Antibacterial compounds

Abstract

The present invention relates to novel antibacterial compounds (1) as defined below, pharmaceutical compositions containing them and their use as antimicrobials. A compound of formula (1): A-L.sub.1-Y-L.sub.2-RB, wherein A is a cyclic group having one of the following formulae (I), (II) and (III), and wherein B is cyclic group having one of the following formulae (IV) to (IX). ##STR00001## ##STR00002##

Claims

1. A compound of formula (1):
A-L.sub.1-Y-L.sub.2-RB(1) wherein A is a cyclic group having the following formulae (I): ##STR00463## wherein G.sub.1 is CH or N; G.sub.2 is CR.sub.1 or N; R.sub.1 is hydrogen atom, halogen atom, cyano or (C.sub.1-3)alkoxy group; L.sub.1 is bond, or (C.sub.1-3)alkylenyl group, optionally substituted with hydroxy group; Y is a ring selected from the group consisting of piperidinyl, piperazinyl, pyrrolidinyl, 1-3 cyclobutyl, 1-3 cyclopentyl, 1-2 cyclopropyl, azetidinyl, azabicyclo-octyl, morpholinyl and cyclohexyl ring, said ring being optionally substituted by one or more substituent selected from the group consisting of hydroxy group, (C.sub.1-3)alkylenyl-OH group, (C.sub.1-3)alkylenyl-O(C.sub.1-3)alkyl group, (C.sub.1-3)alkylenyl-CONRR group, and CONRR group, wherein R and R are hydrogen atom or (C.sub.1-3)alkyl; L.sub.2 is bond, (C.sub.1-3)alkylenyl- group, NR group, NR(C.sub.1-3)alkylenyl group, (C.sub.1-3)alkylenyl-NR group, NR(C.sub.1-3)alkylenyl-NR group, or (C.sub.1-3)alkylenyl-NR (C.sub.1-3)alkylenyl group, said group being optionally substituted with a hydroxy group, wherein R is hydrogen, (C.sub.1-3)alkyl, (C.sub.1-3)alkylenyl-OH, (C.sub.1-3)alkylenyl-O(C.sub.1-3)alkyl, or (C.sub.1-3)alkylenyl-CONRR, wherein R and R are hydrogen atom or (C.sub.1-3)alkyl; R is bond or heterocyclic ring, aliphatic or aromatic, having 5 members containing one or more nitrogen atoms, optionally substituted with CH.sub.2OH, CH.sub.2CN, CN or CONRR, wherein R and R are hydrogen atom or (C.sub.1-3)alkyl; and B is cyclic group having the following formulae (V): ##STR00464## wherein G.sub.6 is CH or N; G.sub.7 is CH or N; and R.sub.2 is hydrogen atom or halogen atom, with the proviso that when Y is piperidinyl ring or piperazinyl ring, if A is represented by formula (I) and B is represented by formula (V), then G.sub.2 is only N, or an addition salt with a pharmaceutically acceptable organic or inorganic acid or base, an enantiomer, an N-oxide, or a quaternary ammonium salt of said compound of formula (1).

2. The compound according to claim 1, wherein R.sub.1 is a halogen atom, a cyano group, or an alkoxy group having from 1 to 2 carbon atoms.

3. The compound according to claim 2, wherein R.sub.1 is selected from the group consisting of a fluorine atom, a chlorine atom, a cyano group and a methoxy group.

4. The compound according to claim 1, wherein L.sub.1 is a bond or a methylene group (CH.sub.2).

5. The compound according to claim 1, wherein L.sub.2 is a bond, a (C.sub.1-3)alkylenyl group, NR group, NR(C.sub.1-3)alkylenyl group, (C.sub.1-3)alkylenyl-NR group, NR(C.sub.1-3)alkylenyl-NR group, or (C.sub.1-3)alkylenyl-NR(C.sub.1-3)alkylenyl group, said group being optionally substituted with one or more hydroxy group.

6. The compound according to claim 5, wherein L.sub.2 is a bond, a (C.sub.1-2)alkylenyl group, NR group, NR(C.sub.1-2)alkylenyl group, (C.sub.1-2)alkylenyl-NR group, NR(C.sub.1-2)alkylenyl-NR group, or (C.sub.1-2)alkylenyl-NR(C.sub.1-2)alkylenyl group, said group being optionally substituted with one hydroxy group.

7. The compound according to claim 1, wherein R is a bond or an aromatic heterocyclic ring having 5 members containing one or more nitrogen atoms, optionally substituted with CH.sub.2CN or CN.

8. The compound according to claim 7, wherein R is a bond, a 1H-imidazol-4-yl group, or a 1H-pyrrol-2-yl group, optionally substituted with CH.sub.2CN or CN.

9. The compound according to claim 1, wherein A is a cyclic group having one of the following formulae: ##STR00465##

10. The compound according to claim 1, wherein Y is a ring having one of the following formulae: ##STR00466##

11. The compound according to claim 1, wherein B is a cyclic group having one of the following formulae: ##STR00467##

12. A pharmaceutical composition, comprising at least one compound, salt thereof with a pharmaceutically acceptable organic or inorganic acid or with a pharmaceutically acceptable organic or inorganic base, enantiomer, quaternary ammonium salt, or N-oxide according to claim 1, and at least one inert pharmaceutically acceptable excipient.

13. A method for treating a bacterial infection, comprising administering an effective amount of a compound, salt with a pharmaceutically acceptable organic or inorganic acid or with a pharmaceutically acceptable organic or inorganic base, enantiomer, quaternary ammonium salt, or N-oxide according to claim 1 to a patient in need thereof.

14. The method according to claim 13, wherein said bacterial infection is selected from the group consisting of a skin infection, a mucosal infection, a gynecological infection, a respiratory tract infection (RTI), a CNS infections, a gastro-intestinal infection, a bone infection, a cardiovascular infection, a sexually transmitted infection, and a urinary tract infection.

15. The compound according to claim 1, wherein Y is a ring having one of the following formulae: ##STR00468##

16. The method of according to claim 13, wherein said bacterial infection is an E. coli infection or an S. aureus infection.

17. The method of according to claim 13, wherein said bacterial infection is an E. coli infection.

18. The method of according to claim 13, wherein said bacterial infection is an S. aureus infection.

Description

EXAMPLES

(1) List of the abbreviations used in the synthetic pathways described hereinafter:

(2) Boc: tert-butyl carbamate

(3) cHex cyclohexane

(4) CV column volume

(5) DBU: 1,5-diazabiciclo[5.4.0]undec-5-ene

(6) DCM: dichloromethane

(7) DIPEA N,N-diisopropylethylamine

(8) DME: 1,2-dimethoxyethane

(9) DMF: N,N-dimethylformamide

(10) Et.sub.2O: diethyl ether

(11) EtOAc: ethylacetate

(12) MS: mass spectroscopy

(13) TEA: triethylamine

(14) TFA: trifluoroacetic acid

(15) THF: tetrahydrofuran

(16) Pd/C: palladium on activated charcoal

(17) Pd(OH).sub.2/C: palladium hydroxide on activated charcoal

(18) r.t.: room temperature

(19) UPLC: Ultra High Performance Liquid Chromatography

(20) Preparation of Compound 157

(21) Compound 157 was prepared as described herein below.

Step 1Synthesis of methyl 5-fluoro-2-hydroxybenzoate

(22) ##STR00290##

(23) To a stirred solution of 5-fluoro salicylic acid (25 g, 160 mmol) in MeOH (250 mL), conc. sulfuric acid (20 mL) was added slowly at 0 C. The resulting reaction mixture was refluxed for 48 h then was concentrated under reduced pressure and the resulting crude was basified to pH 8.0 with sat. NaHCO.sub.3. The mixture was neutralized by 1.5 N HCl solution and extracted with EtOAc. The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and concentrated to afford methyl 5-fluoro-2-hydroxybenzoate as a light brown liquid (22.8 g, 83% yield). GC-MS (AcqMethod HP-1MS.M): 170.1 (M). .sup.1H NMR (400 MHz, DMSO-d6): ppm 10.29 (s, 1H), 7.51-7.49 (m, 1H), 7.42-7.41 (m, 1H), 7.03-7.01 (m, 1H), 3.89 (s, 3H).

Step 2Synthesis of 5-fluoro-2-hydroxybenzamide

(24) ##STR00291##

(25) A mixture of methyl 5-fluoro-2-hydroxybenzoate (22 g, 129 mmol) and methanolic ammonia (250 mL) was heated at 50 C. in an autoclave for 10 h. The reaction mixture was concentrated under reduced pressure, the resulting crude was codistilled with toluene and dried to give 5-fluoro-2-hydroxybenzamide as a brown solid (18.5 g, 92% yield). LC-MS m/z: 154.0 (MH.sup.+). .sup.1H NMR (400 MHz, DMSO-d6): ppm 12.74 (s, 1H), 8.40 (s, 1H), 8.03 (s, 1H), 7.73-7.71 (m, 1H), 7.31-7.29 (m, 1H), 6.91-6.90 (m, 1H).

Step 3Synthesis of 6-fluoro-2H-1,3-benzoxazine-2,4(3H)-dione

(26) ##STR00292##

(27) To a stirred solution of 5-fluoro-2-hydroxybenzamide (8.0 g, 51.6 mmol) in dry THF (80 mL), 1,1-carbonyldiimidazole (10.9 g, 67.09 mmol) was added at 0 C. The mixture was stirred at room temperature for 14 h then was concentrated under reduced pressure. The resulting crude was treated with MeOH and washed with diethyl ether. The resulting white solid was dried and used in the next step without further purification (5.1 g, 55% yield, white solid). LC-MS m/z: 180.0 (MH.sup.+). .sup.1H NMR (400 MHz, DMSO-d6): ppm 12.19 (s, 1H), 7.68-7.67 (m, 2H), 7.50-7.48 (m, 1H).

Step 4Synthesis of 4-chloro-6-fluoro-2H-1,3-benzoxazin-2-one

(28) ##STR00293##

(29) To a stirred solution of 6-fluoro-2H-1,3-benzoxazine-2,4(3H)-dione (0.5 g, 2.76 mmol) in dry 1,2-dichloroethane (2.5 mL), phosphorous pentachloride (0.69 g, 3.31 mmol) was added at 0 C. The resulting mixture was refluxed for 6 h then was concentrated under reduced pressure. DCM (15 mL) was added to the resulting crude, washed with water (2 mL) and dried over anhydrous Na.sub.2SO.sub.4. The solvent was concentrated under reduced pressure to afford 4-chloro-6-fluoro-2H-1,3-benzoxazin-2-one as an off-white solid (0.46 g, 84% yield). .sup.1H NMR (400 MHz, DMSO-d6): ppm 7.69-7.67 (m, 2H), 7.50-7.49 (m, 1H).

Step 5Synthesis of tert-butyl 4-(3-chloro-2-hydroxypropyl)piperazine-1-carboxylate

(30) ##STR00294##

(31) To a solution of tert-butyl piperazine-1-carboxylate (6.0 g, 32.2 mmol) in ethanol (100 mL), epichlorohydrine (14.8 g, 161.0 mmol) was added and the solution was stirred for 15 h at room temperature. After completion of the reaction, the mixture was concentrated under reduced pressure and the resulting crude product (colorless thick liquid, 8.9 g, 99% yield) was used as such in the following step. LC-MS m/z: 279.2 (M+1). .sup.1HNMR (400 MHz, DMSO-d6): 1.40 (s, 9H), 2.27-2.42 (m, 6H), 3.21-3.32 (m, 4H), 3.52-3.56 (m, 1H), 3.62-3.67 (m, 1H), 3.79-3.89 (m, 1H), 5.04 (d, J=4.9 Hz, 1H).

Step 6Synthesis of tert-butyl 4-(3-azido-2-hydroxypropyl) piperazine-1-carboxylate

(32) ##STR00295##

(33) To a solution of tert-butyl 4-(3-chloro-2-hydroxypropyl)piperazine-1-carboxylate (8.9 g, 32 mmol) in DMF (80 mL) sodium azide (3.12 g, 48 mmol) was added. The mixture was stirred at 85 C. for 5 h then was diluted with ethyl acetate (300 mL) and washed with water. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The resulting crude was purified on silica gel (60-120 mesh) column chromatography (0-10% methanol in dichloromethane) to obtain tert-butyl 4-(3-azido-2-hydroxypropyl)piperazine-1-carboxylate (8.2 g, 89% yield) as a pale yellow thick liquid. LC-MS: 286.2 (M+1). .sup.1HNMR (400 MHz, DMSO-d6): 1.40 (s, 9H), 2.29-2.42 (m, 6H), 3.15-3.20 (m, 1H), 3.24-3.32 (m, 5H), 3.79-3.82 (m, 1H), 5.04 (d, J=4.9 Hz, 1H).

Step 7Synthesis of tert-butyl 4-(3-amino-2-hydroxypropyl) piperazine-1-carboxylate

(34) ##STR00296##

(35) To a solution of tert-butyl 4-(3-azido-2-hydroxypropyl)piperazine-1-carboxylate (1.5 g, 5.2 mmol) in ethanol (40 mL), 10% palladium on carbon (0.15 g) was added and the mixture was stirred under hydrogen atmosphere at room temperature for 3 h. The reaction mixture was filtered through a celite bed and the filtrate was concentrated under reduced pressure to obtain tert-butyl 4-(3-amino-2-hydroxypropyl) piperazine-1-carboxylate (1.25 g, 85% yield) as a pale yellow color solid. LC-MS m/z: 260.6 (M+1). .sup.1HNMR (300 MHz, DMSO-d6): 1.39 (s, 9H), 2.22-2.43 (m, 8H), 2.58-2.66 (m, 2H), 3.48-3.52 (m, 4H), 3.88-3.90 (m, 1H).

Step 8Synthesis of tert-butyl 4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4-yl)amino)-2-hydroxypropyl)piperazine-1-carboxylate

(36) ##STR00297##

(37) To a stirred solution of 4-chloro-6-fluoro-2H-benzo[e][1,3]oxazin-2-one (0.89 g, 4.5 mmol) and DIPEA (2.9 g, 22.5 mmol) in acetonitrile (50 mL), tert-butyl 4-(3-amino-2-hydroxypropyl)piperazine-1-carboxylate (1.39 g, 5.4 mmol) was added at room temperature. The mixture was stirred at the same temperature overnight. The solid was filtered, washed with water and dried. The crude product was purified on silica gel column chromatography (0-20% methanol in dichloromethane) to obtain the title compound (0.4 g, 21% yield) as an off-white solid. LC-MS m/z: 423.2 (M+1). .sup.1HNMR (300 MHz, DMSO-d6): 1.38 (s, 9H), 2.27-2.50 (m, 6H), 3.28-3.39 (m, 4H), 3.66-3.71 (m, 1H), 3.98 (br. S, 1H), 4.97 (br s, 1H), 7.35-7.40 (m, 1H), 7.59-7.65 (m, 1H) 8.07-8.10 (d, J=6.3 Hz, 1H), 9.10-9.11 (m, 1H).

Step 9Synthesis of 6-fluoro-4-((2-hydroxy-3-(piperazin-1-yl)propyl)amino)-2Hbenzo[e][1,3]oxazin-2-one Hydrochloride

(38) ##STR00298##

(39) A solution of tert-butyl 4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4yl) amino-2-hydroxypropyl)piperazine-1-carboxylate (100 mg, 0.023 mmol) in HCl.dioxane (4.5 N solution, 5 mL) was stirred for 4 h at room temperature. The reaction mixture was concentrated under reduced pressure and the resulting product (off-white solid, 100 mg, quant. yield) was used as such in the next step.

Step 10Synthesis of 6-fluoro-4-({2-hydroxy-3-[4-(2,7-naphthyridin-1-yl)piperazin-1-yl]propyl}amino)-2H-1,3-benzoxazin-2-one (Compound 157)

(40) ##STR00299##

(41) To a solution of 6-fluoro-4-((2-hydroxy-3-(piperazin-1-yl)propyl)amino)-2Hbenzo[e][1,3]oxazin-2-one hydrochloride (200 mg, 0.56 mmol) and TEA (0.18 mL, 0.85 mmol) in DMF (2 mL), 1-chloro-2,7-naphthyridine (60 mg, 0.37 mmol) was added. The reaction mixture was stirred for 8 h at 80 C. then was cooled and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography (5 to 15% MeOH in DCM) to give the title compound as a light brown solid (35 mg, 14% yield). LC-MS m/z: 451.2 (M+1). .sup.1HNMR (DMSO d6): 2.49-2.78 (m, 4H), 3.07-3.31 (m, 6H), 3.02-3.04 (m, 2H), 4.37-4.38 (m, 1H), 6.08 (s, 1H) 7.32-7.49 (m, 2H), 7.48-7.49 (m, 1H), 7.65-7.71 (m, 1H), 8.12-8.18 (m, 1H), 8.31-8.32 (m, 1H), 8.64-8.66 (m, 1H), 9.32-9.43 (m, 1H), 9.93-9.94 (m, 1H).

(42) Preparation of Compound 160

(43) Compound 160 was prepared as described herein below.

Step 1Synthesis of tert-butyl [1-(7-fluoroisoquinolin-1-yl)piperidin-4-yl]carbamate

(44) ##STR00300##

(45) Potassium carbonate (2.9 g, 21 mmol) was added to a stirred solution of 1-chloro-7-fluoroisoquinoline (2.6, 14 mmol) and tert-butyl piperidin-4-ylcarbamate (5.2 g, 28 mmol) in DMSO (20 mL) at room temperature. The resulting mixture was stirred at 120 C. overnight then was allowed to cool to room temperature and partitioned between EtOAc (300 mL) and water (300 mL). The organic layer was separated, washed with 1M citric acid solution (100 mL) and brine (70 mL) and dried over sodium sulfate. The solvents were evaporated under reduced pressure and the resulting residue was purified by flash chromatography on silica gel (SNAP 100, from Cy to Cy/Ethyl acetate 8:2) to obtain tert-butyl [1-(7-fluoroisoquinolin-1-yl)piperidin-4-yl]carbamate (4.3 g, 12.4 mmol, 88% yield). LC-MS (MH.sup.+)=346.5

Step 2Synthesis of 1-(7-fluoroisoquinolin-1-yl)piperidin-4-amine

(46) ##STR00301##

(47) TFA (10 mL) was added to a solution of tert-butyl 4-(7-fluoroisoquinolin-1-yl)piperazine-1-carboxylate (4.3 g, 12.4 mmol) in dichloromethane (30 mL) and the resulting mixture was stirred overnight at room temperature. The volatiles were evaporated under reduced pressure, the residue was dissolved in dichloromethane (20 mL) and evaporated under reduced pressure. The resulting residue was dissolved in MeOH and loaded onto a preconditioned SCX cartridge (50 g). The SCX was eluted with MeOH and a 2M solution of ammonia in methanol. The basic fraction was evaporated under reduced pressure to give 3.1 g (Y=quant.) of 1-(7-fluoroisoquinolin-1-yl)piperidin-4-amine. LC-MS (MH.sup.+)=246.3

Step 3Synthesis of 2-(pyridin-2-yl)-1H-imidazole-4-carbaldehyde

(48) ##STR00302##

(49) To a suspension of [2-(pyridin-2-yl)-1H-imidazol-4-yl]methanol (1 g, 5.7 mmol) in dry DCM (30 mL) Dess-Martin Periodinane (3.1 g, 1.3 eq) was added portionwise. The cloudy mixture was stirred 1.5 h at room temperature then was treated with sat. NaHCO.sub.3 and 10% Na.sub.2S.sub.2O.sub.3 solution. After stirring for 30 min. the organic phase was separated, dried over Na.sub.2SO.sub.4, filtered and evaporated to recover 730 mg of crude 2-(pyridin-2-yl)-1H-imidazole-4-carbaldehyde, that was progressed without further purification and characterization.

Step 4Synthesis of 1-(7-fluoroisoquinolin-1-yl)-N-{[2-(pyridin-2-yl)-1H-imidazol-4-yl]methyl}piperidin-4-amine (Formate Salt, Compound 160)

(50) ##STR00303##

(51) 2-(pyridin-2-yl)-1H-imidazole-4-carbaldehyde (100 mg, 0.57 mmol) was dissolved in dry DCM (20 mL) and treated with 1-(7-fluoroisoquinolin-1-yl)piperidin-4-amine (140 mg, 0.57 mmol) and 2 drops of acetic acid. After stirring for 30 min. NaBH(OAc).sub.3 (180 mg, 0.86 mmol) was added in one portion and the reaction mixture was stirred overnight. The reaction was partitioned between DCM and sat. NaHCO.sub.3. The organic phase was evaporated and purified by reversed phase chromatography (H2O/MeCN+0.1% HCOOH from 100/0 to 85/15) to afford 1-(7-fluoroisoquinolin-1-yl)-N-{[2-(pyridin-2-yl)-1H-imidazol-4-yl]methyl}piperidin-4-amine as formate salt (56 mg, 0.125 mmol, 21.9% yield). LC-MS (MH.sup.+)=403.4. .sup.1H NMR (500 MHz, METHANOL-d.sub.4) ppm 1.95-2.08 (m, 2H), 2.35 (dd, J=11.74, 1.47 Hz, 2H), 3.06 (t, J=12.00 Hz, 2H), 3.40-3.50 (m, 1H), 3.86 (d, J=12.00 Hz, 2H), 4.34 (s, 2H), 7.37-7.41 (m, 1H), 7.41-7.47 (m, 2H), 7.55 (td, J=8.80, 2.45 Hz, 1H), 7.75 (dd, J=10.27, 2.45 Hz, 1H), 7.86-7.99 (m, 2H), 8.06-8.11 (m, 2H), 8.44 (s, 1H), 8.63 (d, J=4.40 Hz, 1H).

