Substituted indoline derivatives as dengue viral replication inhibitors

Abstract

The present invention concerns substituted indoline compounds, methods to prevent or treat dengue viral infections by using said compounds and also relates to said compounds for use as a medicine, more preferably for use as a medicine to treat or prevent dengue viral infections. The present invention furthermore relates to pharmaceutical compositions or combination preparations of the compounds, to the compositions or preparations for use as a medicine, more preferably for the prevention or treatment of dengue viral infections. The invention also relates to processes for preparation of the compounds.

Claims

1. A compound having formula (I) ##STR00068## a stereoisomeric form, a pharmaceutically acceptable salt, a solvate or a polymorph thereof, wherein R.sub.1 is CF.sub.3 or OCF.sub.3; R.sub.2 is H, F or OCH.sub.3; R.sub.3 is H or CH.sub.3; and wherein the compound is selected from the group consisting of: ##STR00069## ##STR00070## ##STR00071##

2. A pharmaceutical composition comprising a compound according to claim 1 or its stereoisomeric form, or a pharmaceutically acceptable salt, solvate or polymorph thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.

3. A method of treating a Dengue viral infection in a subject comprising administering to the subject the compound according to claim 1, or its stereoisomeric form, or a pharmaceutically acceptable salt, solvate or polymorph thereof, or a pharmaceutical composition thereof in an amount effective to treat a Dengue viral infection in a subject.

4. A method of inhibiting replication of a Dengue virus in a biological sample or in a patient comprising contacting the virus with the compound according to claim 1, or a stereoisomeric form, or a pharmaceutically acceptable salt, solvate or polymorph thereof comprising a mono- or di-substituted indole group, in an amount effective to inhibit replication of a Dengue virus in a biological sample or patient.

5. The method according to claim 3 further comprising contacting the Dengue virus with an additional therapeutic agent.

6. The method of claim 5, wherein the additional therapeutic agent is an antiviral agent, a Dengue vaccine, or both.

7. The method according to claim 4 further comprising co-administering an additional therapeutic agent.

8. The method of claim 7, wherein the additional therapeutic agent is an antiviral agent, a Dengue vaccine, or both.

9. The compound as claimed in claim 1 wherein the compound is selected from the group consisting of ##STR00072## ##STR00073## ##STR00074## or a pharmaceutically acceptable salt, solvate or polymorph thereof.

10. A pharmaceutical composition comprising a compound according to claim 9, or a pharmaceutically acceptable salt, solvate or polymorph thereof together with one or more pharmaceutically acceptable excipients, diluents or carriers.

11. A method of treating a Dengue viral infection in a subject comprising administering to the subject the compound according to claim 9 or its stereoisomeric form, or a pharmaceutically acceptable salt, solvate or polymorph thereof, or a pharmaceutical composition thereof, in an amount effective to treat a Dengue viral infection in a subject.

12. A method of inhibiting replication of a Dengue virus in a biological sample or in a patient comprising contacting the virus with the compound according to claim 9, or a stereoisomeric form, a pharmaceutically acceptable salt, a solvate or a polymorph thereof comprising a mono- or di-substituted indole group, in an amount effective to inhibit replication of a Dengue virus in a biological sample or patient.

13. The method according to claim 11 further comprising co-administering an additional therapeutic agent.

14. The method of claim 13, wherein the additional therapeutic agent is an antiviral agent, aDengue vaccine, or both.

15. The method according to claim 12 further comprising contacting the Dengue virus with an additional therapeutic agent.

16. The method of claim 15, wherein the additional therapeutic agent is an antiviral agent, a Dengue vaccine, or both.

Description

EXAMPLES

(1) LC/MS Methods

(2) High Performance Liquid Chromatography (HPLC) measurement was performed using a LC pump, a diode-array (DAD) or a UV detector and a column as specified in the respective methods. If necessary, additional detectors were included (see table of methods below).

(3) Flow from the column was brought to the Mass Spectrometer (MS) which was configured with an atmospheric pressure ion source. It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time . . . ) in order to obtain ions allowing the identification of the compound's nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software.

(4) Compounds are described by their experimental retention times (R.sub.t) and ions. If not specified differently in the table of data, the reported molecular ion corresponds to the [M+H].sup.+ (protonated molecule) and/or [MH].sup. (deprotonated molecule). In case the compound was not directly ionizable the type of adduct is specified (i.e. [M+NH.sub.4].sup.+, [M+HCOO].sup., etc. . . . ). For molecules with multiple isotopic patterns (Br, Cl), the reported value is the one obtained for the lowest isotope mass. All results were obtained with experimental uncertainties that are commonly associated with the method used.

(5) Hereinafter, SQD means Single Quadrupole Detector, MSD Mass Selective Detector, RT room temperature, BEH bridged ethylsiloxane/silica hybrid, DAD Diode Array Detector, HSS High Strength silica.

(6) LC/MS Method codes (Flow expressed in mL/min; column temperature (T) in C.; Run time in minutes)

(7) TABLE-US-00001 Run Method Flow time code Instrument Column Mobile phase Gradient Col T (min) LC-A Waters: Acquity UPLC - DAD-Quattro Micro Waters BEH C18 (1.7 m, 2.1 100 mm) A: 95% CH.sub.3COONH.sub.4 7 mM/5% CH.sub.3CN, B: CH.sub.3CN 84.2% A for 0.49 min, to 10.5% A in 2.18 min, held for 1.94 min, back to 84.2% A in 0.73 min, held for 0.73 min. $\frac{0.343\mathrm{mL}/\min }{40^{}C.}$ 6.2 LC-B Waters: Acquity H Clsss - DAD and SQD2TM Waters BEH C18 (1.7 m, 2.1 100 mm) A: 95% CH.sub.3COONH.sub.4 7 mM/5% CH.sub.3CN, B: CH.sub.3CN 84.2% A/15.8% B to 10.5% A in 2.18 min, held for 1.96 min, back to 84.2% A/15.8% B in 0.73 min, held for 0.49 min. $\frac{0.343\mathrm{mL}/\min }{40^{}C.}$ 6.1
SFC/MS Methods

(8) The SFC measurement was performed using an Analytical Supercritical fluid chromatography (SFC) system composed by a binary pump for delivering carbon dioxide (CO.sub.2) and modifier, an autosampler, a column oven, a diode array detector equipped with a high-pressure flow cell standing up to 400 bars. If configured with a Mass Spectrometer (MS) the flow from the column was brought to the (MS). It is within the knowledge of the skilled person to set the tune parameters (e.g. scanning range, dwell time . . . ) in order to obtain ions allowing the identification of the compound's nominal monoisotopic molecular weight (MW). Data acquisition was performed with appropriate software.

(9) Analytical SFC/MS Methods (Flow expressed in mL/min; column temperature (T) in C.; Run time in minutes, Backpressure (BPR) in bars.

(10) TABLE-US-00002 Method Flow/ Run time/ code column mobile phase gradient Col T BPR SFC-A Daicel A:CO.sub.2 40% 3/35 7/100 Chiralcel OD B: MeOH B hold column (5 m, 7 min 150 4.6 mm) SFC-B Daicel A:CO.sub.2 40% 3.5/35 3/103 Chiralcel OD-3 B: EtOH B hold column (3 m, (+0.3% iPrNH.sub.2) 3 min 100 4.6 mm) SFC-C Daicel A:CO.sub.2 40% 3.5/35 3/103 Chiralpak IA B: EtOH B hold column (5 m, (+0.3% iPrNH.sub.2) 7 min 150 4.6 mm) SFC-D Daicel A:CO.sub.2 40% 3/35 7/100 Chiralcel OJ-H B: EtOH B hold column (5 m, (+0.3% iPrNH.sub.2) 7 min 150 4.6 mm) SFC-E Daicel A:CO.sub.2 30% 3.5/35 3/103 Chiralpak IC-3 B: iPrOH B hold column (3 m, (+0.3% iPrNH.sub.2) 5 min 100 4.6 mm) SFC-F Daicel A:CO.sub.2 30% 3/35 7/100 Chiralcel OD B: EtOH B hold column (5 m, (+0.3% iPrNH.sub.2) 7 min 150 4.6 mm) SFC-G Daicel A:CO.sub.2 50% 3/35 7/100 Chiralpak IA B: iPrOH B hold column (5 m, (+0.3% iPrNH.sub.2) 7 min 150 4.6 mm) SFC-H Daicel A:CO.sub.2 30% 3/35 7/100 Chiralpak IA B: EtOH B hold column (5 m, (+0.3% iPrNH.sub.2) 7 min 150 4.6 mm) SFC-I Daicel A:CO.sub.2 50% 3.5/35 Chiralpak IA B: EtOH B hold column (5 m, (+0.3% iPrNH.sub.2) 7 min 150 4.6 mm) SFC-J Daicel A:CO.sub.2 40% 3/35 7/100 Chiralpak IC B: EtOH B hold column (5 m, (+0.3% iPrNH.sub.2) 7 min 150 4.6 mm)
Melting Points

(11) Values are either peak values or melt ranges, and are obtained with experimental uncertainties that are commonly associated with this analytical method.

(12) DSC823e (Indicated as DSC)

(13) For a number of compounds, melting points were determined with a DSC823e (Mettler-Toledo). Melting points were measured with a temperature gradient of 10 C./minute. Maximum temperature was 300 C.

(14) Optical Rotations:

(15) Optical rotations were measured on a Perkin-Elmer 341 polarimeter with a sodium lamp and reported as follows: [] (A, c g/100 ml, solvent, T C.). [].sub..sup.T=(100)/(lc): where l is the path length in dm and c is the concentration in g/100 ml for a sample at a temperature T ( C.) and a wavelength A (in nm). If the wavelength of light used is 589 nm (the sodium D line), then the symbol D might be used instead. The sign of the rotation (+ or ) should always be given. When using this equation the concentration and solvent are always provided in parentheses after the rotation. The rotation is reported using degrees and no units of concentration are given (it is assumed to be g/100 ml).

Example 1: Synthesis of 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 1) and Chiral Separation into Enantiomers 1A and 1B

(16) ##STR00007##
Synthesis of Intermediate 1a:

(17) A mixture of 2-(4-chloro-2-methoxyphenyl)acetic acid [CAS 170737-95-8] (1.55 g, 7.75 mmol) and 6-(trifluoromethyl)indoline [CAS 181513-29-1] (1.45 g, 7.75 mmol), HOBt (2.78 g, 11.6 mmol), EDC (2.23 g, 11.6 mmol) and triethylamine (2.15 mL, 15.5 mmol) in CH.sub.2Cl.sub.2 (40 mL) was stirred at room temperature for 12 h. Water was added and the layers were separated. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 m, 120 g, heptane/EtOAc 80/20). The pure fractions were combined and evaporated to dryness to give 2-(4-chloro-2-methoxyphenyl)-1-(6-(trifluoromethyl)-indolin-1-yl)ethanone 1a (1.66 g).

(18) Synthesis of Intermediate 1b:

(19) At 78 C., under a N.sub.2 flow, LiHMDS 1M in THF (8.98 mL, 8.98 mmol) was added dropwise to a mixture of 2-(4-chloro-2-methoxyphenyl)-1-(6-(trifluoromethyl)-indolin-1-yl)ethanone 1a (1.66 g, 4.49 mmol) in THF (18 mL). The mixture was stirred for 15 min at 78 C. and a solution of NBS (879 mg, 4.94 mmol) in THF (7 mL) was added dropwise. After stirring for 2 h at 78 C., the reaction was quenched by the addition of a saturated aqueous solution of NH.sub.4Cl. The mixture was extracted with EtOAc, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 m, 40 g, heptane/EtOAc 80/20). The pure fractions were combined and evaporated to dryness to give 2-bromo-2-(4-chloro-2-methoxyphenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 1b (1.23 g).

(20) Synthesis of Compound 1 and Chiral Separation into Enantiomers 1A and 1B:

(21) A mixture of 2-bromo-2-(4-chloro-2-methoxyphenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 1b (1.2 g, 2.68 mmol), 2-(3-amino-5-methoxyphenoxy)ethanol [CAS 725237-16-1] (735 mg, 4.01 mmol) and triethylamine (558 L, 4.01 mmol) in CH.sub.3CN (50 mL) was stirred at 70 C. for 6 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with 1N HCl. The organic phase was separated, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 97.5/2.5). The pure fractions were combined and evaporated to dryness to give racemic 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)-ethanone (Compound 1, 900 mg) after crystallization from CH.sub.3CN. This batch was combined with 2 other batches (total amount: 1.84 g). The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralcel OD-H 5 m 25030 mm, Mobile phase: 55% CO.sub.2, 45% MeOH). The first eluted enantiomer was further purified by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness to give, after solidification in petroleum ether/CH.sub.3CN/diisopropyl ether, Enantiomer 1A (540 mg). The second eluted enantiomer was further purified by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness to give, after solidification in petroleum ether/CH.sub.3CN/diisopropyl ether, Enantiomer 1B (560 mg).

(22) Compound 1:

(23) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.24 (m, 2H) 3.59-3.67 (m, 5H) 3.78-3.87 (m, 2H) 3.90 (s, 3H) 3.98-4.07 (m, 1H) 4.33-4.42 (m, 1H) 4.79 (br t, J=4.7 Hz, 1H) 5.60 (d, J=8.5 Hz, 1H) 5.76 (s, 1H) 5.87 (br s, 2H) 6.44 (br d, J=8.5 Hz, 1H) 7.03 (dd, J=8.2, 1.9 Hz, 1H) 7.15 (d, J=1.6 Hz, 1H) 7.32 (d, J=8.5 Hz, 1H) 7.39 (d, J=7.9 Hz, 1H) 7.46 (d, J=7.9 Hz, 1H) 8.36 (s, 1H) LC/MS (method LC-A): R.sub.t 3.35 min, MH.sup.+ 551

(24) Melting point: 194 C.

