Substituted indoline derivatives as dengue viral replication inhibitors

Abstract

The present invention concerns substituted indoline derivatives, 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 of formula (I), including any stereochemically isomeric form thereof, ##STR00053## wherein R.sup.1 is fluoro, R.sup.2 is —CH.sub.2CH.sub.2OH, R.sup.3 is trifluoromethyl, and R.sup.4 is hydrogen, or R.sup.1 is fluoro, R.sup.2 is —CH.sub.2CH.sub.2OH, R.sup.3 is trifluoromethyl, and R.sup.4 is methoxy, or R.sup.1 is fluoro, R.sup.2 is —CH.sub.2CH.sub.2OH, R.sup.3 is trifluoromethoxy, and R.sup.4 is hydrogen, or R.sup.1 is chloro, R.sup.2 is —CH.sub.2CH.sub.2OH, R.sup.3 is trifluoromethyl, and R.sup.4 is hydrogen, or R.sup.1 is chloro, R.sup.2 is —CH.sub.2CH.sub.2OH, R.sup.3 is trifluoromethyl, and R.sup.4 is methoxy, or R.sup.1 is chloro, R.sup.2 is —CH.sub.2CH.sub.2OH, R.sup.3 is trifluoromethoxy, and R.sup.4 is hydrogen, or R.sup.1 is chloro, R.sup.2 is —(CH.sub.2).sub.3COOH, R.sup.3 is trifluoromethyl, and R.sup.4 is hydrogen, or R.sup.1 is chloro, R.sup.2 is —(CH.sub.2).sub.3COOH, R.sup.3 is trifluoromethyl, and R.sup.4 is methoxy, or R.sup.1 is chloro, R.sup.2 is —(CH.sub.2).sub.3COOH, R.sup.3 is trifluoromethoxy, and R.sup.4 is hydrogen; or a pharmaceutically acceptable salt, solvate or polymorph thereof.

2. The compound according to claim 1 wherein said compound is selected from the group consisting of: ##STR00054## ##STR00055## ##STR00056##

3. The compound according to claim 1 wherein said compound has the (+) specific rotation.

4. The compound according to claim 1 wherein said compound is selected from the group consisting of: ##STR00057## ##STR00058## ##STR00059##

5. A pharmaceutical composition comprising the compound according to claim 1 together with one or more pharmaceutically acceptable excipients, diluents or carriers.

6. The pharmaceutical composition according to claim 5 which comprises a second or further active ingredient.

7. The pharmaceutical composition according to claim 6 wherein the second or further active ingredient is an antiviral agent.

8. A method of treating Dengue infection or a disease associated with Dengue infection, comprising administering to a subject in need thereof an effective amount of the compound according to claim 1.

9. The method according to claim 8 wherein the Dengue infection is infection by viruses of the DENV-1, DENV-2, DENV-3 or DENV-4 strain.

10. A method of inhibiting Dengue virus replication in an animal cell, comprising administering to a subject in need thereof an effective amount of the compound according to claim 1.

Description

EXAMPLES

(1) LC/MS Methods

(2) The 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 [M−H].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, CI), 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: Waters: BEH ® A: 95% 84.2% A for 0.49 min, 0.343 mL/min 6.2 Acquity ® C18 (1.7 μm, CH.sub.3COONH.sub.4 to 10.5% A in 2.18 min, 40° C. UPLC ®-DAD- 2.1 × 100 mm) 7 mM/5% held for 1.94 min, Quattro CH.sub.3CN, back to 84.2% A Micro ™ B: CH.sub.3CN in 0.73 min, held for 0.73 min. LC-B Waters: Waters: BEH ® A: 95% 84.2% A/15.8% B to 0.343 mL/min 6.1 Acquity ® H- C18 (1.7 μm, CH.sub.3COONH.sub.4 10.5% A in 2.18 min, 40° C. Class-DAD 2.1 × 100 mm) 7 mM/5% held for 1.96 min, and SQD2TM CH.sub.3CN, back to 84.2% B: CH.sub.3CN A/15.8% B in 0.73 min, held for 0.49 min. LC-C Waters: Waters: UPLC A: 0.1% 50% A to 10% A in 0.5 mL/min 5 Acquity ® HSS C18 (1.8 μm, HCOOH 3.5 min, held for 1.5 min. 40° C. UPLC ®-DAD- 2.1 × 50 mm) B: CH.sub.3CN Acquity ® TQ detector LC-D Waters: Waters: BEH A: 10 mM From 95% A to 5% A 0.8 mL/min 2 Acquity ® C18 (1.7 μm, CH.sub.3COONH.sub.4 in in 1.3 min, held for 55° C. UPLC ®-DAD- 2.1 × 50 mm) 95% H.sub.2O + 5% 0.7 min. SQD CH.sub.3CN B: CH.sub.3CN

(8) SFC/MS Methods

(9) 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.

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

(11) TABLE-US-00002 Flow Run time Method code column mobile phase gradient Col T BPR SFC-A Daicel Chiralpak ® A: CO.sub.2 20% B hold 7 min, 3 7 AD-H column (5 μm, B: EtOH (+0.3% 150 × 4.6 mm) iPrNH.sub.2) 35 100 SFC-B Regis Whelk A: CO.sub.2 50% B hold 3 min, 3.5 3 O1 ®(S,S) column (3 μm, B: EtOH (+0.3% 100 × 4.6 mm) iPrNH.sub.2) 35 103 SFC-C Daicel Chiralpak ® A: CO.sub.2 30% B hold 7 min, 3 7 AD-H column (5 μm, B: EtOH (+0.3% 150 × 4.6 mm) iPrNH.sub.2) 35 100 SFC-D Daicel Chiralpak ® A: CO.sub.2 40% B hold 3 min, 3.5 3 IC-3 column (3 μm, B: EtOH (+0.3% 100 × 4.6 mm) iPrNH.sub.2) 35 103 SFC-E Daicel Chiralpak ® IA A: CO.sub.2 30% B hold 7 min, 3.5 7 column (5 μm, 150 × B: iPrOH (+0.3% 4.6 mm) iPrNH.sub.2) 35 103 SFC-F Daicel Chiralpak ® IC A: CO.sub.2 30% B hold 7 min, 3 7 column (5 μm, 150 × B: MeOH 4.6 mm) 35 100 SFC-G Daicel Chiralpak ® IC- A: CO.sub.2 40% B hold 5 min, 3.5 5 3 column (3 μm, 100 × B: iPrOH (+0.3% 4.6 mm) iPrNH.sub.2) 35 103 SFC-H Daicel Chiralpak ® IC A: CO.sub.2 25% B hold 7 min, 3 7 column (5 μm, 150 × B: EtOH/iPrOH 4.6 mm) 50/50 (+0.3% iPrNH.sub.2) 35 100 SFC-I Daicel Chiralpak ® A: CO.sub.2 10%-50% B in 6 min, 2.5 9.5 AS3 column (3.0 μm, B: EtOH hold 3.5 min 150 × 4.6 mm) (+0.2% iPrNH.sub.2 40 110 +3% H.sub.2O)

(12) Melting Points

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

(14) DSC823e (Indicated as DSC)

(15) 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.

(16) Optical Rotations:

(17) Optical rotations were measured on a Perkin-Elmer 341 polarimeter with a sodium lamp and reported as follows: [α].sup.o (λ, c g/100 ml, solvent, T° C.).

(18) [α].sub.λ.sup.T=(100α)/(l×c): 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 λ (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).

Abbreviations Used in Experimental Part

(19) TABLE-US-00003 (M + H).sup.+ protonated molecular ion aq. aqueous Boc tert-butyloxycarbonyl Boc.sub.2O di-tert-butyl dicarbonate br broad CH.sub.3CN acetonitrile CHCl.sub.3 chloroform CH.sub.2Cl.sub.2 dichloromethane CH.sub.3OH methanol CO.sub.2 carbon dioxide d doublet DCM dichloromethane DIEA diisopropylethylamine DIPE diisopropyl ether DMA dimethylacetamide DMAP 4-dimethylaminopyridine DME 1,2-dimethoxyethane DMF dimethylformamide DMSO dimethyl sulfoxide eq. equivalent Et.sub.2O diethyl ether Et.sub.3N triethylamine EtOAc ethyl acetate EtOH ethanol H.sub.2O water H.sub.2SO.sub.4 sulfuric acid HATU O-(7-aza-1H-benzotriazol-1- yl)-N,N,N′,N′-tetramethyl- uronium hexafluorophosphate- CAS [148893-10-1] HCl hydrochloric acid HPLC high performance liquid chromatography iPrNH.sub.2 isopropylamine iPrOH 2-propanol K.sub.2CO.sub.3 potassium carbonate LiAlH.sub.4 lithium aluminium hydride m/z mass-to-charge ratio Me methyl MeOH methanol MgSO.sub.4 magnesium sulfate min minute(s) N.sub.2 nitrogen Na.sub.2CO.sub.3 sodium carbonate Na.sub.2SO.sub.4 sodium sulfate NaBH.sub.4 sodium borohydride NaHCO.sub.3 sodium bicarbonate NaOH sodium hydroxide NH.sub.4Cl ammonium chloride q quartet rt or RT room temperature s singlet t triplet tBuOK potassium tert-butanolaat TEA triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TMSCl trimethylsilyl chloride

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

(20) ##STR00006## ##STR00007## ##STR00008##

Synthesis of Intermediate 1a

(21) A solution of 4-fluoro-2-methoxyphenylacetic acid [CAS 886498-61-9] (10 g, 54.30 mmol) in EtOH (200 mL) and H.sub.2SO.sub.4 (2 mL) was heated under reflux for 12 h. Water was added and the mixture was concentrated under reduced pressure to half of the original volume. Ice was added, the solution was basified with K.sub.2CO.sub.3 and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO.sub.4, filtered, and the solvent was concentrated under reduced pressure to give ethyl 2-(4-fluoro-2-methoxyphenyl)acetate 1a (11.6 g). The compound was used in the next step without further purification.