(52) Preparation of Compound 164

(53) Compound 164 was prepared as described herein below.

Step 1Synthesis of 8-chloro-2,7-naphthyridine 2-oxide

(54) ##STR00304##

(55) To a solution of 1-chloro-2,7-naphthyridine (0.15 g, 0.9 mmol) in DCM, m-CPBA (55%, 472 mg, 1.4 mmol) was added. The mixture was stirred 3 h at room temperature then the organic layer was washed with saturated potassium carbonate solution, water, brine solution and dried over sodium sulfate. The solvent was removed under reduced pressure to give 8-chloro-2,7-naphthyridine 2-oxide (120 mg, 73%) as a pale yellow solid. LC-MS m/z: 181.0 (M+1). .sup.1HNMR (400 MHz, DMSO d6): 7.94 (d, J=5.6 Hz, 1H), 8.11 (d, J=7.12 Hz, 1H), 8.43-8.39 (m, 1H), 8.49-8.46 (m, 1H), 8.95 (s, 1H).

Step 2Synthesis of 8-(4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4-yl)amino)-2-hydroxypropyl)piperazin-1-yl)-2,7-naphthyridine 2-oxide (Compound 164)

(56) ##STR00305##

(57) The synthesis was performed according to the procedure described for compound 157 (step 10) using 8-chloro-2,7-naphthyridine 2-oxide (23% yield). LC-MS m/z: 467.0 (M+1). .sup.1HNMR (400 MHz, DMSO-d6): 2.74-2.60 (m, 5H), 3.16-3.09 (m, 1H), 3.42-3.21 (m, 5H), 3.72-3.70 (m, 1H), 4.06-4.05 (m, 1H), 5.01 (d, J=4.4 Hz, 1H), 7.39-7.36 (m, 2H), 7.65-7.60 (m, 1H), 7.91 (d, J=6.8 Hz, 1H), 8.13-8.10 (m, 1H), 8.18 (d, J=5.6 Hz, 1H), 8.31 (d, J=7.2 Hz, 1H), 8.64 (s, 1H), 9.14-9.12 (m, 1H).

(58) Preparation of compound 165

(59) Compound 165 was prepared as described herein below.

Step 1Synthesis of 4-(3-((6-fluoro-2-oxo-2H-benzo[e][1,3]oxazin-4-yl)amino)-2-hydroxypropyl)-1-(2,7-naphthyridin-1-yl)piperazine 1-oxide (Compound 165)

(60) ##STR00306##

(61) To a solution of compound 157 (30 mg, 0.06 mmol) in IPA (3 mL), magnesium monoperoxyphthalate hexahydrate (32 mg, 0.06 mmol) was added at room temperature. The reaction mixture was refluxed for 6 h then was cooled and concentrated under reduced pressure. The resulting crude was purified by flash column chromatography (8 to 10% MeOH/NH.sub.3 in DCM) to give the title compound (3.5 mg, 11.2% yield). LC-MS m/z: 465.2 (M-1). .sup.1HNMR (CDCl.sub.3+MeOH-d4): 1.28 (t, J=6.8 Hz, 3H), 3.35-3.29 (m, 2H), 3.71-3.56 (m, 5H), 3.86-3.78 (m, 2H), 4.03-3.98 (m, 2H), 4.61-4.59 (m, 1H), 7.22-7.20 (m, 2H), 7.35-7.30 (m, 1H), 7.58-7.56 (m, 1H), 7.70 (d, J=6 Hz, 1H), 8.22 (d, J=5.6 Hz, 1H), 8.53 (d, J=4.8 Hz, 1H), 9.31 (s, 1H).

(62) Preparation of Compounds 180 and 181

(63) Compounds 180 and 181 were prepared as described herein below.

Synthesis of 4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde

(64) ##STR00307##

(65) The title intermediate was prepared according to the procedure described for the synthesis of 8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde (compound 193, steps 1-4) starting from 4-hydroxy-2H-1-benzopyran-2-one. LC-MS m/z: 212.0 (MH.sup.). .sup.1HNMR (DMSO d6): 12.61 (s, 1H), 9.76 (s, 1H), 8.38-8.36 (m, 1H), 7.70 (d, J=2.0 Hz, 1H), 7.61 (d, J=1.6, 1H), 7.43-7.41 (m, 2H).

Step 1Synthesis of tert-butyl 3-hydroxypyrrolidine-1-carboxylate

(66) ##STR00308##

(67) To a stirred solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (5.2 g, 26.9 mmol) in MeOH/THF 1:1 (50 mL), sodium borohydride (2.05 g, 53.9 mmol) was added portionwise at 0 C. The reaction mixture was stirred for 40 min at room temperature then was quenched with ice. The solvent was concentrated under reduced pressure, the resulting residue was diluted with EtOAc (100 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate and concentrated to afford the title compound (5.1 g, 98% yield) as a mixture of isomers. .sup.1HNMR (CDCl.sub.3): 4.86 (d, J=4.8 Hz, 1H), 4.20 (s, 1H), 2.25-2.23 (m, 3H), 3.11-3.01 (m, 1H), 1.83-1.81 (m, 2H), 1.39 (s, 9H).

Step 2Synthesis of tert-butyl 3-[(methanesulfonyl)oxy]pyrrolidine-1-carboxylate

(68) ##STR00309##

(69) To a stirred solution of tert-butyl 3-hydroxypyrrolidine-1-carboxylate (mixture of isomers, 5.1 g, 27.2 mmol) in DCM (75 mL), TEA (5.9 mL, 42.7 mmol) was added at room temperature. The mixture was cooled to 0 C. then methane sulfonyl chloride (2.62 mL, 32.7 mmol) was added dropwise. The resulting solution was stirred at room temperature for 1 h then was diluted with DCM (100 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over sodium sulfate and concentrated to afford the title compound (7.15 g, 99% yield) as a mixture of isomers, that was progressed without any further purification. .sup.1HNMR (CDCl.sub.3): 5.30-5.27 (m, 1H), 3.60-3.58 (m, 4H), 3.05 (s, 3H), 2.19-2.16 (m, 1H), 2.13-2.12 (m, 1H), 1.39 (s, 9H).

Step 3Synthesis of tert-butyl 3-cyanopyrrolidine-1-carboxylate

(70) ##STR00310##

(71) To a stirred solution of tert-butyl 3-[(methanesulfonyl)oxy]pyrrolidine-1-carboxylate (mixture of isomers, 6.1 g, 23.0 mmol) in DMF (40 mL) NaCN (3.38 g, 69.0 mmol) was added. The reaction mixture was stirred at 100 C. for 16 h then was cooled to room temperature and diluted with EtOAc (75 mL). The organic layer was washed with water (240 mL), dried over sodium sulfate and concentrated. The resulting crude was purified by column chromatography (silica gel: 230-400, 25% EtOAc in pet. ether) to afford the title compound (3.1 g, 15.8 mmol, 69% yield, colorless liquid) as a mixture of isomers. .sup.1HNMR (CDCl.sub.3): 3.69-3.59 (m, 4H), 3.12-3.09 (m, 1H), 2.27-2.25 (m, 2H), 1.48 (s, 9H).

Step 4Synthesis of tert-butyl 3-(aminomethyl)pyrrolidine-1-carboxylate

(72) ##STR00311##

(73) To a stirred solution of tert-butyl 3-cyanopyrrolidine-1-carboxylate (mixture of isomers, 3.1 g, 15.8 mmol) and TEA (1.3 mL, 9.4 mmol) in EtOH (90 mL) Raney nickel (2.04 g, 23.7 mmol) was added under nitrogen. The reaction mixture was stirred at room temperature for 16 h under 30 psi hydrogen pressure then was filtered through celite washing with ethanol (50 mL). The solution was concentrated under reduced pressure to afford the title compound (2.6 g, 82% yield, yellow liquid) as a mixture of isomers. LC-MS (ELSD) m/z: 201.2 (M+H+). .sup.1HNMR (CDCl.sub.3): 3.49-3.41 (m, 4H), 3.00-2.99 (m, 1H), 2.73 (m, 2H), 2.23-2.22 (m, 1H), 2.00 (m, 1H), 1.46 (s, 9H).

Step 5Synthesis of tert-butyl 3-({[(benzyloxy)carbonyl]amino}methyl)pyrrolidine-1-carboxylate

(74) ##STR00312##

(75) To a stirred solution of tert-butyl 3-(aminomethyl)pyrrolidine-1-carboxylate (mixture of isomers, 2.5 g, 12.4 mmol) in 1,4 dioxane (50 mL) a saturated sodium carbonate solution (14 mL) was added at room temperature. The reaction mixture was cooled to 0-5 C. then benzyl chloroformate (50% in toluene, 5.5 mL, 16 mmol) was slowly added. After stirring for 5 h at room temperature the reaction mixture was diluted with water (50 mL) and extracted with EtOAc (360 mL). The combined organic layers were washed with brine (50 mL), dried over sodium sulfate and evaporated under vacuum. The crude was purified by column chromatography (Silica gel: 200-400, 12-15% EtOAc in pet. ether) to afford the title compound (2.4 g, 57.5% yield, colorless liquid) as a mixture of isomers. LC-MS m/z: 235.1 (M+H.sup.+-Boc). .sup.1HNMR (CDCl.sub.3): 7.36-7.27 (m, 5H), 5.11 (s, 2H), 4.87-4.85 (m, 1H), 3.50-3.05 (m, 6H), 3.00-2.97 (m, 1H), 2.35-2.20 (m, 1H), 2.05-1.97 (m, 1H), 1.46 (s, 9H).

Step 6Synthesis of benzyl [(pyrrolidin-3-yl)methyl]carbamate Hydrochloride

(76) ##STR00313##

(77) To a stirred solution of tert-butyl 3-({[(benzyloxy)carbonyl]amino}methyl)pyrrolidine-1-carboxylate (mixture of isomers, 2.4 g, 7.1 mmol) in 1,4-dioxane (30 mL), HCl (4 N solution in 1,4-dioxane, 30 mL) was added at 0-5 C. The reaction mixture was stirred for 2 h at room temperature then was concentrated under reduced pressure. The resulting crude was treated with petroleum ether, filtered and dried under reduced pressure to afford the title compound (1.7 g, crude) as a mixture of isomers, that was progressed without any further purification. LC-MS m/z: 235.2 (MH.sup.+).

Step 7Synthesis of benzyl {[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}carbamate

(78) ##STR00314##

(79) To a stirred solution of benzyl [(pyrrolidin-3-yl)methyl]carbamate hydrochloride (mixture of isomers, 957 mg, 3.5 mmol) in NMP (15 mL) TEA (1.1 g, 11.0 mmol) was added. After stirring for 15 min at room temperature, 1-chloro-7-fluoroisoquinoline (400 mg, 2.2 mmol) was added. The reaction mixture was stirred at 140 C. for 24 h then was cooled, concentrated under reduced pressure and diluted with EtOAc (50 mL). The organic phase was washed with water (225 mL) and brine (20 mL), dried over sodium sulfate and evaporated under vacuum. The resulting crude was purified by column chromatography (silica gel: 200-400 mesh, 30-40% EtOAc in pet. ether) to afford the title compound (500 mg, 60% yield) as a mixture of isomers. LC-MS m/z: 380.2 (M+H).

Step 8Synthesis of 1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine

(80) ##STR00315##

(81) To a stirred solution of benzyl {[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}carbamate (mixture of isomers, 500 mg, 1.3 mmol) in methanol (50 mL), 10% Pd/C (100 mg, 50% wet) was added. The reaction mixture was stirred under hydrogen atmosphere for 3 h then was filtered onto a celite bed washing with methanol (50 mL). The filtrate was concentrated under reduced pressure to afford the title compound (310 mg, 1.3 mmol, 97% yield) as a mixture of isomers. LC-MS m/z: 246.1 (M+H).

Step 9Synthesis of the enantiomers of 2-[({[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}amino)methyl][1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Compounds 180 and 181)

(82) ##STR00316##

(83) To a stirred solution of 1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine (mixture of isomers, 320 mg, 1.3 mmol) and 4-oxo-1, 4-dihydrochromeno [4, 3-b] pyrrole-2-carbaldehyde (330 mg, 1.56 mmol) in DCE (5 mL) and DMF (2 mL), AcOH (0.1 mL) was added at room temperature. After stirring for 20 h at 50 C. the mixture was cooled to room temperature and sodium triacetoxy borohydride (553 mg, 2.6 mmol) was added. The reaction mixture was stirred for 3 h at room temperature then was diluted with water (30 mL) and extracted with DCM (330 mL). The combined organic layers were washed with water (220 mL) and brine (20 mL), dried over sodium sulfate and concentrated under reduced pressure. The resulting crude was purified by flash chromatography (silica gel: 200-400, 3-5% MeOH in DCM) to afford the isomeric mixture of 2-[({[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}amino)methyl][1]benzopyrano[4,3-b]pyrrol-4(1H)-one. The mixture was purified by chiral HPLC to afford the 2 enantiomers.

(84) Compound 180 (stereochemistry not assigned): 29.2 mg, 5% yield. LC-MS m/z: 443.1 (M+H). .sup.1H NMR (400 MHz, DMSO-d6): 12.56 (brs, 1H), 8.06 (d, J=7.56 Hz, 1H), 7.93-7.80 (m, 3H), 7.55-7.50 (m, 1H), 7.45-7.36 (m, 2H), 7.34-7.32 (m, 1H), 7.05 (d, J=5.6 Hz, 1H), 6.55 (s, 1H), 3.85-3.69 (m, 5H), 3.58 (t, J=9.6 Hz, 1H), 2.66-2.50 (m, 2H), 2.43-2.41 (m, 2H), 2.09-2.06 (m, 1H), 1.70-1.68 (m, 1H).

(85) Compound 181 (stereochemistry not assigned): 21.4 mg, 3.7% yield. LC-MS m/z: 443.1 (M+H+). .sup.1H NMR (400 MHz, DMSO-d6): 12.46 (brs, 1H), 8.05 (d, J=7.76 Hz, 1H), 7.93-7.82 (m, 3H), 7.53 (t, J=8.5 Hz, 1H), 7.46-7.33 (m, 3H), 7.05 (d, J=5.2 Hz, 1H), 6.54 (s, 1H), 3.82-3.71 (m, 5H), 3.59 (t, J=9.08 Hz, 1H), 3.01-3.00 (m, 2H), 2.66-2.61 (m, 1H), 2.43-2.32 (m, 1H), 2.07-2.05 (m, 1H), 1.70-1.68 (m, 1H).

(86) Preparation of Compounds 182 and 183

(87) Compounds 182 and 183 were prepared as described herein below.

Step 1Synthesis of Tert-butyl 2-{[(methanesulfonyl)oxy]methyl}morpholine-4-carboxylate

(88) ##STR00317##

(89) The title intermediate was prepared as a mixture of isomers according to the procedure described for the synthesis of tert-butyl 3-[(methanesulfonyl)oxy]pyrrolidine-1-carboxylate (see compounds 180-181, step 2) using tert-butyl 2-(hydroxymethyl)morpholine-4-carboxylate (92% yield). LC-MS (ELSD) m/z: 196.0 (M+H.sup.+-Boc). .sup.1HNMR (CDCl.sub.3): 4.25 (d, J=4.8 Hz, 2H), 3.93-3.73 (m, 3H), 3.69-3.67 (m, 1H), 3.58-3.55 (m, 1H), 3.10 (s, 3H), 2.98-2.85 (m, 1H), 2.80-2.75 (m, 1H), 1.43 (s, 9H).

Step 2Synthesis of Tert-butyl 2-(azidomethyl)morpholine-4-carboxylate

(90) ##STR00318##

(91) To a stirred solution of tert-butyl 2-{[(methanesulfonyl)oxy]methyl}morpholine-4-carboxylate (mixture of isomers, 2.5 g, 8.46 mmol) in DMF (25 mL), sodium azide (4.64 g, 42.3 mmol) and sodium iodide (253 mg, 1.6 mmol) were added at room temperature. The reaction mixture was stirred at 60 C. for 20 h then was diluted with water (20 mL) and extracted with EtOAc (250 mL). The organic layer was washed with brine (50 mL), dried over sodium sulfate and concentrated under vacuum. The resulting crude was purified by column chromatography (silica gel: 200-400, 20% EtOAc in pet. ether) to afford the title compound (1.8 g, 87% yield) as a mixture of isomers. LC-MS (ELSD) m/z: 143.2 (M+H-Boc). .sup.1HNMR (CDCl.sub.3): 4.05-3.85 (m, 3H), 3.65-3.45 (m, 2H), 3.35-3.00 (m, 2H), 3.00-2.90 (m, 1H), 2.89-2.79 (m, 1H), 1.45 (s, 9H).

Step 3Synthesis of Tert-butyl 2-(aminomethyl)morpholine-4-carboxylate

(92) ##STR00319##

(93) The title intermediate was prepared as a mixture of isomers according to the procedure described for the synthesis of 1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine (see compounds 180-181, step 8) using tert-butyl 2-(azidomethyl)morpholine-4-carboxylate (93% yield). LC-MS (ELSD): m/z 217.3 (M+H).

Step 4Synthesis of Tert-butyl 2-({[(benzyloxy)carbonyl]amino}methyl)morpholine-4-carboxylate

(94) ##STR00320##

(95) The title intermediate was prepared as a mixture of isomers according to the procedure described for the synthesis of tert-butyl 3-({[(benzyloxy)carbonyl]amino}methyl)pyrrolidine-1-carboxylate (see compounds 180-181, step 5) using tert-butyl 2-(aminomethyl)morpholine-4-carboxylate (54% yield). LC-MS m/z: 251.3 (M+H.sup.+-Boc); .sup.1HNMR (CDCl.sub.3): 7.39-7.33 (m, 5H), 5.13 (s, 2H), 3.88-3.86 (m, 3H), 3.73-3.71 (m, 1H), 3.55-3.47 (m, 3H), 3.18-2.88 (m, 1H), 2.68-2.65 (m, 1H), 1.48 (s, 9H).

Step 5Synthesis of Benzyl [(morpholin-2-yl)methyl]carbamate Hydrochloride

(96) ##STR00321##

(97) The title intermediate was prepared as a mixture of isomers according to the procedure described for the synthesis of benzyl [(pyrrolidin-3-yl)methyl]carbamate hydrochloride (see compounds 180-181, step 6) using tert-butyl 2-({[(benzyloxy)carbonyl]amino}methyl)morpholine-4-carboxylate (99% yield). LC-MS m/z: 251.1 (M+H.sup.+).

Step 6Synthesis of Benzyl {[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methyl}carbamate

(98) ##STR00322##

(99) The title intermediate was prepared as a mixture of isomers according to the procedure described for the synthesis of benzyl {[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methyl}carbamate (see compounds 180-181, step 7) using benzyl [(morpholin-2-yl)methyl]carbamate hydrochloride (37% yield). LC-MS m/z: 396.2 (M+H.sup.+).

Step 7Synthesis of 1-[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methanamine

(100) ##STR00323##

(101) The title intermediate was prepared as a mixture of isomers according to the procedure described for the synthesis of 1-[1-(7-fluoroisoquinolin-1-yl)pyrrolidin-3-yl]methanamine (see compounds 180-181, step 8) using benzyl {[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methyl}carbamate (84% yield). LC-MS m/z: 262.1 (M+H.sup.+).

Step 8Synthesis of the Enantiomers of 2-[({[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methyl}amino)methyl][1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Compounds 182 and 183)

(102) ##STR00324##

(103) Compounds 182 and 183 were prepared according to the procedure described for compounds 180 and 181 (step 9) using the isomeric mixture of 1-[4-(7-fluoroisoquinolin-1-yl)morpholin-2-yl]methanamine.

(104) Compound 182 (stereochemistry not assigned): 9.9 mg, 2.6% yield. LC-MS m/z: 459.2 (M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6): 12.35 (brs, 1H), 8.11 (d, J=5.6 Hz, 1H), 8.04-7.99 (m, 2H), 7.76 (d, J=10.2 Hz, 1H), 7.66-7.62 (m, 1H), 7.47-7.32 (m, 4H), 6.53 (s, 1H), 3.96-3.83 (m, 5H), 3.64-3.48 (m, 2H), 3.01 (t, J=10.2 Hz, 1H), 2.82-2.76 (m, 1H), 2.70-2.67 (m, 2H).

(105) Compound 183 (stereochemistry not assigned): 12.9 mg, 3.3% yield. LC-MS m/z: 459.2 (M+H.sup.+). .sup.1H NMR (400 MHz, DMSO-d6): 12.50 (brs, 1H), 8.12 (d, J=5.6 Hz, 1H), 8.05-8.00 (m, 2H), 7.79-7.76 (m, 1H), 7.68-7.63 (m, 1H), 7.48-7.34 (m, 4H), 6.57 (s, 1H), 3.97-3.86 (m, 5H), 3.64-3.61 (m, 1H), 3.52-3.49 (m, 1H), 3.03-3.00 (m, 1H), 2.83-2.51 (m, 3H).