(25) Enantiomer 1A:

(26) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.16-3.30 (m, 2H) 3.57-3.67 (m, 5H) 3.79-3.88 (m, 2H) 3.90 (s, 3H) 4.03 (td, J=10.3, 7.1 Hz, 1H) 4.37 (td, J=10.1, 6.6 Hz, 1H) 4.79 (t, J=5.4 Hz, 1H) 5.61 (d, J=8.5 Hz, 1H) 5.77 (t, J=1.9 Hz, 1H) 5.87 (br s, 2H) 6.44 (d, J=8.8 Hz, 1H) 7.03 (dd, J=8.2, 2.2 Hz, 1H) 7.15 (d, J=1.9 Hz, 1H) 7.32 (d, J=8.2 Hz, 1H) 7.39 (d, J=7.9 Hz, 1H) 7.46 (d, J=7.6 Hz, 1H) 8.37 (s, 1H) LC/MS (method LC-A): R.sub.t 3.34 min, MH.sup.+ 551 [].sub.D.sup.20: 26.5 (c 0.3091, DMF)

(27) Chiral SFC (method SFC-A): R.sub.t 1.45 min, MH.sup.+ 551, chiral purity 100%.

(28) Enantiomer 1B:

(29) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.20-3.30 (m, 2H) 3.58-3.68 (m, 5H) 3.80-3.88 (m, 2H) 3.91 (s, 3H) 4.03 (td, J=10.2, 7.1 Hz, 1H) 4.38 (td, J=10.2, 6.6 Hz, 1H) 4.80 (t, J=5.5 Hz, 1H) 5.61 (d, J=8.8 Hz, 1H) 5.77 (t, J=2.0 Hz, 1H) 5.88 (br s, 2H) 6.45 (d, J=8.5 Hz, 1H) 7.04 (dd, J=8.2, 1.9 Hz, 1H) 7.15 (d, J=1.9 Hz, 1H) 7.32 (d, J=8.2 Hz, 1H) 7.39 (d, J=7.6 Hz, 1H) 7.47 (d, J=7.9 Hz, 1H) 8.37 (s, 1H) LC/MS (method LC-A): R.sub.t 3.34 min, MH.sup.+ 551

(30) [].sub.D.sup.20: +28.8 (c 0.2845. DMF)

(31) Chiral SFC (method SFC-A): R.sub.t 3.64 min, MH.sup.+ 551, chiral purity 100%.

Example 2.1: Synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 2)

(32) ##STR00008##
Synthesis of Intermediate 2a:

(33) A mixture of 2-(4-chloro-2-fluorophenyl)acetic acid [CAS 194240-75-0] (504 mg, 2.67 mmol) and 6-(trifluoromethyl)indoline [CAS 181513-29-1] (500 mg, 2.67 mmol), HOBt (541 mg, 4 mmol), EDCI (768 mg, 4 mmol) and triethylamine (743 L, 5.34 mmol) in CH.sub.2Cl.sub.2 (6 mL) was stirred at room temperature for 12 h. Water was added and the layers were separated. The organic layer was washed with water, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give 2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 2a (1.04 g). The compound was used as such in the next step.

(34) Synthesis of intermediate 2b:

(35) At 78 C., under a N.sub.2 flow, LiHMDS 1M in THF (5.5 mL, 5.5 mmol) was added dropwise to a solution of 2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 2a (980 mg, 2.74 mmol) in THF (8 mL). The reaction mixture was stirred for 15 min at 78 C. and a solution of NBS (536 mg, 3.01 mmol) in THF (5 mL) was added dropwise. After stirring for 2 h at 78 C., the reaction was quenched by the addition of a saturated aqueous solution of NH.sub.4Cl. The mixture was extracted with EtOAc, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure to give 2-bromo-2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 2b (1.3 g). The compound was used as such in the next step.

(36) Synthesis of Compound 2:

(37) A mixture of 2-bromo-2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethyl)indolin-1-yl)-ethanone 2b (500 mg, 1.15 mmol), 2-(3-amino-5-methoxyphenoxy)ethanol [CAS 725237-16-1] (315 mg, 1.72 mmol) and triethylamine (296 L, 1.72 mmol) in CH.sub.3CN (5 mL) was stirred at 50 C. for 24 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with 1N HCl. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 m, 24 g, heptane/EtOAc gradient 70/30 to 50/50). The pure fractions were combined and evaporated to dryness to give 2-(4-chloro-2-fluoro-phenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethyl)-indolin-1-yl)ethanone (Compound 2, 136 mg) after crystallization from Et.sub.2O.

(38) Compound 2:

(39) .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 3.20-3.28 (m, 2H) 3.57-3.69 (m, 5H) 3.79-3.91 (m, 2H) 4.03-4.13 (m, 1H) 4.39-4.49 (m, 1H) 4.78 (br s, 1H) 5.71 (d, J=9.1 Hz, 1H) 5.77-5.82 (m, 1H) 5.93 (d, J=2.0 Hz, 2H) 6.59 (d, J=9.1 Hz, 1H) 7.32 (dd, J=8.3, 1.8 Hz, 1H) 7.38-7.51 (m, 4H) 8.36 (s, 1H) LC/MS (method LC-A): R.sub.t 3.36 min, MH.sup.+ 539 Melting point: 168 C.

Example 2.2: Synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 2) and Chiral Separation into Enantiomers 2A and 2B

(40) ##STR00009##
Synthesis of Intermediate 2c:

(41) At 78 C., under a N.sub.2 flow, LiHMDS 1M in THF (38.5 mL, 38.5 mmol) was added dropwise to a mixture of methyl 2-(4-chloro-2-fluorophenyl)acetate [CAS 917023-04-2] (3.9 g, 19.3 mmol) in THF (120 mL). A solution of TMSCl (3.9 mL, 30.8 mmol) in THF (30 mL) was added dropwise. The mixture was stirred for 15 min at 78 C. and a solution of NBS (3.77 g, 21.2 mmol) in THF (50 mL) was added dropwise. After stirring for 2 h at 78 C., the reaction was quenched by the addition of a saturated aqueous solution of NH.sub.4Cl. The mixture was extracted with EtOAc, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure to give methyl 2-bromo-2-(4-chloro-2-fluorophenyl)acetate 2c (5.4 g). The compound was used as such in the next step.

(42) Synthesis of intermediate 2d:

(43) A mixture of methyl 2-bromo-2-(4-chloro-2-fluorophenyl)acetate 2c (4 g, 12.8 mmol), 2-(3-amino-5-methoxyphenoxy)ethanol [CAS 725237-16-1] (2.1 g, 11.6 mmol), triethylamine (2.4 mL, 17.4 mmol) in CH.sub.3CN (80 mL) was stirred at 50 C. for 12 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with water. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness to give methyl 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetate 2d (2.1 g).

(44) Synthesis of intermediate 2e:

(45) A mixture of methyl 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetate 2d (1 g, 2.6 mmol) and LiOH (330 mg, 7.8 mmol) in THF/water (1/1) (40 mL) was stirred at room temperature for 1 h. The mixture was diluted with water. The aqueous layer was slowly acidified with 3N HCl and extracted with EtOAc. The combined organic layers were dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 2e (2.7 g). The compound was used as such in the next step.

(46) Synthesis of Compound 2 and chiral separation into Enantiomers 2A and 2B:

(47) HATU (1.85 g, 4.87 mmol) was added to a mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1] (607 mg, 3.24 mmol), 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 2e (1.2 g, 3.24 mmol) and diisopropylethylamine (1.6 mL, 9.74 mmol) in DMF (35 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water and the precipitate was filtered off. The solids were washed with water and taken up in EtOAc. The organic solution was washed with a 10% solution of K.sub.2CO.sub.3 in water and brine, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). A second purification was performed via achiral SFC (Stationary phase: 2-Ethylpyridine 5 m 15030 mm, Mobile phase: 70% CO.sub.2, 30% MeOH) to afford 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)-ethanone (Compound 2, 550 mg) as a racemic mixture. This batch was combined with another batch (total amount: 950 mg). The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 70% CO.sub.2, 30% iPrOH (+0.3% iPrNH.sub.2)) to give, after solidification in petroleum ether/diisopropyl ether, the first eluted Enantiomer 2A (384 mg) and the second eluted Enantiomer 2B (375 mg).

(48) Enantiomer 2A:

(49) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.20-3.29 (m, 2H) 3.60-3.68 (m, 5H) 3.85 (dq, J=10.6, 5.2 Hz, 2H) 4.08 (td, J=10.1, 7.3 Hz, 1H) 4.40-4.48 (m, 1H) 4.80 (t, J=5.5 Hz, 1H) 5.71 (d, J=8.8 Hz, 1H) 5.80 (t, J=2.0 Hz, 1H) 5.93 (d, J=1.9 Hz, 2H) 6.61 (d, J=8.8 Hz, 1H) 7.33 (dd, J=8.2, 1.9 Hz, 1H) 7.38-7.51 (m, 4H) 8.36 (s, 1H)

(50) LC/MS (method LC-A): R.sub.t 3.34 min, MH.sup.+ 539

(51) [].sub.D.sup.20: 26.4 (c 0.2691, DMF)

(52) Chiral SFC (method SFC-B): R.sub.t 0.83 min, MH.sup.+ 539, chiral purity 100%.

(53) Enantiomer 2B:

(54) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.21-3.29 (m, 2H) 3.60-3.68 (m, 5H) 3.85 (tq, J=10.4, 5.0 Hz, 2H) 4.04-4.12 (m, 1H) 4.40-4.48 (m, 1H) 4.80 (t, J=5.5 Hz, 1H) 5.71 (d, J=8.8 Hz, 1H) 5.78-5.81 (m, 1H) 5.93 (d, J=1.6 Hz, 2H) 6.61 (d, J=8.8 Hz, 1H) 7.33 (dd, J=8.5, 1.9 Hz, 1H) 7.39-7.52 (m, 4H) 8.36 (s, 1H)

(55) LC/MS (method LC-A): R.sub.t 3.34 min, MH.sup.+ 539

(56) [].sub.D.sup.20: +27.3 (c 0.2564, DMF)

(57) Chiral SFC (method SFC-B): R.sub.t 1.69 min. MH.sup.+ 539, chiral purity 99.07%.

Example 3.1: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 3) and Chiral Separation into Enantiomers 3A and 3B

(58) ##STR00010##
Synthesis of Intermediate 3a:

(59) Benzoyl peroxide (5 mg) was added to a mixture of methyl 2-(4-chlorophenyl)-acetate [CAS 52449-43-1] (5.0 g, 29.7 mmol) and NBS (4.82 g, 27.1 mmol) in CH.sub.3CN (80 mL). The mixture was heated under reflux for 48 h and the solvent was evaporated under reduced pressure. The mixture was taken up in cyclohexane/EtOAc 80/20 and the precipitate was filtered off and discarded (succinimide). The filtrate was concentrated under reduced pressure to give methyl 2-bromo-2-(4-chlorophenyl)acetate 3a (7.2 g). The compound was used as such in the next step.

(60) Synthesis of Intermediate 3b:

(61) A mixture of methyl 2-bromo-2-(4-chlorophenyl)acetate 3a (6 g, 3.80 mmol), 3-(2-(tert-butoxy)ethoxy)-5-methoxyaniline [CAS 1428973-39-0] (4.63, 4.05 mmol), triethylamine (4.04 mL, 29.0 mmol) in CH.sub.3CN (30 mL) was stirred at 50 C. for 12 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with water. The organic phase was separated, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 m, 40 g, heptane/EtOac 90/10). The pure fractions were combined and evaporated to dryness to give methyl 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)-acetate 3b (4.8 g).

(62) Synthesis of intermediate 3c:

(63) A mixture of methyl 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)acetate 3b (4.8 g, 11.4 mmol) and LiOH (1.43 mg, 34.1 mmol) in THF/water (1/1) (50 mL) was stirred at room temperature for 1 h. The mixture was diluted with water. The aqueous layer was slowly acidified with 3N HCl and extracted with EtOAc. The combined organic layers were dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)acetic acid 3c (4.6 g). The compound was used as such in the next step.

(64) Synthesis of intermediate 3d:

(65) HATU (2.10 g, 5.52 mmol) was added to a mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8] (747 mg, 3.68 mmol), 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxy-phenyl)amino)-2-(4-chlorophenyl)acetic acid 3c (1.5 g, 3.68 mmol) and diisopropylethylamine (1.82 mL, 11.03 mmol) in DMF (30 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water and extracted with EtOAc. The organic layer was separated, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 m, 40 g, heptane/EtOAc 85/15). The pure fractions were combined and evaporated to dryness to give 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chloro-phenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 3d (1.33 g).

(66) Synthesis of Compound 3 chiral separation into Enantiomers 3A and 3B:

(67) A mixture of 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chloro-phenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 3d (1.33 g, 2.24 mmol) in 4M HCl in dioxane (25 mL) was stirred at room temperature for 18 h. The mixture was diluted with water and was basified with K.sub.2CO.sub.3. The aqueous layer was extracted with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH 99/1/0.1). The pure fractions were combined and evaporated to dryness to afford 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 3 (1 g) as a racemic mixture. The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 55% CO.sub.2, 40% EtOH (+0.3% iPrNH.sub.2), 5% CH.sub.2Cl.sub.2). The enantiomers were further separated via preparative chiral SFC (Stationary phase: Chiralcel OD-H 5 m 25030 mm, Mobile phase: 50% CO.sub.2, 50% EtOH (+0.3% iPrNH.sub.2)) to give, after solidification in petroleum ether/diisopropyl ether, the first eluted Enantiomer 3A (294 mg) and the second eluted Enantiomer 3B (244 mg).

(68) Compound 3:

(69) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.08-3.25 (m, 2H) 3.58-3.68 (m, 5H) 3.79-3.89 (m, 2H) 4.00-4.11 (m, 1H) 4.47-4.57 (m, 1H) 4.79 (t, J=5.4 Hz, 1H) 5.56 (br d, J=8.8 Hz, 1H) 5.76 (s, 1H) 5.94 (s, 2H) 6.47 (br d, J=8.5 Hz, 1H) 7.01 (br d, J=7.9 Hz, 1H) 7.34 (d, J=8.2 Hz, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.55 (br d, J=8.2 Hz, 2H) 8.03 (br s, 1H)

(70) LC/MS (method LC-A): R.sub.t 3.36 min, MH.sup.+ 537

(71) Melting point: 162 C.

