cyanoacetyl)pyrrolidine-2-carboxylate (2) (196 mg, 1 mg) and 3,4-dihydroxy-5-nitrobenzaldehyde (183 mg, 1 mmol) in MeOH (5 mL) was added CH.sub.3COONH.sub.4 (770 mg, 10 mmol) at rt. The reaction solution was stirred at 60 C. overnight and monitored by TLC until all the starting material was consumed completely. Then the reaction mixture was cooled to r.t. and the solvent was eliminated under reduced pressure, then the Sat. aq. NaCl (100 mL) was added. The aqueous solution was extracted by EA for three times (50 mL*3). The organic layer was concentrated in vacuo to afford crude product which was purified by silica chromatograph chromatography to afford the desired product (43 mg, 12%). MS [M+H].sup.+ calcd for C.sub.16H.sub.16N.sub.3O.sub.7362.3, found 362.3.

Step 3: Synthesis of (S,E)-1-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acryloyl)pyrrolidine-2-carboxylic acid (4)

(188) A solution of (S,E)-methyl 2-(2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylamido)-3-hydroxypropanoate (3) (43 mg, 0.12 mmol) in THF/H.sub.2O (2 mL/1 mL) was added LiOH (8 mg, 0.18 mmol) at 0 C. The resulting suspension was stirred for 1 hour at 0 C. and was quenched by adding aqueous HCl. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product (6 mg, 15%). 1H NMR (400 MHz, DMSO) 7.25 (t, J=7.4 Hz, 1H), 7.21-7.09 (m, 2H), 2.30 (s, 2H), 2.07 (s, 4H). MS [M+H].sup.+ calcd for C.sub.15H.sub.14N.sub.3O.sub.7 348.3, found 347.8

(189) Compound 347N:

(190) 347N was prepared in single synthetic step from 2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (1, compound 347), according to the following procedure:

(191) ##STR00189##

Step 1: Synthesis of 2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide (2)

(192) A solution of 2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-oxopropanamide (350 mg, 1 mmol) in DCM (5 mL) was added HMDS (0.5 mL) at rt, then stirred for 48 h. After the reaction was completed, the reaction mixture was concentrated in vacuo to afford the crude product, further purification by Prep-HPLC afforded the desired product as yellow solid (60 mg, 19%). MS [M+H].sup.+ calcd for C.sub.14H.sub.16N.sub.4O.sub.5 321.1, found 321.1.

(193) Compound 661N:

(194) 661N was prepared in one synthetic step from (Z)-3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile, according to the following procedure:

(195) ##STR00190##

Step 1: Synthesis of 3-amino-3-(3,4-dihydroxy-5-nitrophenyl)-2-(piperidine-1-carbonyl)acrylonitrile (2)

(196) A solution of 3-(3,4-dihydroxy-5-nitrophenyl)-3-hydroxy-2-(piperidine-1-carbonyl)acrylonitrile (20 mg, 0.06 mmol) in DCM (5 mL) was added HMDS (1 mL) and stirred at rt for 72 h. And then the reaction mixture was concentrated in vacuo to afford the crude product, which was further purified by HPLC to afford the desired product as a yellow solid (1 mg, 5%). MS [MH]+ calcd for C.sub.15H.sub.16N.sub.4O.sub.5 333.3, found 333.1. 1H NMR (400 MHz, DMSO) 8.21 (s, 1H), 7.66 (s,1H), 6.74 (s, 1H), 1.25-1.65 (m, 6H), 1.23 (s, 1H).

(197) Compound 691N:

(198) 691N was prepared in four synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(199) ##STR00191##

Step 1: Synthesis of (Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide (3)

(200) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(201) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 2-cyano-N-ethylacetamide (0.23 g, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound 3 as an yellow solid (265 mg, 36%).

Step 2: Synthesis of (Z)-3-chloro-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide (4)

(202) A solution of (Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-ethylacrylamide (176 mg, 0.5 mmol) in DCM (5 mL) was added PCl.sub.5 (104 mg, 0.5 mmol) at 0 C. The resulting suspension was stirred at overnight until all the start materials were consumed detected by LC-MS. Then the reaction mixture was allowed to cool to room temperature and used in the next step without further purification.