Synthesis of Intermediate 1b

(22) Boron tribromide (109.3 mL, 109.3 mmol) was added dropwise to a cooled (−30° C.) solution of ethyl 2-(4-fluoro-2-methoxyphenyl)acetate 1a (11.6 g, 54.7 mmol) in CH.sub.2Cl.sub.2 (300 mL). The reaction was stirred at −20° C. for 1 h, and quenched with CH.sub.3OH. The pH was adjusted to 8 by adding a saturated water solution of NaHCO.sub.3. The solution was extracted with CH.sub.2Cl.sub.2 and the combined organic layers were dried over MgSO.sub.4, filtered, and the solvent was concentrated under reduced pressure to give ethyl 2-(4-fluoro-2-hydroxyphenyl)acetate 1b (10.8 g). The compound was used in the next step without further purification.

Synthesis of Intermediate 1c

(23) To a mixture of ethyl 2-(4-fluoro-2-hydroxyphenyl)acetate 1b (1.24 g, 6.26 mmol) and cesium carbonate (4.08 g, 12.5 mmol) in DMF (20 mL) was added benzyl 2-bromoethyl ether [CAS 1462-37-9] (1.61 g, 7.51 mmol). The reaction mixture was stirred at room temperature overnight. Water was added and the reaction mixture was extracted with EtOAc. The organic phase was 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 CH.sub.2Cl.sub.2 (15% to 100%) in heptane to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-fluorophenyl)acetate 1c (1.55 g).

Synthesis of Intermediate 1d

(24) To a cooled (−78° C.) solution of 1M lithium bis(trimethylsilyl)amide in THF (4.51 mL, 4.51 mmol) was added a solution of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-fluorophenyl)acetate 1c (0.750 g, 2.26 mmol) in THF (4 mL). After 1 h at −78° C., chlorotrimethylsilane (0.458 mL, 3.61 mmol) was added. The reaction mixture was stirred at −78° C. for 15 min. N-Bromosuccinimide (0.482 g, 2.71 mmol) was added and stirring was continued at −40° C. for 2 h. The reaction mixture was poured out into H.sub.2O and extracted twice with EtOAc. The organic phases were combined, dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-fluorophenyl)-2-bromoacetate 1d (0.920 g) which was used in the next step without further purification.

Synthesis of Intermediate 1e

(25) A mixture of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-fluorophenyl)-2-bromoacetate d (0.920 g, 2.24 mmol) and 3-methoxy-5-(methylsulfonyl)aniline [CAS 62606-02-4](1.35 g, 6.71 mmol) in CH.sub.3CN (5 mL) and THF (5 mL) was stirred at 60° C. overnight. The reaction mixture was diluted with EtOAc and washed with 1N HCl. The organic phase was washed with 1N HCl, an aqueous saturated NaHCO.sub.3 solution, 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 (5% to 50%) in heptane to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl) phenyl)amino)acetate 1e (0.870 g).

Synthesis of Intermediate 1f

(26) A mixture of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 1e (0.868 g, 1.63 mmol) and 10% palladium on carbon (0.180 g) in EtOAc (30 mL) was stirred overnight at room temperature under H.sub.2 atmosphere. The reaction mixture was filtered through Celite®. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (30% to 100%) in heptane to give quantitatively ethyl 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 1f.

Synthesis of Intermediate 1g

(27) To a solution of ethyl 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 1f (0.910 g, 2.06 mmol) in THF (6 mL), MeOH (6 mL) and H.sub.2O (6 mL) was added lithium hydroxide monohydrate (0.432 g, 10.3 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was partially concentrated under reduced pressure to remove THF and MeOH. The residual aqueous solution was acidified with 1N HCl and extracted with CH.sub.2Cl.sub.2. The organic phase was dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to give 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 1g (0.736 g) which was used in the next step without further purification.

Synthesis of Compound 1 and Chiral Separation into Enantiomers 1A and 1B

(28) Method 1:

(29) To a solution of 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 1g (0.200 g, 0.484 mmol) in DMF (4 mL) were added HATU (0.184 g, 0.484 mmol), triethylamine (0.267 mL, 1.94 mmol) and 6-(trifluoromethyl)indoline [CAS 181513-29-1] (0.091 g, 0.484 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with EtOAc and washed with 1N HCl. The organic phase was washed with an aqueous saturated NaHCO.sub.3 solution, 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 (2% to 40%) in CH.sub.2Cl.sub.2. The fractions containing the desired product were combined and concentrated under reduced pressure. The residue was purified by preparative TLC using a mixture of EtOAc (50%) in CH.sub.2Cl.sub.2 as eluent. Subsequent purification by preparative HPLC (Column: X-Bridge® C18—5 μm 100×19 mm, mobile phase: pH 10 NH.sub.4OAc solution in H.sub.2O, CH.sub.3CN) furnished 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 1, 0.043 g) as a racemic mixture.

(30) Method 2:

(31) To a solution of 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 1g (0.300 g, 0.726 mmol) in Me-THF (5.4 mL) under N.sub.2 flow, were added 6-(trifluoromethyl)indoline [CAS 181513-29-1](0.136 g, 0.726 mmol), N-diisopropylethylamine (264 μL, 1.596 mmol) and propylphosphonic anhydride (653 μL, 1.09 mmol). The reaction was stirred at room temperature for 16 h. The mixture was poured out into water and extracted with EtOAc. The combined organic layers were washed with a 10% solution of K.sub.2CO.sub.3 in water and with water. The organic solution was dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. This fraction (0.47 g) was combined with a second batch (total amount: 0.585 g) and purified by flash chromatography on silica gel (15-40 μM, 24 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and concentrated under reduced pressure to provide 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 1, 0.160 g) as a racemic mixture.

(32) The Enantiomers of Compound 1 (160 mg) were separated via Preparative Chiral SFC (Stationary phase: Chiralpak® AD-H 5 μm 250×20 mm, Mobile phase: 75% CO.sub.2, 25% EtOH (+0.3% iPrNH.sub.2)). The first eluted product (72 mg) was solidified in heptane/diisopropyl ether to give Enantiomer 1A (50 mg). The second eluted product (80 mg) was solidified in heptane/diisopropyl ether to give Enantiomer 1B (43 mg).

(33) Compound 1:

(34) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.22 (m, 2H) 3.62-3.92 (m, 5H) 3.97-4.22 (m, 3H) 4.46 (m, 1H) 4.98 (br. s., 1H) 5.82 (d, J=7.9 Hz, 1H) 6.56 (s, 1H) 6.62 (s, 1H) 6.80 (t, J=7.7 Hz, 1H) 6.92 (s, 1H) 6.95-7.11 (m, 2H) 7.29-7.53 (m, 3H) 8.39 (s, 1H)

(35) LC-MS (method LC-C): R.sub.t 1.37 min, MH.sup.+ 583

(36) Enantiomer 1A:

(37) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.14-3.29 (m, 2H) 3.73 (s, 3H) 3.75-3.78 (m, 1H) 3.78-3.89 (m, 1H) 3.98-4.23 (m, 3H) 4.37-4.55 (m, 1H) 4.97 (t, J=5.4 Hz, 1H) 5.82 (d, J=8.2 Hz, 1H) 6.56 (s, 1H) 6.62 (s, 1H) 6.79 (dt, J=2.2, 8.5 Hz, 1H) 6.91 (s, 1H) 6.98-7.04 (m, 2H) 7.32-7.43 (m, 2H) 7.46 (d, J=7.9 Hz, 1H) 8.39 (s, 1H).

(38) LC/MS (method LC-A): R.sub.t 3.04 min, MH.sup.+ 583

(39) [α].sub.D.sup.20: −49.6° (c 0.25, DMF)

(40) Chiral SFC (method SFC-A): R.sub.t 2.76 min, MH.sup.+ 583, chiral purity 100%.

(41) Enantiomer 1B:

(42) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.14-3.29 (m, 2H) 3.73 (s, 3H) 3.75-3.78 (m, 1H) 3.78-3.89 (m, 1H) 3.98-4.23 (m, 3H) 4.37-4.55 (m, 1H) 4.97 (t, J=5.4 Hz, 1H) 5.82 (d, J=8.2 Hz, 1H) 6.56 (s, 1H) 6.62 (s, 1H) 6.79 (dt, J=2.2, 8.5 Hz, 1H) 6.91 (s, 1H) 6.98-7.04 (m, 2H) 7.32-7.43 (m, 2H) 7.46 (d, J=7.9 Hz, 1H) 8.39 (s, 1H).

(43) LC/MS (method LC-A): R.sub.t 3.04 min, MH.sup.+ 583

(44) [α].sub.D.sup.20: +51.7° (c 0.23, DMF)

(45) Chiral SFC (method SFC-A): R.sub.t 4.16 min, MH.sup.+ 583, chiral purity 100%.

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

(46) ##STR00009## ##STR00010## ##STR00011##

Synthesis of Intermediate 2a

(47) To a mixture of ethyl 2-(4-fluoro-2-hydroxyphenyl)acetate 1b (10.6 g, 53.5 mmol) and cesium carbonate (34.8 g, 106.9 mmol) in DMF (200 mL) at 10° C. was added (2-bromoethoxy)(tert-butyl)dimethylsilane [CAS 86864-60-0] (13.8 mL, 64.2 mmol). The reaction mixture was stirred at room temperature overnight. H.sub.2O was added and the reaction mixture was extracted with EtOAc. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μM, 40 g, heptane/EtOAc 80/20). The pure fractions were combined and the solvent was removed under reduced pressure to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)acetate 2a (17.7 g).

Synthesis of Intermediate 2b

(48) To a 1M lithium bis(trimethylsilyl)amide solution in THF (28.05 mL, 28.05 mmol) cooled at −78° C. was added a solution of ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)acetate 2a (5 g, 14.03 mmol) in THF (30 mL). After stirring for 1 h at −78° C., chlorotrimethylsilane (2.85 mL, 22.44 mmol) was added. The reaction mixture was stirred at −78° C. for 15 min. N-Bromosuccinimide (3 g, 16.83 mmol) in THF (30 mL) was added and stirring was continued at −55° C. for 2 h. The reaction mixture was poured out into H.sub.2O and extracted twice with EtOAc. The organic phases were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give ethyl 2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)acetate 2b (6.57 g) which was used in the next step without further purification.