(106) Preparation of Compound 193

(107) Compound 193 was prepared as described herein below.

Step 1Synthesis of 4-chloro-6-fluoro-2-oxo-2H-1-benzopyran-3-carbaldehyde

(108) ##STR00325##

(109) POCl.sub.3 (25.8 mL) was added to DMF (40 mL) in one portion at 0 C. The resulting mixture was heated at 50 C. for 0.5 h then a solution of 6-fluoro-4-hydroxycoumarin (10 g, 55.5 mmol) in DMF (30 mL) was added at 50 C. The reaction mixture was further heated at 60 C. overnight. The mixture was concentrated in vacuo, treated with toluene (250 mL) and evaporated. DCM (400 mL) was added, the resulting mixture was poured onto ice and stirred for 10 min keeping the temperature around 0 C. The organic phase was separated, dried and evaporated in vacuo to obtain 12.5 g of the crude product. The title intermediate was progressed into the next step without further purification and characterization.

Step 2Synthesis of Ethyl 8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carboxylate

(110) ##STR00326##

(111) 4-chloro-6-fluoro-2-oxo-2H-chromene-3-carbaldehyde (12.5 g, crude material) and glycine ethyl ester hydrochloride (8.1 g, 58.3 mmol) were suspended in absolute ethanol (120 mL). TEA (3 eq.) was added at 0 C. then the resulting mixture was stirred at the same temperature for 1 hour and at 80 C. for 24 hours. The mixture was concentrated in vacuo, dissolved in DCM (400 mL) and washed with sat. NaHCO.sub.3 (200 mL). The organic phase was dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The crude material was treated with EtOH (10 mL) to obtain the title compound (7.9 g, 28.7 mmol, 51% yield over two steps). LC-MS (MH.sup.+)=276.0

Step 3Synthesis of 8-fluoro-2-(hydroxymethyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

(112) ##STR00327##

(113) 8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carboxylate (7.9 g, 28.7 mmol) was dissolved in dry THF (240 mL). The solution was chilled to 10 C. then LiAlH.sub.4 (1 M solution in THF, 38.2 mL) was slowly added. The reaction mixture was stirred at 0 C. for 4 hours then was quenched by adding Na.sub.2SO.sub.4.10H.sub.2O. The inorganic salts were filtered off and the solvents were evaporated to recover the title product (3.2 g, 13.7 mmol, 48% yield), that was progressed without any further purification. LC-MS (MH.sup.+)=234.1

Step 4Synthesis of 8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde

(114) ##STR00328##

(115) To a solution of 8-fluoro-2-(hydroxymethyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (1.2 g, 5.15 mmol) in DMSO (12 mL), Dess-Martin periodinane (2.4 g, 5.66 mmol) was added. The mixture was stirred at rt for 30 min then an aqueous solution of sat. NaHCO.sub.3/10% Na.sub.2S.sub.2O.sub.3 1:1 was added. The precipitate was filtered, washed with water and treated with MeCN (4 mL) and diethyl ether (10 mL) to obtain the title intermediate (1.1 g, 4.76 mmol, 92% yield). LC-MS (MH.sup.+)=232.1

Step 5Synthesis of 3-amino-5-fluoropyridin-2(1H)-one

(116) ##STR00329##

(117) 5-Fluoro-3-nitropyridin-2-ol (10 g, 63.3 mmol) was dissolved in ethanol (300 mL), 10% Pd/C (1.8 g) was added and the mixture was stirred at room temperature under atmospheric pressure of hydrogen for 2 h. Pd/C was removed by filtration and the solvent was evaporated in vacuum to obtain the title compound as an off-white solid (7.5 g, 58.5 mmol, Y=92%). LC-MS (MH.sup.+)=129.0

Step 6Synthesis of Tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

(118) ##STR00330##

(119) 3-amino-5-fluoropyridin-2(1H)-one (3.7 g, 28.9 mmol) and tert-butyl (4-oxocyclohexyl)carbamate (8 g, 37.6 mmol) were dissolved in DMF (213 mL). TFA (18.4 mL, 240 mmol) was added dropwise followed by sodium triacetoxyborohydride (9.19 g, 43.4 mmol). The mixture was stirred at room temperature for 1.5 h then the reaction was quenched with sat. NaHCO.sub.3. The mixture was extracted with ethyl acetate, dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure. The residue was purified by flash chromatography (SNAP C-18 400 g, from 100% water+0.1% formic acid to 50:50 water+0.1% formic acid/acetonitrile+0.1% formic acid) to give the title compound as a formate salt. This material was dissolved in ethyl acetate, washed with sat. NaHCO.sub.3 sat. sol., dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure to give the free base of target compound as mixture of isomers (5.8 g, 17.8 mmol, 60% yield). LC-MS (MH.sup.+)=326.3

Step 7Synthesis of Tert-butyl [4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

(120) ##STR00331##

(121) To a suspension of tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (5.8 g, 17.8 mmol) and potassium carbonate (9.9 g, 71.3 mmol) in DMF (110 mL) chloroacetyl chloride (3.1 mL, 39.2 mmol) was added. The mixture was stirred at room temperature for 30 minutes then was heated to 80 C. for 5 h. The mixture was cooled to 0 C. then sat. NaHCO.sub.3 was added (50 mL) followed by ethyl acetate (200 mL). The organic phase was separated, dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The crude product was purified by Silica gel column (from 100% of cy to cy/ethyl acetate 50:50) to obtain the title compound (3.79 g, 10.4 mmol, 58% yield) as a mixture of isomers. LC-MS (MH.sup.+)=366.4

Step 8Synthesis of 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

(122) ##STR00332##

(123) tert-butyl [4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate (3.79 g, 10.4 mmol) was dissolved in DCM (70 mL), TFA (10 mL) was added at 0 C. then the mixture was stirred at rt for 2 h. The solvent was evaporated in vacuo and the residue was purified by SCX column and then by preparative HPLC purification under basic conditions (0.1% v/v ammonia aqueous solution/acetonitrile) to afford the trans diastereoisomer (1.15 g, 4.3 mmol, 41% yield). LC-MS (MH.sup.+)=266.0

Step 9Synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 193)

(124) ##STR00333##

(125) 8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde (158 mg, 0.41 mmol) and 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (100 mg, 0.37 mmol) were suspended in dry dichloromethane (20 mL). 2 drops of acetic acid were added. The mixture was stirred for 2 hours at 50 C. then NaBH(OAc).sub.3 (195 mg, 0.925 mmol) was added in one portion. The mixture was stirred at room temperature for 3 h then was partitioned between DCM (50 mL) and a sat. NaHCO.sub.3 (20 mL). The organic phase was dried over Na.sub.2SO.sub.4, filtered and evaporated in vacuo. The crude product was purified by SCX column, the resulting free base was dissolved in DCM/MeOH (20:1, 12 mL), the mixture was cooled to 0 C. and HCl (1 M solution in diethyl ether) was added. After stirring at room temperature for 10 min the solvent was evaporated in vacuo and the solid was treated with diethyl ether and dried to afford 170 mg (0.29 mmol, 78% yield) of the title product. LC-MS (MH.sup.+)=481.3. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.55-1.73 (m, 2H), 1.86 (d, J=10.74 Hz, 2H), 2.26 (d, J=11.51 Hz, 2H), 2.34-2.47 (m, 2H), 3.24 (br. s., 1H), 4.03-4.17 (m, 1H), 4.37 (br. s., 2H), 4.72 (s, 2H), 6.95 (s, 1H), 7.37 (td, J=8.77, 2.96 Hz, 1H), 7.53 (dd, J=9.10, 4.60 Hz, 1H), 7.82-7.90 (m, 2H), 7.97 (dd, J=9.92, 2.47 Hz, 1H), 9.33 (br. s., 2H), 13.40 (br. s., 1H).

(126) Preparation of Compound 194

(127) Compound 194 was prepared as described herein below.

Step 1Synthesis of 8-fluoro-4-oxo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde

(128) ##STR00334##

(129) 8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde (see compound 193, 3.5 g, 15.2 mmol) was dissolved in dry THF (120 mL) and cooled to 0 C. TEA (4.2 mL, 30.3 mmol) and SEM-Cl (3.2 mL, 18.2 mmol) were added. After stirring 45 min at the same temperature, a saturated solution of NaHCO.sub.3 was added at 0 C. followed by ethyl acetate. The organic phase was separated, washed with sat. NH.sub.4Cl and brine, dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The crude material was purified by Si-column SNAP 100 eluting with cyclohexane/ethyl acetate from 95:5 to 7:3. The resulting residue was treated with diethyl ether and filtered to give the title compound (860 mg, 2.4 mmol, 16% yield). LC-MS (MH.sup.+)=362.3

Step 2Synthesis of 8-fluoro-2-[(E)-2-methoxyethenyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

(130) ##STR00335##

(131) A solution of (methoxymethyl)triphenylphosphonium chloride (1.2 g, 3.6 mmol) in THF (20 mL) was cooled to 78 C. LiHMDS (1 M solution in THF, 3.6 mL) was added and the mixture was allowed to warm to 0 C. The mixture was cooled again to 78 C. and a solution of 8-fluoro-4-oxo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1,4-dihydro[1]benzopyrano[4,3-b]pyrrole-2-carbaldehyde (645 mg, 1.8 mmol) in THF (10 mL) was added dropwise. After stirring for 2 hours at 78 C. sat. NH.sub.4Cl was added, the resulting mixture was diluted with ethyl acetate and the organic phase was separated, dried and evaporated in vacuo. The crude material was purified by Si-column eluting with cy to cy/ethyl acetate 7:3 to obtain the title intermediate (600 mg, 1.5 mmol, 83% yield) as a mixture of cis/trans isomers. LC-MS (MH.sup.+)=390.4

Step 3Synthesis of (8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehyde

(132) ##STR00336##

(133) TFA (1.5 mL) was added to a solution of 8-fluoro-2-[(E)-2-methoxyethenyl]-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (100 mg, 0.26 mmol) in DCM (6 mL). The resulting mixture was stirred at 40 C. for 3 hours then was diluted with toluene (60 mL) and evaporated under vacuo to afford the title intermediate, that was progressed without further purification. LC-MS (MH.sup.+)=246.3

Step 4Synthesis of Tert-butyl 4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]piperidine-1-carboxylate

(134) ##STR00337##

(135) The synthesis was performed according to the procedure described for the preparation of intermediate tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (see compound 193, step 6) using tert-butyl 4-oxopiperidine-1-carboxylate (51% yield). LC-MS (MH.sup.+)=312.3

Step 5Synthesis of tert-butyl 4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate

(136) ##STR00338##

(137) The synthesis was performed according to the procedure described for the preparation of intermediate tert-butyl [4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate (see compound 193, step 7) using tert-butyl 4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]piperidine-1-carboxylate (46% yield). LC-MS (MH.sup.+)=352.3

Step 6Synthesis of 7-fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

(138) ##STR00339##

(139) The synthesis was performed according to the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl 4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate (87% yield). LC-MS (MH.sup.+)=252.3

Step 7Synthesis of 8-fluoro-2-{2-[4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Compound 194)

(140) ##STR00340##

(141) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using (8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehyde and 7-fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (39% yield). LC-MS (MH.sup.+)=481.3. .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 1.70 (d, J=9.74 Hz, 2H), 2.18 (t, J=11.40 Hz, 2H), 2.44-2.61 (m, 2H), 2.65-2.73 (m, 2H), 2.90 (t, J=7.48 Hz, 2H), 3.02 (d, J=11.39 Hz, 2H), 3.95-4.08 (m, 1H), 4.71 (s, 2H), 6.50 (s, 1H), 7.29 (td, J=8.71, 3.02 Hz, 1H), 7.47 (dd, J=8.70, 4.53 Hz, 1H), 7.75-7.93 (m, 3H), 12.39 (br. s., 1H).

(142) Preparation of Compound 197

(143) Compound 197 was prepared as described herein below.

Step 1Synthesis of Tert-butyl 4-[(6-methoxy-3-nitropyridin-2-yl)amino]piperidine-1-carboxylate

(144) ##STR00341##

(145) A mixture of tert-butyl 4-aminopiperidine-1-carboxylate (2.34 g, 11.7 mmol), 2-chloro-6-methoxy-3-nitropyridine (3 g, 10.6 mmol) and K.sub.2CO.sub.3 (1.47 g, 10.6 mmol) in MeCN (60 mL) and DMF (15 mL) was heated at 60 C. for 3 h. The mixture was filtered and concentrated in vacuo. The residue was suspended in diethyl ether/water 1:1, the organic phase was dried over MgSO.sub.4 and concentrated. The residue was purified by flash chromatography (silica-gel) to afford the title compound (2.3 g, 6.5 mmol, 61% yield). LC-MS (MH.sup.+)=353.3

Step 2Synthesis of Tert-butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate

(146) ##STR00342##

(147) tert-Butyl 4-[(6-methoxy-3-nitropyridin-2-yl)amino]piperidine-1-carboxylate (2.3 g, 6.5 mmol) was dissolved in ethyl acetate/ethanol 1:1 (60 mL), Pd/C (10% wt, 0.2 g) was added and the mixture was stirred at room temperature under atmospheric pressure of hydrogen for 24 h. Pd/C was removed by filtration and the solvent was evaporated in vacuo. The resulting residue was purified by Si-column eluting with cy to cy/ethyl acetate 1:1 to obtain the title product (1.2 g, 3.7 mmol, 57% yield). LC-MS (MH.sup.+)=323.4

Step 3Synthesis of Tert-butyl 4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate

(148) ##STR00343##

(149) tert-Butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (1.2 g, 3.7 mmol) was dissolved in DMF/MeCN 1:2 (30 mL), potassium carbonate (513 mg, 3.7 mmol) was added followed by ethyl bromoacetate (0.43 mL, 3.8 mmol). The mixture was stirred at room temperature overnight then ethyl acetate was added followed by water. The organic phase was separated, washed with brine, dried and evaporated in vacuo. The crude material was purified by Si-column eluting with cy to cy/ethyl acetate 7:3 to obtain the title intermediate (1.4 g, 3.4 mmol, 92% yield). LC-MS (MH.sup.+)=409.5

Step 4Synthesis of Tert-butyl 4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate

(150) ##STR00344##

(151) A solution of tert-butyl 4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate (500 mg, 1.2 mmol) and cat. acetic acid (0.1 mL) in toluene (25 mL) was refluxed overnight. The reaction mixture was concentrated, the residue was dissolved in DCM (20 mL) and treated with MnO.sub.2 (2 g) at room temperature for 2 hours. The solid was filtered and the solvent was evaporated in vacuo. The crude material was purified by Si-column eluting with cy to cy/ethyl acetate 8:2 to obtain the title compound (137 mg, 0.38 mmol, 32% yield). LC-MS (M-Na.sup.+)=383.3

Step 5Synthesis of 6-methoxy-4-(piperidin-4-yl)pyrido[2,3-b]pyrazin-3(4H)-one

(152) ##STR00345##

(153) The synthesis was performed according to the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl 4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate (98% yield). LC-MS (MH.sup.+)=261.3

Step 6Synthesis of 8-fluoro-2-{2-[4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

(154) ##STR00346##

(155) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using (8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehyde and 6-methoxy-4-(piperidin-4-yl)pyrido[2,3-b]pyrazin-3(4H)-one (11% yield). LC-MS (MH.sup.+)=490.5. .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 1.62 (d, J=10.43 Hz, 2H), 2.13 (t, J=11.05 Hz, 2H), 2.66-2.77 (m, 2H), 2.85-3.01 (m, 4H), 3.12 (d, J=11.39 Hz, 2H), 3.92 (s, 3H), 5.25 (br. s., 1H), 6.52 (s, 1H), 6.84 (d, J=8.64 Hz, 1H), 7.29 (td, J=8.71, 3.02 Hz, 1H), 7.48 (dd, J=9.06, 4.53 Hz, 1H), 7.81 (dd, J=8.92, 3.02 Hz, 1H), 8.07 (s, 1H), 8.12 (d, J=8.64 Hz, 1H), 12.40 (br. s., 1H).

(156) Preparation of Compound 200

(157) Compound 200 was prepared as described herein below.

Step 1Synthesis of 3-chloro-6-methoxypyrazin-2-amine

(158) ##STR00347##

(159) To a solution of 6-methoxypyrazin-2-amine (3 g, 24 mmol) in dry DMF (35 mL), N-chlorosuccinimide (3.2 g, 24 mmol) was added. The reaction mixture was stirred at room temperature for 16 h then was poured into ice and brine (130 mL). The aqueous solution was extracted with ethyl acetate (3120 mL), the organic phase was washed with 5% solution of LiCl, dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure. The crude was purified by SNAP-340-NH (cyclohexane/ethyl acetate from 95:5 up to 8:2) and by SNAP100-SiOH (eluting with DCM) to afford the title intermediate (1.98 g, 12.5 mmol, 52% yield). LC-MS (MH.sup.+)=160.1

Step 2Synthesis of Tert-butyl 4-[(3-chloro-6-methoxypyrazin-2-yl)amino]piperidine-1-carboxylate

(160) ##STR00348##

(161) A solution of titanium (IV) chloride (1 M in dichloromethane, 5.8 mL) was added dropwise at 0 C. to a stirred solution of 3-chloro-6-methoxypyrazin-2-amine (1.85 g, 11.6 mmol), tert-butyl 4-oxopiperidine-1-carboxylate (2.55 g, 12.8 mmol) and triethylamine (4.85 mL, 34.8 mmol) in dry dichloromethane (58 mL). The reaction mixture was slowly allowed to warm to room temperature and stirred overnight. To the resulting mixture a solution of sodium cyanoborohydride (2.18 g, 34.8 mmol) in MeOH (23 mL) was then added at room temperature. After 4 hours the reaction mixture was quenched with a 3 M NaOH solution (180 mL). EtOAc (600 mL) was then added and the mixture was filtered onto a Celite pad (10 cm diameter, 3 cm height). The phases were separated and the organic layer was washed with aqueous sodium bicarbonate solution and brine, dried over sodium sulfate and evaporated under reduced pressure. The crude was purified by flash chromatography (Biotage KP-Sil 100 g SNAP cartridge, eluent from 100% DCM to DCM/MeOH 95:5) to give the title compound (1 g, 2.9 mmol, 25% yield). LC-MS (MH.sup.+)=343.4

Step 3Synthesis of Tert-butyl 4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate

(162) ##STR00349##

(163) tert-Butyl 4-[(3-chloro-6-methoxypyrazin-2-yl)amino]piperidine-1-carboxylate (0.764 g, 2.23 mmol), Pd(PtBu.sub.3).sub.3(0.08 g, 0.156 mmol, 7% mol) and ethyl acrylate (0.29 mL, 2.67 mmol) were suspended in triethylamine (5.0 mL). The resulting mixture was stirred for 90 min at 130 C. then was cooled and partitioned between water and EtOAc. The aqueous layer was extracted twice with EtOAc, the combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated under vacuum. The resulting crude was purified by column chromatography on SiO.sub.2 (cyclohexane/EtOAc from 9:1 to 1:1) to afford the title compound (448 mg, 1.1 mmol, 49% yield). LC-MS (MH.sup.+)=407.5

Step 4Synthesis of Tert-butyl 4-(3-methoxy-6-oxopyrido[2,3-b]pyrazin-5(6H)-yl)piperidine-1-carboxylate

(164) ##STR00350##

(165) Intermediate tert-butyl 4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate (0.34 g, 0.85 mmol) was dissolved in MeOH (17 mL), potassium carbonate (234 mg, 1.69 mmol) was added and the mixture was stirred at 65 C. for 24 h. The volatiles were removed under vacuum, the residue was partitioned between EtOAc and H.sub.2O and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine and dried over Na.sub.2SO.sub.4. The solvent was removed under vacuum to afford a crude which was purified by column chromatography (from cyclohexane/EtOAc 7:3 to EtOAc) to give the title compound (177 mg, 0.49 mmol, 58% yield). LC-MS (MH.sup.+)=361.4

Step 5Synthesis of 3-methoxy-5-(piperidin-4-yl)pyrido[2,3-b]pyrazin-6(5H)-one

(166) ##STR00351##

(167) The synthesis was performed according to the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl 4-(3-methoxy-6-oxopyrido[2,3-b]pyrazin-5(6H)-yl)piperidine-1-carboxylate (92% yield). LC-MS (MH.sup.+)=261.3

Step 6Synthesis of 8-fluoro-2-{2-[4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 200)

(168) ##STR00352##

(169) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using (8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehyde and 3-methoxy-5-(piperidin-4-yl)pyrido[2,3-b]pyrazin-6(5H)-one (12% yield). LC-MS (MH.sup.+)=490.4. .sup.1H NMR (500 MHz, METHANOL-d.sub.4) ppm 2.08 (d, J=13.69 Hz, 2H), 3.32-3.43 (m, 6H), 3.55-3.66 (m, 2H), 3.87 (d, J=10.76 Hz, 2H), 4.18 (s, 3H), 5.87 (br. s., 1H), 6.64-6.78 (m, 2H), 7.24 (td, J=8.56, 2.93 Hz, 1H), 7.45 (dd, J=9.29, 4.40 Hz, 1H), 7.64 (dd, J=8.31, 2.93 Hz, 1H), 7.96 (d, J=9.29 Hz, 1H), 8.21 (s, 1H).