(72) Enantiomer 3A:

(73) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.08-3.25 (m, 2H) 3.60-3.68 (m, 5H) 3.79-3.89 (m, 2H) 4.05 (td, J=10.4, 7.3 Hz, 1H) 4.52 (td, J=10.4, 6.3 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.56 (d, J=8.8 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.94 (d, J=1.9 Hz, 2H) 6.47 (d, J=8.5 Hz, 1H) 7.01 (dd, J=8.0, 1.7 Hz, 1H) 7.33 (d, J=8.2 Hz, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.55 (d, J=8.5 Hz, 2H) 8.03 (s, 1H) LC/MS (method LC-A): R.sub.t 3.36 min, MH.sup.+ 537

(74) [].sub.D.sup.20: +51.9 (c 0.2736, DMF)

(75) Chiral SFC (method SFC-C): R.sub.t 2.55 min, MH.sup.+ 537, chiral purity 100%.

(76) Enantiomer 3B:

(77) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.08-3.26 (m, 2H) 3.59-3.68 (m, 5H) 3.80-3.90 (m, 2H) 4.05 (td, J=10.4, 7.3 Hz, 1H) 4.52 (td, J=10.4, 6.3 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.56 (d, J=8.8 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.94 (d, J=1.6 Hz, 2H) 6.47 (d, J=8.5 Hz, 1H) 7.01 (dd, J=8.2, 1.6 Hz, 1H) 7.34 (d, J=8.2 Hz, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.55 (d, J=8.5 Hz, 2H) 8.03 (s, 1H)

(78) LC/MS (method LC-A): R.sub.t 3.36 min, MH.sup.+ 537

(79) [].sub.D.sup.20: 51.1 (c 0.2973, DMF)

(80) Chiral SFC (method SFC-C): R.sub.t 3.56 min. MH.sup.+ 537, chiral purity 99.58%.

Example 3.2: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 3) and Chiral Separation into Enantiomers 3A and 3B

(81) ##STR00011##
Synthesis of Intermediate 3e:

(82) A mixture of methyl 2-bromo-2-(4-chlorophenyl)acetate 3a (19.2 g, 72.9 mmol), 2-(3-amino-5-methoxyphenoxy)ethanol [CAS 725237-16-1] (13.4 g, 72.9 mmol) and triethylamine (15.2 mL, 109.3 mmol) in CH.sub.3CN (115 mL) was heated under reflux for 3 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with 1N HCl. The organic phase was separated, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give crude intermediate 3e (30 g). This fraction was combined with another batch of crude intermediate 3e (total amount: 37 g) and purified by flash chromatography on silica gel (15-40 m, 400 g, heptane/EtOAc 60/40). The pure fractions were combined and evaporated to dryness to give methyl 2-(4-chlorophenyl)-2-((3-(2-hydroxy-ethoxy)-5-methoxyphenyl)amino)acetate 3e (26 g).

(83) Synthesis of Intermediate 3f:

(84) A mixture of methyl 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)-amino)acetate 3e (10 g, 27.3 mmol) and LiOH (3.44 g, 82.0 mmol) in THF/water (1/1) (200 mL) was stirred at room temperature for 2 h. The mixture was diluted with water. The aqueous layer was slowly acidified with 3N HCl and extracted with EtOAc. The organic layers were washed with water, separated, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 3f (9.5 g). The compound was used as such in the next step.

(85) Synthesis of Compound 3 and Chiral Separation into Enantiomers 3A and 3B:

(86) Under N.sub.2 flow at 5 C., propylphosphonic anhydride (2.56 mL, 4.26 mmol) was added dropwise to a mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8](577 mg, 2.84 mmol), 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)acetic acid 3f (1.3 g, 3.70 mmol) and diisopropylethylamine (1.03 mL, 6.25 mmol) in 2-Me-THF (30 mL). The mixture was stirred at room temperature for 4 h. Water was added and the mixture was extracted with EtOAc. The organic layer was washed with a 10% solution of K.sub.2CO.sub.3 in water and then with water, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel, (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99/1). The pure fractions were combined and evaporated to dryness, to give 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 3, 800 mg) as a racemic mixture. This fraction was combined with another batch (total amount: 1.4 g) and crystallized from diisopropyl ether to give 1.03 g of Compound 3. The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 55% CO.sub.2, 45% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer was further purified via reverse phase chromatography (stationary phase: X-bridge-C-18 10 m 30150 mm, mobile phase: 0.2% NH.sub.4HCO.sub.3/CH.sub.3CN gradient 60/40 to 0/100) to give Enantiomer 3A (312 mg). The second eluted Enantiomer 3B (436 mg) was not further purified.

Example 4: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 4) and Chiral Separation into Enantiomers 4A and 4B

(87) ##STR00012##
Synthesis of Intermediate 4a:

(88) HATU (2.24 g, 5.88 mmol) was added to a mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1] (734 mg, 3.92 mmol), 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxy-phenyl)amino)-2-(4-chlorophenyl)acetic acid 3c (1.6 g, 3.92 mmol) and diisopropylethylamine (1.95 mL, 11.8 mmol) in DMF (30 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water and EtOAc. The organic layer was separated, washed with a 10% solution of K.sub.2CO.sub.3 in water, washed with brine, dried over MgSO.sub.4, filtered, and the solvent was concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (15-40 m, 40 g, heptane/EtOAc 85/15). The pure fractions were combined and the solvent was concentrated under reduced pressure to give 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chloro-phenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4a (1.38 g).

(89) Synthesis of Compound 4 and Chiral Separation into Enantiomers 4A and 4B:

(90) 2-((3-(2-(tert-Butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4a (1.38 g, 2.39 mmol) was mixed with 4M HCl in dioxane (25 mL) stirred at room temperature for 18 h. The mixture was diluted with water and basified with K.sub.2CO.sub.3. The aqueous layer was extracted with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH 99/1/0.1). The pure fractions were combined and evaporated to dryness to afford 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 4, 1.08 g) as a racemic mixture. The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralcel OJ-H 5 m 25020 mm, Mobile phase: 60% CO.sub.2, 40% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (413 mg) was solidified in heptane/to give Enantiomer 4A (327 mg). The second eluted enantiomer (410 mg) was solidified in heptane/diisopropyl ether to give Enantiomer 4B (330 mg).

(91) Compound 4:

(92) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.16-3.32 (m, 2H) 3.59-3.68 (m, 5H) 3.79-3.90 (m, 2H) 4.03 (td, J=10.4, 6.9 Hz, 1H) 4.53 (td, J=10.3, 6.5 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.58 (d, J=8.5 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.95 (s, 2H) 6.44 (d, J=8.5 Hz, 1H) 7.37-7.41 (m, 1H) 7.43-7.49 (m, 3H) 7.56 (d, J=8.5 Hz, 2H) 8.38 (s, 1H)

(93) LC/MS (method LC-A): R.sub.t 3.31 min. MH.sup.+ 521

(94) Melting point: 160 C.

(95) Enantiomer 4A:

(96) .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 3.15-3.29 (m, 2H) 3.58-3.70 (m, 5H) 3.79-3.89 (m, 2H) 4.03 (td, J=10.4, 7.6 Hz, 1H) 4.53 (td, J=10.2, 6.3 Hz, 1H) 4.78 (br s, 1H) 5.57 (d, J=9.1 Hz, 1H) 5.74-5.80 (m, 1H) 5.95 (d, J=1.0 Hz, 2H) 6.43 (br d, J=8.6 Hz, 1H) 7.36-7.41 (m, 1H) 7.42-7.49 (m, 3H) 7.56 (d, J=8.6 Hz, 2H) 8.38 (s, 1H)

(97) LC/MS (method LC-B): R.sub.t 3.03 min, MH.sup.+ 521

(98) [].sub.D.sup.20: +52.0 (c 0.3036, DMF)

(99) Chiral SFC (method SFC-D): R.sub.t 1.82 min, MH.sup.+ 521, chiral purity 100%.

(100) Enantiomer 4B:

(101) .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 3.18-3.29 (m, 2H) 3.59-3.70 (m, 5H) 3.84 (m, 2H) 3.97-4.10 (m, 1H) 4.46-4.59 (m, 1H) 4.78 (br s, 1H) 5.57 (br d, J=8.6 Hz, 1H) 5.76 (s, 1H) 5.95 (s, 2H) 6.43 (br d, J=8.6 Hz, 1H) 7.35-7.50 (m, 4H) 7.56 (br d, J=8.6 Hz, 2H) 8.38 (s, 1H)

(102) LC/MS method LC-B): R.sub.t 3.03 min, MH.sup.+ 521

(103) [].sub.D.sup.20: 51.8 (c 0.3418, DMF)

(104) Chiral SFC (method SFC-D): R.sub.t 3.17 min, MH.sup.+ 521, chiral purity 99.56%.

Example 5: Synthesis of 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 5) and Chiral Separation into Enantiomers 5A and 5B

(105) ##STR00013##
Synthesis of Intermediate 5a:

(106) A mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8] (1 g, 4.92 mmol), 2-(4-chloro-2-methoxyphenyl)acetic acid [CAS 170737-95-8] (1.09 g, 5.41 mmol), HATU (2.81 g, 7.38 mmol) and diisopropylethylamine (2.44 mL, 14.8 mmol) in DMF (10 mL) was stirred at room temperature for 12 h. Water and EtOAc were added and the layers were separated. The organic layer was washed with water, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by chromatography on silica gel (15-40 m, 80 g, heptane/EtOAc 85/15). The pure fractions were combined and the solvent was concentrated under reduced pressure to give, after crystallization from CH.sub.3CN/heptane, 2-(4-chloro-2-methoxyphenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 5a (1.53 g).

(107) Synthesis of Intermediate 5b:

(108) At 78 C., under a N.sub.2 flow, LiHMDS 1M in THF (7.93 mL, 7.93 mmol) was added dropwise to a mixture of 2-(4-chloro-2-methoxyphenyl)-1-(6-(trifluoromethoxy)-indolin-1-yl)ethanone 5a (1.53 g, 3.97 mmol) in THF (12 mL). The mixture was stirred for 15 min at 78 C. and a solution of NBS (776 mg, 4.36 mmol) in THF (10 mL) was added dropwise. After stirring for 2 h at 78 C., the reaction was quenched by the addition of a saturated aqueous solution of NH.sub.4Cl. The mixture was extracted with EtOAc, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give 2-bromo-2-(4-chloro-2-methoxy-phenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 5b (1.70 g). The compound was used as such in the next step.

(109) Synthesis of Compound 5 and Chiral Separation into Enantiomers 5A and 5B:

(110) A mixture of 2-bromo-2-(4-chloro-2-methoxyphenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 5b (1.37 g, 2.95 mmol), 2-(3-amino-5-methoxyphenoxy)ethanol [CAS 725237-16-1] (810 mg, 4.42 mmol) and diisopropylethylamine (762 L, 4.42 mmol) in CH.sub.3CN (20 mL) was stirred at 50 C. for 8 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with 1N HC. The organic phase was separated, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2/MeOH 98.5/1.5). The pure fractions were combined and evaporated to dryness to give, after crystallization from CH.sub.3CN, 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 5, 500 mg) as a racemic mixture. This batch was combined with another batch (total amount: 903 mg). The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IC 5 m 25030 mm, Mobile phase: 65% CO.sub.2, 35% iPrOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (453 mg) was solidified in petroleum ether/diisopropyl ether to give Enantiomer 5A (355 mg). The second eluted enantiomer (436 mg) was solidified in petroleum ether/diisopropyl ether to give Enantiomer 5B (342 mg).

(111) Compound 5:

(112) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.11-3.23 (m, 2H) 3.59-3.67 (m, 5H) 3.79-3.87 (m, 2H) 3.90 (s, 3H) 4.01-4.09 (m, 1H) 4.32-4.41 (m, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.59 (d, J=8.5 Hz, 1H) 5.76 (s, 1H) 5.87 (br s, 2H) 6.46 (br d, J=8.8 Hz, 1H) 6.98-7.06 (m, 2H) 7.14 (d, J=1.6 Hz, 1H) 7.30-7.35 (m, 2H) 8.02 (s, 1H)

(113) LC/MS (method LC-A): R.sub.t 3.38 min, MH.sup.+ 567

(114) Melting point: 162 C.

(115) Enantiomer 5A:

(116) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.10-3.25 (m, 2H) 3.59-3.67 (m, 5H) 3.78-3.88 (m, 2H) 3.90 (s, 3H) 4.04 (td, J=10.3, 7.1 Hz, 1H) 4.37 (td, J=10.2, 6.8 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.59 (d, J=8.5 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.84-5.89 (m, 2H) 6.46 (d, J=8.5 Hz, 1H) 6.99-7.05 (m, 2H) 7.14 (d, J=2.2 Hz, 1H) 7.31 (d, J=8.2 Hz, 1H) 7.34 (d, J=8.2 Hz, 1H) 8.02 (s, 1H)

(117) LC/MS (method LC-A): R.sub.t 3.39 min, MH.sup.+ 567

(118) [].sub.D.sup.20: +31.1 (c 0.2736, DMF)

(119) Chiral SFC (method SFC-E): R.sub.t 2.02 min, MH.sup.+ 567, chiral purity 100%.

(120) Enantiomer 5B:

(121) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.09-3.25 (m, 2H) 3.60-3.67 (m, 5H) 3.78-3.87 (m, 2H) 3.90 (s, 3H) 4.04 (td, J=10.2, 6.9 Hz, 1H) 4.37 (td, J=10.2, 6.8 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.59 (d, J=8.5 Hz, 1H) 5.76 (t, J=1.9 Hz, 1H) 5.87 (br s, 2H) 6.47 (d, J=8.5 Hz, 1H) 6.99-7.05 (m, 2H) 7.14 (d, J=1.9 Hz, 1H) 7.31 (d, J=8.2 Hz, 1H) 7.34 (d, J=8.2 Hz, 1H) 8.02 (s, 1H)

(122) LC/MS (method LC-A): R.sub.t 3.39 min, MH.sup.+ 567

(123) [].sub.D.sup.20: 31.0 (c 0.2773, DMF)

(124) Chiral SFC (method SFC-E): R.sub.t 3.00 min. MH.sup.+ 567, chiral purity 100%.