Synthesis of Intermediate 2c

(49) A mixture of ethyl 2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)acetate 2b (3 g, 6.89 mmol), 3-methoxy-5-(methylsulfonyl)aniline [CAS 62606-02-4] (2.08 g, 10.3 mmol) and diisopropylethylamine (2.37 mL, 13.8 mmol) in CH.sub.3CN (60 mL) was stirred at 50° C. overnight. The reaction mixture was concentrated under reduced pressure. The residue was taken up with EtOAc and washed with 0.5N HCl and water. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μM, 120 g, heptane/EtOAc 90/10 to 80/20) to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 2c (2.6 g).

Synthesis of Intermediate 2d

(50) Lithium hydroxide monohydrate (205 mg, 4.8 mmol) was added portionwise to a solution of ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 2c (2.227 g, 4.09 mmol) in THF/CH.sub.3OH/H.sub.2O (1/1/1) (100 mL) at 10° C. The reaction was stirred at room temperature for 4 h, and diluted with water. After cooling to 0° C., the solution was slowly acidified to pH 6 with 0.5 N HCl, and extracted with EtOAc. The organic layer was dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 2d (2 g). The compound was used in the next step without further purification.

Synthesis of Intermediate 2e

(51) 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 (27.35 g, 243.7 mmol) in DMF (100 mL) at −10° C. After addition, the purple solution was maintained at −10° C. for 1 h. 500 mL of ice-water and 500 mL of 6N HCl were added and the precipitate was filtered off, washed with water and dried under reduced pressure to afford 40.4 g of 2-(5-methoxy-2-nitro-4-(trifluoromethyl)phenyl)acetonitrile 2e (used as such in the next step).

Synthesis of Intermediate 2f

(52) A solution of 2-(5-methoxy-2-nitro-4-(trifluoromethyl)phenyl)acetonitrile 2e (26 g, 99.9 mmol) in ethanol/water (9/1) (500 mL) and AcOH (5.2 mL) was hydrogenated for 1 h at a pressure of 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 solvent mixture of CH.sub.2Cl.sub.2 and CH.sub.3OH. The filtrate was concentrated under reduced pressure. The residue was filtered through a glass filter charged with silica 60-200 μm 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 2f (15.6 g).

Synthesis of Intermediate 2g

(53) At 0° C., BH.sub.3-Pyridine (23.5 mL, 232.4 mmol) was added dropwise to a solution of 5-methoxy-6-(trifluoromethyl)-1H-indole 2f (10 g, 46.5 mmol) in EtOH (60 mL). 6N HCl (140 mL) was slowly added while maintaining the temperature below 10° C. The mixture was stirred at 0° C. for 2 h. Water (200 mL) was added and the mixture was basified to pH 8-9 with a concentrated aqueous solution of NaOH (the reaction temperature was kept 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 2g (9 g).

Synthesis of Intermediate 2h

(54) To a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 2d (1 g, 1.90 mmol) in DMF (10 mL) were added HATU (1.08 g, 2.84 mmol), diisopropylethylamine (940 μL, 5.69 mmol) and 5-methoxy-6-(trifluoromethyl)indoline 2g (412 mg, 1.90 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic layer was washed with 1N HCl, water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 2h (1.36 g, purity by LC: 70%). The crude compound was used directly in the next reaction step.

Synthesis of Compound 2 and Chiral Separation into Enantiomers 2A and 2B

(55) A solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 2h (1.29 g, 1.77 mmol) in HCl 4M in dioxane (30 mL) and dioxane (100 mL) was stirred at room temperature for 1 h. The solvent was removed under reduced pressure. EtOAc and a 10% aqueous solution of K.sub.2CO.sub.3 were added. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μm, 80 g, CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH 99/1/0.1) to give, after crystallization from CH.sub.3CN, 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 2, 595 mg) as a racemate. The Enantiomers of Compound 2 (560 mg) were separated via Preparative Chiral SFC (Stationary phase: Whelk O1 (S,S)® 5 μm 250×21.1 mm, Mobile phase: 50% CO.sub.2, 50% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (288 mg) was crystallized from CH.sub.3CN/diisopropyl ether to give Enantiomer 2A (240 mg). The second eluted enantiomer (293 mg) was crystallized from CH.sub.3CN/diisopropyl ether to give Enantiomer 2B (232 mg).

(56) Compound 2:

(57) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.09 (s, 3H) 3.12-3.29 (m, 2H) 3.73 (s, 3H) 3.74-3.80 (m, 1H) 3.80-3.90 (m, 4H) 4.02 (td, J=10.4, 7.2 Hz, 1H) 4.05-4.17 (m, 2H) 4.42 (td, J=10.4, 6.2 Hz, 1H) 4.97 (t, J=5.7 Hz, 1H) 5.79 (d, J=8.20 Hz, 1H) 6.56 (s, 1H) 6.61 (s, 1H) 6.78 (td, J=8.51, 2.52 Hz, 1H) 6.90 (s, 1H) 6.95-7.04 (m, 2H) 7.24 (s, 1H) 7.37 (dd, J=8.67, 6.78 Hz, 1H) 8.35 (s, 1H)

(58) LC-MS (method LC-A): R.sub.t 3.02 min, MH.sup.+ 613

(59) Melting point: 215° C.

(60) Enantiomer 2A:

(61) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.09 (s, 3H) 3.12-3.29 (m, 2H) 3.73 (s, 3H) 3.74-3.80 (m, 1H) 3.80-3.90 (m, 4H) 4.02 (td, J=10.4, 7.2 Hz, 1H) 4.05-4.17 (m, 2H) 4.42 (td, J=10.4, 6.2 Hz, 1H) 4.97 (t, J=5.7 Hz, 1H) 5.79 (d, J=8.20 Hz, 1H) 6.56 (s, 1H) 6.61 (s, 1H) 6.78 (td, J=8.51, 2.52 Hz, 1H) 6.90 (s, 1H) 6.95-7.04 (m, 2H) 7.24 (s, 1H) 7.37 (dd, J=8.67, 6.78 Hz, 1H) 8.35 (s, 1H)

(62) LC/MS (method LC-A): R.sub.t 3.00 min, MH.sup.+ 613

(63) [α].sub.D.sup.20: +53.5° (c 0.2392, DMF)

(64) Chiral SFC (method SFC-B): R.sub.t 1.43 min, MH.sup.+ 613, chiral purity 100%.

(65) Melting point: 204° C.

(66) Enantiomer 2B:

(67) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.09 (s, 3H) 3.12-3.29 (m, 2H) 3.73 (s, 3H) 3.74-3.80 (m, 1H) 3.80-3.90 (m, 4H) 4.02 (td, J=10.4, 7.2 Hz, 1H) 4.05-4.17 (m, 2H) 4.42 (td, J=10.4, 6.2 Hz, 1H) 4.97 (t, J=5.7 Hz, 1H) 5.79 (d, J=8.20 Hz, 1H) 6.56 (s, 1H) 6.61 (t, J=1.73 Hz, 1H) 6.78 (td, J=8.51, 2.52 Hz, 1H) 6.90 (s, 1H) 6.95-7.04 (m, 2H) 7.24 (s, 1H) 7.37 (dd, J=8.67, 6.78 Hz, 1H) 8.35 (s, 1H)

(68) LC/MS (method LC-A): R.sub.t 3.00 min, MH.sup.+ 613

(69) [α].sub.D.sup.20: −56.5° (c 0.255, DMF)

(70) Chiral SFC (method SFC-B): R.sub.t 1.72 min, MH.sup.+ 613, chiral purity 99.8%.

(71) Melting point: 206° C.

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

(72) ##STR00012##

Synthesis of Intermediate 3a

(73) To a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 2d (1 g, 1.90 mmol) in DMF (10 mL) were added HATU (1.08 g, 2.84 mmol), diisopropylethylamine (940 μL, 5.69 mmol) and 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (385 mg, 1.90 mmol). The reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K.sub.2CO.sub.3 in water, a saturated solution of NaCl in water, water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 3a (1.32 g). The crude compound was used without purification in the next reaction step.

Synthesis of Compound 3 and Chiral Separation into Enantiomers 3A and 3B

(74) A solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-fluorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 3a (1.17 g, 1.64 mmol) in HCl 4M in dioxane (3.3 mL) and dioxane (50 mL) was stirred at room temperature for 1h. The solvent was removed by evaporation under reduced pressure. EtOAc and a 10% solution of K.sub.2CO.sub.3 in water were added. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μm, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5) to give 2-(4-fluoro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 3, 508 mg) as a racemate. An analytical sample of Compound 3 was solidified from CH.sub.3CN/diisopropyl ether (35 mg). The remaining amount was used to separate the enantiomers of Compound 3 via Preparative Chiral SFC (Stationary phase: Chiralpak® AD-H 5 μm 250×20 mm, Mobile phase: 70% CO.sub.2, 30% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (166 mg) was solidified from heptane/diisopropyl ether to give Enantiomer 3A (130 mg). The second eluted enantiomer (165 mg) was solidified in heptane/diisopropyl ether to give Enantiomer 3B (110 mg).