(170) Preparation of Compound 201

(171) Compound 201 was prepared as described herein below.

Step 1Synthesis of Tert-butyl {trans-4-[(6-methoxy-3-nitropyridin-2-yl)amino]cyclohexyl}carbamate

(172) ##STR00353##

(173) To a solution of tert-butyl (trans-4-aminocyclohexyl)carbamate (1.7 g, 8 mmol) and TEA (1.1 mL, 8 mmol) in acetonitrile (53 mL) 2-chloro-6-methoxy-3-nitropyridine (1.5 g, 7.95 mmol) was added. The reaction mixture was heated at 90 C. overnight then was cooled, filtered, and the filtrate was concentrated. The residue was treated with 50 mL of hot ethyl acetate, filtered and concentrated. The residue was dissolved in DCM and washed with sat. NH.sub.4Cl. The organic phase was dried over Na.sub.2SO.sub.4 and concentrated to afford the title compound (1.5 g, 6.7 mmol, 84% yield). LC-MS (MH.sup.+)=367.4

Step 2Synthesis of Tert-butyl {trans-4-[(3-amino-6-methoxypyridin-2-yl)amino]cyclohexyl}carbamate

(174) ##STR00354##

(175) The title compound was prepared according to the procedure described for the synthesis of tert-butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (see compound 197, step 2) using tert-butyl {trans-4-[(6-methoxy-3-nitropyridin-2-yl)amino]cyclohexyl}carbamate (94% yield). LC-MS (MH.sup.+)=337.4

Step 3Synthesis of Ethyl N-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-6-methoxypyridin-3-yl]glycinate

(176) ##STR00355##

(177) The title compound was prepared according to the procedure described for the synthesis of tert-butyl 4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate (see compound 197, step 3) using tert-butyl {trans-4-[(3-amino-6-methoxypyridin-2-yl)amino]cyclohexyl}carbamate (62% yield). LC-MS (MH.sup.+)=423.5

Step 4Synthesis of Tert-butyl [trans-4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)cyclohexyl]carbamate

(178) ##STR00356##

(179) The title compound was prepared according to the procedure described for the synthesis of tert-butyl 4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate (see compound 197, step 4) using ethyl N-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-6-methoxypyridin-3-yl]glycinate (51% yield). LC-MS (M-Na.sup.+)=397.5

Step 5Synthesis of 4-(trans-4-aminocyclohexyl)-6-methoxypyrido[2,3-b]pyrazin-3(4H)-one

(180) ##STR00357##

(181) The title compound was prepared according to the procedure described for the synthesis of 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [trans-4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)cyclohexyl]carbamate (87% yield). LC-MS (M-Na.sup.+)=275.3

Step 6Synthesis of 8-fluoro-2-({[trans-4-(6-methoxy-3-oxopyrido[2,3-b]pyrazin-4(3H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 201)

(182) ##STR00358##

(183) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 4-(trans-4-aminocyclohexyl)-6-methoxypyrido[2,3-b]pyrazin-3(4H)-one (17% yield). LC-MS (MH.sup.+)=490.3. .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 1.21-1.32 (m, 2H), 1.67 (d, J=10.27 Hz, 2H), 2.10 (d, J=11.98 Hz, 2H), 2.41-2.56 (m, 1H), 2.72 (br. s., 2H), 3.83-3.96 (m, 5H), 4.96-5.49 (m, 1H), 6.58 (s, 1H), 6.83 (d, J=8.56 Hz, 1H), 7.29 (td, J=8.80, 2.93 Hz, 1H), 7.48 (dd, J=9.05, 4.65 Hz, 1H), 7.95 (dd, J=9.05, 2.93 Hz, 1H), 8.00-8.07 (m, 1H), 8.10 (d, J=8.56 Hz, 1H), 8.22 (s, 1H), 12.61 (br. s, 2H).

(184) Preparation of Compound 202

(185) Compound 202 was prepared as described herein below.

Step 1Synthesis of N-(6-chloropyridin-2-yl)-2,2-dimethylpropanamide

(186) ##STR00359##

(187) To a solution of 2-amino-6-chloropyridine (10.7 g, 83.5 mmol) in toluene (103 mL) NaHCO.sub.3 (14 g, 167 mmol) and pivaloyl chloride (15.4 mL, 125.2 mmol) were added at 0 C. The resulting mixture was stirred at room temperature for 5 hours then the suspension was filtered and the solid was washed with DCM. The filtrates were concentrated under vacuum then heptane (22 mL) was added and the resulting mixture was concentrated. The solid was filtered, washed with heptane (15 mL) and dried under vacuum to afford the title intermediate (15.4 g, 72.4 mmol, 87% yield). LC-MS (MH.sup.+)=213.2

Step 2Synthesis of N-(6-chloro-3-formylpyridin-2-yl)-2,2-dimethylpropanamide

(188) ##STR00360##

(189) To a solution of N-(6-chloropyridin-2-yl)-2,2-dimethylpropanamide (10.6 g, 50 mmol) in THF (100 mL) n-butyllithium (2.5 M solution in hexane, 50 mL, 125 mmol) was added. The resulting mixture was stirred at 20 C. for 3 h. After the addiction of DMF (4 mL) the reaction mixture was allowed to warm to room temperature and then was quenched with 0.5 M HCl. Ethyl acetate was added, the organic phase was washed with water, sat. K.sub.2CO.sub.3 and with brine. The crude material was purified by Si-column eluting with cy to cy/ethyl acetate 7:3 to obtain the title product (6.7 g, 27.8 mmol, 56% yield). LC-MS (MH.sup.+)=241.3

Step 3Synthesis of Tert-butyl 3-[6-chloro-2-(2,2-dimethylpropanamido)pyridin-3-yl]-3-hydroxypropanoate

(190) ##STR00361##

(191) An oven-dried flask was charged with THF (80 mL) and diisopropylamine (8.2 mL, 58.4 mmol). The solution was cooled to 78 C. then n-butyllithium (2.5 M solution in hexane, 23.3 mL, 58.3 mmol) was added. The mixture was stirred for 15 min then a solution of tert-butyl acetate (7.8 mL, 58.3 mmol) in THF (2 mL) was added. After stirring at 78 C. for 20 min a solution of N-(6-chloro-3-formylpyridin-2-yl)-2,2-dimethylpropanamide (6.7 g, 27.8 mmol) in THF (5 mL) was added. The mixture was allowed to warm to room temperature then was poured into sat. NH.sub.4Cl. The resulting mixture was extracted with ethyl acetate and the organic phase was dried and evaporated in vacuo to obtain the title compound (9.4 g, 26.4 mmol, 95% yield). LC-MS (MH.sup.+)=357.3

Step 4Synthesis of 7-chloro-1,8-naphthyridin-2(1H)-one

(192) ##STR00362##

(193) Intermediate tert-butyl 3-[6-chloro-2-(2,2-dimethylpropanamido)pyridin-3-yl]-3-hydroxypropanoate (4 g, 11 mmol) was dissolved in 1 M HCl/dioxane 1:1 (50 mL) and refluxed overnight. Ice was added to the mixture and the resulting solid was filtered and dried to afford the title product (1.7 g, 9.4 mmol, 86% yield), that was used without any further purification. LC-MS (MH.sup.+)=181.1

Step 5Synthesis of 7-methoxy-1,8-naphthyridin-2(1H)-one

(194) ##STR00363##

(195) To a suspension of 7-chloro-1,8-naphthyridin-2(1H)-one (700 mg, 3.9 mmol) in MeOH (15 mL), NaOMe (25% solution in MeOH, 20 mL) was added. The resulting solution was stirred at reflux for 15 h then the solvent was removed in vacuo. Water (100 mL) and EtOAc (80 mL) were added, the phases were separated and the aqueous layer was extracted with EtOAc (880 mL). The combined organic layers were washed with brine (50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give the title compound (630 mg, 3.6 mmol, 92% yield). LC-MS (MH.sup.+)=177.2

Step 6Synthesis of Tert-butyl 4-(7-methoxy-2-oxo-1,8-naphthyridin-1(2H)-yl)piperidine-1-carboxylate

(196) ##STR00364##

(197) Cs.sub.2CO.sub.3 (828 mg, 2.6 mmol) and tert-butyl 4-bromopiperidine-1-carboxylate (1.35 g, 5.1 mmol) were sequentially added to a solution of 7-methoxy-1,8-naphthyridin-2(1H)-one (150 mg, 0.85 mmol) in DMF (15 mL). The reaction mixture was stirred at 50 C. overnight then was partitioned between EtOAc and water. The organic phases were dried over Na.sub.2SO.sub.4, filtered and evaporated in vacuo. The crude material was purified by silica-gel chromatography (cyclohexane/EtOAc from 100:0 to 50:50) to afford the title product (70 mg, 0.2 mmol, 22% yield). LC-MS (MH.sup.+)=360.4

Step 7Synthesis of 7-methoxy-1-(piperidin-4-yl)-1,8-naphthyridin-2(1H)-one

(198) ##STR00365##

(199) The title intermediate was prepared according to the procedure described for the synthesis of 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl 4-(7-methoxy-2-oxo-1,8-naphthyridin-1(2H)-yl)piperidine-1-carboxylate (72% yield). LC-MS (MH.sup.+)=260.3

Step 8Synthesis of 8-fluoro-2-{2-[4-(7-methoxy-2-oxo-1,8-naphthyridin-1(2H)-yl)piperidin-1-yl]ethyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 202)

(200) ##STR00366##

(201) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using (8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)acetaldehyde and 7-methoxy-1-(piperidin-4-yl)-1,8-naphthyridin-2(1H)-one (11% yield). LC-MS (MH.sup.+)=489.3. .sup.1H NMR (500 MHz, METHANOL-d.sub.4) ppm 1.95-2.11 (m, 2H), 3.30-3.40 (m, 6H), 3.55-3.63 (m, 2H), 3.81-3.88 (m, 2H), 4.08 (s, 3H), 6.03 (br. s., 1H), 6.50 (d, J=9.39 Hz, 1H), 6.69 (s, 1H), 6.75 (d, J=8.41 Hz, 1H), 7.22 (td, J=8.71, 2.93 Hz, 1H), 7.43 (dd, J=9.10, 4.40 Hz, 1H), 7.62 (dd, J=8.61, 2.93 Hz, 1H), 7.83 (d, J=9.39 Hz, 1H), 7.98 (d, J=8.61 Hz, 1H).

(202) Preparation of Compounds 204 and 205

(203) Compounds 204 and 205 were prepared as described herein below.

Step 1Synthesis of Tert-butyl {4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate

(204) ##STR00367##

(205) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (see compound 193, step 6) using 2-bromo-5-fluoropyridin-3-amine (98% yield). LC-MS (MH.sup.+)=388.2

Step 2Synthesis of Ethyl (2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate

(206) ##STR00368##

(207) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl 4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate (see compound 200, step 3) using tert-butyl {4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate (66% yield). LC-MS (MH.sup.+)=408.5

Step 3Synthesis of Tert-butyl 4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate

(208) ##STR00369##

(209) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl 4-(3-methoxy-6-oxopyrido[2,3-b]pyrazin-5(6H)-yl)piperidine-1-carboxylate (see compound 200, step 4) using ethyl (2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate (17% yield). LC-MS (MH.sup.+)=374.3

Step 4Synthesis of 1-(4-aminocyclohexyl)-7-methoxy-1,5-naphthyridin-2(1H)-one

(210) ##STR00370##

(211) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl 4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate (73% yield). LC-MS (MH.sup.+)=274.1

Step 5Synthesis of 8-fluoro-2-({[trans-4-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 204) and 8-fluoro-2-({[cis-4-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 205)

(212) ##STR00371##

(213) The title compounds were prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-(4-aminocyclohexyl)-7-methoxy-1,5-naphthyridin-2(1H)-one as a mixture of isomers.

(214) A purification by preparative HPLC (A=0.1% ammonia aqueous solution, B=acetonitrile, form 98:2 A:B to 100% B) followed by treatment with HCl under the previously described conditions afforded the trans (20% yield) and cis (15% yield) diastereoisomers.

(215) Compound 204: LC-MS (MH.sup.+)=489.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.62-1.91 (m, 4H), 2.20-2.40 (m, 2H), 2.67 (br. s., 2H), 3.28 (br. s., 1H), 4.01 (s, 3H), 4.34-4.86 (m, 3H), 6.60 (d, J=9.59 Hz, 1H), 6.95 (d, J=1.57 Hz, 1H), 7.38 (td, J=8.75, 3.03 Hz, 1H), 7.53 (dd, J=9.10, 4.50 Hz, 1H), 7.63 (br. s., 1H), 7.78-7.92 (m, 2H), 8.32 (d, J=2.15 Hz, 1H), 9.33 (br. s., 2H), 13.41 (br. s., 1H).

(216) Compound 205: LC-MS (MH.sup.+)=489.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.65 (d, J=11.74 Hz, 2H), 2.00 (m, J=13.89 Hz, 2H), 2.17 (d, J=14.28 Hz, 2H), 2.76 (m, J=12.13 Hz, 2H), 3.40-3.56 (m, 1H), 4.01 (s, 3H), 4.39-4.51 (m, 2H), 4.59-4.92 (m, 1H), 6.62 (d, J=9.59 Hz, 1H), 6.99 (d, J=1.76 Hz, 1H), 7.38 (td, J=8.75, 3.03 Hz, 1H), 7.53 (dd, J=9.10, 4.60 Hz, 1H), 7.67 (s, 1H), 7.78-7.92 (m, 2H), 8.33 (d, J=2.15 Hz, 1H), 9.23 (br. s., 2H), 13.49 (s, 1H).

(217) Preparation of Compound 206

(218) Compound 206 was prepared as described herein below.

Step 1Synthesis of Tert-butyl [4-(5-cyano-2-iodoanilino)cyclohexyl]carbamate

(219) ##STR00372##

(220) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (see compound 193, step 6) using 3-amino-4-iodobenzonitrile (24% yield). LC-MS (MH.sup.+)=442.2

Step 2Synthesis of Ethyl (2E)-3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]prop-2-enoate

(221) ##STR00373##

(222) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl 4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate (see compound 200, step 3) using tert-butyl [4-(5-cyano-2-iodoanilino)cyclohexyl]carbamate (88% yield). LC-MS (MH.sup.+)=414.4

Step 3Synthesis of Ethyl 3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoate

(223) ##STR00374##

(224) Ethyl (2E)-3-{2-[(4-{[(tert-butoxy)carbonyl]amino}cyclohexyl)amino]-4-cyanophenyl}prop-2-enoate (37.9 g, 91.7 mmol) was dissolved in ethyl acetate (379 mL), 10% Pd/C (7.6 g) was added and the mixture was stirred under hydrogen atmosphere (1 atm) overnight. The catalyst was removed by filtration and the solvent was evaporated in vacuo to obtain the title intermediate as a mixture of isomers (35.7 g, 85.9 mmol, Y=94%, LC-MS (MH.sup.+)=416.4

Step 4Synthesis of 3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoic Acid

(225) ##STR00375##

(226) Ethyl 3-{2-[(4-{[(tert-butoxy)carbonyl]amino}cyclohexyl)amino]-4-cyanophenyl}propanoate (43.5 g, 0.11 mol) was dissolved in THF/H.sub.2O (455/136 mL), LiOH.H.sub.2O (8.8 g, 0.21 mol) was added and the mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo, the residue was treated with acetonitrile, filtered and concentrated in vacuo. The crude title product (mixture of isomers) was progressed without any further purification.

Step 5Synthesis of Tert-butyl [trans-4-(7-cyano-2-oxo-3,4-dihydroquinolin-1(2H)-yl)cyclohexyl]carbamate

(227) ##STR00376##

(228) 3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoic acid (0.11 mol from previous step) was dissolved in DMF (910 mL), TEA (30.5 mL, 0.22 mol) was added and the mixture was cooled to 0 C. HATU (41.6 g, 0.11 mol) was added at the same temperature and the mixture was allowed to slowly reach room temperature. After stirring overnight the mixture was partitioned between water (1 L) and EtOAc (500 mL). The organic phase was washed with brine and dried over Na.sub.2SO.sub.4. The volatiles were removed under vacuum and the crude residue was purified by Si-column (cyclohexane/ethyl acetate from 8:2 to 1:1) to obtain the title trans diastereoisomer (14.4 g, 39 mmol, 35% yield over two steps). LC-MS (MH.sup.+)=370.2

Step 6Synthesis of Tert-butyl [trans-4-(7-cyano-2-oxoquinolin-1(2H)-yl)cyclohexyl]carbamate

(229) ##STR00377##

(230) tert-Butyl [trans-4-(7-cyano-2-oxo-3,4-dihydroquinolin-1(2H)-yl)cyclohexyl]carbamate (13.7 g 37.2 mmol) was dissolved in DCE (274 mL), N-bromosuccinimide (8.6 g, 48.4 mmol) and 2,2-azobis(2-methylpropionitrile) (917 mg, 5.6 mmol) were added and the mixture was stirred at 80 C. for 4 h. Water (100 mL) was added, the organic phase was separated, washed with brine (80 mL) and evaporated in vacuo to obtain the crude title product that was progressed without any further purification. LC-MS (MH.sup.+)=368.3

Step 7Synthesis of 1-(trans-4-aminocyclohexyl)-2-oxo-1,2-dihydroquinoline-7-carbonitrile

(231) ##STR00378##

(232) The title intermediate was prepared according to the procedure described for the synthesis of 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [trans-4-(7-cyano-2-oxoquinolin-1(2H)-yl)cyclohexyl]carbamate (quant. yield). LC-MS (MH.sup.+)=268.2

Step 8Synthesis of 1-(trans-4-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}cyclohexyl)-2-oxo-1,2-dihydroquinoline-7-carbonitrile (Hydrochloride, Compound 206)

(233) ##STR00379##

(234) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-(trans-4-aminocyclohexyl)-2-oxo-1,2-dihydroquinoline-7-carbonitrile (12% yield). LC-MS (MH.sup.+)=483.3. .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 1.66-1.90 (m, 4H), 2.19-2.40 (m, 2H), 2.61-2.82 (m, 2H), 3.08-3.42 (m, 1H), 4.40 (br. s., 2H), 4.64 (br. s., 1H), 6.70 (d, J=8.51 Hz, 1H), 6.96 (br. s., 1H), 7.37 (td, J=8.71, 2.88 Hz, 1H), 7.53 (dd, J=9.13, 4.60 Hz, 1H), 7.66 (d, J=8.10 Hz, 1H), 7.85-7.93 (m, 2H), 7.96 (d, J=9.61 Hz, 1H), 8.40 (br. s., 1H), 9.44 (br. s., 1H), 13.53 (br. s., 1H).

(235) Preparation of Compound 207

(236) Compound 207 was prepared as described herein below.

Step 1Synthesis of Tert-butyl {4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate

(237) ##STR00380##

(238) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (see compound 193, step 6) using 2-bromo-5-fluoropyridin-3-amine (98% yield). LC-MS (MH.sup.+)=388.2

Step 2Synthesis of ethyl (2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate

(239) ##STR00381##

(240) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl 4-({3-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]-6-methoxypyrazin-2-yl}amino)piperidine-1-carboxylate (see compound 200, step 3) using tert-butyl {4-[(2-bromo-5-fluoropyridin-3-yl)amino]cyclohexyl}carbamate (66% yield). LC-MS (MH.sup.+)=408.5

Step 3Synthesis of Ethyl 3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]propanoate

(241) ##STR00382##

(242) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate ethyl 3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoate (see compound 206, step 3) using ethyl (2E)-3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]prop-2-enoate (97% yield). LC-MS (MH.sup.+)=410.5

Step 4Synthesis of Tert-butyl [trans-4-(7-fluoro-2-oxo-3,4-dihydro-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate

(243) ##STR00383##

(244) 3-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]propanoate (1.8 g, 4.5 mmol) was dissolved in THF (20 mL), t-BuONa (0.87 g, 9 mmol) was added and the mixture was stirred at 50 C. for 4 h. Ethyl acetate was added followed by water. The organic phase was separated, dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The crude was purified by Si-column (cy to cy/ethyl acetate 1:1) to obtain 180 mg of the title trans diastereoisomer (0.18 g, 0.5 mmol, 11% yield). LC-MS (MH.sup.+)=364.4

Step 5Synthesis of Tert-butyl [trans-4-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate

(245) ##STR00384##

(246) The title compound was prepared following the procedure described for the synthesis of intermediate tert-butyl [trans-4-(7-cyano-2-oxoquinolin-1(2H)-yl)cyclohexyl]carbamate (see compound 206, step 6) using tert-butyl [trans-4-(7-fluoro-2-oxo-3,4-dihydro-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate (96% yield). LC-MS (MH.sup.+)=362.2

Step 6Synthesis of 1-(trans-4-aminocyclohexyl)-7-fluoro-1,5-naphthyridin-2(1H)-one

(247) ##STR00385##

(248) The title compound was obtained following the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [trans-4-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]carbamate (98% yield). LC-MS (MH.sup.+)=262.2

Step 7Synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 207)

(249) ##STR00386##

(250) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-(trans-4-aminocyclohexyl)-7-fluoro-1,5-naphthyridin-2(1H)-one (18% yield). LC-MS (MH.sup.+)=477.2; .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.28-1.46 (m, 2H), 1.56-1.70 (m, 2H), 1.93-2.12 (m, 2H), 2.57 (br. s., 2H), 3.90 (s, 2H), 4.45 (br. s., 1H), 6.59 (s, 1H), 6.72 (d, J=9.29 Hz, 1H), 7.28 (td, J=8.80, 2.93 Hz, 1H), 7.46 (dd, J=9.05, 4.65 Hz, 1H), 7.86 (d, J=9.29 Hz, 1H), 7.94 (dd, J=8.80, 2.93 Hz, 1H), 8.18 (s, 1H), 8.28 (d, J=9.78 Hz, 1H), 8.52 (d, J=1.96 Hz, 1H), 12.62 (br. s., 1H).