Example 6.1: Synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 6)

(125) ##STR00014##
Synthesis of Compound 6:

(126) HATU (1.54 g, 4.06 mmol) was added to a mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8] (550 mg, 2.70 mmol), 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 2e (1 g, 2.70 mmol) and diisopropylethylamine (1.34 mL, 8.11 mmol) in DMF (30 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water. The precipitate was filtered off, and washed with water. The solid was dissolved in EtOAc, washed with a 10% solution of K.sub.2CO.sub.3 in water and then with brine. The organic layer was dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5) to afford 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoro-methoxy)indolin-1-yl)ethanone (Compound 6, 500 mg). An analytical sample of Compound 6 was obtained by crystallized from diisopropyl ether.

(127) Compound 6:

(128) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.14-3.22 (m, 2H) 3.58-3.68 (m, 5H) 3.80-3.90 (m, 2H) 4.05-4.15 (m, 1H) 4.38-4.47 (m, 1H) 4.80 (t, J=5.5 Hz, 1H) 5.70 (d, J=9.1 Hz, 1H) 5.79 (t, J=1.9 Hz, 1H) 5.93 (d, J=1.9 Hz, 2H) 6.63 (d, J=9.1 Hz, 1H) 7.03 (dd, J=8.2, 1.6 Hz, 1H) 7.31-7.37 (m, 2H) 7.42-7.51 (m, 2H) 8.02 (s, 1H)

(129) LC/MS (method LC-A): R.sub.t 3.39 min, MH.sup.+ 555

(130) Melting point: 166 C.

Example 6.2: Synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 6) and Chiral Separation into Enantiomers 6A and 6B

(131) ##STR00015##
Synthesis of Intermediate 6a:

(132) HATU (7.02 g, 18.5 mmol) was added to a mixture of 6-(trifluoromethoxy)indoline [CAS 953906-76-8] (2.5 g, 12.31 mmol), 2-(4-chloro-2-fluorophenyl)acetic acid [CAS 194240-75-0] (2.32 g, 12.3 mmol) and diisopropylethylamine (6.1 mL, 36.9 mmol) in DMF (100 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water and the precipitate was filtered off and washed with water. The residue was taken up with EtOAc and the organic layer was washed with a 10% solution of K.sub.2CO.sub.3 in water, washed with brine, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The crude product was crystallized from diisopropyl ether to give 2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6a (4 g).

(133) Synthesis of Intermediate 6b:

(134) At 78 C., under a N.sub.2 flow, LiHMDS 1M in THF (21.4 mL, 21.4 mmol) was added dropwise to a mixture of 2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6a (4 g, 10.7 mmol) in THF (60 mL). The mixture was stirred for 15 min at 78 C. and a solution of NBS (2.1 g, 11.8 mmol) in THF (40 mL) was added dropwise. After stirring for 2 h at 78 C., the reaction was quenched with a saturated aqueous solution of NH.sub.4Cl. The mixture was extracted with EtOAc, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give 2-bromo-2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6b (4.8 g). The compound was used as such in the next step.

(135) Synthesis of Compound 6 and chiral separation into Enantiomers 6A and 6B:

(136) A mixture of 2-bromo-2-(4-chloro-2-fluorophenyl)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6b (4.8 g, 10.6 mmol), 2-(3-amino-5-methoxyphenoxy)ethanol [CAS 725237-16-1] (2.3 g, 12.7 mmol) and diisopropylethylamine (2.2 mL, 12.7 mmol) in CH.sub.3CN (200 mL) was stirred at 70 C. for 72 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with 1N HCl and water. The organic phase was separated, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness to give racemic 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 6, 3 g) after crystallization from CH.sub.3CN/diisopropyl ether. The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralcel OD-H 5 m 25030 mm, Mobile phase: 60% CO.sub.2, 40% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (1.45 g) was solidified by trituration with MeOH/water to give Enantiomer 6A (1.409 g). The second eluted enantiomer (1.41 g) was solidified by trituration with MeOH/water to give Enantiomer 6B (1.37 g).

(137) Enantiomer 6A:

(138) .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 3.13-3.21 (m, 2H) 3.60-3.68 (m, 5H) 3.78-3.91 (m, 2H) 4.04-4.14 (m, 1H) 4.37-4.48 (m, 1H) 4.77 (t, J=5.6 Hz, 1H) 5.69 (d, J=9.1 Hz, 1H) 5.80 (s, 1H) 5.93 (d, J=1.5 Hz, 2H) 6.60 (br d, J=9.1 Hz, 1H) 7.02 (br d, J=8.1 Hz, 1H) 7.30-7.38 (m, 2H) 7.41-7.51 (m, 2H) 8.02 (s, 1H)

(139) LC/MS (method LC-A): R.sub.t 3.41 min, MH.sup.+ 555

(140) []D20: 25.9 (c 0.27, DMF)

(141) Chiral SFC (method SFC-F): R.sub.t 4.08 min, MH.sup.+ 555, chiral purity 100%.

(142) Enantiomer 6B:

(143) .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 3.13-3.20 (m, 2H) 3.59-3.68 (m, 5H) 3.79-3.91 (m, 2H) 4.04-4.14 (m, 1H) 4.38-4.49 (m, 1H) 4.77 (t, J=5.6 Hz, 1H) 5.69 (d, J=9.1 Hz, 1H) 5.80 (s, 1H) 5.93 (d, J=2.0 Hz, 2H) 6.60 (d, J=8.6 Hz, 1H) 7.02 (br d, J=9.1 Hz, 1H) 7.29-7.38 (m, 2H) 7.42-7.50 (m, 2H) 8.02 (s, 1H)

(144) LC/MS (method LC-A): R.sub.t 3.41 min, MH.sup.+ 555

(145) [].sub.D.sup.20: +23.3 (c 0.27, DMF)

(146) Chiral SFC (method SFC-F): R.sub.t 2.25 min, MH.sup.+ 555, chiral purity 99.42%.

Example 7.1: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 7)

(147) ##STR00016##
Synthesis of Intermediate 7a:

(148) A mixture of 1-methoxy-4-nitro-2-(trifluoromethyl)benzene [CAS 654-76-2] (24.5 g, 110.8 mmol) and 4-chlorophenoxyacetonitrile [CAS 3598-13-8] (20.4 g, 121.9 mmol) in DMF (100 mL) was added dropwise over 30 min to a stirred solution of tBuOK (27.4 g, 243.7 mmol) in DMF (100 mL) at 10 C. After addition, the purple solution was maintained at 10 C. for 1 h. Ice-water (500 mL) and 6N HCl (500 mL) were added and the precipitate was filtered off, washed with water and dried under vacuum to afford 2-(5-methoxy-2-nitro-4-(trifluoromethyl)-phenyl)acetonitrile 7a (40.4 g) which was used as such in the next step.

(149) Synthesis of Intermediate 7b:

(150) A solution of 2-(5-methoxy-2-nitro-4-(trifluoromethyl)phenyl)acetonitrile 7a (26 g, 99.9 mmol) in ethanol/water (9/1) (500 mL) and AcOH (5.2 mL) was hydrogenated for 1 h under pressure (3.5 Bar) with 10% Pd/C (15.3 g) as the catalyst. The reaction mixture was filtered through a pad of Celite and the filter cake was washed with a mixture of CH.sub.2Cl.sub.2 and CH.sub.3OH. The combined filtrates were concentrated under reduced pressure. The crude residue was filtered through a pad of silica (60-200 m) using heptane/EtOAc 80/20 as the eluent. The fractions containing the expected compound were combined and the solvent was concentrated under reduced pressure to give 5-methoxy-6-(trifluoromethyl)-1H-indole 7b (15.6 g).

(151) Synthesis of Intermediate 7c:

(152) At 0 C., BH.sub.3Pyridine (23.5 mL, 232.4 mmol) was added dropwise to a solution of 5-methoxy-6-(trifluoromethyl)-1H-indole 7b (10 g, 46.5 mmol) in EtOH (60 mL). 6N HCl (140 mL) was slowly added while maintaining the reaction temperature below 10 C. The mixture was stirred at 0 C. for 2 h. Water (200 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water, while keeping the reaction temperature below 20 C. The precipitate was filtered off, washed with water (twice) and co-evaporated under reduced pressure with toluene to give 5-methoxy-6-(trifluoromethyl)indoline 7c (9 g).

(153) Synthesis of Intermediate 7d:

(154) HATU (0.84 g, 2.21 mmol) was added to a mixture of 5-methoxy-6-(trifluoro-methyl)indoline 7c (320 mg, 1.47 mmol), 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxy-phenyl)amino)-2-(4-chlorophenyl)acetic acid 3c (631 mg, 1.55 mmol) and diisopropylethylamine (731 L, 4.42 mmol) in DMF (18 mL). The reaction mixture was stirred at room temperature for 12 h. The reaction was diluted with water and EtOAc. The organic layer was separated, washed with a 10% solution of K.sub.2CO.sub.3 in water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5) to give 2-((3-(2-(tert-butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)-1-(5-methoxy-6-(trifluoromethyl)-indolin-1-yl)ethanone 7d (839 mg).

(155) Synthesis of Compound 7:

(156) 2-((3-(2-(tert-Butoxy)ethoxy)-5-methoxyphenyl)amino)-2-(4-chlorophenyl)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 7d (1.15 g, 1.89 mmol) was added to 4M HCl in dioxane (20 mL) and the mixture was stirred at room temperature for 18 h. The mixture was diluted with water and was basified with K.sub.2CO.sub.3. The aqueous layer was extracted with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH 99/1/0.1). The pure fractions were combined and evaporated to dryness to afford 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)-indolin-1-yl)ethanone (Compound 7, 915 mg). An analytical sample of Compound 7 was obtained by crystallized from CH.sub.3CN.

(157) Compound 7:

(158) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.15-3.29 (m, 2H) 3.60-3.68 (m, 5H) 3.79-3.90 (m, 5H) 3.96-4.07 (m, 1H) 4.51 (td, J=10.4, 6.0 Hz, 1H) 4.78 (t, J=5.0 Hz, 1H) 5.54 (d, J=8.5 Hz, 1H) 5.76 (s, 1H) 5.95 (s, 2H) 6.40 (br d, J=8.5 Hz, 1H) 7.23 (s, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.56 (d, J=8.5 Hz, 2H) 8.34 (s, 1H)

(159) LC/MS (method LC-A): R.sub.t 3.21 min, MH.sup.+ 551

(160) Melting point: 188 C.

Example 7.2: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 7) and Chiral Separation into Enantiomers 7A and 7B

(161) ##STR00017##
Synthesis of Compound 7 and Chiral Separation into Enantiomers 7A and 7B:

(162) Under N.sub.2 flow, at 5 C., propylphosphonic anhydride (4.15 mL, 6.91 mmol) was added dropwise to a mixture of 5-methoxy-6-(trifluoromethyl)indoline 7c (1 g, 4.60 mmol), 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 3f (1.94 g, 5.53 mmol) and diisopropylethylamine (1.67 mL, 10.1 mmol) in DMF (20 mL). The mixture was stirred at room temperature for 7 h. Water was added and the mixture was extracted with EtOAc. The organic layer was washed with a 10% solution of K.sub.2CO.sub.3 in water, and then with water. The organic layer was dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Purification was carried out by flash chromatography on silica gel, (15-40 m, 90 g, CH.sub.2Cl.sub.2/MeOH 99/1). The pure fractions were combined and evaporated to dryness, to give 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 7, 2.17 g) as a racemic mixture. The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 45% CO.sub.2, 55% iPrOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (980 mg) was crystallized from MeOH to afford Enantiomer 7A (711 mg). The second eluted enantiomer (1.08 g) was further purified by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness (950 mg) to afford, after crystallization from MeOH, Enantiomer 7B (770 mg).

(163) Enantiomer 7A:

(164) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.15-3.31 (m, 2H) 3.60-3.68 (m, 5H) 3.79-3.90 (m, 5H) 3.95-4.04 (m, 1H) 4.51 (td, J=10.4, 6.3 Hz, 1H) 4.80 (t, J=5.4 Hz, 1H) 5.54 (d, J=8.5 Hz, 1H) 5.76 (s, 1H) 5.95 (s, 2H) 6.41 (d, J=8.5 Hz, 1H) 7.24 (s, 1H) 7.44 (d, J=8.2 Hz, 2H) 7.56 (d, J=8.5 Hz, 2H) 8.34 (s, 1H)

(165) LC/MS (method LC-A): R.sub.t 3.22 min, MH.sup.+ 551

(166) []D20: 45.2 (c 0.314, DMF)

(167) Chiral SFC (method SFC-G): R.sub.t 2.35 min, MH.sup.+ 551, chiral purity 100%.

(168) Melting point: 112 C.

(169) Enantiomer 7B:

(170) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.15-3.31 (m, 2H) 3.60-3.68 (m, 5H) 3.79-3.90 (m, 5H) 3.95-4.05 (m, 1H) 4.51 (td, J=10.3, 6.5 Hz, 1H) 4.80 (br t, J=5.0 Hz, 1H) 5.54 (d, J=8.8 Hz, 1H) 5.76 (s, 1H) 5.95 (s, 2H) 6.41 (d, J=8.8 Hz, 1H) 7.24 (s, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.56 (d, J=8.5 Hz, 2H) 8.34 (s, 1H)

(171) LC/MS (method LC-A): R.sub.t 3.21 min, MH.sup.+ 551

(172) []D20: +43.8 (c 0.27, DMF)

(173) Chiral SFC (method SFC-G): R.sub.t 3.84 min, MH.sup.+ 551, chiral purity 100%.

(174) Melting point: 112 C.

Example 8.1: Synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 8)

(175) ##STR00018##
Synthesis of Compound 8:

(176) HATU (308 mg, 0.81 mmol) was added to a mixture of 5-methoxy-6-(trifluoro-methyl)indoline 7c (117 mg, 0.54 mmol), 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 2e (200 mg, 0.54 mmol) and diisopropylethylamine (0.267 mL, 1.61 mmol) in DMF (10 mL). The reaction mixture was stirred at room temperature for 12 h. The reaction was diluted with water, causing precipitation. The precipitate was filtered off and washed with water. The solid was dissolved in EtOAc. The organic layer was washed with a 10% solution of K.sub.2CO.sub.3 in water and with brine, dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The residue was crystallized from Et.sub.2O/diisopropyl ether to give 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 8, 35 mg).