(75) Compound 3:

(76) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.11-3.25 (m, 2H) 3.73 (s, 3H) 3.74-3.79 (m, 1H) 3.79-3.88 (m, 1H) 3.96-4.19 (m, 3H) 4.45 (dt, J=6.3, 10.4 Hz, 1H) 4.95 (t, J=5.52 Hz, 1H) 5.81 (d, J=8.51 Hz, 1H) 6.57 (s, 1H) 6.62 (t, J=1.89 Hz, 1H) 6.80 (td, J=8.43, 2.36 Hz, 1H) 6.91 (s, 1H) 6.96-7.05 (m, 3H) 7.28-7.46 (m, 2H) 8.05 (s, 1H)

(77) LC-MS (method LC-A): R.sub.t 3.15 min, MH.sup.+ 599

(78) Enantiomer 3A:

(79) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.11-3.25 (m, 2H) 3.73 (s, 3H) 3.74-3.79 (m, 1H) 3.79-3.88 (m, 1H) 3.96-4.19 (m, 3H) 4.45 (dt, J=6.3, 10.4 Hz, 1H) 4.97 (t, J=5.52 Hz, 1H) 5.81 (d, J=8.51 Hz, 1H) 6.57 (s, 1H) 6.62 (t, J=1.89 Hz, 1H) 6.80 (td, J=8.43, 2.36 Hz, 1H) 6.91 (s, 1H) 6.96-7.11 (m, 3H) 7.28-7.46 (m, 2H) 8.05 (s, 1H)

(80) LC/MS (method LC-A): R.sub.t 3.13 min, MH.sup.+ 599

(81) [α].sub.D.sup.20: −59.0° (c 0.2542, DMF)

(82) Chiral SFC (method SFC-C): R.sub.t 1.87 min, MH.sup.+ 599, chiral purity 100%.

(83) Enantiomer 3B:

(84) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.11-3.25 (m, 2H) 3.73 (s, 3H) 3.74-3.79 (m, 1H) 3.79-3.88 (m, 1H) 3.96-4.19 (m, 3H) 4.45 (dt, J=6.3, 10.4 Hz, 1H) 4.97 (t, J=5.52 Hz, 1H) 5.81 (d, J=8.51 Hz, 1H) 6.57 (s, 1H) 6.62 (t, J=1.89 Hz, 1H) 6.80 (td, J=8.43, 2.36 Hz, 1H) 6.91 (s, 1H) 6.96-7.11 (m, 3H) 7.28-7.46 (m, 2H) 8.05 (s, 1H)

(85) LC/MS (method LC-A): R.sub.t 3.13 min, MH.sup.+ 599

(86) [α].sub.D.sup.20: +56.8° (c 0.2467, DMF)

(87) Chiral SFC (method SFC-C): R.sub.t 2.34 min, MH.sup.+ 599, chiral purity 100%.

Example 4 (Method 1): Synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 4)

(88) ##STR00013## ##STR00014##

Synthesis of Intermediate 4a

(89) To a mixture of ethyl 2-(4-chloro-2-hydroxyphenyl)acetate [CAS 1261826-30-5](2.82 g, 3.28 mmol) and cesium carbonate (8.56 g, 26.3 mmol) in DMF (50 mL) was added benzyl 2-bromoethyl ether [CAS 1462-37-9] (2.29 g, 14.5 mmol). The reaction mixture was stirred at room temperature for 24 h. H.sub.2O was added and the reaction mixture was extracted with EtOAc. The organic phase was 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 (2% to 20%) in heptane to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetate 4a (4.17 g).

Synthesis of Intermediate 4b

(90) To a cooled (−78° C.) solution of 1M lithium bis(trimethylsilyl)amide in THF (11.0 mL, 11.0 mmol) was added a solution of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetate 4a (1.82 g, 5.22 mmol) in THF (9 mL). After stirring for 1 h at −78° C., chlorotrimethylsilane (1.1 mL, 8.67 mmol) was added. The reaction mixture was stirred at −78° C. for 15 min. N-Bromosuccinimide (1.11 g, 8.67 mmol) was added and stirring was continued at −78° C. for 2 h. The reaction mixture was poured out into H.sub.2O and extracted with EtOAc. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromoacetate 4b (2.23 g) which was used in the next step without further purification.

Synthesis of Intermediate 4c

(91) To a solution of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromoacetate 4b (2.23 g, 5.22 mmol) in CH.sub.3CN (22.5 mL) and THF (22.5 mL) was added 3-methoxy-5-(methylsulfonyl)aniline [CAS 62606-02-4] (3.12 g, 15.5 mmol). The reaction mixture was stirred at 60° C. overnight. The reaction mixture was concentrated under reduced pressure. The residue was partitioned between EtOAc and 1N HCl. The aqueous phase was 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 using a gradient of EtOAc (0% to 40%) in heptane to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl) phenyl)amino)acetate 4c (1.57 g).

Synthesis of Intermediate 4d

(92) A mixture of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 4c (1.57 g, 2.86 mmol) and 10% palladium on carbon (0.320 g) in EtOAc (40 mL) was stirred overnight at room temperature under H.sub.2 atmosphere. The reaction mixture was filtered through Celite®. The filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (30% to 100%) in heptane to give ethyl 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino) acetate 4d (1.13 g).

Synthesis of Intermediate 4e

(93) To a solution of ethyl 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 4d (1.14 g, 2.49 mmol) in THF (8 mL), MeOH (8 mL) and H.sub.2O (8 mL) was added lithium hydroxide monohydrate (0.522 g, 12.5 mmol). The reaction mixture was stirred at room temperature for 3 h. 1N HCl and EtOAc were added and the phases were separated. The aqueous phase was extracted with EtOAc. The organic phases were combined, washed with brine, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give quantitatively 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 4e which was used in the next step without further purification.

Synthesis of Compound 4

(94) To a solution of 6-trifluroromethylindoline [CAS 181513-29-1] (0.200 g, 1.07 mmol), 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 4e (0.478 g, 1.11 mmol) and triethylamine (0.593 mL, 4.28 mmol) in DMF (10 mL) was added HATU (0.406 g, 1.07 mmol). The reaction mixture was stirred overnight. The reaction mixture was diluted with H.sub.2O and was extracted with ethyl acetate. The organic phase was washed with 1N HCl, an aqueous saturated NaHCO.sub.3 solution and brine, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (60% to 70%) in heptane. The fractions containing the desired product were combined and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel using a gradient of EtOAc (0% to 25%) in CH.sub.2Cl.sub.2 to give 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethan-1-one (Compound 4, 0.162 g) as a racemic mixture.

(95) Compound 4:

(96) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ ppm 3.11 (s, 3H) 3.22 (m, 2H) 3.67-3.88 (m, 5H) 4.00-4.22 (m, 3H) 4.44 (m, 1H) 4.98 (t, J=5.5 Hz, 1H) 5.83 (d, J=8.3 Hz, 1H) 6.56 (s, 1H) 6.63 (s, 1H) 6.92 (s, 1H) 7.04 (m, 2H) 7.17 (m, 1H) 7.31-7.50 (m, 3H) 8.38 (s, 1H)

(97) LC-MS (method LC-C): R.sub.t 1.89 min, MH.sup.+ 599

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

(98) ##STR00015## ##STR00016##

Synthesis of Intermediate 4f

(99) To a solution of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetate 4a (4.17 g, 12.0 mmol) in a mixture of EtOH (80 mL) and THF (40 mL) was added 0.5N NaOH (72 mL, 36.0 mmol). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was partially concentrated under reduced pressure to remove the organic solvents. The residue was acidified to pH 2-3 with 1N HCl and the mixture was extracted with EtOAc. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetic acid 4f (3.83 g).

Synthesis of Intermediate 4g

(100) A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetic acid 4f (7.12 g, 22.2 mmol) in thionyl chloride (50 mL, 689 mmol) was stirred at room temperature for 18 h. The reaction mixture was concentrated under reduced pressure and co-evaporated with toluene to give 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetyl chloride 4g (7.53 g) which was used in the next step without further purification.

Synthesis of Intermediate 4h

(101) A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)acetyl chloride 4g (5.29 g, 15.6 mmol) in CH.sub.3CN (50 mL) was added dropwise under N.sub.2-atm to a stirring mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1] (2.92 g, 15.6 mmol) and sodium bicarbonate (1.44 g, 17.1 mmol) in CH.sub.3CN (50 mL). The reaction mixture was stirred at room temperature for 65 h and poured out into water (500 mL). The product was extracted (2×) with Et.sub.2O. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and evaporated under reduced pressure. The residue solidified upon standing. The product was stirred up in diisopropyl ether (25 mL), filtered off, washed (3×) with diisopropyl ether, and dried under vacuum at 45° C. to provide 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-1-(6-(trifluoromethyl)indol in-1-yl)ethanone 4h (6.97 g).

Synthesis of Intermediate 4i

(102) A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4h (1.0 g, 2.04 mmol) in 2-Me-THF (100 mL) was stirred under N.sub.2-flow and cooled to −78° C. A solution of 1M lithium bis(trimethylsilyl)amide in THF (4.08 mL, 4.08 mmol) was added dropwise and the resulting mixture was stirred at −78° C. for 15 minutes. Chlorotrimethylsilane (417 μL, 3.27 mmol) was added dropwise and the mixture was stirred at −78° C. for 15 minutes. A solution of N-bromosuccinimide (400 mg, 2.25 mmol) in 2-Me-THF (25 mL) was added dropwise and the reaction mixture was stirred at −78° C. for 50 min. An aqueous saturated solution of NH.sub.4Cl (40 mL) was added at once, and the resulting mixture was stirred without cooling until the temperature reached 0° C. Water (10 mL) was added and the layers were separated. The organic layer was dried over MgSO.sub.4, filtered, evaporated under reduced pressure, and co-evaporated with CH.sub.3CN to provide 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromo-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4i (1.16 g). The product was used without further purification in the next step.

Synthesis of Intermediate 4j

(103) To a stirred solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromo-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4i (1.16 g, 2.04 mmol) in CH.sub.3CN (50 mL) under N.sub.2-atm were added 3-methoxy-5-(methylsulfonyl)aniline [CAS 62606-02-4](0.82 g, 4.08 mmol), and diisopropylethylamine (703 μL, 4.08 mmol) and the reaction mixture was stirred at 60° C. for 65 h. The mixture was cooled to room temperature and poured out into stirring H.sub.2O (250 mL). The product was extracted (2×) with Et.sub.2O. The combined organic layers were dried over MgSO.sub.4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica (40 g) using a gradient of heptane/EtOAc/EtOH 100/0/0 to 40/45/15. The desired fractions were combined and the solvent was evaporated under reduced pressure on a Rotavapor® to a residual volume of 35 mL. The product crystallized upon standing. The precipitate was filtered off, washed (3×) with EtOAc/heptane 1/1, and dried under vacuum at 45° C. to provide 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4j (870 mg).