(251) Preparation of Compound 208

(252) Compound 208 was prepared as described herein below.

Step 1Synthesis of Tert-butyl [trans-4-(5-methoxy-2-nitroanilino)cyclohexyl]carbamate

(253) ##STR00387##

(254) A suspension of 2-chloro-4-methoxy-1-nitrobenzene (2.0 g, 10.7 mmol), N-Boc-trans-1,4-cyclohexanediamine (2.3 g, 10.7 mmol) and triethylamine (1.8 mL, 12.8 mmol) in acetonitrile (80 mL) was heated at 130 C. in a sealed tube for 3 days. The mixture was cooled to room temperature, the volatiles were removed under vacuum and the crude was purified by column chromatography (from cyclohexane/EtOAc 85:15 to cyclohexane/EtOAc/MeOH 6:3:1) to afford the title compound (1.2 g, 3.3 mmol, 31% yield). LC-MS (MH.sup.+)=366.4

Step 2Synthesis of Tert-butyl [trans-4-(2-amino-5-methoxyanilino)cyclohexyl]carbamate

(255) ##STR00388##

(256) The title compound was prepared according to the procedure described for the synthesis of tert-butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (see compound 197, step 2) using tert-butyl [trans-4-(5-methoxy-2-nitroanilino)cyclohexyl]carbamate and MeOH as solvent (94% yield). LC-MS (MH.sup.+)=336.4

Step 3Synthesis of Ethyl N-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-methoxyphenyl]glycinate

(257) ##STR00389##

(258) The title compound was prepared according to the procedure described for the synthesis of tert-butyl 4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate (see compound 197, step 3) using tert-butyl [trans-4-(2-amino-5-methoxyanilino)cyclohexyl]carbamate (63% yield). LC-MS (MH.sup.+)=422.5

Step 4Synthesis of Tert-butyl [trans-4-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]carbamate

(259) ##STR00390##

(260) The title compound was prepared according to the procedure described for the synthesis of tert-butyl 4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate (see compound 197, step 4) using ethyl N-[2-({trans-4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-methoxyphenyl]glycinate (59% yield). LC-MS (MH.sup.+)=374.4

Step 5Synthesis of 1-(trans-4-aminocyclohexyl)-7-methoxyquinoxalin-2(1H)-one

(261) ##STR00391##

(262) The title compound was prepared according to the procedure described for the synthesis of 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [trans-4-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]carbamate (96% yield). LC-MS (MH.sup.+)=274.3

Step 6Synthesis of 8-fluoro-2-({[trans-4-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 208)

(263) ##STR00392##

(264) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-(trans-4-aminocyclohexyl)-7-methoxyquinoxalin-2(1H)-one (20% yield). LC-MS (MH.sup.+)=489.2. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.31-1.50 (m, 2H), 1.69 (d, J=10.63 Hz, 2H), 2.07 (d, J=10.85 Hz, 2H), 2.52-2.70 (m, 3H), 3.91 (s, 3H), 3.94 (s, 2H), 4.58 (br. s., 1H), 6.62 (s, 1H), 7.01 (dd, J=8.82, 2.36 Hz, 1H), 7.20 (br. s., 1H), 7.30 (td, J=8.74, 3.01 Hz, 1H), 7.47 (dd, J=9.10, 4.60 Hz, 1H), 7.74 (d, J=8.88 Hz, 1H), 7.88-8.00 (m, 2H), 8.18 (s, 1H), 12.72 (br. s., 1H).

(265) Preparation of Compounds 209 and 210

(266) Compounds 209 and 210 were prepared as described herein below.

Step 1Synthesis of Ethyl N-(5-fluoro-3-nitropyridin-2-yl)glycinate

(267) ##STR00393##

(268) The synthesis was performed according to the procedure described for the preparation of intermediate tert-butyl 4-({3-[(2-ethoxy-2-oxoethyl)amino]-6-methoxypyridin-2-yl}amino)piperidine-1-carboxylate (see compound 197, step 3) using N-(5-fluoro-3-nitropyridin-2-yl)glycinate (95% yield). LC-MS (MH.sup.+)=244.2

Step 2Synthesis of Tert-butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate

(269) ##STR00394##

(270) The synthesis was performed according to the procedure described for the preparation of intermediate tert-butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (see compound 197, step 2) using 5-fluoro-3-nitropyridin-2-amine (47% yield). LC-MS (MH.sup.+)=214.0

Step 3Synthesis of Ethyl N-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]glycinate

(271) ##STR00395##

(272) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (see compound 193, step 6) using tert-butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (69% yield). LC-MS (MH.sup.+)=411.4

Step 4Synthesis of Tert-butyl [4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate

(273) ##STR00396##

(274) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl 4-(6-methoxy-3-oxo-2,3-dihydropyrido[2,3-b]pyrazin-4(1H)-yl)piperidine-1-carboxylate (see compound 197, step 4) using ethyl N-[3-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-5-fluoropyridin-2-yl]glycinate (43% yield). LC-MS (MH.sup.+)=363.4

Step 5Synthesis of 1-(4-aminocyclohexyl)-7-fluoropyrido[2,3-b]pyrazin-2(1H)-one

(275) ##STR00397##

(276) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate (94% yield). LC-MS (MH.sup.+)=263.1

Step 6Synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 209) and 8-fluoro-2-({[cis-4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 210)

(277) ##STR00398##

(278) The title compounds were prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-(4-aminocyclohexyl)-7-fluoropyrido[2,3-b]pyrazin-2(1H)-one as a mixture of isomers.

(279) A purification by preparative HPLC (A=0.1% ammonia aqueous solution, B=acetonitrile, form 98:2 A:B to 100% B) afforded the trans (10% yield) and cis (6% yield) diastereoisomers.

(280) Compound 209: LC-MS (MH.sup.+)=478.4. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.41 (m, J=11.80 Hz, 2H), 1.70 (d, J=11.29 Hz, 2H), 2.06 (d, J=10.29 Hz, 2H), 2.51-2.58 (m, 2H), 2.67 (br. s., 1H), 3.96 (s, 2H), 4.47-4.79 (m, 1H), 6.63 (s, 1H), 7.30 (td, J=8.78, 3.01 Hz, 1H), 7.48 (dd, J=9.03, 4.52 Hz, 1H), 7.95 (dd, J=9.03, 3.01 Hz, 1H), 8.21 (s, 1H), 8.29 (s, 1H), 8.45 (d, J=11.04 Hz, 1H), 8.58 (d, J=2.26 Hz, 1H).

(281) Compound 210: LC-MS (MH.sup.+)=478.4. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.50 (d, J=9.39 Hz, 2H), 1.67 (t, J=12.91 Hz, 2H), 1.95 (d, J=14.09 Hz, 2H), 2.69-2.80 (m, 2H), 2.92 (br. s., 1H), 3.90 (s, 2H), 4.92 (br. s., 1H), 6.65 (s, 1H), 7.21-7.31 (m, 1H), 7.45 (dd, J=9.00, 4.70 Hz, 1H), 7.87 (dd, J=9.00, 3.13 Hz, 1H), 8.19 (s, 1H), 8.33 (s, 1H), 8.47 (d, J=10.56 Hz, 1H), 8.54 (d, J=2.35 Hz, 1H).

(282) Preparation of Compound 211

(283) ##STR00399##

(284) Compound 8-fluoro-2-({[trans-4-(7-fluoro-2-oxoquinoxalin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 211) was prepared according to the procedure described for the synthesis of compound 208, starting from 2,4-difluoro-1-nitrobenzene.

(285) LC-MS (MH.sup.+)=477.4. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.32-1.50 (m, 2H), 1.68 (d, J=10.56 Hz, 2H), 2.05 (d, J=11.35 Hz, 2H), 2.37-2.64 (m, 3H), 3.93 (s, 2H), 4.56 (br. s., 1H), 6.62 (s, 1H), 7.24 (td, J=8.41, 2.35 Hz, 1H), 7.29 (td, J=8.71, 2.93 Hz, 1H), 7.47 (dd, J=9.00, 4.70 Hz, 1H), 7.86 (dd, J=8.80, 6.46 Hz, 2H), 7.95 (dd, J=9.19, 2.93 Hz, 1H), 8.06 (s, 1H), 8.23 (s, 1H), 10.48-13.49 (m, 2H).

(286) Preparation of Compounds 212 and 213

(287) Compounds 212 and 213 were prepared as described herein below.

Step 1Synthesis of Tert-butyl [4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate

(288) ##STR00400##

(289) 200 mg (0.55 mmol) of tert-butyl [4-(7-fluoro-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate, prepared as previously described (step 4 in the synthesis of compounds 209 and 210, mixture of isomers), was dissolved in MeOH (10 mL). NaOMe (25% wt solution in MeOH, 25 mL) was added and the mixture was stirred at 50 C. for 3 h. The solvent was evaporated in vacuo, DCM was added and the solution was washed with sat. NaHCO.sub.3. The organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to obtain 210 mg (0.55 mmol, quant. yield) of the title product, that was progressed without any further purification. LC-MS (MH.sup.+)=375.4

Step 2Synthesis of 1-(4-aminocyclohexyl)-7-methoxypyrido[2,3-b]pyrazin-2(1H)-one

(290) ##STR00401##

(291) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]carbamate (82% yield). LC-MS (MH.sup.+)=275.2

Step 3Synthesis of 8-fluoro-2-({[trans-4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 212) and 8-fluoro-2-({[cis-4-(7-methoxy-2-oxopyrido[2,3-b]pyrazin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 213)

(292) ##STR00402##

(293) The title compounds were prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-(4-aminocyclohexyl)-7-methoxypyrido[2,3-b]pyrazin-2(1H)-one as a mixture of the isomers.

(294) A purification by preparative HPLC (A=0.1% ammonia aqueous solution, B=acetonitrile, form 98:2 A:B to 100% B) followed by the formation of the hydrochloride salt under standard conditions afforded the trans (43% yield) and cis (10% yield) diastereoisomers.

(295) Compound 212: LC-MS (MH.sup.+)=490.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.69-1.79 (m, 2H), 1.84 (d, J=10.98 Hz, 2H), 2.25-2.34 (m, 2H), 2.56-2.71 (m, 2H), 3.17-3.35 (m, 1H), 4.03 (s, 3H), 4.37-4.45 (m, 2H), 4.67 (br. s., 1H), 6.96 (d, J=1.65 Hz, 1H), 7.38 (td, J=8.78, 3.02 Hz, 1H), 7.53 (dd, J=9.19, 4.53 Hz, 1H), 7.69-7.81 (m, 1H), 7.88 (dd, J=8.65, 2.88 Hz, 1H), 8.16 (s, 1H), 8.35 (d, J=2.47 Hz, 1H), 9.42 (br. s., 2H), 13.54 (br. s., 1H).

(296) Compound 213: LC-MS (MH.sup.+)=490.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.69 (d, J=10.96 Hz, 2H), 2.01 (m, J=13.59 Hz, 2H), 2.18 (d, J=14.03 Hz, 2H), 2.66-2.80 (m, 2H), 3.49 (br. s., 1H), 4.03 (s, 3H), 4.40-4.45 (m, 2H), 4.62-4.78 (m, 1H), 6.99 (d, J=1.75 Hz, 1H), 7.38 (td, J=8.77, 3.07 Hz, 1H), 7.53 (dd, J=9.21, 4.38 Hz, 1H), 7.76 (d, J=2.19 Hz, 1H), 7.87 (dd, J=8.33, 3.07 Hz, 1H), 8.18 (s, 1H), 8.36 (d, J=2.19 Hz, 1H), 9.25 (br. s., 2H), 13.49 (s, 1H).

(297) Preparation of Compounds 214 and 215

(298) Compounds 214 and 215 were prepared as described herein below.

Step 1Synthesis of (aminooxy)(6-hydroxy-5-nitropyridin-3-yl)methanone

(299) ##STR00403##

(300) 6-hydroxy-5-nitropyridine-3-carboxylic acid (3.3 g, 17.9 mmol) was dissolved in DMF (16 mL). CDI (3.2 g, 19.7 mmol) was added portionwise at room temperature. The resulting mixture was stirred for 2 hours at 60 C. then was cooled to room temperature. 30% aqueous ammonia (23 mL) was added and the resulting mixture was stirred for 20 min. The precipitate was filtered, washed with water (5 mL) and dried to give the title product (2.85 g, 15.5 mmol, 86% yield), that was progressed without any further purification. LC-MS (MH.sup.+)=184.1

Step 2Synthesis of 6-hydroxy-5-nitropyridine-3-carbonitrile

(301) ##STR00404##

(302) Phosphorus(V) oxychloride (0.66 mL, 7.1 mmol) was added to DMF (11 mL) at 0 C. and the resulting mixture was stirred at the same temperature for 15 min. Intermediate (aminooxy)(6-hydroxy-5-nitropyridin-3-yl)methanone (1.1 g, 6.0 mmol) was added portionwise. The reaction mixture was heated at 50 C. for 1 hour then was cooled to 0 C. Water (12 mL) was added, the precipitate was collected by filtration and washed with additional water (6 mL). Acetonitrile was added to the solid and evaporated under vacuum several times to remove residual water. The obtained title compound (0.74 g, 4.5 mmol, 75% yield) was progressed without any further purification. LC-MS (MH.sup.+)=166.1

Step 3Synthesis of 5-amino-6-hydroxypyridine-3-carbonitrile

(303) ##STR00405##

(304) The title compound was prepared according to the procedure described for the synthesis of tert-butyl 4-[(3-amino-6-methoxypyridin-2-yl)amino]piperidine-1-carboxylate (see compound 197, step 2) using 6-hydroxy-5-nitropyridine-3-carbonitrile and MeOH/DMF as solvents (96% yield). LC-MS (MH.sup.+)=136.1

Step 4Synthesis of Tert-butyl {4-[(5-cyano-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

(305) ##STR00406##

(306) The title compound was obtained as a mixture of isomers following the procedure described for the preparation of intermediate tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (see compound 193, step 6) using 5-amino-6-hydroxypyridine-3-carbonitrile (15% yield). LC-MS (MH.sup.+)=333.3

Step 5Synthesis of Tert-butyl [4-(7-cyano-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

(307) ##STR00407##

(308) DIPEA (4.0 mL, 23.4 mmol) and chloroacetyl chloride (934 L, 11.7 mmol) were added to a stirred solution of tert-butyl {4-[(5-cyano-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (390 mg, 1.17 mmol) in DMF (24 mL). The reaction mixture was heated at 150 C. in a sealed flask for 1 hour then was cooled and partitioned between EtOAc and a 1/1 mixture of brine and sat. NaHCO.sub.3. The aqueous layer was extracted three times with EtOAc and the combined organic layers were washed with brine. The volatiles were removed under vacuum and the residue was purified by column chromatography on silica gel (from cyclohexane/EtOAc 85:15 to cyclohexane/EtOAc/MeOH 6:3:1). A further purification through column chromatography on NHSiO.sub.2 (cyclohexane/EtOAc from 6:4 to 0:100) afforded the title product as a mixture of isomers (208 mg, 1 mmol, 48% yield). LC-MS (MH.sup.+)=373.3

Step 6Synthesis of 1-(4-aminocyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrile

(309) ##STR00408##

(310) The title intermediate was prepared as a mixture of isomers according to the procedure described for the synthesis of 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [4-(7-cyano-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate (98% yield). LC-MS (MH.sup.+)=273.3

Step 7Synthesis of 1-(trans-4-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}cyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrile (formate salt, compound 214) and 1-(cis-4-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}cyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrile (Formate Salt, Compound 215)

(311) ##STR00409##

(312) The title compounds were prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-(4-aminocyclohexyl)-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-carbonitrile as mixture of isomers.

(313) A purification by preparative HPLC (A=0.1% ammonia aqueous solution, B=acetonitrile, form 98:2 A:B to 100% B) followed by the formation of the formate salts under standard conditions afforded the trans (21% yield) and cis (15% yield) diastereoisomers.

(314) Compound 214: LC-MS (MH.sup.+)=488.2. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.35 (q, J=11.74 Hz, 2H), 1.75 (d, J=10.96 Hz, 2H), 2.06 (d, J=11.35 Hz, 2H), 2.25-2.40 (m, 2H), 2.61-2.71 (m, 1H), 3.99 (s, 2H), 4.04-4.14 (m, 1H), 4.83 (s, 2H), 6.65 (s, 1H), 7.30 (td, J=8.71, 2.93 Hz, 1H), 7.47 (dd, J=9.00, 4.70 Hz, 1H), 7.95 (dd, J=9.00, 3.13 Hz, 1H), 8.21 (s, 1H), 8.31 (d, J=1.56 Hz, 1H), 8.35 (d, J=1.96 Hz, 1H), 12.35-13.58 (m, 1H).

(315) Compound 215: LC-MS (MH.sup.+)=488.2. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.46 (d, J=10.27 Hz, 2H), 1.57 (t, J=13.69 Hz, 2H), 1.86 (d, J=13.69 Hz, 2H), 2.51-2.61 (m, 2H), 2.81 (br. s., 1H), 3.86 (s, 2H), 4.29 (t, J=11.98 Hz, 1H), 4.87 (s, 2H), 6.61 (s, 1H), 7.28 (td, J=8.80, 2.93 Hz, 1H), 7.46 (dd, J=9.05, 4.65 Hz, 1H), 7.87 (dd, J=9.05, 3.18 Hz, 1H), 8.17 (s, 1H), 8.30 (d, J=1.96 Hz, 1H), 8.35 (d, J=1.96 Hz, 1H), 12.58 (br. s., 1H).

(316) Preparation of Compound 216

(317) ##STR00410##

(318) Compound 8-fluoro-2-({[trans-4-(7-fluoro-2-oxoquinolin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 216) was prepared according to the procedure described for the synthesis of compound 206, starting from 5-fluoro-2-iodoaniline.

(319) LC-MS (MH.sup.+)=476.1. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.43-1.79 (m, 4H), 2.18 (br. s., 2H), 2.57-2.77 (m, 2H), 2.80-3.13 (m, 1H), 4.19 (br. s., 2H), 4.32-5.63 (m, 1H), 6.47 (d, J=5.87 Hz, 1H), 6.81 (br. s., 1H), 7.13 (td, J=8.44, 2.20 Hz, 1H), 7.34 (td, J=8.56, 2.93 Hz, 1H), 7.51 (dd, J=9.05, 4.65 Hz, 1H), 7.66-7.81 (m, 2H), 7.86 (d, J=9.29 Hz, 1H), 7.91 (dd, J=8.80, 2.93 Hz, 1H), 8.32-10.31 (m, 1H), 13.13 (br. s., 1H).

(320) Preparation of Compound 217

(321) Compound 217 was prepared as described herein below.

Step 1Synthesis of Diethyl 1-[(diphenylmethylidene)amino]-4-oxocyclohexane-1,3-dicarboxylate

(322) ##STR00411##

(323) Ethyl N-(diphenylmethylidene)glycinate (10 g, 37 mmol) was dissolved in THF (100 mL) and the solution was cooled at 0 C. under a N.sub.2 atmosphere. t-BuOK (20.9 g, 187 mmol) was added and the mixture was stirred at 0 C. for 20 min. Then ethyl acrylate (20.3 mL, 187 mmol) was added dropwise and the reaction mixture was stirred at 0 C. for 3 h. A saturated solution of NH.sub.4Cl was added, the organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue containing the title intermediate was progressed into the next step without further purification and characterization.