(177) Compound 8:

(178) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.19-3.28 (m, 2H) 3.59-3.69 (m, 5H) 3.85 (m, 5H) 3.99-4.08 (m, 1H) 4.37-4.47 (m, 1H) 4.80 (t, J=5.4 Hz, 1H) 5.68 (br d, J=9.1 Hz, 1H) 5.79 (s, 1H) 5.93 (s, 2H) 6.58 (br d, J=9.1 Hz, 1H) 7.26 (s, 1H) 7.33 (br d, J=7.6 Hz, 1H) 7.42-7.52 (m, 2H) 8.33 (s, 1H)

(179) LC/MS (method LC-A): R.sub.t 3.27 min, MH.sup.+ 569

(180) Melting point: 176 C.

Example 8.2: Synthesis of 2-(4-chloro-2-fluorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 8) and Chiral Separation into Enantiomers 8A and 8B

(181) ##STR00019##
Synthesis of intermediate 8a:

(182) HATU (2.9 g, 7.6 mmol) was added to a mixture of 5-methoxy-6-(trifluoromethyl)-indoline 7c (1.1 g, 5.06 mmol), 2-(4-chloro-2-fluorophenyl)acetic acid [CAS 194240-75-0] (1.05 g, 5.57 mmol) and diisopropylethylamine (2.51 mL, 15.2 mmol) in DMF (30 mL). The reaction mixture was stirred at room temperature for 12 h. The mixture was diluted with water, causing precipitation. The precipitate was filtered off and washed with water. The residue was taken up with EtOAc and the organic solution was washed with a 10% solution of K.sub.2CO.sub.3 in water and then with brine. The organic solution was dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure. The crude residue was purified by flash chromatography on silica gel (15-40 m, 80 g, heptane/EtOAc 90/10 to 60/40). The pure fractions were combined and the solvent was evaporated under reduced pressure to give 2-(4-chloro-2-fluorophenyl)-1-(5-methoxy-6-(trifluoromethyl)-indolin-1-yl)ethanone 8a (1.8 g).

(183) Synthesis of Intermediate 8b:

(184) At 78 C., under a N.sub.2 flow, LiHMDS 1M in THF (9.3 mL, 9.3 mmol) was added dropwise to a mixture of 2-(4-chloro-2-fluorophenyl)-1-(5-methoxy-6-(trifluoro-methyl)indolin-1-yl)ethanone 8a (1.8 g, 4.65 mmol) in THF (25 mL). TMSCl (0.7 mL, 0.86 mmol) was added dropwise. The mixture was stirred for 15 min at 78 C. and a solution of NBS (1 g, 5.57 mmol) in THF (15 mL) was added dropwise. After stirring for 2 h at 78 C., the reaction was quenched by the addition of a saturated aqueous solution of NH.sub.4Cl. The mixture was extracted with EtOAc. The organic layer was dried over MgSO.sub.4, filtered, and the solvent was evaporated under reduced pressure to give 2-bromo-2-(4-chloro-2-fluorophenyl)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 8b (2.1 g). The compound was used as such in the next step.

(185) Synthesis of Compound 8 and Chiral Separation into Enantiomers 8A and 8B:

(186) A mixture of 2-bromo-2-(4-chloro-2-fluorophenyl)-1-(5-methoxy-6-(trifluoromethyl)-indolin-1-yl)ethanone 8b (2.1 g, 4.5 mmol), 2-(3-amino-5-methoxyphenoxy)-ethanol [CAS 725237-16-1] (0.99 g, 5.4 mmol) and diisopropylethylamine (1.16 mL, 6.75 mmol) in CH.sub.3CN (80 mL) was stirred at 70 C. for 72 h. The mixture was concentrated under reduced pressure, diluted with EtOAc and washed with 1N HCl and water. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. The compound was purified by flash chromatography on silica gel (15-40 m, 80 g, CH.sub.2Cl.sub.2). The pure fractions were combined and evaporated to dryness to give racemic 2-(4-chloro-2-fluoro-phenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 8, 850 mg) after crystallization from CH.sub.3CN. This fraction was combined with another batch (total amount: 1.3 g). The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 70% CO.sub.2, 30% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (680 mg) was solidified by trituration with CH.sub.3CN to give Enantiomer 8A (590 mg). The second eluted enantiomer (630 mg) was solidified by trituration with CH.sub.3CN to give Enantiomer 8B (569 mg).

(187) Enantiomer 8A:

(188) .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 3.19-3.27 (m, 2H) 3.59-3.69 (m, 5H) 3.78-3.91 (m, 5H) 3.98-4.08 (m, 1H) 4.35-4.47 (m, 1H) 4.77 (t, J=5.6 Hz, 1H) 5.67 (d, J=9.1 Hz, 1H) 5.79 (t, J=1.8 Hz, 1H) 5.93 (d, J=2.0 Hz, 2H) 6.55 (d, J=9.1 Hz, 1H) 7.25 (s, 1H) 7.31 (dd, J=8.3, 1.8 Hz, 1H) 7.42-7.50 (m, 2H) 8.32 (s, 1H)

(189) LC/MS (method LC-A): R.sub.t 3.26 min, MH.sup.+ 569

(190) [].sub.D.sup.20: 28.9 (c 0.225, DMF)

(191) Chiral SFC (method SFC-H): R.sub.t 4.51 min, MH.sup.+ 569, chiral purity 100%.

(192) Enantiomer 8B:

(193) .sup.1H NMR (400 MHz, DMSO-d.sub.6) ppm 3.19-3.27 (m, 2H) 3.59-3.68 (m, 5H) 3.80-3.90 (m, 5H) 3.98-4.09 (m, 1H) 4.35-4.47 (m, 1H) 4.77 (t, J=5.3 Hz, 1H) 5.67 (d, J=9.1 Hz, 1H) 5.79 (t, J=1.8 Hz, 1H) 5.93 (d, J=1.5 Hz, 2H) 6.54 (d, J=8.6 Hz, 1H) 7.25 (s, 1H) 7.31 (dd, J=8.6, 2.0 Hz, 1H) 7.42-7.50 (m, 2H) 8.32 (s, 1H)

(194) LC/MS (method LC-A): R.sub.t 3.26 min, MH.sup.+ 569

(195) [].sub.D.sup.20: +25.7 (c 0.2333, DMF)

(196) Chiral SFC (method SFC-H): R.sub.t 5.81 min, MH.sup.+ 569, chiral purity 100%.

Example 9: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 9) and Chiral Separation into Enantiomers 9A and 9B

(197) ##STR00020## ##STR00021##
Synthesis of Intermediate 9a:

(198) To a solution of 2-methyl-4-(trifluoromethoxy)aniline [CAS 86256-59-9] (10.0 g, 52.3 mmol) in dioxane (20 mL) was added trifluoroacetic anhydride (8 mL, 57.2 mmol). The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was partitioned between EtOAc and 1N HCl. The phases were separated. The organic phase was washed with a saturated solution of NaHCO.sub.3 in water, H.sub.2O and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to afford 14.7 g of 2,2,2-trifluoro-N-(2-methyl-4-(trifluoromethoxy)phenyl)acetamide 9a as a white powder. The compound was used in the next step without further purification.

(199) Synthesis of Intermediate 9c:

(200) To acetic anhydride (11.4 mL, 61.1 mmol), cooled at 0 C. was added dropwise 70% nitric acid (3.9 mL). 2,2,2-Trifluoro-N-(2-methyl-4-(trifluoromethoxy)phenyl)-acetamide 9a (5 g, 17.4 mmol) was added portionwise and the reaction mixture was heated at 55 C. for 12 h. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with H.sub.2O. The organic phase was washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was dissolved in methanol (46 mL). 2M K.sub.2CO.sub.3 (23 mL, 46 mmol) was added and the reaction mixture was heated at 70 C. for 4 h. More 2M K.sub.2CO.sub.3 (10 mL, 20 mmol) was added and the reaction mixture was heated at 70 C. for 12 h. The reaction mixture was partially concentrated under reduced pressure to remove methanol. The residue was extracted with EtOAc. The organic phase was washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (20% to 50%) in heptane to afford 3.6 g of 2-methyl-6-nitro-4-(trifluoromethoxy)aniline 9c as a yellow solid.

(201) Synthesis of Intermediate 9d:

(202) To a solution of 2-methyl-6-nitro-4-(trifluoromethoxy)aniline 9c (1.8 g, 7.69 mmol) in acetic acid (10.9 mL) was added dropwise a solution of sodium nitrite (0.806 g, 11.7 mmol) in H.sub.2SO.sub.4/H.sub.2O (2 mL, 1/1). The reaction mixture was stirred at room temperature for 30 min. H.sub.2O (22 mL) and urea (0.802 g, 13.4 mmol) were added. After 10 min at room temperature, a solution of potassium iodide (1.7 g, 10.2 mmol) in H.sub.2O (11 mL) was added dropwise. The reaction mixture was stirred at room temperature for 30 min. The yellow solid was filtered off, washed with H.sub.2O and dried to give 2.4 g of 2-iodo-1-methyl-3-nitro-5-(trifluoromethoxy)benzene 9d.

(203) Synthesis of Intermediate 9e:

(204) To a solution of 2-iodo-1-methyl-3-nitro-5-(trifluoromethoxy)benzene 9d (3.5 g, 10.0 mmol) in EtOH (30 mL) was added a solution of NH.sub.4Cl (2.7 g, 49.9 mmol) in H.sub.2O (30 mL). The reaction mixture was heated at 50 C. Iron (2.6 g, 46.9 mmol) was added and the reaction mixture was heated under reflux for 40 min. After cooling to room temperature, the reaction mixture was filtered through Celite. The solids were washed with EtOH. The filtrate was partially concentrated under reduced pressure to remove EtOH. The residue was partitioned between EtOAc and a saturated solution of NaHCO.sub.3 in water. The phases were separated. The organic phase was washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 25%) in heptane to afford 2.9 g of 2-iodo-3-methyl-5-(trifluoromethoxy)aniline 9e as a yellow oil.

(205) Synthesis of Intermediate 9f:

(206) A solution of 2-iodo-3-methyl-5-(trifluoromethoxy)aniline 9e (2.9 g, 9.1 mmol) in triethylamine (23 mL) was degassed with argon for 15 min. Dichlorobis(triphenylphosphine)palladium(II) (0.327 g, 0.47 mmol), copper(I) iodide (0.164 g, 0.86 mmol) and trimethylsilylacetylene (1.8 mL, 13.1 mmol) were added. The reaction mixture was heated at 65 C. for 12 h. After cooling to room temperature, the reaction mixture was diluted with H.sub.2O and extracted with EtOAc (3). The organic phases were combined, washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 20%) in heptane to afford 2.6 g of 3-methyl-5-(trifluoromethoxy)-2-((trimethylsilyl)-ethynyl)aniline 9f as an orange oil.

(207) Synthesis of Intermediate 9g:

(208) To a solution of 3-methyl-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 9f (2.7 g, 9.3 mmol) in NMP (27 mL) was added tBuOK (3.1 g, 27.8 mmol). The reaction mixture was heated at 80 C. for 4 h. After cooling to room temperature, the reaction mixture was diluted with H.sub.2O and extracted with EtOAc (2). The organic phases were combined, washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 20%) in heptane to afford 1.7 g of 4-methyl-6-(trifluoromethoxy)-1H-indole 9g as an orange oil.

(209) Synthesis of Intermediate 9h:

(210) At 0 C., BH.sub.3Pyridine (1.2 mL, 11.6 mmol) was added dropwise to a solution of 4-methyl-6-(trifluoromethoxy)-1H-indole 9g (0.5 g, 2.32 mmol) in EtOH (3 mL). 6N HCl (6 mL) was slowly added dropwise while maintaining the reaction temperature below 10 C. The mixture was stirred at 0 C. for 3 h. Water (12 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20 C.). The mixture was extracted with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure to give 450 mg of 4-methyl-6-(trifluoromethoxy)indoline 9h.

(211) Synthesis of Compound 9 and Chiral Separation into Enantiomers 9A and 9B:

(212) Under N.sub.2 flow at 5 C., propylphosphonic anhydride (1.87 mL, 3.11 mmol) was added dropwise to a mixture of 4-methyl-6-(trifluoromethoxy)indoline 9h (450 mg, 2.07 mmol), 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)-amino)acetic acid 3f (729 mg, 2.07 mmol) and diisopropylethylamine (753 L, 4.56 mmol) in DMF (20 mL). The mixture was stirred at room temperature for 7 h. Water was added and the mixture was extracted with EtOAc. The organic layer was washed with a solution of K.sub.2CO.sub.3 10% in water, then with water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Purification was performed by flash chromatography on silica gel, (15-40 m, 24 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness, to give racemic 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(4-methyl-6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 9, 411 mg). The enantiomers of Compound 9 were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 50% CO.sub.2, 50% iPrOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (180 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 9A (121 mg). The second eluted enantiomer (180 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 9B (132 mg).

(213) Enantiomer 9A:

(214) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 2.20 (s, 3H) 2.98-3.17 (m, 2H) 3.59-3.69 (m, 5H) 3.79-3.90 (m, 2H) 4.05 (td, J=10.6, 6.9 Hz, 1H) 4.52 (td, J=10.5, 6.1 Hz, 1H) 4.77 (t, J=5.5 Hz, 1H) 5.55 (d, J=8.8 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.94 (d, J=1.9 Hz, 2H) 6.43 (d, J=8.8 Hz, 1H) 6.88 (s, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.55 (d, J=8.5 Hz, 2H) 7.88 (s, 1H)

(215) LC/MS (method LC-A): R.sub.t 3.48 min, MH.sup.+ 551

(216) [].sub.D.sup.20: 40.6 (c 0.2067, DMF)

(217) Chiral SFC (method SFC-G): R.sub.t 2.08 min, MH.sup.+ 551, chiral purity 100%.