Step 3: Synthesis of (Z)-3-amino-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide (5)

(203) A solution of (Z)-3-chloro-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide (150 mg, 0.4 mmol) in DCM was added a saturated solution of NH.sub.3 in ACN at 0 C., and then the reaction solution was stirred at 0 C. continuously until all the starting materials were consumed completely. The reaction was quenched by the addition of H.sub.2O and stirred for 30 min. The aqueous phase was extracted with ethyl acetate. The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product.

Step 4: Synthesis of (Z)-3-amino-2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N-ethylacrylamide (6)

(204) A solution of (Z)-3-amino-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N-ethylacrylamide (67 mg, 0.2 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (1 mL, 1 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product as yellow solid (5 mg, 9%). 1H NMR (400 MHz, DMSO) 10.53 (s, 2H), 9.44 (s, 1H), 8.02 (s, 2H), 7.63 (d, J=2.0 Hz, 1H), 7.36 (d, J=2.0 Hz, 1H), 3.32-3.25 (m, 2H), 1.14 (t, J=7.1 Hz, 3H). MS [M+H].sup.+ calcd for C.sub.12H.sub.13N.sub.4O.sub.5 293.2, found 292.8.

(205) Compound 692N:

(206) 692N was prepared in four synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(207) ##STR00192##

Step 1: Synthesis of (Z)-2-cyano-N-(cyclopropylmethyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylamide (3)

(208) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(209) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 2-cyano-N-cyclopropylacetamide (250 mg, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the title compound 3 as an yellow solid(165 mg, 23%).

Step 2: Synthesis of (Z)-3-chloro-2-cyano-N-(cyclopropylmethyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide (4)

(210) A solution of (Z)-2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-N-propylacrylamide (165 mg, 0.5 mmol) in DCM (5 mL) was added PCl.sub.5 (104 mg, 0.5 mmol) at 0 C. The resulting suspension was stirred at overnight until all the start materials were consumed detected by LC-MS. Then the reaction mixture was allowed to cool to room temperature and used in the next step without further purification.

Step 3: Synthesis of (Z)-3-amino-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide (5)

(211) A solution of (Z)-3-chloro-2-cyano-N-(cyclopropylmethyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide (164 mg, 0.5 mmol) in DCM was added a saturated solution of NH.sub.3 in ACN at 0 C., and then the reaction solution was stirred at 0 C. continuously until all the starting materials were consumed completely. The reaction was quenched by the addition of H.sub.2O and stirred for 30 min. The aqueous phase was extracted with ethyl acetate. The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product which was used in the next step without further purification.

Step 4: Synthesis of (Z)-3-amino-2-cyano-N-cyclopropyl-3-(3,4-dihydroxy-5-nitrophenyl)acrylamide (6)

(212) A solution of (Z)-3-amino-2-cyano-N-cyclopropyl-3-(3,4-dimethoxy-5-nitrophenyl)acrylamide (66 mg, 0.2 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (1 mL, 1 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product. (7 mg, 12%). 1H NMR (400 MHz, DMSO) 10.52 (s, 2H), 9.53 (s, 1H), 7.63 (d, J=2.0 Hz, 1H), 7.35 (d, J=2.0 Hz, 1H), 2.61-2.55 (m, 1H), 0.86-0.81 (m, 2H), 0.66-0.61 (m, 2H). MS [M+H].sup.+ calcd for C.sub.13H.sub.13N.sub.4O.sub.5 305.3, found 304.9

(213) Compound 697N:

(214) 697N was prepared in two synthetic steps from (E)-methyl 5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl)pyrazine-2-carboxylate, according to the following procedure:

(215) ##STR00193##

Step 1: Synthesis of (E)-methyl 5-(2-chloro-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate (2)

(216) A solution of (E)-methyl 5-(1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)-2-hydroxyvinyl) pyrazine-2-carboxylate (150 mg, 0.39 mmol) in 1,2-dichloroethane (25 mL) was added PCl.sub.5 (83 mg, 0.40 mmol) at 0 C. The resulting suspension was stirred at overnight until all the start materials were consumed detected by LC-MS. Then the reaction mixture was allowed to cool to room temperature and used in the next step without further purification.

Step 2: Synthesis of (E)-methyl 5-(2-amino-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate (3)

(217) A solution of (E)-methyl 5-(2-chloro-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl) pyrazine-2-carboxylate (180 mg, 0.45 mmol) in 1,2-dichloroethane (25 mL) was added a saturated solution of NH.sub.3 in ACN at 0 C., and then the reaction solution was stirred at 0 C. continuously until all the starting materials were consumed completely. The reaction was quenched by the addition of H.sub.2O and stirred for 30 min. The aqueous phase was extracted with ethyl acetate. The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product which was used in the next step without further purification.