Step 2Synthesis of Diethyl 1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1,3-dicarboxylate

(324) ##STR00412##

(325) The residue from step 1 was dissolved in THF (80 mL) and treated with HCl (2 M solution, 50 mL). Ethyl acetate was added, the organic layer was discarded and the aqueous phase was basified with K.sub.2CO.sub.3 (pH 8). THF was added followed by di-tert-butyl dicarbonate (1 eq.) and the resulting mixture was stirred at room temperature overnight. Ethyl acetate was added, the organic phase was separated, dried over Na.sub.2SO.sub.4 and evaporated in vacuo. The crude material was purified by Si-column (cy to cy/EtOAc 1:1) to obtain 2.8 g (7.8 mmol, 21% yield over two steps) of the title product. LC-MS (MH.sup.+)=358.4

Step 3Synthesis of Ethyl 1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1-carboxylate

(326) ##STR00413##

(327) Diethyl 1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1,3-dicarboxylate (300 mg, 0.84 mmol) was dissolved in DMSO (1 mL), sodium chloride (200 mg, 3.4 mmol) and water (0.05 mL) were added and the reaction mixture was stirred at 150 C. for 4 h. The mixture was cooled and diluted with ethyl acetate. The organic layer was washed with water, dried over MgSO.sub.4 and concentrated under reduced pressure. The residue was purified by Si-column (cy to cy/EtOAc 7:3) to obtain 185 mg (0.64 mmol, 76% yield) of the title product. LC-MS (MH.sup.+)=286.3

Step 4Synthesis of Ethyl Trans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylate

(328) ##STR00414##

(329) The synthesis was performed according to the procedure described for the preparation of intermediate tert-butyl {4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (see compound 193, step 6) using ethyl 1-[(tert-butoxycarbonyl)amino]-4-oxocyclohexane-1-carboxylate. A purification by C-18 chromatography (from water+0.1% formic acid to water+0.1% formic acid/acetonitrile+0.1% formic acid 8:2) provided the title compound (25% yield). LC-MS (MH.sup.+)=398.4

Step 5Synthesis of Trans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylic Acid

(330) ##STR00415##

(331) The synthesis was performed according to the procedure described for the preparation of intermediate 3-[2-({4-[(tert-butoxycarbonyl)amino]cyclohexyl}amino)-4-cyanophenyl]propanoic acid (see compound 206, step 4) using ethyl trans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylate (98% yield). LC-MS (MH.sup.)=368.4

Step 6Synthesis of Tert-butyl {trans-1-carbamoyl-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

(332) ##STR00416##

(333) Intermediate trans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylic acid (340 mg, 0.9 mmol) was dissolved in DMF (20 mL). DIPEA (0.8 mL, 4.6 mmol) and ammonium chloride (199 mg, 3.7 mmol) were added and the mixture was cooled to 0 C. HATU (426 mg, 1.1 mmol) was added at the same temperature and the mixture was allowed to slowly reach room temperature. After 6 hours water was added, the mixture was extracted with ethyl acetate, the organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The resulting crude material was purified by Si-column (from DCM to DCM/MeOH 8:2) to obtain the title compound (233 mg, 0.63 mmol, 68% yield). LC-MS (MH.sup.)=369.3

Step 7Synthesis of Tert-butyl [trans-1-carbamoyl-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

(334) ##STR00417##

(335) The synthesis was performed according to the procedure described for the preparation of intermediate tert-butyl [4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate (see compound 193, step 7) using tert-butyl {trans-1-carbamoyl-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (47% yield). LC-MS (MH.sup.+)=409.2

Step 8Synthesis of Trans-1-amino-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexane-1-carboxamide

(336) ##STR00418##

(337) The synthesis was performed according to the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using tert-butyl [trans-1-carbamoyl-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate (62% yield). LC-MS (MH.sup.+)=309.2

Step 9Synthesis of Trans-1-{[(8-fluoro-4-oxo-1,4-dihydro[1]benzopyrano[4,3-b]pyrrol-2-yl)methyl]amino}-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexane-1-carboxamide (Formate Salt, Compound 217)

(338) ##STR00419##

(339) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using trans-1-amino-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexane-1-carboxamide (10% yield). LC-MS (MH.sup.+)=524.3. .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 1.53 (td, J=13.21, 3.91 Hz, 2H), 1.57-1.66 (m, 2H), 2.24 (d, J=12.23 Hz, 2H), 2.41-2.56 (m, 2H), 3.72 (s, 2H), 4.27-4.54 (m, 1H), 4.74 (s, 2H), 6.58 (s, 1H), 7.27-7.33 (m, 2H), 7.48 (dd, J=9.05, 4.65 Hz, 1H), 7.53 (s, 1H), 7.82 (dd, J=10.03, 2.69 Hz, 1H), 7.86 (d, J=2.45 Hz, 1H), 7.94 (dd, J=9.05, 3.18 Hz, 1H), 8.18 (s, 1H), 12.40 (br. s., 1H).

(340) Preparation of Compound 219

(341) ##STR00420##

(342) Compound 8-fluoro-2-({[trans-4-(7-methoxy-2-oxoquinolin-1(2H)-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 219) was prepared according to the procedure described for the synthesis of compound 206, starting from 2-bromo-5-methoxyaniline.

(343) LC-MS (MH.sup.+)=488.3. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.32-1.48 (m, 2H), 1.64 (d, J=10.74 Hz, 2H), 2.09 (d, J=8.11 Hz, 2H), 2.53-2.76 (m, 3H), 3.87 (s, 3H), 3.95 (s, 2H), 4.38 (br. s., 1H), 6.32 (d, J=7.78 Hz, 1H), 6.62 (s, 1H), 6.90 (dd, J=8.61, 2.03 Hz, 1H), 7.11 (d, J=1.75 Hz, 1H), 7.30 (td, J=8.74, 3.01 Hz, 1H), 7.48 (dd, J=9.10, 4.71 Hz, 1H), 7.62 (d, J=8.66 Hz, 1H), 7.74 (d, J=9.32 Hz, 1H), 7.96 (dd, J=9.10, 2.96 Hz, 1H), 8.18 (s, 1H), 12.83 (br. s., 1H).

(344) Preparation of Compound 220

(345) Compound 220 was prepared as described herein below.

Step 1Synthesis of Tert-butyl {trans-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]-1-(hydroxymethyl)cyclohexyl}carbamate

(346) ##STR00421##

(347) 1.0 g (2.5 mmol) of intermediate ethyl trans-1-[(tert-butoxycarbonyl)amino]-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexane-1-carboxylate, prepared as described in step 4 of the synthesis of compound 217, was dissolved in THF (20 mL). The solution was cooled to 10 C. then LiAlH.sub.4 (1 M in THF, 2.5 mL, 2.5 mmol) was added dropwise. After stirring for 2 h the reaction was quenched by adding Na.sub.2SO.sub.4.10H.sub.2O. The mixture was filtered and concentrated in vacuo. The crude material was dissolved in ethyl acetate and washed with sat. NH.sub.4Cl. The organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to obtain the title product (614 mg, 1.8 mmol, 69% yield). LC-MS (MH.sup.+)=356.2

Step 2Synthesis of Tert-butyl {trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate

(348) ##STR00422##

(349) To a solution of tert-butyl {trans-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]-1-(hydroxymethyl)cyclohexyl}carbamate (344 mg, 0.96 mmol) in DMF (5 mL), imidazole (165 mg, 2.14 mmol) was added followed by TBDMSCl (581 mg, 3.87 mmol). The mixture was stirred at room temperature overnight then ethyl acetate was added followed by water. The organic phase was separated, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude material was purified by Si-column (from cy to cy/ethyl acetate 1:1) to obtain the title compound (300 mg, 0.64 mmol, 67% yield). LC-MS (MH.sup.+)=470.4

Step 3Synthesis of Tert-butyl [trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

(350) ##STR00423##

(351) To a suspension of tert-butyl {trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-[(5-fluoro-2-hydroxypyridin-3-yl)amino]cyclohexyl}carbamate (253 mg, 0.54 mmol) and potassium carbonate (224 mg, 1.62 mmol) in DMF (5.2 mL) chloroacetyl chloride (52 L, 0.65 mmol) was added. The mixture was stirred at 80 C. for 6 h then was cooled to 0 C. Sat. NaHCO.sub.3 was added followed by ethyl acetate. The organic phase was separated, dried over Na.sub.2SO.sub.4 and the solvents were evaporated in vacuo to afford the title intermediate (220 mg, 0.43 mmol, 80% yield), that was progressed without any further purification. LC-MS (MH.sup.+)=510.4

Step 4Synthesis of 1-[trans-4-amino-4-(hydroxymethyl)cyclohexyl]-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

(352) ##STR00424##

(353) To a stirred solution of tert-butyl [trans-1-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate (220 mg, 0.43 mmol) in MeOH (1.7 mL) HCl (4 M in dioxane, 2.74 mL) was added. The mixture was stirred at room temperature for 4 h then was concentrated under reduced pressure. The residue was purified by SCX column to give the title compound (98 mg, 0.33 mmol, 77% yield). LC-MS (MH.sup.+)=296.1

Step 5Synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)-1-(hydroxymethyl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Hydrochloride, Compound 220)

(354) ##STR00425##

(355) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 1-[trans-4-amino-4-(hydroxymethyl)cyclohexyl]-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (30% yield). LC-MS (MH.sup.+)=511.2. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.66-1.96 (m, 4H), 2.02-2.19 (m, 2H), 2.46 (br. s., 2H), 3.93 (s, 2H), 4.00 (br. s., 1H), 4.36 (br. s., 2H), 4.73 (s, 2H), 6.92 (d, J=1.97 Hz, 1H), 7.37 (td, J=8.77, 3.07 Hz, 1H), 7.52 (dd, J=9.21, 4.60 Hz, 1H), 7.77-8.03 (m, 3H), 9.04 (br. s., 2H), 13.49 (br. s., 1H).

(356) Preparation of Compound 221

(357) Compound 221 was prepared as described herein below.

Step 1Synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

(358) ##STR00426##

(359) 1-(4-aminocyclohexyl)-7-fluoro-1H,2H,3H-pyrido[2,3-b][1,4]oxazin-2-one (133 mg, 0.5 mmol, see step 8 in the synthesis of compound 193) was suspended in dry dichloroethane (20 mL). 8-fluoro-4-oxo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H,4H-chromeno[4,3-b]pyrrole-2-carbaldehyde (200 mg, 0.55 mmol, see step 1 in the synthesis of compound 194) was added at room temperature followed by glacial acetic acid (catalytic, 2 drops). The reaction mixture was stirred at 50 C. for 4 hours then was cooled down to room temperature. Sodium triacetoxyborohydride (264 mg, 1.25 mmol) was added and the resulting suspension was stirred overnight at room temperature. The reaction mixture was partitioned between dichloromethane and a saturated aqueous NaHCO.sub.3 solution. The two layers were separated and the organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was purified by flash chromatography (Silica NH, 28 g cartridge, cyclohexane/ethyl acetate from 70:30 to 50:50) to afford the title compound (127 mg, 0.2 mmol, 41% yield) as an off-white solid. LC-MS (MH.sup.+)=611.3

Step 2Synthesis of 2-({(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-8-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one

(360) ##STR00427##

(361) To a solution of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (56 mg, 0.09 mmol) in dry dichloroethane (2 mL) 2-[(tert-butyldimethylsilyl)oxy]acetaldehyde (0.034 mL, 0.18 mmol) and glacial acetic acid (1 drop) were added. The reaction mixture was heated at 40 C. for 1 h then NaBH(OAc).sub.3 (38 mg, 0.18 mmol) was added and the resulting mixture was stirred overnight. The mixture was partitioned between dichloromethane and a saturated aq. solution of NaHCO.sub.3. The two layers were separated and the organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude material was eluted through a 1 g SCX cartridge with methanol and a 2 N solution of NH.sub.3 in methanol. Evaporation of the solvents afforded the title intermediate (50 mg, 0.065 mmol, 72% yield), which was progressed into the next step without any further purification. LC-MS (MH.sup.+)=769.6

Step 3Synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl](2-hydroxyethyl)amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Formate Salt, Compound 221)

(362) ##STR00428##

(363) The synthesis was performed according to the procedure described for the preparation of intermediate 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (see compound 193, step 8) using 2-({(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)-8-fluoro-1-{[2-(trimethylsilyl)ethoxy]methyl}[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (77% yield). LC-MS (MH.sup.+)=525.3. .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 1.58-2.45 (m, 8H), 2.83-2.91 (m, 2H), 3.52 (br. s., 2H), 3.88-4.17 (m, 4H), 4.64-4.72 (m, 2H), 6.68 (br. s., 1H), 7.26 (td, J=8.71, 2.93 Hz, 1H), 7.44 (dd, J=9.00, 4.70 Hz, 1H), 7.74 (dd, J=9.98, 2.54 Hz, 1H), 7.80 (d, J=2.74 Hz, 1H), 7.92 (dd, J=9.00, 3.13 Hz, 1H), 8.12 (s, 1H).

(364) Preparation of Compound 301

(365) Compound 301 was prepared as described herein below.

Step 1Synthesis of Ethyl 3-(3-hydroxypyridin-2-yl)-3-oxopropanoate

(366) ##STR00429##

(367) 1-(3-Hydroxypyridin-2-yl)ethan-1-one (2.50 g, 18.2 mmol) in a mixture of diethyl carbonate (20 mL) and toluene (20 mL) was treated portionwise with NaH (60%, 3.3 g, 82 mmol) and stirred at RT for 1 h. A further portion of toluene (10 mL) was added and stirring continued for a further 5 h. The reaction mixture was added to a mixture of sat. aq. NH.sub.4Cl and EtOAc. The aqueous phase was extracted with EtOAc and the combined organic fractions were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. The residue was purified by FCC (0-10% EtOAc in DCM) to afford the title compound (3.27 g, 86% yield) as a pale yellow oil. LC-MS (MH.sup.+)=210. .sup.1H NMR (400 MHz, CDCl.sub.3): 11.28 (s, 1H), 8.22 (dd, J=1.4, 4.2 Hz, 1H), 7.43 (dd, J=4.2, 8.5 Hz, 1H), 7.35 (dd, J=1.4, 8.5 Hz, 1H), 4.22 (s, 2H), 4.21 (q, J=7.1 Hz, 2H), 1.26 (t, J=7.1 Hz, 3H).

Step 2Synthesis of 4-hydroxy-2H-pyrano[3,2-b]pyridin-2-one

(368) ##STR00430##

(369) Ethyl 3-(3-hydroxypyridin-2-yl)-3-oxopropanoate (3.27 g, 15.6 mmol) in xylene (100 mL) was treated with NaH (60%, 63 mg, 1.6 mmol). The reaction mixture was heated to reflux and some solvent (15 mL) was distilled off. Heating under reflux was continued for 7 h. After cooling to RT the solid was collected by filtration, washed with toluene and dried under vacuum to afford the title compound (2.16 g, 85% yield) as a brown solid. LC-MS (MH.sup.+)=164. .sup.1H NMR (400 MHz, DMSO-d6): 8.55 (dd, J=1.2, 4.4 Hz, 1H), 7.80 (dd, J=1.2, 8.4 Hz, 1H), 7.64 (dd, J=4.4, 8.4 Hz, 1H), 5.58 (s, 1H).

Step 3Synthesis of 4-chloro-2-oxo-2H-pyrano[3,2-b]pyridine-3-carbaldehyde

(370) ##STR00431##

(371) Phosphorus oxychloride (1.7 mL, 18 mmol) was added to an ice-cooled mixture of 4-hydroxy-2H-pyrano[3,2-b]pyridin-2-one (1.00 g, 6.13 mmol) and DMF (5 mL). The mixture was stirred at RT for 10 min then heated at 60 C. for 1.5 h. The reaction was cooled to RT, poured onto ice and extracted with DCM (3). The combined organic extracts were washed with water, then dried (Na.sub.2SO.sub.4) and evaporated to afford the title compound (1.69 g, quantitative yield) as a brown solid. LC-MS (M-Cl+H.sub.2O.sup.+)=192. .sup.1H NMR (400 MHz, CDCl.sub.3): 10.42 (s, 1H), 8.81 (dd, J=1.4, 4.3 Hz, 1H), 7.76 (dd, J=1.4, 8.5 Hz, 1H), 7.69 (dd, J=4.3, 8.5 Hz, 1H).

Step 4Synthesis of 4-oxo-1,4-dihydropyrrolo[2,3:4,5]pyrano[3,2-b]pyridine-2-carbaldehyde

(372) ##STR00432##

(373) 4-Chloro-2-oxo-2H-pyrano[3,2-b]pyridine-3-carbaldehyde (76%, 0.513 g, 1.86 mmol) was dissolved in ethanol (10 mL) and treated with aminoacetaldehyde dimethylacetal (0.215 g, 2.05 mmol) followed by Et.sub.3N (0.54 mL, 3.9 mmol). The reaction was stirred at 70 C. for 50 min. The reaction mixture was allowed to cool, toluene was added and then evaporated. The residue was partitioned between EtOAc and water. The aqueous phase was extracted with ethyl acetate. The combined organic extracts were washed with brine, dried (Na.sub.2SO.sub.4) and evaporated. The resulting solid was dissolved in AcOH (20 mL). Water (0.8 mL) was added and the reaction was stirred at 110 C. for 50 min. The reaction mixture was allowed to cool, toluene was added and evaporated (2). The residue was purified by FCC (EtOAc, then 10% MeOH in DCM) to afford the title compound (0.248 g, 62% yield) as a yellow solid. LC-MS (MH.sup.+)=215.

Step 5Synthesis of 2-(trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)pyrrolo[2,3:4,5]pyrano[3,2-b]pyridin-4(1H)-one (Compound 301)

(374) ##STR00433##

(375) The title compound was prepared according to the procedure described for the synthesis of 8-fluoro-2-({[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (compound 193, step 9) using 4-oxo-1,4-dihydropyrrolo[2,3:4,5]pyrano[3,2-b]pyridine-2-carbaldehyde (75% yield). LC-MS (MH.sup.+)=464.3. .sup.1H NMR (400 MHz, DMSO-d6): 8.54 (dd, J=1.3, 4.6 Hz, 1H), 7.88-7.83 (m, 3H), 7.50 (dd, J=4.6, 8.4 Hz, 1H), 6.63 (s, 1H), 4.68 (s, 2H), 4.06-3.99 (m, 1H), 3.85 (s, 2H), 2.48-2.41 (m, 1H), 2.34-2.24 (m, 2H), 1.99-1.93 (m, 2H), 1.72-1.67 (m, 2H), 1.30-1.20 (m, 2H).

(376) Preparation of Compound 302

(377) Compound 302 was prepared as described herein below.

Step 1Synthesis of 2-chloro-5-methoxypyridin-3-ol

(378) ##STR00434##

(379) 2-Chloro-5-methoxypyridin-3-yl boronic acid (1.0 g, 5.35 mmol) was suspended in methanol (20 mL) and cooled to 0 C. To the resulting suspension was added dropwise 50% aq. hydrogen peroxide (2.5 mL) and the reaction mixture was stirred for 5 hours at room temperature. The reaction was cooled to 0 C. and treated with saturated aqueous sodium thiosulphate (5 mL), followed by water (50 mL) and extracted with dichloromethane. The combined organic extracts were evaporated to dryness. The resulting residue was purified by flash column chromatography, eluting with 0 to 100% ethyl acetate/iso-hexane, to afford the title compound (506 mg, 60% yield). .sup.1H NMR (400 MHz, DMSO): 10.75 (s, 1H), 7.67 (d, J=2.8 Hz, 1H), 6.97 (d, J=2.8 Hz, 1H), 3.84 (s, 3H).

Step 2Synthesis of Tert-butyl [trans-4-(2-chloroacetamido)cyclo hexyl]carbamate

(380) ##STR00435##

(381) tert-Butyl (trans-4-aminocyclohexyl)carbamate (10.7 g, 50 mmol) was dissolved in DCM (200 mL), cooled to 0 C. and treated with triethylamine (6.1 g, 60.4 mmol). Chloroacetyl chloride (5.65 g, 50 mmol) was then added dropwise over 10-15 minutes and the reaction was left to stir overnight at room temperature. The reaction was quenched with water (100 mL), the phases separated and the combined DCM fractions dried over MgSO.sub.4, filtered and evaporated. The resulting residue was washed with iso-hexane, filtered and dried in vacuum to give the title compound (11.12 g, 76% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): 6.37 (d, J=7.3 Hz, 1H), 4.40-4.39 (m, 1H), 4.02 (s, 2H), 3.79-3.69 (m, 1H), 3.50-3.43 (m, 1H), 2.08-1.98 (m, 4H), 1.45 (s, 9H), 1.42-1.20 (m, 4H).

Step 3Synthesis of Tert-butyl [trans-4-(7-methoxy-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate

(382) ##STR00436##

(383) 2-Chloro-5-methoxypyridin-3-ol (506 mg, 3.18 mmol) was dissolved in DMF (20 mL) and tert-butyl [trans-4-(2-chloroacetamido)cyclohexyl]carbamate (926 mg, 1 eq) and Cs.sub.2CO.sub.3 (2.5 g, 2.4 eq) added. The reaction mixture was heated at 120 C. for 18 h. After cooling to room temperature, the mixture was filtered and evaporated to dryness. The residue was purified by flash column chromatography eluting with 0 to 100% ethyl acetate/iso-hexane to afford the title compound (756 mg, 63% yield). LC-MS (MH.sup.+)=378. .sup.1H NMR (400 MHz, DMSO): 7.63 (d, J=2.5 Hz, 1H), 7.39 (d, J=2.5 Hz, 1H), 6.85 (d, J=7.1 Hz, 1H), 4.68 (s, 2H), 4.16-4.06 (m, 1H), 3.90 (s, 3H), 2.47-2.34 (m, 2H), 1.90 (d, J=11.1 Hz, 2H), 1.77 (d, J=10.1 Hz, 2H), 1.44 (s, 11H).