(218) Enantiomer 9B:

(219) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 2.20 (s, 3H) 2.98-3.17 (m, 2H) 3.59-3.67 (m, 5H) 3.79-3.90 (m, 2H) 4.05 (td, J=10.6, 6.9 Hz, 1H) 4.52 (td, J=10.5, 6.1 Hz, 1H) 4.77 (t, J=5.5 Hz, 1H) 5.55 (d, J=8.5 Hz, 1H) 5.76 (t, J=1.9 Hz, 1H) 5.94 (d, J=1.9 Hz, 2H) 6.43 (d, J=8.8 Hz, 1H) 6.88 (s, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.55 (d, J=8.5 Hz, 2H) 7.88 (s, 1H)

(220) LC/MS (method LC-A): R.sub.t 3.48 min, MH.sup.+ 551

(221) [].sub.D.sup.20 +42.6 (c 0.2392, DMF)

(222) Chiral SFC (method SFC-G): R.sub.t 3.34 min, MH.sup.+ 551, chiral purity 99.7%.

Example 10: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(4-methyl-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 10) and Chiral Separation into Enantiomers 10A and 10B

(223) ##STR00022##
Synthesis of Intermediate 10a:

(224) Pd/C (10%) (1.18 g) was added to a solution of 1-benzyl-4-methyl-6-(trifluoro-methyl)indoline [CAS 1156512-79-6] (11.8 g, 40.5 mmol) in AcOH (11.8 mL) and MeOH (118 mL). The reaction was stirred at room temperature for 12 h under H.sub.2 atmosphere. The mixture was filtered through a pad of Celite and concentrated under reduced pressure. The residue was taken up with CH.sub.2Cl.sub.2, washed with water, brine, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (heptane/EtOAc 9/1). The pure fractions were combined and the solvent was evaporated to dryness to give 8.2 g of 4-methyl-6-(trifluoromethyl)indoline 10a.

(225) Synthesis of Compound 10 and Chiral Separation into Enantiomers 10A and 10B:

(226) A mixture of 4-methyl-6-(trifluoromethyl)indoline 10a (515 mg, 2.56 mmol), 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 3f (900 mg, 2.56 mmol), diisopropylethylamine (1.27 mL, 7.67 mmol) and HATU (1.46 g, 3.84 mmol) in DMF (7 mL) was stirred at room temperature for 18 h. Water was added and the mixture was extracted with EtOAc. The organic layer was washed with water (several times), dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Purification was performed by flash chromatography on silica gel, (15-40 m, 40 g, heptane/EtOAc 50/50). This fraction was combined with another batch (total amount: 640 mg) and further purified by reverse phase chromatography (Stationary phase: X-Bridge C18 m 30150 mm, Mobile phase: gradient from 60% NH.sub.4HCO.sub.3 0.2%, 40% CH.sub.3CN to 0% NH.sub.4HCO.sub.3 0.2%, 100% CH.sub.3CN). The pure fractions were combined and evaporated to dryness, to give racemic 2-(4-chlorophenyl)-2-((3-(2-hydroxy-ethoxy)-5-methoxyphenyl)amino)-1-(4-methyl-6-(trifluoromethyl)indolin-1-yl)-ethanone (Compound 10, 425 mg). The enantiomers of Compound 10 were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 50% CO.sub.2, 50% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (180 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 10A (145 mg). The second eluted enantiomer (170 mg) was solidified from heptane/diisopropyl ether to afford Enantiomer 10B (113 mg).

(227) Enantiomer 10A:

(228) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 2.25 (s, 3H) 3.05-3.24 (m, 2H) 3.60-3.68 (m, 5H) 3.80-3.90 (m, 2H) 4.04 (td, J=10.6, 6.9 Hz, 1H) 4.54 (td, J=10.5, 6.1 Hz, 1H) 4.78 (t, J=5.5 Hz, 1H) 5.57 (d, J=8.5 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.95 (d, J=0.9 Hz, 2H) 6.42 (d, J=8.5 Hz, 1H) 7.25 (s, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.56 (d, J=8.5 Hz, 2H) 8.23 (s, 1H)

(229) LC/MS (method LC-A): R.sub.t 3.43 min, MH.sup.+ 535

(230) [].sub.D.sup.20: 46.2 (c 0.2275, DMF)

(231) Chiral SFC (method SFC-1): R.sub.t 2.26 min, MH.sup.+ 535, chiral purity 100%.

(232) Enantiomer 10B:

(233) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 2.25 (s, 3H) 3.05-3.24 (m, 2H) 3.59-3.68 (m, 5H) 3.80-3.91 (m, 2H) 4.04 (td, J=10.5, 7.1 Hz, 1H) 4.54 (td, J=10.4, 6.3 Hz, 1H) 4.78 (t, J=5.5 Hz, 1H) 5.57 (d, J=8.5 Hz, 1H) 5.76 (t, J=2.0 Hz, 1H) 5.95 (d, J=0.9 Hz, 2H) 6.42 (d, J=8.8 Hz, 1H) 7.25 (s, 1H) 7.44 (d, J=8.5 Hz, 2H) 7.56 (d, J=8.2 Hz, 2H) 8.23 (s, 1H)

(234) LC/MS (method LC-A): R.sub.t 3.43 min, MH.sup.+ 535

(235) [].sub.D.sup.20: +43.0 (c 0.2092, DMF)

(236) Chiral SFC (method SFC-1): R.sub.t 3.61 min. MH.sup.+ 535, chiral purity 100%.

Example 11: Synthesis of 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 11) and Chiral Separation into Enantiomers 11A and 11B

(237) ##STR00023##
Synthesis of Intermediate 11a:

(238) At 78 C., under a N.sub.2 flow, LiHMDS 1.5 M in THF (6.2 mL, 9.32 mmol) was added dropwise to a mixture of methyl 2-(4-chloro-2-methoxyphenyl)acetate [CAS 193290-23-2] (1 g, 4.66 mmol) in THF (30 mL). A solution of TMSCl (0.95 mL, 7.45 mmol) in THF (10 mL) was added dropwise. The mixture was stirred for 15 min at 78 C. and NBS (0.912 g, 5.13 mmol) in THF (10 mL) was added dropwise. After stirring for 2 h at 78 C., the reaction was quenched with a saturated solution of NH.sub.4Cl. The mixture was extracted with EtOAc, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure to give methyl 2-bromo-2-(4-chloro-2-methoxyphenyl)acetate 11a (1.4 g). The compound was used as such in the next step.

(239) Synthesis of Intermediate 11b:

(240) A mixture of methyl 2-bromo-2-(4-chloro-2-methoxyphenyl)acetate 11a (0.5 g, 1.71 mmol), 2-(3-amino-5-methoxyphenoxy)ethanol [CAS 725237-16-1] (328 mg, 1.79 mmol), trimethylamine (355 L, 2.56 mmol) in CH.sub.3CN (10 mL) was stirred at 50 C. for 12 h. The mixture was diluted with EtOAc and washed with water. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99/1). The pure fractions were combined and evaporated to dryness to give methyl 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetate 11b (510 mg).

(241) Synthesis of Intermediate 11c:

(242) Methyl 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)-amino)acetate 11b (1.2 g, 3.03 mmol) and LiOH (382 mg, 9.10 mmol) in THF/water (1/1) (20 mL) was stirred at room temperature for 1 h. The mixture was diluted with water. The aqueous layer was slowly acidified with 3N HCl and extracted with EtOAc. The combined organic layers were dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure to give 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 11c (1.12 g). The compound was used as such in the next step.

(243) Synthesis of Compound 11 and Chiral Separation into Enantiomers 11A and 11B:

(244) HATU (0.692 g, 1.82 mmol) was added to a mixture of 5-methoxy-6-(trifluoro-methyl)indoline 7c (264 mg, 1.21 mmol), 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)acetic acid 11c (520 mg, 1.36 mmol) and diisopropylethylamine (0.602 mL, 3.64 mmol) in DMF (14 mL). The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with water and EtOAc. The organic layer was separated, washed with a 10% solution of K.sub.2CO.sub.3 in water and then with brine. The organic layer was dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography on silica gel (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fraction were combined and evaporated to dryness to afford racemic 2-(4-chloro-2-methoxyphenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 11, 398 mg). This batch was combined with another batch (total amount: 535 mg). The enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IA 5 m 25020 mm, Mobile phase: 70% CO.sub.2, 30% iPrOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (240 mg) was crystallized from CH.sub.3CN/diisopropyl ether to give Enantiomer 11A (194 mg). The second eluted enantiomer (240 mg) was crystallized from CH.sub.3CN/diisopropyl ether to give Enantiomer 11B (189 mg).

(245) Compound 11:

(246) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.19-3.29 (m, 2H) 3.59-3.69 (m, 5H) 3.78-3.88 (m, 5H) 3.91 (s, 3H) 3.95-4.04 (m, 1H) 4.31-4.41 (m, 1H) 4.80 (t, J=5.4 Hz, 1H) 5.59 (br d, J=8.5 Hz, 1H) 5.73-5.78 (m, 1H) 5.87 (br s, 2H) 6.41 (br d, J=8.5 Hz, 1H) 7.03 (dd, J=8.2, 1.3 Hz, 1H) 7.15 (d, J=1.3 Hz, 1H) 7.25 (s, 1H) 7.33 (d, J=8.2 Hz, 1H) 8.33 (s, 1H)

(247) LC/MS (method LC-A): R.sub.t 3.22 min, MH.sup.+ 581

(248) Melting point: 224 C.

(249) Enantiomer 11A:

(250) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.16-3.30 (m, 2H) 3.60-3.68 (m, 5H) 3.77-3.88 (m, 5H) 3.90 (s, 3H) 3.99 (td, J=10.2, 7.3 Hz, 1H) 4.35 (td, J=10.2, 6.6 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.58 (d, J=8.5 Hz, 1H) 5.73-5.78 (m, 1H) 5.87 (s, 2H) 6.41 (d, J=8.8 Hz, 1H) 7.02 (dd, J=8.2, 1.9 Hz, 1H) 7.14 (d, J=1.9 Hz, 1H) 7.24 (s, 1H) 7.32 (d, J=8.5 Hz, 1H) 8.33 (s, 1H)

(251) LC/MS (method LC-A): R.sub.t 3.25 min, MH.sup.+ 581

(252) []D20: 31.4 (c 0.274, DMF)

(253) Chiral SFC (method SFC-H): R.sub.t 3.60 min, MH.sup.+ 581, chiral purity 100%.

(254) Melting point: 175 C.

(255) Enantiomer 11B:

(256) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.17-3.30 (m, 2H) 3.59-3.68 (m, 5H) 3.78-3.88 (m, 5H) 3.91 (s, 3H) 3.99 (td, J=10.2, 7.3 Hz, 1H) 4.35 (td, J=10.2, 6.6 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.58 (d, J=8.5 Hz, 1H) 5.73-5.78 (m, 1H) 5.87 (s, 2H) 6.41 (d, J=8.8 Hz, 1H) 7.02 (dd, J=8.2, 1.9 Hz, 1H) 7.14 (d, J=1.9 Hz, 1H) 7.24 (s, 1H) 7.32 (d, J=8.2 Hz, 1H) 8.33 (s, 1H)

(257) LC/MS (method LC-A): R.sub.t 3.25 min, MH.sup.+ 581

(258) []D20: +29.4 (c 0.272, DMF)

(259) Chiral SFC (method SFC-H): R.sub.t 4.96 min, MH.sup.+ 581, chiral purity 100%.

(260) Melting point: 175 C.

Example 12: Synthesis of 2-(4-chlorophenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)ethanone (Compound 12) and Chiral Separation into Enantiomers 12A and 12B

(261) ##STR00024##
Synthesis of Intermediate 12a:

(262) At 0 C., BH.sub.3Pyridine (10.5 mL, 103.5 mmol) was added dropwise to a solution of 5-fluoro-6-(trifluoromethyl)-1H-indole [CAS 1493800-10-4] (7 g, 34.5 mmol) in EtOH (45 mL). 6N HCl (105 mL) was slowly added dropwise while maintaining the reaction temperature below 10 C. The mixture was stirred at 0 C. for 3 h. Water (210 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20 C.). The mixture was extracted with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure. Purification was performed by flash chromatography on silica gel, (15-40 m, 120 g, heptane/EtOAc 85/15). The pure fractions were combined and evaporated to dryness to give 3.5 g of 5-fluoro-6-(trifluoromethyl)indoline 12a.

(263) Synthesis of Compound 12 and Chiral Separation into Enantiomers 12A and 12B:

(264) Under N.sub.2 flow, at 5 C., propylphosphonic anhydride (1.76 mL, 2.93 mmol) was added dropwise to a mixture of 5-fluoro-6-(trifluoromethyl)indoline 12a (400 mg, 1.95 mmol), 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)-amino)acetic acid 3f (823 mg, 2.34 mmol) and diisopropylethylamine (709 L, 4.29 mmol) in DMF (30 mL). The mixture was stirred at room temperature for 5 h. Water was added and the precipitate was filtered off and washed with a 10% solution of K.sub.2CO.sub.3 in water. The solid was taken up with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Purification was performed by flash chromatography on silica gel, (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and evaporated to dryness, to give racemic 2-(4-chloro-phenyl)-1-(5-fluoro-6-(trifluoromethyl)indolin-1-yl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)ethanone (Compound 12, 825 mg). Crystallization from CH.sub.3CN/diisopropyl ether afforded Compound 12 (448 mg) as a crystalline fraction. The enantiomers of Compound 12 were separated via Preparative Chiral SFC (Stationary phase: Chiralcel OJ-H 5 m 25020 mm, Mobile phase: 60% CO.sub.2, 40% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (193 mg) was solidified from heptane/diisopropyl ether to give Enantiomer 12A (164 mg). The second eluted enantiomer (190 mg) was solidified from heptane/diisopropyl ether to give Enantiomer 12B (131 mg).

(265) Compound 12:

(266) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.18-3.31 (m, 2H) 3.62 (s, 3H) 3.63-3.67 (m, 2H) 3.80-3.89 (m, 2H) 4.03 (td, J=10.3, 7.4 Hz, 1H) 4.54 (td, J=10.2, 6.3 Hz, 1H) 4.79 (br t, J=5.0 Hz, 1H) 5.57 (d, J=8.5 Hz, 1H) 5.76 (s, 1H) 5.94 (s, 2H) 6.44 (d, J=8.8 Hz, 1H) 7.42-7.48 (m, 3H) 7.55 (d, J=8.2 Hz, 2H) 8.39 (d, J=6.6 Hz, 1H)

(267) LC/MS (method LC-A): R.sub.t 3.37 min, MH.sup.+ 539

(268) Melting point: 130 C.