Step 3: Synthesis of (E)-methyl 5-(2-chloro-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl)pyrazine-2-carboxylate (4)

(218) A solution of (E)-methyl 5-(2-amino-1-cyano-2-(3,4-dimethoxy-5-nitrophenyl)vinyl) pyrazine-2-carboxylate (98 mg, 0.25 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (3 mL, 3 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) gave the desired product as yellow solid (11 mg, 13%). 1H NMR (400 MHz, DMSO) 10.96-11.2 (m, 1H), 10.42 (s, 1H), 8.94 (s, 1H), 8.66 (s, 1H), 7.76 (s, 1H), 6.79 (s, 1H). MS [M+H].sup.+ calcd for C.sub.14H.sub.9N.sub.5O.sub.6 344.0, found 344.0

(219) Compound 701N:

(220) 701N was prepared in four synthetic steps from 3,4-dimethoxy-5-nitrobenzoic acid, according to the following procedure:

(221) ##STR00194##

Step 1: Synthesis of (Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile (3)

(222) Under a nitrogen atmosphere, SOCl.sub.2 (0.38 mL, 5.28 mmol) and anhydrous DMF (0.01 mL, 0.22 mmol) were added to a suspension of 3,4-dimethoxy-5-nitrobenzoic acid (500 mg, 2.2 mmol) in toluene (10 mL) at room temperature. The mixture was heated at 60 C. and stirred for 15 hours. The organic solvent was eliminated by distillation under reduced pressure. More toluene was added and eliminated again. The resulting yellowish solid 3,4-dimethoxy-5-nitrobenzoyl chloride was dissolved in anhydrous THF (5 mL).

(223) Under a nitrogen atmosphere, 60% NaH (0.18 g, 4.4 mmol) was added to the solution of 3-(azetidin-1-yl)-3-oxopropanenitrile (250 mg, 2.0 mmol) in anhydrous THF (5 mL) at 5 C. The resulting suspension was stirred at 5 C. for 15 min and the solution of 3,4-dimethoxy-5-nitrobenzoyl chloride in THF was added over 10 min and stirred for an additional 1 hour at 5 C. The reaction mixture was warmed to 0 C., quenched by the addition of 1N.HCl solution (4 mL) and stirred for 10 min at room temperature, extracted by ethyl acetate (25 mL*2), the organic layers was dried with Na.sub.2SO.sub.4 and concentrated in vacuo to give the compound 3.

Step 2: Synthesis of (Z)-2-(azetidine-1-carbonyl)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile (4)

(224) A solution of (Z)-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxyacrylonitrile (175 mg, 0.5 mmol) in DCM (5 mL) was added PCl.sub.5 (104 mg, 0.5 mmol) at 0 C. The resulting suspension was stirred at overnight until all the start materials were consumed detected by LC-MS. Then the reaction mixture was allowed to cool to room temperature and used in the next step without further purification.

Step 3: Synthesis of (Z)-3-amino-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile (5)

(225) A solution of (Z)-2-(azetidine-1-carbonyl)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile (164 mg, 0.5 mmol) in DCM was added a saturated solution of NH.sub.3 in ACN at 0 C., and then the reaction solution was stirred at 0 C. continuously until all the starting materials were consumed completely. The reaction was quenched by the addition of H.sub.2O and stirred for 30 min. The aqueous phase was extracted with ethyl acetate. The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product which was used in the next step without further purification.

Step 4: Synthesis of (Z)-3-amino-2-(azetidine-1-carbonyl)-3-(3,4-dihydroxy-5-nitrophenyl)acrylonitrile (6)

(226) A solution of (Z)-3-amino-2-(azetidine-1-carbonyl)-3-(3,4-dimethoxy-5-nitrophenyl)acrylonitrile (66 mg, 0.2 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (1 mL, 1 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted with ethyl acetate (30 mL*3). The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC gave the desired product (7 mg, 12%). 1H NMR (400 MHz, DMSO) 10.52 (s, 2H), 9.87 (s, 1H), 7.60 (d, J=2.1 Hz, 1H), 7.35 (d, J=2.1 Hz, 1H), 4.26 (s, 4H), 2.29 (m, 2H). MS [M+H].sup.+ calcd for C.sub.13H.sub.13N.sub.4O.sub.5 305.3, found 305.0