Step 4Synthesis of 1-[trans-4-aminocyclohexyl]-7-methoxy-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (Hydrochloride Salt)

(384) ##STR00437##

(385) tert-butyl [trans-4-(7-methoxy-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]carbamate (756 mg, 2.01 mmol) was dissolved in 1,4 dioxane (15 mL). A 4 N solution of HCl in dioxane (15 mL) was added and the reaction mixture stirred for 1 h at room temperature followed by addition of another 15 mL of a 4 N solution of HCl in dioxane. The reaction mixture was stirred for 1 h at room temperature, diluted with diethyl ether and filtered to yield a white solid. The precipitate was washed with ether and dried in vacuum to give the title compound (675 mg, 96% yield) as HCl salt. .sup.1H NMR (400 MHz, DMSO): 8.16 (d, J=1.6 Hz, 3H), 7.60 (d, J=2.5 Hz, 1H), 7.41 (d, J=2.5 Hz, 1H), 4.64 (s, 2H), 4.13-4.05 (m, 1H), 3.85 (s, 3H), 3.11 (d, J=4.4 Hz, 1H), 2.42-2.32 (m, 2H), 2.05 (d, J=11.4 Hz, 2H), 1.78 (d, J=10.5 Hz, 2H), 1.61-1.50 (m, 2H).

Step 5Synthesis of 8-fluoro-2-({[trans-4-(7-methoxy-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}methyl)[1]benzopyrano[4,3-b]pyrrol-4(1H)-one (Compound 302)

(386) ##STR00438##

(387) 1-[trans-4-aminocyclohexyl]-7-methoxy-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one HCl (307 mg, 878 mol) was dissolved in methanol (10 mL) and treated with triethylamine (354 mg, 4 eq), followed by 3 molecular sieves (500 mg) and 8-fluoro-4-oxo-1,4-dihydrochromeno[4,3-b]pyrrole-2-carbaldehyde (231 mg, 1 eq). The reaction mixture was stirred at 60 C. for 18 h, cooled to room temperature and treated with NaBH.sub.4 (134 mg, 4 eq). The resulting mixture was stirred for 18 h at room temperature then was cooled, filtered through celite and the combined filtrates evaporated. The resulting material was purified by flash column chromatography eluting with 0 to 100% ethyl acetate/iso-hexane followed by 0 to 100% methanol/ethyl acetate to give 123 mg of crude product. This material was triturated with DMF followed by ether, filtered and dried in vacuum to give the title compound (52 mg, 12.1% yield). LC-MS (MH.sup.+)=493. .sup.1H NMR (400 MHz, DMSO): 13.35 (bs, 1H), 9.30 (bs, 1H), 7.88 (dd, J=2.6, 8.5 Hz, 1H), 7.60 (d, J=2.3 Hz, 1H), 7.53 (dd, J=4.5, 9.1 Hz, 1H), 7.44-7.35 (m, 2H), 6.92 (s, 1H), 4.65 (s, 2H), 4.34-4.34 (m, 2H), 4.11 (m, 1H), 3.85 (s, 3H), 3.19 (m, 1H), 2.41 (d, J=11.4 Hz, 2H), 2.23 (m, 2H), 1.87 (d, J=1.3 Hz, 2H), 1.64-1.63 (m, 2H).

(388) Preparation of Compound 303

(389) Compound 303 was prepared as described herein below.

Step 1Synthesis of Tert-butyl 4-(2-chloroacetamido)piperidine-1-carboxylate

(390) ##STR00439##

(391) To a solution of tert-butyl 4-aminopiperidine-1-carboxylate (2.5 g, 12.5 mmol) in dichloromethane (30 mL) and triethylamine (2.25 mL, 16.1 mmol) was added at 0 C. chloroacetyl chloride (1.1 mL, 13.8 mmol). The reaction mixture was allowed to warm to room temperature and stirring continued for 1 hour. The reaction was quenched by addition of a saturated NaHCO.sub.3 solution and extracted with ethyl acetate. The combined organic extracts were evaporated and the residue purified by flash column chromatography eluting with ethyl acetate to afford the title compound (3.5 g, 100% yield). .sup.1H NMR (400 MHz, CDCl3): 6.46 (d, J=6.1 Hz, 1H), 4.08-4.05 (m, 1H), 4.04 (s, 2H), 4.00-3.89 (m, 2H), 2.88 (dd, J=12.2, 12.2 Hz, 2H), 1.96-1.89 (m, 2H), 1.46 (s, 9H), 1.44-1.29 (m, 2H).

Step 2Synthesis of Tert-butyl 4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate

(392) ##STR00440##

(393) tert-butyl 4-(2-chloroacetamido)piperidine-1-carboxylate (2.02 g, 7.31 mmol) and 2-chloro-5-fluoropyridin-3-ol (1.08 g, 7.32 mmol) were dissolved in DMF (70 mL). Caesium carbonate (4.88 g, 15 mmol) was added and the mixture heated to 100 C. for 18 hours. After cooling to room temperature the mixture was partitioned between ethyl acetate and water and the organic phase washed with water. The organic layer was then evaporated onto silica and purified by flash column chromatography eluting with 20 to 100% ethyl acetate/iso-hexane to give the title compound (2.19 g, 85% yield). .sup.1H NMR (400 MHz, CDCl.sub.3): 7.80 (d, J=2.5 Hz, 1H), 7.23 (dd, J=2.7, 9.0 Hz, 1H), 4.70 (s, 2H), 4.43-4.29 (m, 3H), 2.81 (m, 2H), 2.50-2.37 (m, 2H), 1.74 (dd, J=1.5, 11.6 Hz, 2H), 1.50 (s, 9H).

Step 3Synthesis of 7-fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one Hydrogen Chloride

(394) ##STR00441##

(395) tert-butyl 4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)piperidine-1-carboxylate (2.19 g, 6.23 mmol) was dissolved in MeOH (3 mL) and treated with a 2 N solution of HCl in diethyl ether (6 mL) at room temperature for 18 hours. The mixture was evaporated to furnish the title compound (1.6 g) that was used without further purification in the next step. .sup.1H NMR (400 MHz, DMSO): 9.26-9.20 (m, 1H), 8.69-8.67 (m, 1H), 8.02 (dd, J=2.6, 10.1 Hz, 1H), 7.90 (d, J=2.5 Hz, 1H), 4.76 (s, 2H), 4.43-4.34 (m, 1H), 3.37 (d, J=12.3 Hz, 2H), 3.12-3.00 (m, 2H), 2.79-2.66 (m, 2H), 1.91 (d, J=12.7 Hz, 2H).

Step 4Synthesis of 1-[1-(3-amino-2-hydroxypropyl)piperidin-4-yl]-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

(396) ##STR00442##

(397) 7-Fluoro-1-(piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one hydrogen chloride (1.6 g, 6.23 mmol) and 2-(oxiran-2-ylmethyl)isoindoline-1,3-dione (1.28 g, 6.3 mmol) were suspended in acetonitrile (60 mL) and triethylamine (4 mL) was added. The mixture was heated at 70 C. for 20 hours, then cooled to room temperature and evaporated to dryness. The residue (2.8 g, 6.23 mmol) was treated with methylamine (20 mL, 33% in ethanol) in a sealed tube at 50 C. for 3 hours. The mixture was evaporated and purified by reverse phase preparative HPLC to afford the title compound (119 mg). LC-MS (MH.sup.+)=325

Step 5Synthesis of 6-fluoro-2-oxo-2H-1-benzopyran-4-yl trifluoromethanesulfonate

(398) ##STR00443##

(399) To a solution of 6-fluoro-4-hydroxy-2H-chromen-2-one (720 mg, 4 mmol) and triethylamine (1.4 mL, 10 mmol) in dichloromethane (30 mL), trifluoromethane sulfonic anhydride (0.74 mL, 4.4 mmol) was added dropwise at 0 C. under nitrogen. The reaction was allowed to warm to room temperature and stirred overnight. The mixture was then poured into diethyl ether/iso-hexane (1:1, 600 mL) and filtered through a silica plug. The filtrate was evaporated to give the title compound (647 mg, 52% yield) which was used without further purification in the next step.

Step 6Synthesis of 7-fluoro-1-(1-{3-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-2-hydroxypropyl}piperidin-4-yl)-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (Compound 303)

(400) ##STR00444##

(401) 1-(1-(3-Amino-2-hydroxypropyl)piperidin-4-yl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (95 mg, 277 mol) and 6-fluoro-2-oxo-2H-chromen-4-yl trifluoromethane sulfonate (320 mg, 1 mmol) were dissolved in acetonitrile (10 mL) and triethylamine (1 mL) and heated to 80 C. for 2 hours in a sealed tube. The reaction mixture was cooled to room temperature and evaporated to dryness. The crude material was purified by reverse phase preparative HPLC to afford the title compound (25 mg, 18% yield). .sup.1H NMR (400 MHz, DMSO): 8.07 (dd, J=2.8, 10.1 Hz, 1H), 7.91 (m, 2H), 7.74 (dd, J=5.6, 5.6 Hz, 1H), 7.56-7.50 (m, 1H), 7.43 (dd, J=4.8, 9.1 Hz, 1H), 5.38 (s, 1H), 4.98 (d, J=4.5 Hz, 1H), 4.78 (s, 2H), 4.14-4.06 (m, 1H), 3.98-3.93 (m, 1H), 3.44-3.48 (m, 1H), 3.29-3.20 (m, 1H), 3.04 (dd, J=11.5, 25.1 Hz, 2H), 2.67-2.58 (m, 1H), 2.50-2.38 (m, 2H), 2.25 (dd, J=11.7, 15.5 Hz, 2H), 1.74-1.68 (m, 2H).

(402) Preparation of Compound 314

(403) Compound 314 was prepared as described herein below.

Step 1Synthesis of 5-(benzyloxy)-2-(hydroxymethyl)pyridin-4(1H)-one

(404) ##STR00445##

(405) A suspension of 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (53.2 g, 229 mmol) in IMS (75 mL) and aq. NH.sub.4OH solution (400 mL) was stirred at 70 C. for 18 h. The cooled solution was diluted with water (400 mL), cooled to 5 C. and the suspension stirred for 30 min. The solid was filtered and dried under vacuum to leave the title compound as a pale brown solid (42.5 g, 80% yield). LC-MS (MH.sup.+)=232. .sup.1H NMR (300 MHz, DMSO-d6): 11.08 (br s, 1H), 7.51-7.28 (m, 6H), 6.16 (br s, 1H), 5.56 (br s, 1H), 5.01 (s, 2H), 4.34 (s, 2H).

Step 2Synthesis of 5-(benzyloxy)-3-bromo-2-(hydroxymethyl) pyridin-4(1H)-one

(406) ##STR00446##

(407) A suspension of 5-(benzyloxy)-2-(hydroxymethyl)pyridine-4(1H)-one (11.6 g, 50.0 mmol) and NBS (10.2 g, 57.5 mmol) in AcOH (75 mL) was stirred at 40 C. for 1 h. The cooled suspension was filtered, the solid washed with AcOH (25 mL) and dried under vacuum to leave the title compound as a pale yellow solid (13.7 g, 89% yield). LC-MS (MH.sup.+)=310-312. .sup.1H NMR (300 MHz, DMSO-d6): 7.56 (s, 1H), 7.47-7.30 (m, 5H), 5.12 (s, 2H), 4.56 (s, 2H).

Step 3Synthesis of 5-bromo-6-(hydroxymethyl)pyridine-3,4-diol Hydrochloride

(408) ##STR00447##

(409) A suspension of 5-(benzyloxy)-3-bromo-2-(hydroxymethyl)pyridin-4(1H)-one (3.07 g, 9.90 mmol) in water (10 mL) and conc. aq. HCl solution (10 mL) was stirred at 100 C. for 2 h. The mixture was cooled to 0 C., then the suspension filtered. The solid was washed with EtOAc (10 mL) then dried under vacuum to leave a brown solid (1.93 g, 76% yield). LC-MS (MH.sup.+)=220-222. .sup.1H NMR (300 MHz, DMSO-d6): 7.88 (s, 1H), 4.63 (s, 2H), 2.42 (s, 1H).

Step 4Synthesis of (8-bromo-2,3-dihydro[1,4]dioxino[2,3-c]pyridin-7-yl)methanol

(410) ##STR00448##

(411) A suspension of 5-bromo-6-(hydroxymethyl)pyridine-3,4-diol hydrochloride (1.93 g, 7.52 mmol), 1,2-dibromoethane (2.12 g, 11.3 mmol) and K.sub.2CO.sub.3 (1.56 g, 11.3 mmol) in DMF (5 mL) was stirred at 75 C. for 5 h. The cooled suspension was concentrated under vacuum, then suspended in DCM-MeOH (98:2, 25 mL) and filtered through Celite. The filter cake was washed with DCM-MeOH (98:2, 50 mL) then the combined organics were concentrated under vacuum to leave a pale brown crystalline solid (1.30 g, 70% yield). LC-MS (MH.sup.+)=246-248. .sup.1H NMR (300 MHz, DMSO-d6): 8.08 (s, 1H), 4.66 (s, 2H), 4.50-4.46 (m, 2H), 4.35-4.30 (m, 2H).

Step 5Synthesis of 8-bromo-2,3-dihydro[1,4]dioxino[2,3-c]pyridine-7-carbaldehyde

(412) ##STR00449##

(413) A suspension of (8-bromo-2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methanol (1.25 g, 5.08 mmol) and MnO.sub.2 (4.42 g, 51 mmol) in dioxane (25 mL) was stirred at 90 C. for 5 h. The suspension was cooled to RT then was filtered through Celite, and the filter cake washed with warm dioxane (40 C., 25 mL). The combined organics were concentrated under vacuum to leave a pale orange solid (715 mg, 58% yield). .sup.1H NMR (300 MHz, CDCl3): 10.16 (s, 1H), 8.29 (s, 1H), 4.54-4.49 (m, 2H), 4.43-4.39 (m, 2H).

Step 6Synthesis of 9-tert-butyl 8-methyl 2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8,9-dicarboxylate

(414) ##STR00450##

(415) To a solution of 8-bromo-2,3-dihydro-[1,4]dioxino[2,3-c]pyridine-7-carbaldehyde (715 mg, 2.93 mmol) and N-Boc-2-phosphonoglycine trimethyl ester (1.09 g, 3.66 mmol) in DCM (20 mL) at RT was added DBU (558 mg, 3.66 mmol) and the resulting mixture stirred at RT for 30 min. Aq. citric acid solution (10%, 20 mL) was added, then the aq. layer extracted with DCM (20 mL). The combined organics were passed through a hydrophobic frit and concentrated under vacuum to 3 mL volume. FCC (10-35% EtOAc in iso-hexane) gave the intermediate methyl (Z)-3-(8-bromo-2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)-2-((tert-butoxycarbonyl)-amino)acrylate as an off-white solid (835 mg, 69% yield, LC-MS (MH.sup.+)=415-417). N.sub.2 was bubbled through a suspension of the intermediate, K.sub.3PO.sub.4 (862 mg, 4.06 mmol) and 3 MS (400 mg) in t-BuOH (40 mL) at 50 C. for 30 min. BrettPhos Pd G3 (CAS: 1470372-59-8; 92 mg, 0.10 mmol) was added, then the flask evacuated and purged with N.sub.2 thrice. The mixture was stirred at 85 C. for 4.5 h. The cooled suspension was diluted with EtOAc (40 mL) then the mixture filtered through Celite. The filter cake was washed with EtOAc (40 mL), then the combined organics concentrated under vacuum to leave a gum (810 mg). FCC (10-60% EtOAc in iso-hexane) gave a crystalline orange solid (543 mg, 80% yield). LC-MS (MH.sup.+)=335. .sup.1H NMR (300 MHz, CDCl.sub.3): 8.22 (s, 1H), 7.26 (s, 1H), 4.47-4.42 (m, 2H), 4.41-4.36 (m, 2H), 3.93 (s, 3H), 1.64 (s, 9H).

Step 7Synthesis of Methyl 2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carboxylate

(416) ##STR00451##

(417) A solution of 9-(tert-butyl) 8-methyl 2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8,9-dicarboxylate (543 mg, 1.62 mmol) in TFA (2 mL) and DCM (10 mL) was stirred at reflux for 1 h. The cooled solution was concentrated under vacuum, dissolved in MeOH (3 mL), applied to an SCX-2 cartridge (10 g) and washed with MeOH (40 mL). The product was eluted with a 2 M solution of NH.sub.3 in MeOH (40 mL); concentration under vacuum left the title product as a pale yellow solid (346 mg, 91% yield). LC-MS (MH.sup.+)=235. .sup.1H NMR (300 MHz, DMSO-d6): 12.22 (1H, br s), 8.08 (1H, s), 7.12 (1H, s), 4.50-4.44 (2H, m), 4.41-4.35 (2H, m), 3.85 (3H, s).

Step 8Synthesis of (2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridin-8-yl)methanol

(418) ##STR00452##

(419) To a suspension of methyl 2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carboxylate (344 mg, 1.47 mmol) in dry THF (20 mL) was added lithium aluminium hydride (1 M in THF, 2.9 mL, 2.9 mmol) dropwise, at 0 C. under N.sub.2. The suspension was stirred at 0 C. for 5 min and then at RT for 1 h. The suspension was cooled to 0 C., then water (0.12 mL), aq. NaOH solution (15%, 0.12 mmol), water (0.36 mL) and Na.sub.2SO.sub.4 were added sequentially. The suspension was stirred at RT for 30 min, then filtered through Celite. The filter cake was washed with DCM-MeOH (9:1, 50 mL), then the combined organics concentrated under vacuum to leave the title compound as a pale yellow solid (110 mg, 36% yield). LC-MS (MH.sup.+)=207. .sup.1H NMR (300 MHz, DMSO-d6): 11.18 (br s, 1H), 7.86 (s, 1H), 6.28 (s, 1H), 5.16 (t, J=4.6 Hz, 1H), 4.55 (d, J=4.6 Hz, 2H), 4.47-4.42 (m, 2H), 4.34-4.29 (m, 2H).

Step 9Synthesis of 2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carbaldehyde

(420) ##STR00453##

(421) A suspension of (2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridin-8-yl)methanol (108 mg, 0.524 mmol) and MnO.sub.2 (228 mg, 2.62 mmol) in dioxane (5 mL) was stirred at 90 C. for 2 h. The suspension was cooled to 50 C., then filtered through Celite. The filter cake was washed with dioxane (10 mL), then the combined organics concentrated under vacuum to leave the title compound as a yellow solid (84 mg, 79% yield). LC-MS (MH.sup.+)=205. .sup.1H NMR (300 MHz, DMSO-d6): 12.32 (br s, 1H), 9.83 (s, 1H), 8.13 (s, 1H), 7.38 (s, 1H), 4.51-4.45 (m, 2H), 4.43-4.37 (m, 2H).

Step 10Synthesis of 1-[trans-4-{[(2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridin-8-yl)methyl]amino}cyclohexyl]-7-fluoro-1H-pyrido [2,3-b][1,4]oxazin-2(3H)-one (Compound 314)

(422) ##STR00454##

(423) A solution of 1-(trans-4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (106 mg, 0.400 mmol), 2,3-dihydro-9H-[1,4]dioxino[2,3-d]pyrrolo[3,2-b]pyridine-8-carbaldehyde (82 mg, 0.40 mmol), sodium triacetoxyborohydride (340 mg, 1.6 mmol) and AcOH (46 L, 0.80 mmol) in MeCN (10 mL) was stirred at RT for 7 h. The mixture was concentrated under vacuum, suspended in a mixture of water and sat. aq. NaHCO.sub.3 solution (1:1, 10 mL) and extracted with DCM-MeOH (19:1, 210 mL). The combined organics were passed through a hydrophobic frit and concentrated under vacuum to leave an orange-yellow gum. FCC (15 m silica, 2-6% [2M NH.sub.3 in MeOH] in DCM) followed by trituration with warm MeCN (50 C.) provided the title compound as an off-white solid (89 mg, 49% yield). LC-MS (MH.sup.+)=454.3. .sup.1H NMR (400 MHz, DMSO-d6): 11.09 (br s, 1H), 7.86-7.82 (m, 3H), 6.28 (s, 1H), 4.68 (s, 2H), 4.48-4.44 (m, 2H), 4.34-4.31 (m, 2H), 4.01 (tt, J=3.6, 12.1 Hz, 1H), 3.82 (s, 2H), 2.42 (tt, J=3.5, 11.0 Hz, 1H), 2.27 (dq, J=2.9, 12.6 Hz, 2H), 2.14 (br s, 1H), 1.96 (apparent br d, J=11.9 Hz, 2H), 1.69 (apparent br d, J=11.9 Hz, 2H), 1.24 (dq, J=3.4, 12.3 Hz, 2H).

(424) Preparation of Compound 343

(425) Compound 343 was prepared as described herein below.

Step 1Synthesis of [(2S)-3,8-dioxo-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylen-2-yl]methyl Methanesulfonate

(426) ##STR00455##

(427) To an ice-cooled suspension of (2S)-2-(hydroxymethyl)-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylene-3,8-dione (prepared as in WO2009/141398, 0.500 g, 2.28 mmol) in DCM (35 mL) was added Et.sub.3N (0.48 mL, 3.4 mmol), then methanesulfonyl chloride (0.21 mL, 2.7 mmol) was added over 3 min. The mixture was stirred at RT for 45 min then purified directly by FCC (2-10% MeOH in DCM) to give the title compound (0.64 g, 94% yield). LC-MS (MH.sup.+)=298. .sup.1H NMR (400 MHz, DMSO-d6): 7.88 (d, J=9.6 Hz, 1H), 7.79 (s, 1H), 6.27 (d, J=9.6 Hz, 1H), 5.36-5.30 (m, 1H), 4.86 (dd, J=3.5, 11.2 Hz, 1H), 4.62 (dd, J=2.4, 11.2 Hz, 1H), 4.43 (dd, J=10.1, 12.0 Hz, 1H), 4.25 (dd, J=5.1, 12.1 Hz, 1H), 3.21 (s, 3H).