(269) Enantiomer 12A:

(270) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.17-3.32 (m, 2H) 3.62 (s, 3H) 3.64 (q, J=5.6 Hz, 2H) 3.79-3.90 (m, 2H) 4.03 (td, J=10.2, 7.3 Hz, 1H) 4.54 (td, J=10.4, 6.3 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.57 (d, J=8.5 Hz, 1H) 5.76 (s, 1H) 5.94 (s, 2H) 6.44 (d, J=8.8 Hz, 1H) 7.41-7.48 (m, 3H) 7.55 (d, J=8.5 Hz, 2H) 8.39 (d, J=6.3 Hz, 1H)

(271) LC/MS (method LC-A): R.sub.t 3.37 min, MH.sup.+ 539

(272) []D20: +50.2 (c 0.299, DMF)

(273) Chiral SFC (method SFC-D): R.sub.t 1.91 min, MH.sup.+ 539, chiral purity 100%.

(274) Enantiomer 12B:

(275) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.18-3.32 (m, 2H) 3.62 (s, 3H) 3.65 (q, J=5.3 Hz, 2H) 3.78-3.90 (m, 2H) 4.03 (td, J=10.2, 7.3 Hz, 1H) 4.54 (td, J=10.2, 6.3 Hz, 1H) 4.79 (t, J=5.4 Hz, 1H) 5.57 (d, J=8.8 Hz, 1H) 5.76 (s, 1H) 5.94 (s, 2H) 6.44 (d, J=8.5 Hz, 1H) 7.41-7.49 (m, 3H) 7.55 (d, J=8.2 Hz, 2H) 8.39 (d, J=6.6 Hz, 1H)

(276) LC/MS (method LC-A): R.sub.t 3.38 min. MH.sup.+ 539

(277) [].sub.D.sup.20: 51.0 (c 0.3, DMF)

(278) Chiral SFC (method SFC-D): R.sub.t 3.93 min, MH.sup.+ 539, chiral purity 99.52%.

Example 13: Synthesis of 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)-1-(5-methoxy-6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 13) and Chiral Separation into Enantiomers 13A and 13B

(279) ##STR00025## ##STR00026##
Synthesis of Intermediate 13a:

(280) A solution of 4-methoxy-3-(trifluoromethoxy)aniline [CAS 647855-21-8] (3.1 g, 15.0 mmol) in toluene (50 mL) was treated with N-bromosuccinimide (2.8 g, 15.7 mmol) at 5 C. and the resulting mixture was stirred at 5-10 C. for 2 h. The mixture was diluted with water and extracted with EtOAc. The combined extracts were dried over MgSO.sub.4, filtered and evaporated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 m, 24 g, heptane/EtOAc 95/5 to 90/10) The pure fractions were combined and evaporated to dryness to give 2-bromo-4-methoxy-5-(trifluoromethoxy)aniline 13a (2.5 g).

(281) Synthesis of Intermediate 13b:

(282) A solution of 2-bromo-4-methoxy-5-(trifluoromethoxy)aniline 13a (2.72 g, 9.51 mmol) in DMF (30 mL) was degassed with N.sub.2 for 15 min. Dichlorobis(triphenylphosphine)palladium(II) (667 mg, 0.95 mmol), copper(I) iodide (362 mg, 1.90 mmol), triethylamine (3.96 mL, 28.53 mmol) and trimethyl-silylacetylene (3.95 mL, 28.5 mmol) were added. The reaction mixture was heated at 70 C. for 12 h under N.sub.2 flow. After cooling to room temperature, the reaction mixture was diluted with H.sub.2O and extracted with EtOAc. The organic phases were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 m, 80 g, heptane/EtOAc 85/15). The pure fractions were combined and evaporated to dryness to give 4-methoxy-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 13b (1.4 g).

(283) Synthesis of Intermediate 13c:

(284) To a solution of 4-methoxy-5-(trifluoromethoxy)-2-((trimethylsilyl)ethynyl)aniline 13b (1.2 g, 3.96 mmol) in NMP (11 mL) under N.sub.2 flow, was added tBuOK (1.33 g, 11.9 mmol) in one portion. The reaction mixture was heated at 80 C. for 4 h, then poured out into ice/water and acidified with 3N HCl until pH 4-5. The reaction mixture was extracted with EtOAc. The organic phases were combined, washed with H.sub.2O, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 m, 40 g, heptane/EtOAc 85/15). The pure fractions were combined and evaporated to dryness to give 5-methoxy-6-(trifluoromethoxy)-1H-indole 13c (490 mg).

(285) Synthesis of Intermediate 13d:

(286) At 0 C., BH.sub.3Pyridine (10.5 mL, 103.82 mmol) was added dropwise to a solution of 5-methoxy-6-(trifluoromethoxy)-1H-indole 13c (8 g, 34.6 mmol) in EtOH (45 mL). 6N HCl (6 mL) was slowly added dropwise while maintaining the temperature below 10 C. The mixture was stirred at 0 C. for 3 h. Water (210 mL) was added and the mixture was basified until pH 8-9 with a concentrated solution of NaOH in water (the reaction temperature was kept below 20 C.). The mixture was extracted with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Toluene was added and the solution was concentrated under reduced pressure to give 7.5 g of 5-methoxy-6-(trifluoromethoxy)indoline 13d.

(287) Synthesis of Compound 13 and Chiral Separation into Enantiomers 13A and 13B:

(288) Under N.sub.2 flow at 5 C., propylphosphonic anhydride (2.1 mL, 3.35 mmol) was added dropwise to a mixture of 5-methoxy-6-(trifluoromethoxy)indoline 13d (552 mg, 2.37 mmol), 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxy-phenyl)amino)acetic acid 3f (1 g, 2.84 mmol) and diisopropylethylamine (861 L, 5.21 mmol) in DMF (40 mL). The mixture was stirred at room temperature for 5 h. Water was added and the precipitate was filtered off and washed with a 10% solution of K.sub.2CO.sub.3 in water. The solid was taken up with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was evaporated under reduced pressure. Purification was performed by flash chromatography on silica gel, (15-40 m, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). Pure fractions were combined and evaporated to dryness, to give, after crystallization from diisopropyl ether, racemic 2-(4-chlorophenyl)-2-((3-(2-hydroxyethoxy)-5-methoxyphenyl)amino)-1-(5-methoxy-6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 13, 970 mg). The enantiomers of Compound 13 were separated via Preparative Chiral SFC (Stationary phase: Chiralpak IC 5 m 25030 mm, Mobile phase: 55% CO.sub.2, 45% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (400 mg) was solidified from heptane/diisopropyl ether to give Enantiomer 13A (332 mg). The second eluted enantiomer (397 mg) was solidified from heptane/diisopropyl ether to give Enantiomer 13B (344 mg).

(289) Compound 13:

(290) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.10-3.26 (m, 2H) 3.62 (s, 3H) 3.65 (q, J=5.4 Hz, 2H) 3.81 (s, 3H) 3.82-3.88 (m, 2H) 4.03 (td, J=10.3, 7.1 Hz, 1H) 4.49 (td, J=10.3, 6.5 Hz, 1H) 4.80 (t, J=5.4 Hz, 1H) 5.53 (d, J=8.8 Hz, 1H) 5.75 (s, 1H) 5.94 (d, J=1.6 Hz, 2H) 6.45 (d, J=8.5 Hz, 1H) 7.20 (s, 1H) 7.44 (d, J=8.2 Hz, 2H) 7.55 (d, J=8.5 Hz, 2H) 8.06 (s, 1H)

(291) LC/MS (method LC-A): R.sub.t 3.30 min, MH.sup.+ 567

(292) Melting point: 165 C.

(293) Enantiomer 13A:

(294) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.10-3.26 (m, 2H) 3.62 (s, 3H) 3.65 (q, J=5.4 Hz, 2H) 3.81 (s, 3H) 3.82-3.88 (m, 2H) 4.03 (td, J=10.3, 7.1 Hz, 1H) 4.49 (td, J=10.3, 6.5 Hz, 1H) 4.80 (t, J=5.4 Hz, 1H) 5.53 (d, J=8.8 Hz, 1H) 5.75 (s, 1H) 5.94 (d, J=1.6 Hz, 2H) 6.45 (d, J=8.5 Hz, 1H) 7.20 (s, 1H) 7.44 (d, J=8.2 Hz, 2H) 7.55 (d, J=8.5 Hz, 2H) 8.06 (s, 1H)

(295) LC/MS (method LC-A): R.sub.t 3.31 min, MH.sup.+ 567

(296) []D20: +43.2 (c 0.285, DMF)

(297) Chiral SFC (method SFC-J): R.sub.t 1.82 min, MH.sup.+ 567, chiral purity 100%.

(298) Enantiomer 13B:

(299) .sup.1H NMR (500 MHz, DMSO-d.sub.6) ppm 3.09-3.26 (m, 2H) 3.61 (s, 3H) 3.64 (q, J=5.3 Hz, 2H) 3.81 (s, 3H) 3.82-3.88 (m, 2H) 4.02 (td, J=10.4, 6.9 Hz, 1H) 4.48 (td, J=10.3, 6.1 Hz, 1H) 4.79 (t, J=5.5 Hz, 1H) 5.53 (d, J=8.5 Hz, 1H) 5.75 (s, 1H) 5.94 (d, J=1.6 Hz, 2H) 6.44 (d, J=8.5 Hz, 1H) 7.20 (s, 1H) 7.43 (d, J=8.2 Hz, 2H) 7.55 (d, J=8.5 Hz, 2H) 8.06 (s, 1H)

(300) LC/MS (method LC-A): R.sub.t 3.31 min MH.sup.+ 567

(301) [].sub.D.sup.20: 45.90 (c 0.301, DMF)

(302) Chiral SFC (method SFC-J): R.sub.t 3.15 min MH.sup.+ 567, chiral purity 98.96%.

(303) TABLE-US-00003 TABLE compounds prepared as described above Compound Structure Optical rotation 1 racemic 1A [].sub.D.sup.20 = 26.5 1B [].sub.D.sup.20 = +28.8 2 0 racemic 2A [].sub.D.sup.20 = 26.4 2B [].sub.D.sup.20 = +27.3 3 racemic 3A [].sub.D.sup.20 = +51.9 3B [].sub.D.sup.20 = 51.1 4 racemic 4A [].sub.D.sup.20 = +52.0 4B [].sub.D.sup.20 = 51.8 5 racemic 5A 0 [].sub.D.sup.20 = +31.1 5B [].sub.D.sup.20 = 31.0 6 racemic 6A [].sub.D.sup.20 = 25.9 6B [].sub.D.sup.20 = +23.3 7 racemic 7A [].sub.D.sup.20 = 45.2 7B [].sub.D.sup.20 = +43.8 8 racemic 8A [].sub.D.sup.20 = 28.9 8B 0 [].sub.D.sup.20 = +25.7 9 racemic 9A [].sub.D.sup.20 = 40.6 9B [].sub.D.sup.20 = +42.6 10 racemic 10A [].sub.D.sup.20 = 46.2 10B [].sub.D.sup.20 = +43.0 11 racemic 11A [].sub.D.sup.20 = 31.4 11B [].sub.D.sup.20 = +29.4 12 0 racemic 12A [].sub.D.sup.20 = +50.2 12B [].sub.D.sup.20 = 51.0 13 racemic 13A [].sub.D.sup.20 = +43.2 13B [].sub.D.sup.20 = 45.9
Antiviral Activity of the Compounds of the Invention
DENV-2 Antiviral Assay

(304) The antiviral activity of all the compounds of the invention was tested the against DENV-2 16681 strain which was labeled with enhanced green fluorescent protein (eGPF). The culture medium consists of minimal essential medium supplemented with 2% of heat-inactivated fetal calf serum, 0.04% gentamycin (50 mg/mL) and 2 mM of L-glutamine. Vero cells, obtained from ECACC, were suspended in culture medium and 25 L was added to 384-well plates (2500 cells/well), which already contain the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 dilution steps of the test compound at 200 times the final concentration in 100% DMSO (200 nL). In addition, each compound concentration is tested in quadruplicate (final concentration range: 25 M-0.000064 M or 2.5 M-0.0000064 M for the most active compounds). Finally, each plate contains wells which are assigned as virus controls (containing cells and virus in the absence of compound), cell controls (containing cells in the absence of virus and compound) and medium controls (containing medium in the absence of cells, virus and compounds). To the wells assigned as medium control, 25 L of culture medium was added instead of Vero cells. Once the cells were added to the plates, the plates were incubated for 30 minutes at room temperature to allow the cells to distribute evenly within the wells. Next, the plates were incubated in a fully humidified incubator (37 C., 5% CO.sub.2) until the next day. Then, DENV-2 strain 16681, labeled with eGFP, was added at a multiplicity of infection (MOI) of 0.5. Therefore, 15 L of virus suspension was added to all the wells containing test compound and to the wells assigned as virus control. In parallel, 15 L of culture medium was added to the medium and cell controls. Next, the plates were incubated for 3 days in a fully humidified incubator (37 C., 5% CO.sub.2). At the day of the read out, the eGFP fluorescence was measured using an automated fluorescence microscope at 488 nm (blue laser). Using an in-house LIMS system, inhibition dose response curves for each compound were calculated and the half maximal effective concentration (EC.sub.50) was determined. Therefore, the percent inhibition (I) for every test concentration is calculated using the following formula: I=100*(S.sub.TS.sub.CC)/(S.sub.VCS.sub.CC); S.sub.T, S.sub.CC and S.sub.VC are the amount of eGFP signal in the test compound, cell control and virus control wells, respectively. The EC.sub.50 represents the concentration of a compound at which the virus replication is inhibited with 50%, as measured by a 50% reduction of the eGFP fluorescent intensity compared to the virus control. The EC.sub.50 is calculated using linear interpolation (Table 1).