(227) Compound 711N:

(228) 711N was prepared in three synthetic steps from (Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile, according to the following procedure:

(229) ##STR00195##

Step 1: Synthesis of (Z)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile (2)

(230) A solution of (Z)-3-(3,4-dimethoxy-5-nitrophenyl)-3-hydroxy-2-(thiazole-4-carbonyl)acrylonitrile (150 mg, 0.42 mmol) in 1,2-dichloroethane (25 mL) was added PCl.sub.5 (86 mg, 0.42 mmol) at 0 C. The resulting suspension was stirred at overnight until all the start materials were consumed detected by LC-MS. Then the reaction mixture was allowed to cool to room temperature and used in the next step without further purification.

Step 2: Synthesis of (Z)-3-amino-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile (3)

(231) A solution of (Z)-3-chloro-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile (185 mg, 0.42 mmol) in 1,2-dichloroethane (25 mL) was added a saturated solution of NH.sub.3 in ACN at 0 C., and then the reaction solution was stirred at 0 C. continuously until all the starting materials were consumed completely. The reaction was quenched by the addition of H.sub.2O and stirred for 30 min. The aqueous phase was extracted with ethyl acetate. The organic layers were combined, washed with brine and dried with Na.sub.2SO.sub.4. The solvent was eliminated under reduced pressure to give the crude product which was used in the next step without further purification. MS [M+H].sup.+ calcd for C.sub.15H.sub.12N.sub.4O.sub.5S 361.0, found 361.0

Step 3: Synthesis of ammonium (Z)-5-(1-amino-2-cyano-3-oxo-3-(thiazol-4-yl)prop-1-enyl)-2-hydroxy-3-nitrophenolate (4)

(232) A solution of (Z)-3-amino-3-(3,4-dimethoxy-5-nitrophenyl)-2-(thiazole-4-carbonyl)acrylonitrile (100 mg, 0.28 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (1 mL, 1 mmol) at 15 C. under nitrogen atmosphere. The resulting red suspension was stirred for 1 hour at 15 C. and allowed to warm to room temperature overnight. The reaction was quenched by the addition of 0.5 N.NH.sub.4OH (2 mL) and stirred for 5 min. The aqueous phase was washed with ethyl acetate. The hydrous layer was eliminated under reduced pressure to give the crude product. Further purification by Prep-HPLC (0.05% FA, MeOH/H.sub.2O) gave the desired product as yellow solid (11 mg, 15%). 1H NMR (400 MHz, DMSO) 8.85 (s, 1H), 7.66 (s, 1H), 7.38 (s, 1H), 6.80-6.99 (m, 1H), 6.66-6.74 (m, 1H). MS [M+H].sup.+ calcd for C.sub.13H.sub.11N.sub.5O.sub.5S 333.0, found 333.0

(233) Compound 347M:

(234) 347M was prepared in three synthetic steps from 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide according to the following procedure:

(235) ##STR00196##

Step 1: Synthesis of 2-cyano-3-(diethylamino)-1-(3,4-dimethoxy-5-nitrophenyl)-3-oxoprop-1-enyl methanesulfonate (2)

(236) A solution of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-hydroxyacrylamide (350 mg, 1 mmol) and Et.sub.3N (202 mg, 2 mmol) in DCM (15 mL) was added MsCl (250 mg, 2 mmol) at 0 C. The reaction solution was stirred at rt for 3 h, then it was concentrated in vacuo to afford the crude product (360 mg), which was used in the next step without further purification.

Step 2: Synthesis of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-(methylamino)acrylamide (3)

(237) A solution of 2-cyano-3-(diethylamino)-1-(3,4-dimethoxy-5-nitrophenyl)-3-oxoprop-1-enyl methanesulfonate (250 mg, 0.7 mmol) and K.sub.2CO.sub.3 (193 mg, 1.4 mmol) in MeCN (10 mL) was added MeNH.sub.2 (1.4 mmol, 0.7 mL, 2N in THF) at rt. The reaction solution was refluxed for 2 h, and then 50 mL of water was added. The aqueous phase was extracted by EA for two times (50 mL2), and then the organic layer was concentrated in vacuo to afford the crude product (210 mg), which was used in the next step without further purification. MS [M+H].sup.+ calcd for C.sub.17H.sub.22N.sub.4O.sub.5 363.1, found 363.1.