Synthesis of Compound 4 and Chiral Separation into Enantiomers 4A and 4B

(104) A solution of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethyl)indolin-1-yl)ethanone 4j (210 mg, 0.28 mmol) in THF (30 mL) was added to a stirring mixture of Pd/C (0.5 g) in EtOAc (10 mL). The mixture was hydrogenated for 10 min at room temperature under atmospheric pressure. The catalyst was removed by filtration over Dicalite® and the solvents were evaporated under reduced pressure. The residue was combined with another batch (total amount: 1.0 g) and purified via Reverse phase HPLC (Stationary phase: Kromasil C18 100A 5 um (Eka Nobel), Mobile phase: Gradient from 50% ammoniunbicarbonate (0.25% in water), 50% acetonitrile to 20% ammoniunbicarbonate (0.25% in water), 80% acetonitrile) yielding Compound 4 (700 mg). The enantiomers of Compound 4 (700 mg) were separated via Normal Phase Chiral separation (Stationary phase: Whelk-O1 (SS) 5 μm with recycling peak shaving technique, Mobile phase: 100% ethanol). The fractions containing the first eluted enantiomer were combined and evaporated under reduced pressure. The residue was further purified by flash chromatography on silica gel (4 g) using a gradient of heptane/EtOAc/EtOH 100/0/0 to 40/45/15. The desired fractions were combined and evaporated, and co-evaporated with MeOH. The residue was triturated at 45° C. in H.sub.2O (4 mL) and MeOH (1 mL), the precipitate was filtered off, washed (3×) with H.sub.2O/MeOH 4/1, and dried under vacuum at 45° C. to provide Enantiomer 4A (197 mg). The fractions containing the second eluted enantiomer were combined and evaporated under reduced pressure. The residue was further purified by flash chromatography on silica gel (4 g) using a gradient of heptane/EtOAc/EtOH 100/0/0 to 40/45/15. The desired fractions were combined and evaporated, and co-evaporated with MeOH/water. The residue was stirred up in H.sub.2O (4 mL) and MeOH (1 mL), the precipitate was filtered off, washed (3×) with H.sub.2O/MeOH 4/1, and dried under vacuum at 45° C. to provide Enantiomer 4B (209 mg).

(105) Enantiomer 4A:

(106) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.16-3.27 (m, 2H) 3.68-3.85 (m, 5H) 4.04-4.20 (m, 3H) 4.44 (td, J=10.2, 6.6 Hz, 1H) 4.94 (t, J=5.6 Hz, 1H) 5.83 (d, J=8.4 Hz, 1H) 6.56 (t, J=2.1 Hz, 1H) 6.63 (t, J=1.8 Hz, 1H) 6.91 (t, J=1.4 Hz, 1H) 6.97-7.08 (m, 2H) 7.17 (d, J=2.0 Hz, 1H) 7.36 (d, J=8.1 Hz, 1H) 7.39 (dd, J=7.9, 0.9 Hz, 1H) 7.43-7.49 (m, 1H) 8.38 (br s, 1H)

(107) LC/MS (method LC-D): R.sub.t 1.17 min, MH.sup.+ 599

(108) [α].sub.D.sup.20: +59.8° (c 0.435, DMF)

(109) Chiral SFC (method SFC-I): R.sub.t 2.84 min, MH.sup.+ 599, chiral purity 100%.

(110) Enantiomer 4B:

(111) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.16-3.26 (m, 2H) 3.70-3.85 (m, 5H) 4.02-4.19 (m, 3H) 4.44 (td, J=10.2, 6.4 Hz, 1H) 4.94 (t, J=5.6 Hz, 1H) 5.83 (d, J=8.4 Hz, 1H) 6.56 (t, J=2.0 Hz, 1H) 6.63 (t, J=1.8 Hz, 1H) 6.91 (t, J=1.7 Hz, 1H) 6.99-7.07 (m, 2H) 7.16 (d, J=2.0 Hz, 1H) 7.36 (d, J=8.1 Hz, 1H) 7.37-7.41 (m, 1H) 7.44-7.48 (m, 1H) 8.38 (s, 1H)

(112) LC/MS (method LC-D): R.sub.t 1.17 min, MH.sup.+ 599

(113) [α].sub.D.sup.20: −56.4° (c 0.47, DMF)

(114) Chiral SFC (method SFC-I): R.sub.t 3.14 min, MH.sup.+ 599, chiral purity 97.0%.

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

(115) ##STR00017## ##STR00018##

Synthesis of Intermediate 5a

(116) To a mixture of ethyl 2-(4-chloro-2-hydroxyphenyl)acetate [CAS 1261826-30-5](5.2 g, 24.2 mmol) and cesium carbonate (15.8 g, 48.5 mmol) in DMF (90 mL) at 10° C. was added (2-bromoethoxy)(tert-butyl)dimethylsilane [CAS 86864-60-0](6.26 mL, 29.1 mmol). The reaction mixture was stirred at room temperature overnight. Water was added and the reaction mixture was extracted with EtOAc. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μm, 80 g, heptane/EtOAc 80/20). The pure fractions were combined and the solvent was removed under reduced pressure to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate 5a (7.8 g).

Synthesis of Intermediate 5b

(117) To a cooled (−78° C.) solution of 1M lithium bis(trimethylsilyl)amide in THF (41.8 mL, 41.8 mmol) was added a solution of ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate 5a (7.8 g, 20.9 mmol) in THF (45 mL). After 1 h at −70° C., chlorotrimethylsilane (4.24 mL, 33.5 mmol) was added. The reaction mixture was stirred at −70° C. for 15 min. N-Bromosuccinimide (4.46 g, 25.1 mmol) in THF (45 mL) was added and stirring was continued at −55° C. for 2 h. The reaction mixture was poured out into H.sub.2O and extracted twice with EtOAc. The organic phases were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give ethyl 2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate 5b (10.1 g) which was used in the next step without further purification.

Synthesis of Intermediate 5c

(118) A mixture of ethyl 2-bromo-2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)acetate 5b (4.75 g, 10.5 mmol), 3-methoxy-5-(methylsulfonyl)aniline [CAS 62606-02-4] (3.17 g, 15.8 mmol) and diisopropylethylamine (3.62 mL, 21.0 mmol) in CH.sub.3CN (90 mL) was stirred at 50° C. overnight. The reaction mixture was concentrated under reduced pressure. The residue was taken up with EtOAc and washed with 0.5N HCl and water. The organic phase was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μm, 120 g, heptane/EtOAc 90/10 to 80/20) to give ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 5c (3.5 g).

Synthesis of Intermediate 5d

(119) Lithium hydroxide monohydrate (513 mg, 12.2 mmol) was added portionwise to a solution of ethyl 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 5c (3.5 g, 6.12 mmol) in THF/CH.sub.3OH/H.sub.2O (1/1/1) (75 mL) at 10° C. The reaction was stirred at room temperature for 2 h, diluted with water and cooled to 0° C. The solution was slowly acidified to pH 6 with 0.5N HCl, and extracted with EtOAc. The organic layer was dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 5d (2.85 g). The compound was used without further purification in the next step.

Synthesis of Intermediate 5e

(120) To a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 5d (1 g, 1.84 mmol) in DMF (10 mL) were added HATU (1.05 g, 2.76 mmol), diisopropylethylamine (913 μL, 5.53 mmol) and 5-methoxy-6-(trifluoromethyl)indoline 2g (412 mg, 1.90 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K.sub.2CO.sub.3 and water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 5e (1.4 g). The compound was used without further purification in the next reaction step.

Synthesis of Compound 5 and Chiral Separation into Enantiomers 5A and 5B

(121) Under N.sub.2 flow at 5° C., HCl 4M in dioxane (4.71 mL, 18.8 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone 5e (1.4 g, 1.88 mmol) in MeOH (25 mL). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0° C., basified with a 10% aqueous solution of K.sub.2CO.sub.3 and extracted with EtOAc. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μm, 40 g, CH.sub.2Cl.sub.2/MeOH 98.5/1.5). The pure fractions were combined and the solvent was removed under reduced pressure to give 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)ethanone (Compound 5, 1.0 g) as a racemate. An analytical sample of Compound 5 was crystallized from MeOH (60 mg). The remaining amount was used to separate the Enantiomers via Preparative Chiral SFC (Stationary phase: Chiralpak® IC 5 μm 250×30 mm, Mobile phase: 70% CO.sub.2, 30% EtOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (400 mg) was solidified from diisopropyl ether to give Enantiomer 5A (351 mg). The second eluted enantiomer (430 mg) was solidified from diisopropyl ether to give Enantiomer 5B (336 mg).

(122) Compound 5:

(123) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.13-3.27 (m, 2H) 3.73 (s, 3H) 3.73-3.78 (m, 1H) 3.78-3.84 (m, 1H) 3.84 (s, 3H) 3.98-4.22 (m, 3H) 4.41 (dt, J=6.1, 10.1 Hz, 1H) 4.95 (t, J=5.6 Hz, 1H) 5.80 (d, J=8.08 Hz, 1H) 6.55 (s, 1H) 6.61 (s, 1H) 6.90 (s, 1H) 6.96-7.05 (m, 2H) 7.16 (d, J=1.52 Hz, 1H) 7.24 (s, 1H) 7.35 (d, J=8.08 Hz, 1H) 8.34 (s, 1H)

(124) LC-MS (method LC-A): R.sub.t 3.15 min, MH.sup.+ 629

(125) Melting point: 220° C.