Step 2Synthesis of 4-chloro-6-fluoro-2H-1-benzopyran-2-one

(428) ##STR00456##

(429) Et.sub.3N (3.5 mL, 25 mmol) was added over 5 min to a suspension of 6-fluoro-4-hydroxy-2H-1-benzopyran-2-one (3.0 g, 16.7 mmol) in POCl.sub.3 (42 mL, 450 mmol). The mixture was stirred at RT for 10 min then heated at reflux for 16 h. The reaction was cooled to RT and then concentrated under reduced pressure. Toluene was added and evaporated. The residue was partitioned between DCM and aq. sodium bicarbonate. The aqueous phase was extracted with DCM, then the combined organic extracts were washed with water, dried over Na.sub.2SO.sub.4 and evaporated. The residue was purified by FCC (2-6% EtOAc in toluene) to give the title compound (3.082 g, 93% yield). LC-MS (MH.sup.+)=199. .sup.1H NMR (400 MHz, CDCl3): 7.57 (dd, J=2.4, 8.2 Hz, 1H), 7.39-7.32 (m, 2H), 6.67 (s, 1H).

Step 3Synthesis of Tert-butyl 4-{2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-1-hydroxyethyl}piperidine-1-carboxylate

(430) ##STR00457##

(431) Et.sub.3N (1.0 mL, 7.2 mmol) was added to a solution of tert-butyl 4-(2-amino-1-hydroxyethyl)piperidine-1-carboxylate (0.818 g, 3.34 mmol) in EtOH (25 mL). 4-chloro-6-fluoro-2H-1-benzopyran-2-one (0.65 g, 3.27 mmol) was added and the mixture was stirred at RT for 5 min then heated at 70 C. for 7.5 h. The reaction mixture was allowed to cool and then evaporated. The residue was purified by FCC (2-8% [2M NH.sub.3 in MeOH] in DCM) to give the title compound (0.859 g, 65% yield). LC-MS (M-Na.sup.+)=429. .sup.1H NMR (400 MHz, DMSO-d6): 8.01 (dd, J=2.9, 10.0 Hz, 1H), 7.55 (br t, J=5.3 Hz, 1H), 7.50-7.45 (m, 1H), 7.37 (dd, J=4.9, 9.1 Hz, 1H), 5.23 (s, 1H), 4.95 (d, J=5.3 Hz, 1H), 4.04-3.91 (m, 2H), 3.62-3.57 (m, 1H), 3.36-3.30 (m, 1H, under water peak), 3.18-3.11 (m, 1H), 2.74-2.53 (m, 2H), 1.77-1.69 (m, 1H), 1.61-1.53 (m, 2H), 1.39 (s, 9H), 1.32-1.13 (m, 2H).

Step 4Synthesis of 6-fluoro-4-{[2-hydroxy-2-(piperidin-4-yl)ethyl]amino}-2H-1-benzopyran-2-one

(432) ##STR00458##

(433) TFA (7.5 mL) was added to an ice-cooled solution of tert-butyl 4-{2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-1-hydroxyethyl}piperidine-1-carboxylate (0.859 g, 2.11 mmol) in DCM (30 mL). The mixture was stirred at RT for 1 h. Toluene was added and evaporated. The residue was dissolved in MeOH (25 mL), applied to an SCX-2 cartridge and washed with MeOH. The product was eluted with a 2 M solution of NH.sub.3 in MeOH and the evaporation gave the title compound (0.502 g, 78% yield). LC-MS (MH.sup.+)=307. .sup.1H NMR (400 MHz, DMSO-d6): 8.03 (dd, J=2.9, 10.1 Hz, 1H), 7.57 (t, J=5.4 Hz, 1H), 7.50-7.45 (m, 1H), 7.37 (dd, J=4.8, 9.1 Hz, 1H), 5.20 (s, 1H), 4.88 (br s, 1H), 3.56-3.51 (m, 1H), 3.18-3.08 (m, 2H), 2.99-2.90 (m, 2H), 2.46-2.37 (m, 2H), 1.70-1.65 (m, 1H), 1.55-1.41 (m, 2H), 1.31-1.13 (m, 2H).

Step 5Synthesis of (2R)-2-[(4-{2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]-1-hydroxyethyl}piperidin-1-yl)methyl]-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylene-3,8-dione (Compound 343, Diastereomeric Mixture)

(434) ##STR00459##

(435) A mixture of (S)-(3,8-dioxo-1,2-dihydro-3H,8H-2a,5,8a-triazaacenaphthylen-2-yl)methyl methane-sulfonate (0.300 g, 1.01 mmol), 6-fluoro-4-{[2-hydroxy-2-(piperidin-4-yl)ethyl]amino}-2H-1-benzopyran-2-one (0.464 g, 1.51 mmol), pyridine (0.33 mL, 4.0 mmol) and i-PrOH (20 mL) was stirred in a sealed vial at 120 C. for 2.5 h. The cooled mixture was evaporated to dryness and purified via FCC (2-14% [2M NH.sub.3 in MeOH] in DCM) to give the title compound (0.167 g, 33% yield) as an off-white solid. LC-MS (MH.sup.+)=508.4. .sup.1H NMR (400 MHz, DMSO-d6): 8.01 (dd, J=2.9, 10.0 Hz, 1H), 7.85 (d, J=9.6 Hz, 1H), 7.73 (s, 1H), 7.52 (t, J=5.0 Hz, 1H), 7.50-7.45 (m, 1H), 7.37 (dd, J=4.9, 9.1 Hz, 1H), 6.25 (d, J=9.5 Hz, 1H), 5.21 (s, 1H), 5.08-5.02 (m, 1H), 4.88 (dd, J=1.0, 5.4 Hz, 1H), 4.33 (dd, J=9.4, 11.8 Hz, 1H), 4.25 (dd, J=5.0, 11.7 Hz, 1H), 3.57-3.51 (m, 1H), 3.32-3.28 (m, 1H), 3.15-3.08 (m, 1H), 3.03-2.95 (m, 2H), 2.78 (dd, J=8.9, 12.6 Hz, 1H), 2.71-2.65 (m, 1H), 2.18-2.00 (m, 2H), 1.75-1.66 (m, 1H), 1.59-1.51 (m, 1H), 1.39-1.18 (m, 3H).

(436) Preparation of Compound 354

(437) Compound 354 was prepared as described herein below.

Step 1Synthesis of tert-butyl (2-{[trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl]amino}ethyl) Carbamate

(438) ##STR00460##

(439) A mixture of trans-1-(4-aminocyclohexyl)-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (296 mg, 1.12 mmol), N-Boc-2-aminoacetaldehyde (178 mg, 1.12 mmol) and 3 MS in MeOH (8 mL) was stirred at RT for 18 h. NaBH.sub.4 (80 mg, 2.1 mmol) was added and the mixture stirred at RT for 24 h. The mixture was filtered through Celite, the filter cake washed with MeOH and DCM, then the combined organics concentrated under vacuum to leave a residue. FCC (0-5% [2N NH.sub.3 in MeOH] in DCM) provided the title intermediate (180 mg, 39% yield). LC-MS (MH.sup.+)=409. .sup.1H NMR (400 MHz, CDCl3): 7.78 (d, J=2.6 Hz, 1H), 7.19 (dd, J=2.5, 9.0 Hz, 1H), 4.89 (br s, 1H), 4.07 (tt, J=3.8, 12.6 Hz, 1H), 3.22 (q, J=5.8 Hz, 2H), 2.77 (t, J=5.9 Hz, 2H), 2.57 (tt, J=3.7, 11.1 Hz, 1H), 2.41 (dq, J=3.3, 12.9 Hz, 2H), 2.10 (apparent br d, J=12.8 Hz, 2H), 1.83 (apparent br d, J=12.8 Hz, 2H), 1.51-1.45 (m, 11H), 1.30-1.16 (m, 2H).

Step 2Synthesis of 1-{trans-4-[(2-aminoethyl)amino]cyclohexyl}-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one

(440) ##STR00461##

(441) A mixture of tert-butyl (2-trans-4-(7-fluoro-2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazin-1-yl)cyclohexyl)amino)ethyl)carbamate (180 mg, 0.44 mmol) in TFA (0.50 mL) and DCM (2 mL) was stirred at RT for 1.5 h. The mixture was applied to an SCX-2 cartridge, washed with MeOH and then eluted with NH.sub.3 in MeOH (2 N); concentration under vacuum left a brown solid (113 mg, 83% yield). LC-MS (MH.sup.+)=309. .sup.1H NMR (400 MHz, CDCl3): 7.78 (d, J=2.6 Hz, 1H), 7.20 (dd, J=2.6, 9.0 Hz, 1H), 4.67 (s, 2H), 4.06 (tt, J=3.9, 12.3 Hz, 1H), 2.83 (dd, J=5.3, 6.2 Hz, 2H), 2.71 (dd, J=5.2, 6.4 Hz, 2H), 2.58 (tt, J=3.7, 11.1 Hz, 1H), 2.43 (dq, J=3.4, 12.9 Hz, 2H), 2.15-2.09 (m, 2H), 1.87-1.81 (m, 2H), 1.25 (ddt, J=3.6, 12.5, 12.3 Hz, 2H).

Step 3Synthesis of 7-fluoro-1-[trans-4-({2-[(6-fluoro-2-oxo-2H-1-benzopyran-4-yl)amino]ethyl}amino)cyclohexyl]-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (Compound 354)

(442) ##STR00462##

(443) To a solution of 1-{trans-4-[(2-aminoethyl)amino]cyclohexyl}-7-fluoro-1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (110 mg, 0.36 mmol) and Et.sub.3N (43 mg, 0.43 mmol) in MeCN (3 mL), 6-fluoro-2-oxo-2H-chromen-4-yl trifluoromethanesulfonate was added and the mixture stirred at RT for 2 h. The mixture was partitioned between water and EtOAc, then the aq. phase extracted with EtOAc. The combined organics were dried (MgSO.sub.4), filtered and concentrated under vacuum to leave the crude product. FCC (15 m silica, 0-3.5% [2M NH.sub.3 in MeOH] in DCM) gave an off-white solid. The solid was triturated with MeCN (1 mL) to leave a wet solid that was dried under vacuum at 60 C. to give the title compound (46 mg, 27% yield). LC-MS (MH.sup.+)=471.3. .sup.1H NMR (400 MHz, DMSO-d6): 7.98 (dd, J=2.9, 10.0 Hz, 1H), 7.87-7.81 (m, 2H), 7.55 (s, 1H), 7.48 (ddd, J=2.9, 8.0, 9.0 Hz, 1H), 7.37 (dd, J=4.8, 9.1 Hz, 1H), 5.25 (s, 1H), 4.70 (s, 2H), 4.04 (tt, J=3.7, 11.9 Hz, 1H), 3.32 (2H, methylene signal under water peak), 2.82 (t, J=6.5 Hz, 2H), 2.50 (1H, methine signal under solvent peak), 2.35 (dq, J=2.9, 12.7 Hz, 2H), 1.94 (d, J=11.9 Hz, 2H), 1.71 (d, J=11.4 Hz, 2H), 1.28-1.16 (m, 2H).

Biological Assays

Example 1

Inhibition of DNA Gyrase and Topo IV in E. coli and S. aureus

(444) The above compounds were tested for the inhibition of the enzyme DNA gyrase in a gyrase supercoiling assay and for the inhibition of the enzyme topoisomerase IV in a decatenation assay, in both Gram positive and Gram negative bacteria, according to the following methods.

(445) Both the assays were carried out according to a set-up method modified from the article to Blanche F, et al. Differential Behaviors of Staphylococcus aureus and Escherichia coli Type II DNA Topoisomerases, Antimicrob. Agents Chemother., 1996, Vol. 40, No. 12 p. 2714-2720.

(446) The compounds were screened at single concentration (200, 100 or 50 M), in duplicate.

(447) Ciprofloxacin and novobiocin were used as reference compounds, at single concentration of 200 and 50 M, respectively.

(448) DNA Gyrase Supercoiling Assay.

(449) Reagents from S. aureus and E. coli Gyrase Supercoiling Assay kits (Inspiralis, UK) were used. A master mix with a total volume sufficient for the number of reactions to perform was prepared with the following reagents: 5 assay buffer, relaxed pBR322 substrate (0.5 g/reaction), RNase-DNase free water. Aliquotes of this mix were dispensed in each tube, then 10 compound stock solutions or vehicle control (DMSO), were added to each reaction tube.

(450) Reaction was started with E. Coli (2 U/reaction) or S. aureus (1 U/reaction) gyrase enzyme addition.

(451) A sample added with an equal volume of dilution buffer was used as negative control (without enzyme).

(452) The reaction tubes were gentle vortexed and incubated 30 minutes at 37 C. Each reaction was stopped by adding 30 l of Stop Buffer and 30 l chloroform/isoamyl alcohol (24/1), briefly vortexed for 5-10 seconds and centrifuged at 20000g for 2 minutes. Samples were loaded onto 1% agarose gel and subjected to electrophoresis for 1 hour at 80V constant voltage in TAE (40 mM Tris-acetate, 2 mM EDTA).

(453) Data acquisition and analysis. Treatment of relaxed pBR322 with DNA gyrase converted the relaxed topoisomers (DNAs of different linking number) to the supercoiled form of the plasmid, which migrates faster on an agarose gel. An upper band might also be visible, which consists of open-circular (nicked) DNA which is present in the relaxed substrate but co-migrates with some of the relaxed topoisomers.

(454) Bands were visualized by ethidium bromide staining (dilution 1:20000) for 30 minutes followed by destaining in distilled water for 10 minutes.

(455) In order to evaluate the compounds activity on the enzyme, the bands of supercoiled DNAs in the gel were photographed by a digital imaging system ImageQuant LAS 4000 (GE Healthcare) according to manufacturer's instructions.

(456) The fluorescent intensity of each band was analyzed by ImageQuant TL software and it was expressed as volume (volume of the uncalibrated quantity of material in the image feature after subtraction of the background intensity by using rolling ball method).

(457) Each band intensity was compared, as percentage, to vehicle sample band intensity, which served as positive control, on the same gel.

(458) Inhibitory activity was expressed as percent of inhibition versus the positive control.

(459) The results are summarized in the following Table 2.

(460) Topoisomerase IV Decatenation Assay

(461) S. aureus and E. coli Topoisomerase IV decatenation kits (Inspiralis, UK) were used. A master mix with a total volume sufficient for the number of reactions to perform was prepared with the following reagents: 5 assay buffer (50 mM HEPES-KOH (pH 7.6), 100 mM potassium glutamate, 10 mM magnesium acetate, 10 mM DTT, 1 mM ATP, 50 g/ml albumin), kDNA substrate (200 ng/reaction), RNase-DNase free water. Aliquots of this mix were dispensed in each tube, then 10 compound stock solutions or vehicle control (DMSO), were added in each reaction tube.

(462) Reaction was started with Topoisomerase IV enzyme (0.5 U/reaction) addition.

(463) A sample added with an equal volume of dilution buffer was used as negative control (without enzyme).

(464) The reaction tubes were gentle vortexed and incubated 30 minutes at 37 C. Each reaction was stopped by adding 30 l of Stop Buffer and 30 l of chloroform/isoamyl alcohol (24/1), briefly vortexed for 5-10 seconds and centrifuged at 20000g for 2 minutes. Samples taken from the upper phase were loaded into 1% agarose gel and subjected to electrophoresis for 1 hour at 80V constant voltage in TAE (40 mM Tris-acetate, 2 mM EDTA).

(465) Data acquisition and analysis. Due to the high molecular mass, kDNA could not enter an agarose gel under normal electrophoresis conditions, but remained in the wells. In the presence of Topo IV topoisomerase mini-circles (2.5 Kb) were released from kDNA by decatenation and were quickly and easily resolved in the gel at relatively high voltages.

(466) Bands were visualized by ethidium bromide staining (dil 1:20000) for 30 minutes followed by destaining in distilled water for 10 minutes.

(467) For single concentration screening assay, in order to evaluate the compounds activity on the enzymes, the bands of decatenated DNAs in the gel were photographed by a digital imaging system ImageQuant LAS 4000 (GE Healthcare) according to manufacturer's instructions.

(468) The fluorescent intensity of each band was analyzed by ImageQuant TL software and it was expressed as volume (volume of the uncalibrated quantity of material in the image feature after subtraction of the background intensity by using rolling ball method).

(469) Each band intensity was compared, as percentage, to vehicle sample band intensity, which served as positive control, on the same gel.

(470) Inhibitory activity was expressed as percent of inhibition versus the positive control.

(471) The results are summarized in the following Table 2.

(472) TABLE-US-00002 TABLE 2 E. coli S. aureus % % inhibition % inhibition % Compound conc. DNA inhibition DNA inhibition No. (M) gyrase Topo IV gyrase Topo IV 157 50 83 100 89 100 160 50 50 60 65 n/a 164 50 100 100 100 100 165 50 70 90 100 100 180 50 58 57 90 50 181 50 53 91 92 50 182 50 61 52 92 50a 183 50 81 77 84 85 193 50 77 50 86 74 194 50 79 50 60 50 197 50 87 63 90 79 200 50 90 100 100 100 201 50 100 100 100 100 202 50 100 100 100 100 204 50 100 100 100 100 205 50 100 100 100 100 206 50 100 100 100 100 207 50 100 100 100 100 208 50 100 100 100 100 209 50 100 100 100 100 210 50 92 86 100 84 211 50 100 100 100 100 212 50 100 100 100 100 213 50 98 97 100 84 214 50 99 100 100 100 215 50 69 n/a 96 n/a 216 50 100 100 100 100 217 50 82 50 100 100 219 50 100 100 100 100 220 50 100 50 100 100 221 50 81 50 100 100 n/a = not active

(473) The above results showed that the exemplified compounds effectively inhibited both DNA gyrase and Topo IV of E. coli, which is a Gram positive bacterium, and/or S. aureus, which is a Gram negative bacterium.

Example 2

Determination of IC50

(474) The compounds that in the above example 1 showed an inhibitory activity were further assayed in concentration-response curve (eight half-log concentrations ranging from 0.1 to 300 M) in order to determine the IC.sub.50.

(475) The supercoiled or decatenated DNA bands obtained as described in Example 1 were analysed as follows.

(476) Bands were analyzed by gel documentation equipment (Syngene, Cambridge, UK) and quantitated using Syngene Gene Tools software. Raw gel data (fluorescent band volumes) collected from Syngene, GeneTools gel analysis software were converted to a percentage of the 100% control (the fully supercoiled or decatenated DNA band). These data were analyzed using SigmaPlot Version 12.3 (2013). The IC50 data were calculated by using the global curve fit non-linear regression tool by selecting the Single, 2 Parameter fit function from the Exponential Decay equation category.

(477) The results are reported in the following table 3.

(478) TABLE-US-00003 TABLE 3 E. coli S. aureus Compound IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 No. DNA gyrase Topo IV DNA gyrase Topo IV 157 2.34 0.64 2.48 1.02 160 n/a 21.5 20.8 n/a 164 3.77 1.78 1.46 0.7 165 5.25 1.24 3.60 1.12 180 0.48 3.78 1.70 0.11 181 0.44 3.67 0.73 0.36 182 0.74 3.21 0.64 0.74 183 0.77 2.48 0.74 0.19 193 0.25 1.38 0.28 2.25 194 0.25 6.06 0.49 0.69 200 0.04 <0.1 <0.1 <0.1 201 <0.1 <0.1 0.19 <0.1 204 0.05 <0.1 0.64 <0.1 205 0.1 0.31 0.17 0.1 206 0.05 <0.1 0.13 <0.1 207 <0.1 <0.1 <0.1 <0.1 208 0.08 0.27 0.11 0.13 209 0.13 1.00 0.3 0.22 210 0.44 13.77 1.3 1.31 211 <0.1 <0.1 <0.1 <0.1 212 0.1 0.17 0.14 0.07 213 0.11 1.00 0.28 0.58 214 0.12 1.03 0.16 0.57 216 0.13 0.25 0.31 0.29 217 1.45 0.69 2.15 2.72 219 0.22 0.22 0.22 0.65 220 0.68 0.74 1.97 2.91 221 0.92 0.67 2.76 2.63

Example 3

Determination of IC50

(479) Compounds 301-303, 314, 343 and 354 were assayed in concentration-response curve (eight half-log concentrations ranging from 0.1 to 300 M) in order to determine the IC.sub.50, following the procedure described in example 2.

(480) The results are reported in the following table 4.

(481) TABLE-US-00004 TABLE 4 E. coli S. aureus Compound IC.sub.50 IC.sub.50 IC.sub.50 IC.sub.50 No. DNA gyrase Topo IV DNA gyrase Topo IV 301 0.46 0.43 0.72 70.3 302 0.59 0.12 0.58 3.26 303 2.67 0.62 9.92 >100 314 0.85 1.14 7.1 >100 343 0.91 1.27 1.98 8.73 354 0.47 0.18 0.35 0.92

(482) The data of Table 3 are comparable to the data of Table 2, and confirmed the activity of Compounds 301-303, 314, 343 and 354 to effectively inhibit both DNA gyrase and Topo IV of E. coli and/or S. aureus.