Step 3: Synthesis of 2-cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-diethyl-3-(methylamino)acrylamide (4)

(238) A solution of 2-cyano-3-(3,4-dimethoxy-5-nitrophenyl)-N,N-diethyl-3-(methylamino)acrylamide (210 mg, 0.58 mmol) in DCM (5 mL) was added 1.0 M solution of BBr.sub.3 in DCM (2 mL, 2 mmol) at 15 C. under nitrogen atmosphere. The reaction solution was stirred at 15 C. for 1 h then at room temperature overnight. The reaction was quenched by the addition of H.sub.2O (2 mL) and stirred for 30 min. The aqueous phase was extracted by ethyl acetate for three times (30 mL3). The organic layer was washed with brine and dried over Na.sub.2SO.sub.4, then it was concentrated in vacuo to afford the crude product, which was purified by Prep-HPLC (0.5% TFA, MeOH/H.sub.2O) to gain the desired product as bright yellow solid (25 mg, 13%). MS .sup.1H NMR (400 MHz, DMSO) 10.69 (s, 2H), 7.43 (s, 1H), 7.20 (s, 1H), 3.40-3.43 (m, 4H), 2.69 (d, J=4.9 Hz, 3H), 1.14 (t, J=6.8 Hz, 13H), 1.14 (t, J=6.8 Hz, 3H). [M+H].sup.+ calcd for C.sub.12H.sub.7N.sub.5O.sub.6S 350.0, found 350.0.

(239) Enzymatic Inhibition.

(240) We measured compound inhibition activity in a demethylation reaction catalyzed by FTO (US2014/0148383A1). The reaction system was incubated at 37 C. for 2 h and stopped by heating at 95 C. for 5 min. ssDNA was digested by nuclease P1 and alkaline phosphatase. The concentrations of N.sup.6-mA and A were analyzed by HPLC-MS/MS. When concentration of substrate and enzyme are 0.5 M and 0.1 M, respectively, the measured IC.sub.50 value of entacapone against FTO is 3 M. The compounds of Tables 1-3 were consistently active, with IC.sub.50's less than 10 M, and most less than 1 M.

(241) TABLE-US-00006 Exemplary IC50 Data of submicromolar representative compounds Compound No. Enzymatic inhibition (IC50) M 687 <1 317 >1 371 ~1 660 <1 382 >1 702 ~1 698 >1 675 >1 394 >1 664 ~1 684 >1 688 >1 713 <1 709 <1 712 ~1 693 ~1 331 ~1 333 >1 318 ~1 365 ~1 366 >1 390 ~1 656 <1 666 <1 315 ~1 319 <1 389 ~1 502 <1 505 <1 395 <1 396 <1 522 ~1 655 <1 518 >1 520 <1 347 <1 351 <1 523 <1 524 ~1 525 <1 503 ~1 359 >1 374 <1 668 <1 661 ~1 673 <1 674 >1 691 <1 692 <1 697 <1 701 <1 711 ~1 715 <1 722 <1 347N <1 661N <1 691N ~1 692N >1 697N <1 701N >1 711N >1 347M ~1

(242) In vivo Anti-Obesity Efficacy.

(243) Male wistar rats (6 weeks) were fed with high-fat diet (45% fat, OpenSource Diets D12451), and compound (100 mg/kg) was administered to 12 randomly selected rats by gavage. After 8 weeks, the mean body weight of drug treatment group was less than that of control group. However, the body-weight-normalized food intakes of the two groups showed no difference. The LDL-c (Low Density Lipoproteincholesterol) in serum of drug treatment group was reduced compared to that of control group, and the adipose and hepatic tissues of rat in drug-treated groups showed reduced size of adipose cells and reduced level of liver steatosis.

(244) Atherosclerosis Model: Ldlr-Deficient Mice.

(245) We measured compound anti-atherosclerosis efficacy using Ldlr.sup./ mice fed western style diet (20% fat, 0.15% cholesterol), compound (100 mg/day) was orally administered by blending with diet. After 8 weeks, the mean lesion area in aortic sinuses of drug treatment group was less than that of the control group.

(246) In Vivo Anti-Obesity Efficacy in Obese Mice.

(247) Male C57BL/6 mice were fed with high-fat diet (45% fat, OpenSource Diets D12451) for 8 weeks. Then obese mice with body weight 20% larger than that of mice fed with normal diet (20 mice) were selected for experiments. Compound (100 mg/kg) was orally administered to 10 randomly selected obese mice by blending with diet. After 13 weeks, the mean body weight gain of drug treatment group was about less than that of the control group.