(126) Enantiomer 5A:

(127) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.13-3.27 (m, 2H) 3.73 (s, 3H) 3.73-3.78 (m, 1H) 3.78-3.84 (m, 1H) 3.84 (s, 3H) 3.98-4.22 (m, 3H) 4.41 (dt, J=6.1, 10.1 Hz, 1H) 4.95 (t, J=5.6 Hz, 1H) 5.80 (d, J=8.08 Hz, 1H) 6.55 (s, 1H) 6.61 (s, 1H) 6.90 (s, 1H) 6.96-7.05 (m, 2H) 7.16 (d, J=1.52 Hz, 1H) 7.24 (s, 1H) 7.35 (d, J=8.08 Hz, 1H) 8.34 (s, 1H)

(128) LC/MS (method LC-A): R.sub.t 3.13 min, MH.sup.+ 629

(129) [α].sub.D.sup.20: −60.4° (c 0.28, DMF)

(130) Chiral SFC (method SFC-D): R.sub.t 1.02 min, MH.sup.+ 629, chiral purity 100%.

(131) Enantiomer 5B:

(132) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 3.10 (s, 3H) 3.13-3.27 (m, 2H) 3.73 (s, 3H) 3.73-3.78 (m, 1H) 3.78-3.84 (m, 1H) 3.84 (s, 3H) 3.98-4.22 (m, 3H) 4.41 (dt, J=6.1, 10.1 Hz, 1H) 4.95 (brt, J=5.6 Hz, 1H) 5.80 (d, J=8.08 Hz, 1H) 6.55 (s, 1H) 6.61 (s, 1H) 6.90 (s, 1H) 6.96-7.05 (m, 2H) 7.16 (d, J=1.52 Hz, 1H) 7.24 (s, 1H) 7.35 (d, J=8.08 Hz, 1H) 8.34 (s, 1H)

(133) LC/MS (method LC-A): R.sub.t 3.15 min, MH.sup.+ 629

(134) [α].sub.D.sup.20: +56.7° (c 0.3, DMF)

(135) Chiral SFC (method SFC-D): R.sub.t 1.22 min, MH.sup.+ 629, chiral purity 99.7%.

Example 6 (Method 1): Synthesis of 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 6)

(136) ##STR00019##

Synthesis of Intermediate 6a

(137) To a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 5d (1.07 g, 1.97 mmol) in DMF (10 mL) were added HATU (1.12 g, 2.95 mmol), diisopropylethylamine (976 μL, 5.91 mmol) and 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (400 mg, 1.97 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with water. The precipitate was filtered off, washed with water and taken up with EtOAc. The organic layer was washed with a 10% solution of K.sub.2CO.sub.3, water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6a (1.36 g). The crude compound was used without purification in the next reaction step.

Synthesis of Compound 6

(138) Under N.sub.2 flow at 5° C., HCl 4M in dioxane (4.66 mL, 18.6 mmol) was added dropwise to a solution of 2-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6a (1.36 g, 1.87 mmol) in MeOH (25 mL). The reaction was stirred at room temperature for 1 h. The mixture was cooled to 0° C., basified with a 10% aqueous solution of K.sub.2CO.sub.3 and extracted with EtOAc. The organic phase was separated, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μm, 40 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5). The pure fractions were combined and the solvent was removed under reduced pressure to give 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 6, 540 mg) as a racemate. An analytical sample of Compound 6 was obtained by crystallization from MeOH (34 mg).

(139) Compound 6:

(140) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 3.07-3.23 (m, 5H) 3.70-3.83 (m, 5H) 4.06-4.19 (m, 3H) 4.42 (td, J=10.23, 6.32 Hz, 1H) 4.92 (t, J=5.31 Hz, 1H) 5.81 (d, J=8.59 Hz, 1H) 6.56 (s, 1H) 6.61 (s, 1H) 6.90 (s, 1H) 6.99-7.05 (m, 3H) 7.16 (d, J=2.02 Hz, 1H) 7.30-7.40 (m, 2H) 8.03 (s, 1H) LC-MS (method LC-A): R.sub.t 3.28 min, MH.sup.+ 615

(141) Melting point: 191° C.

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

(142) ##STR00020## ##STR00021##

Synthesis of Intermediate 6b

(143) To a cooled (−70° C.) solution of 1.5M lithium bis(trimethylsilyl)amide in THF (23 mL, 34.4 mmol) under N.sub.2 flow was added a solution of ethyl 2-(2-(2-(benzyloxy)-ethoxy)-4-chlorophenyl)acetate 4a (6 g, 17.2 mmol) in THF (35 mL). After 1 h at −70° C., chlorotrimethylsilane (3.5 mL, 27.5 mmol) was added. The reaction mixture was stirred at −70° C. for 15 min. N-Bromosuccinimide (3.7 g, 20.6 mmol) in THF (35 mL) was added and stirring was continued at −70° C. for 2 h. The reaction mixture was poured into H.sub.2O and extracted with EtOAc. The organic phases were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromoacetate 6b (8.2 g) which was used in the next step without further purification.

Synthesis of Intermediate 6c

(144) A mixture of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-bromoacetate 6b (7.36 g, 17.2 mmol), 3-methoxy-5-(methylsulfonyl)aniline [CAS 62606-02-4] (5.2 g, 25.8 mmol) and diisopropylethylamine (5.9 mL, 25.8 mmol) in CH.sub.3CN (150 mL) was stirred at 50° C. overnight. The solvent was concentrated under reduced pressure. The residue was diluted with EtOAc and washed with 0.5N HCl and water. The organic layer was dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (15-40 μm, 220 g, CH.sub.2Cl.sub.2/MeOH 99/1). The pure fractions were combined and the solvent was removed under reduced pressure to give ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 6c (5.52 g).

Synthesis of Intermediate 6d

(145) At 10° C., Lithium hydroxide monohydrate (845 mg, 20.1 mmol) was added to a solution of ethyl 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetate 6c (5.52 g, 10.1 mmol) in MeOH/THF/water (1/1/1) (90 mL). The mixture was stirred at room temperature for 2 h. The mixture was diluted with ice water and cooled to 0° C. The resulting mixture was acidified to pH 6-7 with 0.5N HCl and extracted with EtOAc. The organic layers were combined, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to give 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 6d (5.26 g). The compound was used in the next reaction step without further purification.

Synthesis of Intermediate 6e

(146) A mixture of 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (1.85 g, 9.12 mmol), 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 6d (5.69 g, 10.9 mmol), HATU (5.2 g, 13.7 mmol) and diisopropylethylamine (4.52 mL, 27.4 mmol) in DMF (40 mL) was stirred at room temperature for 2 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was taken up with EtOAc, washed with a 10% solution of K.sub.2CO.sub.3 in water, 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 μm, 220 g, heptane/EtOAc 70/30). The pure fractions were combined and concentrated to dryness to give 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6e (5.6 g).

Synthesis of Compound 6 and Chiral Separation into Enantiomers 6A and 6B

(147) A mixture of 2-(2-(2-(benzyloxy)ethoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone 6e (5.6 g, 7.94 mmol) in EtOAc (100 mL) was hydrogenated at atmospheric pressure of H.sub.2 in the presence of Pd/C (10%) (1.7 g, 1.59 mmol) as a catalyst for 6 min (until the end of the H.sub.2 consumption). The reaction was diluted with EtOAc and filtered through a pad of Celite®. The filtrate was concentrated under reduced pressure to give 2-(4-chloro-2-(2-hydroxyethoxy)phenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-1-(6-(trifluoromethoxy)indolin-1-yl)ethanone (Compound 6) as a racemate (4.6 g, crude compound). The Enantiomers of Compound 6 were separated via chiral SFC (Stationary phase: Chiralcel® OJ-H 5 μm 250×20 mm, mobile phase: 80% CO.sub.2, 20% MeOH (+0.3% iPrNH.sub.2)). The first eluted enantiomer (1.96 g) was further purified via chiral SFC (Stationary phase: Chiralpak® IA 5 μm 250×20 mm, mobile phase: 74% CO.sub.2, 26% iPrOH (+0.3% iPrNH.sub.2)), to give after precipitation from heptane/diisopropyl ether, Enantiomer 6A (1.527 g). The second eluted enantiomer (2.10 g) was solidified from heptane/diisopropyl ether to give Enantiomer 6B (1.708 g).

(148) Enantiomer 6A:

(149) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.08-3.18 (m, 5H) 3.70-3.83 (m, 5H) 4.05-4.19 (m, 3H) 4.43 (td, J=10.32, 6.46 Hz, 1H) 4.97 (t, J=5.52 Hz, 1H) 5.82 (d, J=8.20 Hz, 1H) 6.56 (s, 1H) 6.62 (s, 1H) 6.91 (s, 1H) 7.00-7.08 (m, 3H) 7.16 (d, J=1.58 Hz, 1H) 7.34 (d, J=8.20 Hz, 2H) 8.04 (s, 1H)

(150) LC/MS (method LC-A): R.sub.t 3.32 min, MH.sup.+ 615

(151) [α].sub.D.sup.20: +64.3° (c 0.305, DMF)

(152) Chiral SFC (method SFC-E): R.sub.t 2.82 min, MH.sup.+ 615, chiral purity 100%.

(153) Enantiomer 6B:

(154) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 3.08-3.18 (m, 5H) 3.70-3.83 (m, 5H) 4.05-4.19 (m, 3H) 4.43 (td, J=10.32, 6.46 Hz, 1H) 4.97 (t, J=5.52 Hz, 1H) 5.82 (d, J=8.20 Hz, 1H) 6.56 (s, 1H) 6.62 (s, 1H) 6.91 (s, 1H) 7.00-7.08 (m, 3H) 7.16 (d, J=1.58 Hz, 1H) 7.34 (d, J=8.20 Hz, 2H) 8.04 (s, 1H)

(155) LC/MS (method LC-A): R.sub.t 3.31 min, MH.sup.+ 615

(156) [α].sub.D.sup.20: −53.7° (c 0.3, DMF)

(157) Chiral SFC (method SFC-E): R.sub.t 3.34 min, MH.sup.+ 615, chiral purity 95.7%.