(305) In parallel, the toxicity of the compounds was assessed on the same plates. Once the read-out for the eGFP signal was done, 40 L of ATPlite, a cell viability stain, was added to all wells of the 384-well plates. ATP is present in all metabolically active cells and the concentration declines very rapidly when the cells undergo necrosis or apoptosis. The ATPLite assay system is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. The plates were incubated for 10 minutes at room temperature. Next, the plates were measured on a ViewLux. The half maximal cytotoxic concentration (CC.sub.50) was also determined, defined as the concentration required to reduce the luminescent signal by 50% compared to that of the cell control wells. Finally, the selectivity index (SI) was determined for the compounds, which was calculated as followed: SI=CC.sub.50/EC.sub.50.

(306) TABLE-US-00004 TABLE 1 EC.sub.50, CC.sub.50, and SI for the compounds of the invention in the DENV-2 antiviral assay compound # EC.sub.50 (M) N CC.sub.50 (M) N SI N 1 0.0056 4 13 4 2070 4 1A 0.22 3 13 3 50 3 1B 0.0036 6 12 6 3578 6 2 0.0089 4 12 4 1400 4 2A 0.32 3 18 3 46 3 2B 0.0025 3 13 5 3750 3 3 0.0055 4 9.5 4 1760 4 3A 0.0023 4 9.6 7 4530 4 3B 0.59 3 12 3 19 3 4 0.0067 3 9.7 3 1440 3 4A 0.0036 4 12 6 3878 4 4B 1.1 3 14 3 11 3 5 0.0046 3 9.3 3 2040 3 5A 0.0014 4 9.8 4 7000 4 5B 0.76 3 14 4 19 3 6 0.0062 4 14 4 1940 4 6A 0.28 4 17 4 49 4 6B 0.0025 10 12 10 4900 10 7 0.0023 3 11 3 4670 3 7A 0.79 5 21 5 26 5 7B 0.0010 9 12 10 10000 9 8 0.0044 3 >25 3 >6100 3 8A 0.46 6 >25 6 >81 6 8B 0.0019 7 76 7 >49900 7 9A 1.0 3 13 3 12 3 9B 0.0014 3 11 3 8440 3 10A 1.3 4 11 4 8.4 4 10B 0.0022 3 11 3 5470 3 11 0.0023 3 22 3 8650 3 11A 0.33 3 >25 3 >94 3 11B 0.0016 3 >25 3 >33700 3 12 0.011 3 11 3 966 3 12A 0.0023 3 12 3 5020 3 12B 0.40 3 11 3 27 3 13 0.0049 3 13 3 2630 3 13A 0.0035 3 12 3 3970 3 13B 0.28 3 >25 3 >123 3 N = the number of independent experiments in which the compounds were tested.
Tetravalent Reverse Transcriptase Quantitative-PCR (RT-qPCR) Assay

(307) The antiviral activity of the compounds of the invention was tested against DEN-1 strain TC974 #666 (NCPV), DENV-2 strain 16681, DENV-3 strain H87 (NCPV) and DENV-4 strain H241 (NCPV) in a RT-qPCR assay. Therefore, Vero cells were infected with either DEN-, or -2, or -3, or -4 in the presence or absence of test compounds. At day 3 post-infection, the cells were lysed and cell lysates were used to prepare cDNA of both a viral target (the 3UTR of DENV; Table 2) and a cellular reference gene (-actin, Table 2). Subsequently, a duplex real time PCR was performed on a Lightcycler480 instrument. The generated Cp value is inversely proportional to the amount of RNA expression of these targets. Inhibition of DENV replication by a test compound results in a shift of Cp's for the 3UTR gene. On the other hand, if a test compound is toxic to the cells, a similar effect on -actin expression will be observed. The comparative Cp method is used to calculate EC.sub.50, which is based on the relative gene expression of the target gene (3UTR) normalized with the cellular housekeeping gene (-actin). In addition, CC.sub.50 values are determined based on the Cp values acquired for the housekeeping gene -actin.

(308) TABLE-US-00005 TABLE2 Primersandprobesusedforthereal-time, quantitativeRT-PCR. Primer/probe Target Sequence.sup.a,b F3utr258 DENV 5-CGGTTAGAGGAGAC 3-UTR CCCTC-3 R3utr425 DENV 5-GAGACAGCAGGATC 3-UTR TCTGGTC-3 P3utr343 DENV -5-AAGGACTAG- 3-UTR ZEN-AGGTTAGAGGAGA CCCCCC-3- Factin743 -actin 5-GGCCAGGTCATCAC CATT-3 Ractin876 -actin 5-ATGTCCACGTCACA CTTCATG-3 Pactin773 -actin -5-TTCCGCTGC- -CCTGAGGCTCTC- 3- .sup.aReporter dyes (FAM, HEX) and quenchers (ZEN and IABkFQ) elements are indicated in bold and italics. .sup.bThe nucleotide sequence of the primers and probes were selected from the conserved region in the 3UTR region of the dengue virus genome, based on the alignment of 300 nucleotide sequences of the four dengue serotypes deposited in Genbank (Gong et al., 2013, Methods Mol Biol, Chapter 16).

(309) The culture medium consisted of minimal essential medium supplemented with 2% of heat-inactivated fetal calf serum, 0.04% gentamycin (50 mg/mL) and 2 mM of L-glutamine. Vero cells, obtained from ECACC, were suspended in culture medium and 75 L/well was added in 96-well plates (10000 cells/well), which already contain the antiviral compounds. Typically, these plates contain a 5-fold serial dilution of 9 dilution steps of the test compound at 200 times the final concentration in 100% DMSO (500 nL; final concentration range: 25 M-0.000064 M or 2.5 M-0.0000064 M for the most active compounds). In addition, each plate contains wells which are assigned as virus controls (containing cells and virus in the absence of compound) and cell controls (containing cells in the absence of virus and compound). Once the cells were added in the plates, the plates were incubated in a fully humidified incubator (37 C., 5% CO.sub.2) until the next day. Dengue viruses serotype-1, 2, 3 and 4 were diluted in order to obtain a Cp of 22-24 in the assay. Therefore, 25 L of virus suspension was added to all the wells containing test compound and to the wells assigned as virus control. In parallel, 25 L of culture medium was added to the cell controls. Next, the plates were incubated for 3 days in a fully humidified incubator (37 C., 5% CO.sub.2). After 3 days, the supernatant was removed from the wells and the cells were washed twice with ice-cold PBS (100 L). The cell pellets within the 96-well plates were stored at 80 C. for at least 1 day. Next, RNA was extracted using the Cells-to-CT lysis kit, according to the manufacturer's guideline (Life Technologies). The cell lysates can be stored at 80 C. or immediately used in the reverse transcription step.

(310) In preparation of the reverse transcription step, mix A (table 3A) was prepared and 7.57 L/well was dispensed in a 96-well plate. After addition of 5 L of the cell lysates, a five minute denaturation step at 75 C. was performed (table 3B). Afterwards, 7.43 L of mix B was added (table 3C) and the reverse transcription step was initiated (table 3D) to generate cDNA.

(311) Finally, a RT-qPCR mix was prepared, mix C (table 4A), and 22.02 L/well was dispensed in 96-well LightCycler qPCR plates to which 3 L of cDNA was added and the qPCR was performed according to the conditions in table 4B on a LightCycler 480.

(312) Using the LightCycler software and an in-house LIMS system, dose response curves for each compound were calculated and the half maximal effective concentration (EC.sub.50) and the half maximal cytotoxic concentration (CC.sub.50) were determined (Tables 5-8).

(313) TABLE-US-00006 TABLE 3 cDNA synthesis using Mix A, denaturation, Mix B and reverse transcription. A Mix A Plates 8 Reaction Samples 828 Vol. (l) 20 Concentration Volume for (l) Mix Item Unit Stock Final 1 sample x samples Milli-Q H.sub.2O 7.27 6019.56 R3utr425 M 20 0.27 0.15 124.20 Ractin876 M 20 0.27 0.15 124.20 Volume mix/well (l) 7.57 Cell lysates 5.00 B Denaturation step: Step Temp Time Denaturation 75 C. 5 Hold 4 C. hold C Mix B Samples 864 Concentration Volume for (l) Mix Item Unit Stock Final 1 sample x samples Expand HIFI X 10.00 1.00 2.00 1728.0 buffer 2 MgCl.sub.2 mM 25.00 3.50 2.80 2419.2 dNTPs mM 10.00 1.00 2.00 1728.0 Rnase inhibitor U/l 40.00 1.00 0.50 432.0 Expand RT U/l 50.00 0.33 0.13 112.3 Total Volume Mix (l) 7.43 D Protocol cDNA synthesis Step Temp Time Rev transc 42 C. 30 Denaturation 99 C. 5 Hold 4 C. hold

(314) TABLE-US-00007 TABLE 4 qPCR mix and protocol. A Mix C Reaction Samples 833 Vol. (l) 25 Concentration Volume for (l) Mix Item Unit Stock Final 1 sample x samples H.sub.2O PCR 7.74 6447.42 grade Roche Roche 2 MM X 2 1 12.50 10412.50 mix F3utr258 M 20 0.3 0.38 316.54 R3utr425 M 20 0.3 0.38 316.54 P3utr343 M 20 0.1 0.13 108.29 Factin743 M 20 0.3 0.38 316.54 Ractin876 M 20 0.3 0.38 316.54 Pactin773 M 20 0.1 0.13 108.29 Volume Mix/Tube (l) 22.02 cDNA 3.00 B Protocol qPCR3 Step Temp Time Ramp rate preincub/denat 95 C. 10 min 4.4 Denaturation 95 C. 10 sec 4.4 40 cycles annealing 58 C. 1 min 2.2 Elongation 72 C. 1 sec 4.4 Cooling 40 C. 10 sec 1.5

(315) TABLE-US-00008 TABLE 5 EC.sub.50, CC.sub.50, and SI for the compounds against serotype 1 in the RT-qPCR assays Protocol A RT-qPCR serotype 1 TC974#666 compound# EC.sub.50 (M) N CC.sub.50 (M) N SI N 1B 0.0044 5 5.1 4 1110 4 2B 0.0024 3 4.8 2 2670 2 3A 0.0036 3 4.5 3 1210 3 4A 0.0039 3 4.4 3 1270 3 4B 0.70 3 >2.5 4 >4.6 3 5A 0.0026 4 4.7 4 1650 4 6B 0.0031 10 4.8 10 1590 10 7B 0.0051 3 4.1 3 802 3 8B 0.0081 3 >2.5 3 >454 3 9B 0.0027 3 >2.5 3 >1800 3 10B 0.0017 3 >2.5 3 >1810 3 11B 0.0078 3 >2.5 3 >352 3 12A 0.0033 3 >2.5 2 >934 2 13A 0.0063 3 >2.5 2 >416 2 N = the number of independent experiments in which the compounds were tested.

(316) TABLE-US-00009 TABLE 6 EC.sub.50, CC.sub.50, and SI for the compounds against serotype 2 in the RT-qPCR assays Protocol A RT-qPCR serotype 2 16681 compound# EC.sub.50 (M) N CC.sub.50 (M) N SI N 1B 0.0036 4 3.8 4 1100 4 2B 0.0032 3 5.3 2 1880 2 3A 0.0022 4 9.5 4 4860 4 4A 0.0043 3 3.4 3 894 3 4B 1.0 3 13 3 17 3 5A 0.0030 3 7.6 3 2680 3 6B 0.0020 11 7.9 11 4400 11 7B 0.00096 4 5.0 4 5780 4 8B 0.0023 4 14 4 5510 4 9B 0.0012 3 12 3 11900 3 10B 0.0023 3 10 3 2670 3 11B 0.0020 3 16 3 4840 3 12A 0.0044 3 >2.5 3 .794 3 13A 0.0015 3 >2.5 3 >4280 3 N = the number of independent experiments in which the compounds were tested.

(317) TABLE-US-00010 TABLE 7 EC.sub.50, CC.sub.50, and SI for the compounds against serotype 3 in the RT-qPCR assays Protocol A RT-qPCR serotype 3 H87 compound# EC.sub.50 (M) N CC.sub.50 (M) N SI N 1B 0.056 5 2.4 4 46 4 2B 0.029 3 3.6 2 156 2 3A 0.029 3 4.0 3 169 3 4A 0.027 3 3.6 3 179 3 4B >1.9 3 8.6 4 7.6 1 5A 0.020 3 4.3 3 214 3 6B 0.028 10 4.5 9 253 9 7B 0.030 3 3.7 2 155 2 8B 0.050 3 >2.5 3 >83 3 9B 0.016 3 >2.5 3 >191 3 10B 0.016 3 >2.5 3 >199 3 11B 0.034 3 >2.5 3 >139 3 12A 0.040 3 >2.5 3 >67 3 13A 0.043 3 >2.5 3 >78 3 N = the number of independent experiments in which the compounds were tested.

(318) TABLE-US-00011 TABLE 8 EC50, CC50, and SI for the compounds against serotype 4 in the RT-qPCR assays Protocol A RT-qPCR serotype 4 H241 compound# EC.sub.50 (M) N CC.sub.50 (M) N SI N 1B 0.25 4 2.9 4 12 4 2B 0.17 3 5.9 2 33 2 3A 0.19 4 6.2 3 32 3 4A 0.21 3 2.2 3 11 3 4B >2.5 3 9.4 4 NA NA 5A 0.21 3 3.8 3 16 3 6B 0.14 11 4.1 10 27 10 7B 0.12 4 3.1 3 25 3 8B 0.12 4 9.0 4 98 4 9B 0.11 3 2.9 3 23 3 10B 0.16 3 >2.0 3 >13 3 11B 0.28 3 7.6 3 20 3 12A 0.24 3 6.1 3 24 3 13A 0.27 3 2.6 3 >9.4 3 N = the number of independent experiments in which the compounds were tested. NA = not approved.

Prior Art Examples

(319) Compounds (56) and (170) disclosed in WO-2013/045516 have been tested in an analogous DENV-2 antiviral assay as the compounds of the present invention and their reported activity is listed below.

(320) TABLE-US-00012 compound (56) of WO-2013/045516 compound (170) of WO-2013/045516

(321) TABLE-US-00013 TABLE 9 EC.sub.50, CC.sub.50, and SI for compounds (56) and (170) disclosed in the DENV-2 antiviral assay compound# EC.sub.50 (M) CC.sub.50 (M) SI (56) of WO-2013/045516 0.45 >139 >312 (170) of WO-2013/045516 0.44 26 58