(248) In Vivo Anti-Diabetic Efficacy in Genetically Diabetic db/db Mice.

(249) Compound (100 mg/kg) was orally administered to 11 randomly selected male db/db mice by blending with normal diet, while the other 9 male db/db mice fed with normal diet as control group. After 20 weeks, the mean fasting plasma glucose of drug treatment group was significantly lower than that of control group (*p-value<0.05).

(250) From our results herein and further data we determine that preferred anti-weight gain, anti-obesity and anti-obesity related disease, such as anti-diabetes, effective human dosages of the compounds should be 0.1-10, 0.5-10, 0.5-5, 0.5-2.5, 0.5-1, 1-10, 1-5, 1-2.5, 2-10, or 2-5 g/day.

(251) COMT Inhibition Assay.

(252) We measured compound COMT inhibitory activity by reaction kinetic model. The test article was diluted with assay buffer to desired concentration. The COMT enzyme was also diluted with assay buffer. Then 5 L diluted test article, 5 L diluted COMT and 5 L Esculetin were added into plate and incubated for 5 mM at 37 C., sealed with TopSeal-A 384, Clear Adhesive (PE). Then 5 L AdoMet was added into the plate. The reaction system contains 1U COMT enzyme, test article, 4 M Esculetin, 0.6 mM AdoMet, 50 mM K.sub.3PO.sub.4, and 10 mM MgCl.sub.2. Read plate by using kinetics model (Excitation at 360 nm & emission at 460 nm). The inhibition was calculated from the slope.

(253) Rat Liver Microsome Stability Assay.

(254) Compound stability was tested in rat liver microsome. Microsome working solution contains rat liver microsome 1 mg/ml, test compound 2 M, NADPH 1 mM in 0.05 M Phosphate buffer (pH=7.4). This reaction system was incubated at 37 C. At each time point of 0, 5, 15, 30 and 60 mM, an aliquot of 15 L was transferred into another tube with 200 L quenching solution (Internal standard (Terfenadine) 5 ng/ml in Methanol). Vortex, Centrifuge at 3,500 rpm for 12 min, Transfer 100 L of supernatant to 96 well plate for LC-MS/MS analysis and dilute sample with MeOH: H2O (1:1) if necessary.

(255) TABLE-US-00007 Result of Selectivity and Rat Liver Microsome Selectivity Rat Liver Microsome Compound FTO (M) COMT(M) Index* T (min) CLint (L/min/mg protein) Entacapone 3 0.06 1.00 39.2 17.7 347 0.5 0.551 55.10 6931 0.100 315 1 70% at 200 nM; 2.24 310 19% at 50 nM 351 0.75 80% at 200 nM; 77.0 9.00 33% at 50 nM 396 0.25 0.108 21.60 33.2 20.9 523 0.5 0.105 10.50 239 2.90 395 0.5 74% at 200 nM; 55.0 12.6 36% at 50 nM 525 0.5 74% at 200 nM; 63.6 10.9 39% at 50 nM 390 1 36% at 200 nM; 2.61 266 12% at 50 nM 503 1.5 24% at 200 nM; 1386 0.50 9% at 50 nM 331 1 54% at 200 nM; 93.7 7.40 25% at 50 nM 374 0.5 0.065 6.50 1155 0.600 371 1 71% at 200 nM; 16.1 43.0 33% at 50 nM 661 1 26% at 200 nM; 315 2.20 12% at 50 nM 655 0.3 0.024 4.00 433 1.60 697 0.75 33% at 200 nM; 630 1.10 3 at 50 nM 687 1 0.033 1.65 2.44 284 701 0.75 3.56 237.33 NA NA Entacapone 3 0.06 1.00 39.2 17.7 347N 0.5 0.214 21.40 141 4.90 371 1 71% at 200 nM; 16.1 43.0 33% at 50 nM 661N 0.75 64% at 200 nM; 67.3 10.3 35% at 50 nM Entacapone 3 0.06 1.00 39.2 17.7 347M 1 0.162 8.10 56.8 12.2 *Selectivity Index is the normalized COMT/FTO value based on Entacapone's value and was calculated as the following equation: (COMT.sub.compound/FTO.sub.compound)/(COMT.sub.entacapone/FTO.sub.entacapone)

(256) It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.