Example 7: Synthesis of 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)-amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1-yl)ethyl)phenoxy)butanoic acid (Compound 7) and Chiral Separation into Enantiomers 7A and 7B

(158) ##STR00022## ##STR00023##

Synthesis of Intermediate 7a

(159) To a suspension of ethyl 2-(4-chloro-2-hydroxyphenyl)acetate [CAS 1261826-30-5](8.5 g, 39.6 mmol), Cs.sub.2CO.sub.3 (25.8 g, 79.2 mmol) in DMF (130 mL) at 10° C. was added dropwise tert-butyl 4-bromobutanoate [CAS 110611-91-1] (7 mL, 39.6 mmol). The mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc and water. The layers were separated. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 μm, 120 g, heptane/EtOAc 90/10). The pure fractions were combined and concentrated to dryness to give tert-butyl 4-(5-chloro-2-(2-ethoxy-2-oxoethyl)phenoxy)butanoate 7a (12.7 g).

Synthesis of Intermediate 7b

(160) A flask was charged with LiHMDS 1.5 M in THF (23.5 mL, 35.3 mmol) under N.sub.2 flow and was cooled to −78° C. A solution of tert-butyl 4-(5-chloro-2-(2-ethoxy-2-oxoethyl)phenoxy)butanoate 7a (6.3 g, 17.6 mmol) in THF (60 mL) was added dropwise and the mixture was stirred at −78° C. for 15 min. Chlorotrimethylsilane (3.6 mL, 28.3 mmol) was added. After 15 min at −78° C., N-bromosuccinimide (3.77 g, 21.2 mmol) in THF (40 mL) was added and the mixture was stirred at −70° C. for 1 h. The reaction was quenched with water and extracted with EtOAc. The organic layer was washed with water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure to yield tert-butyl 4-(2-(1-bromo-2-ethoxy-2-oxoethyl)-5-chlorophenoxy)butanoate 7b (7.6 g). The compound was used in the next reaction step without further purification.

Synthesis of Intermediate 7c

(161) To a solution of tert-butyl 4-(2-(1-bromo-2-ethoxy-2-oxoethyl)-5-chlorophenoxy)butanoate 7b (7.6 g, 17.4 mmol) in CH.sub.3CN (140 mL) at room temperature, was added diisopropylethylamine (4.8 mL, 27.9 mmol) and then 3-methoxy-5-(methylsulfonyl)aniline [CAS 62606-02-4] (4.2 g, 20.9 mmol). The mixture was stirred at 65° C. for 24 h. The mixture was diluted with EtOAc, then washed with HCl 0.5 N (twice) and water. The organic layer was dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 μm, 120 g, heptane/EtOAc 85/15 to 70/30). The pure fractions were combined and concentrated to dryness to give tert-butyl 4-(5-chloro-2-(2-ethoxy-1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxoethyl)phenoxy)butanoate 7c (7.3 g).

Synthesis of Intermediate 7d

(162) Tert-butyl 4-(5-chloro-2-(2-ethoxy-1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxoethyl)phenoxy)butanoate 7c (7.3 g, 13.1 mmol) and lithium hydroxide monohydrate (1.65 g, 39.4 mmol) in THF/water (1/1) (180 mL) was stirred at room temperature for 3 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 concentrated under reduced pressure to give 2-(2-(4-(tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 7d (6.9 g). The product was used in the next reaction step without further purification.

Synthesis of Intermediate 7e

(163) A mixture of 6-(trifluoromethyl)indoline [CAS 181513-29-1] (390 mg, 2.08 mmol), 2-(2-(4-(tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 7d (1.1 g, 2.08 mmol), HATU (1.2 g, 3.12 mmol) and diisopropylethylamine (1 mL, 6.25 mmol) in DMF (40 mL) was stirred at room temperature for 2 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was taken up with EtOAc, washed with an aqueous solution of K.sub.2CO.sub.3 10%, water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 μm, 80 g, CH.sub.2Cl.sub.2/MeOH 99.5/0.5) to give, after crystallization from CH.sub.3CN, tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1-yl)ethyl)phenoxy)butanoate 7e (700 mg).

Synthesis of Compound 7 and Chiral Separation into Enantiomers 7A and 7B

(164) A solution of tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1-yl)ethyl)phenoxy)butanoate 7e (0.6 g, 0.143 mmol) in HCl 4M in dioxane (6 ml) was stirred at 5° C. for 3 h and at room temperature for 8 h. The solvent was removed under reduced pressure and the product was crystallized from diisopropyl ether to yield 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxo-2-(6-(trifluoromethyl)indolin-1-yl)ethyl)phenoxy)butanoic acid (Compound 7, 530 mg) as a racemate. The Enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak® IC 5 μm 250×30 mm, Mobile phase: 65% CO.sub.2, 35% MeOH). The first eluted enantiomer (264 mg) was crystallized from CH.sub.3CN/diisopropyl ether to give

(165) Enantiomer 7A (207 mg). The second eluted enantiomer (269 mg) was crystallized from CH.sub.3CN/diisopropyl ether to give Enantiomer 7B (212 mg).

(166) Compound 7:

(167) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 1.90-2.09 (m, 2H) 2.31-2.43 (m, 2H) 3.12 (s, 3H) 3.17-3.28 (m, 2H) 3.74 (s, 3H) 3.88-4.07 (m, 1H) 4.07-4.15 (m, 2H) 4.35-4.45 (m, 1H) 5.73 (br d, J=7.88 Hz, 1H) 6.55 (br s, 1H) 6.64 (br s, 1H) 6.90 (br s, 1H) 7.04 (br s, 2H) 7.16 (br s, 1H) 7.31 (br d, J=7.88 Hz, 1H) 7.39 (br d, J=7.25 Hz, 1H) 7.46 (br d, J=7.25 Hz, 1H) 8.39 (br s, 1H) 12.12 (br s, 1H)

(168) LC-MS (method LC-A): R.sub.t 2.73 min, MH.sup.+ 641

(169) Melting point: 210° C.

(170) Enantiomer 7A:

(171) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.99 (dq, J=13.26, 6.86 Hz, 2H) 2.30-2.46 (m, 2H) 3.10 (s, 3H) 3.15-3.37 (m, 2H) 3.74 (s, 3H) 3.95-4.06 (m, 1H) 4.07-4.17 (m, 2H) 4.34-4.43 (m, 1H) 5.72 (d, J=8.08 Hz, 1H) 6.54 (s, 1H) 6.63 (s, 1H) 6.89 (s, 1H) 6.99-7.05 (m, 2H) 7.14 (d, J=1.52 Hz, 1H) 7.31 (d, J=8.08 Hz, 1H) 7.38 (d, J=7.58 Hz, 1H) 7.45 (d, J=8.08 Hz, 1H) 8.38 (s, 1H) 12.09 (br s, 1H)

(172) LC/MS (method LC-A): R.sub.t 2.73 min, MH.sup.+ 641

(173) [α].sub.D.sup.20: −49.8° (c 0.225, DMF)

(174) Chiral SFC (method SFC-F): R.sub.t 3.13 min, no MS response, chiral purity 100%.

(175) Melting point: 182° C.

(176) Enantiomer 7B:

(177) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.99 (dq, J=13.26, 6.86 Hz, 2H) 2.30-2.46 (m, 2H) 3.10 (s, 3H) 3.15-3.37 (m, 2H) 3.74 (s, 3H) 3.95-4.06 (m, 1H) 4.07-4.17 (m, 2H) 4.34-4.43 (m, 1H) 5.72 (d, J=8.08 Hz, 1H) 6.54 (s, 1H) 6.63 (s, 1H) 6.89 (s, 1H) 6.99-7.05 (m, 2H) 7.14 (d, J=1.52 Hz, 1H) 7.31 (d, J=8.08 Hz, 1H) 7.38 (d, J=7.58 Hz, 1H) 7.45 (d, J=8.08 Hz, 1H) 8.38 (s, 1H) 12.09 (br s, 1H)

(178) LC/MS (method LC-A): R.sub.t 2.73 min, MH.sup.+ 641

(179) [α].sub.D.sup.20: +49.3° (c 0.2333, DMF)

(180) Chiral SFC (method SFC-F): R.sub.t 4.34 min, no MS response, chiral purity 100%.

(181) Melting point: 180° C.

Example 8: Synthesis of 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)-phenyl)amino)-2-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)phenoxy)-butanoic acid (Compound 8) and Chiral Separation into Enantiomers 8A and 8B

(182) ##STR00024##

Synthesis of Intermediate 8a

(183) A mixture of 5-methoxy-6-(trifluoromethyl)indoline 2g (617 mg, 2.84 mmol), 2-(2-(4-(tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 7d (1.5 g, 2.84 mmol), HATU (1.62 g, 4.26 mmol) and diisopropylethylamine (1.4 mL, 8.5 mmol) in DMF (60 mL) was stirred at room temperature for 12 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was taken up with EtOAc, washed with an aqueous solution of K.sub.2CO.sub.3 10%, water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 μm, 120 g, heptane/EtOAc 60/40) to give, after crystallization from petroleum ether/diisopropyl ether, tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)phenoxy)butanoate 8a (1.36 g).

Synthesis of Compound 8 and Chiral Separation into Enantiomers 8A and 8B

(184) A solution of tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)phenoxy)butanoate 8a (1.36 g, 1.87 mmol) in HCl 4M in dioxane (12 ml) was stirred at 5° C. for 3 h and at room temperature for 14 h. The precipitate was filtered off and washed with dioxane/diisopropyl ether to yield 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-(5-methoxy-6-(trifluoromethyl)indolin-1-yl)-2-oxoethyl)phenoxy)butanoic acid (Compound 8, 1.2 g) as a racemate (contaminated with 2.2% of intermediate 8a). A small fraction (40 mg) was further purified via achiral SFC (Stationary phase: 2-ethylpyridine 6 μm 150×21.2 mm, mobile phase: 60% CO.sub.2, 40% iPrOH) to yield, after crystallization from CH.sub.3CN/diisopropyl ether, 28 mg of compound 8. The remaining amount of Compound 8 was used to separate the enantiomers via Preparative Chiral SFC (Stationary phase: Chiralpak® IC 5 μm 250×30 mm, Mobile phase: 60% CO.sub.2, 40% MeOH). The first eluted enantiomer (340 mg) was solidified in petroleum ether/diisopropyl ether to give Enantiomer 8A (285 mg). The second eluted enantiomer (334 mg) was solidified in petroleum ether/diisopropyl ether to give

(185) Enantiomer 8B (210 mg).

(186) Compound 8:

(187) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.95-2.08 (m, 2H) 2.32-2.44 (m, 2H) 3.08-3.27 (m, 5H) 3.73 (s, 3H) 3.84 (s, 3H) 3.92-4.00 (m, 1H) 4.12 (br d, J=3.54 Hz, 2H) 4.32-4.40 (m, 1H) 5.69 (br d, J=8.08 Hz, 1H) 6.54 (br s, 1H) 6.62 (s, 1H) 6.87 (s, 1H) 6.98-7.04 (m, 2H) 7.14 (s, 1H) 7.22 (s, 1H) 7.31 (d, J=8.08 Hz, 1H) 8.34 (s, 1H) 12.07 (br s, 1H)

(188) LC-MS (method LC-A): R.sub.t 2.74 min, MH.sup.+ 671

(189) Melting point: 232° C.

(190) Enantiomer 8A:

(191) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 1.95-2.07 (m, 2H) 2.35-2.47 (m, 2H) 3.11 (s, 3H) 3.15-3.31 (m, 2H) 3.74 (s, 3H) 3.85 (s, 3H) 3.91-4.02 (m, 1H) 4.06-4.19 (m, 2H) 4.37 (td, J=10.25, 6.31 Hz, 1H) 5.70 (d, J=8.20 Hz, 1H) 6.54 (s, 1H) 6.63 (s, 1H) 6.88 (s, 1H) 7.02 (d, J=8.20 Hz, 2H) 7.12-7.17 (m, 1H) 7.23 (s, 1H) 7.31 (d, J=8.20 Hz, 1H) 8.34 (s, 1H) 12.13 (br s, 1H)

(192) LC/MS (method LC-A): R.sub.t 2.75 min, MH.sup.+ 671

(193) [α].sub.D.sup.20: −52.9° (c 0.28, DMF)

(194) Chiral SFC (method SFC-G): R.sub.t 2.50 min, MH.sup.+ 671, chiral purity 100%.

(195) Enantiomer 8B:

(196) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 1.95-2.07 (m, 2H) 2.35-2.47 (m, 2H) 3.11 (s, 3H) 3.15-3.31 (m, 2H) 3.74 (s, 3H) 3.85 (s, 3H) 3.91-4.02 (m, 1H) 4.06-4.19 (m, 2H) 4.37 (td, J=10.25, 6.31 Hz, 1H) 5.70 (d, J=8.20 Hz, 1H) 6.54 (s, 1H) 6.63 (s, 1H) 6.88 (s, 1H) 7.02 (d, J=8.20 Hz, 2H) 7.12-7.17 (m, 1H) 7.23 (s, 1H) 7.31 (d, J=8.20 Hz, 1H) 8.34 (s, 1H) 11.44 (br s, 1H)

(197) LC/MS (method LC-A): R.sub.t 2.73 min, MH.sup.+ 671

(198) [α].sub.D.sup.20: +46.4° (c 0.28, DMF)

(199) Chiral SFC (method SFC-G): R.sub.t 3.31 min, MH.sup.+ 671, chiral purity 100%.

Example 9: Synthesis of 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)-amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)butanoic acid (Compound 9) and Chiral Separation into Enantiomers 9A and 9B

(200) ##STR00025##

Synthesis of Intermediate 9a

(201) A mixture of 6-(trifluoromethoxy)indoline [CAS 959235-95-1] (577 mg, 2.84 mmol), 2-(2-(4-(tert-butoxy)-4-oxobutoxy)-4-chlorophenyl)-2-((3-methoxy-5-(methylsulfonyl)phenyl)amino)acetic acid 7d (1.5 g, 2.84 mmol), HATU (1.62 g, 4.26 mmol) and diisopropylethylamine (1.4 mL, 8.5 mmol) in DMF (60 mL) was stirred at room temperature for 12 h. The mixture was diluted with water. The precipitate was filtered off and washed with water. The precipitate was taken up with EtOAc, washed with an aqueous solution of K.sub.2CO.sub.3 10%, water, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. Purification was performed by flash chromatography on silica gel (15-40 μm, 120 g, heptane/EtOAc 60/40) to give, after crystallization from petroleum ether/diisopropyl ether, tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)butanoate 9a (1.02 g).

Synthesis of Compound 9 and Chiral Separation into Enantiomers 9A and 9B

(202) A solution of tert-butyl 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)butanoate 9a (1.02 g, 1.43 mmol) in HCl 4M in dioxane (10 ml) was stirred at 5° C. for 3 h and at room temperature for 12 h. The precipitate was filtered off and washed with dioxane/diisopropyl ether to yield 4-(5-chloro-2-(1-((3-methoxy-5-(methylsulfonyl)phenyl)amino)-2-oxo-2-(6-(trifluoromethoxy)indolin-1-yl)ethyl)phenoxy)butanoic acid (Compound 9, 930 mg, 0.78 equiv. HCl, 0.08 equiv. H.sub.2O, 0.162 equiv. dioxane (determined by titration)) as a racemate. The Enantiomers were separated via Preparative Chiral SFC (Stationary phase: Chiralpak® IC 5 μm 250×30 mm, Mobile phase: 70% CO.sub.2, 30% EtOH/iPrOH (50/50)). The first eluted enantiomer was stirred up in a mixture of 1N HCl and EtOAc. The organic layer was separated, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. The compound was crystallized from CH.sub.3CN/diisopropyl ether to give Enantiomer 9A (145 mg). The second eluted enantiomer was stirred up in a mixture of 1N HCl and EtOAc. The organic layer was separated, dried over MgSO.sub.4, filtered and the solvent was concentrated under reduced pressure. The compound was crystallized from CH.sub.3CN/diisopropyl ether to give Enantiomer 9B (156 mg).

(203) Compound 9:

(204) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 1.99 (dq, J=13.71, 7.05 Hz, 2H) 2.32-2.46 (m, 2H) 3.08-3.20 (m, 5H) 3.74 (s, 3H) 4.00 (td, J=10.23, 7.33 Hz, 1H) 4.07-4.15 (m, 2H) 4.38 (td, J=10.23, 6.82 Hz, 1H) 5.70 (s, 1H) 6.54 (s, 1H) 6.63 (s, 1H) 6.88 (s, 1H) 6.95-7.09 (m, 2H) 7.14 (d, J=1.52 Hz, 1H) 7.30 (br d, J=8.08 Hz, 1H) 7.33 (br d, J=8.59 Hz, 1H) 8.03 (s, 1H)

(205) LC-MS (method LC-A): R.sub.t 2.87 min, MH.sup.+ 657

(206) Melting point: 173° C.

(207) Enantiomer 9A:

(208) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 1.91-2.06 (m, 2H) 2.32-2.44 (m, 2H) 3.09-3.23 (m, 5H) 3.74 (s, 3H) 3.98-4.16 (m, 3H) 4.39 (td, J=10.17, 6.78 Hz, 1H) 5.71 (d, J=8.20 Hz, 1H) 6.55 (s, 1H) 6.63 (s, 1H) 6.89 (s, 1H) 7.00-7.08 (m, 2H) 7.15 (s, 1H) 7.30 (d, J=8.20 Hz, 1H) 7.34 (d, J=8.20 Hz, 1H) 8.04 (s, 1H) 12.11 (br s, 1H)

(209) LC/MS (method LC-A): R.sub.t 2.86 min, MH.sup.+ 657

(210) [α].sub.D.sup.20: −56.5° (c 0.255, DMF)

(211) Chiral SFC (method SFC-H): R.sub.t 4.85 min, MH.sup.+ 657, chiral purity 100%.

(212) Melting point: 154° C.

(213) Enantiomer 9B:

(214) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ ppm 1.91-2.06 (m, 2H) 2.32-2.44 (m, 2H) 3.09-3.23 (m, 5H) 3.74 (s, 3H) 3.98-4.16 (m, 3H) 4.39 (td, J=10.17, 6.78 Hz, 1H) 5.71 (d, J=8.20 Hz, 1H) 6.55 (s, 1H) 6.63 (s, 1H) 6.89 (s, 1H) 7.00-7.08 (m, 2H) 7.15 (s, 1H) 7.30 (d, J=8.20 Hz, 1H) 7.34 (d, J=8.20 Hz, 1H) 8.04 (s, 1H) 12.11 (br s, 1H)

(215) LC/MS (method LC-A): R.sub.t 2.86 min, MH.sup.+ 657

(216) [α].sub.D.sup.20: +55.3° (c 0.302, DMF)

(217) Chiral SFC (method SFC-H): R.sub.t 6.34 min, MH.sup.+ 657, chiral purity 100%.

(218) Melting point: 155° C.

(219) TABLE-US-00004 TABLE compounds prepared as described above Compound Structure Optical rotation 1 racemic 1A [α].sub.D.sup.20 = −49.6° 1B [α].sub.D.sup.20 = +51.7° 2 racemic 2A 0 [α].sub.D.sup.20 = +53.5° 2B [α].sub.D.sup.20 = −56.5° 3 racemic 3A [α].sub.D.sup.20 = −59.0° 3B [α].sub.D.sup.20 = +56.8° 4 racemic 4A [α].sub.D.sup.20 = +59.8° 4B [α].sub.D.sup.20 = −56.4° 5 racemic 5A [α].sub.D.sup.20 = −60.4° 5B 0 [α].sub.D.sup.20 = +56.7° 6 racemic 6A [α].sub.D.sup.20 = +64.3° 6B [α].sub.D.sup.20 = −53.7° 7 racemic 7A [α].sub.D.sup.20 = −49.8° 7B [α].sub.D.sup.20 = +49.3° 8 racemic 8A [α].sub.D.sup.20 = −52.9° 8B [α].sub.D.sup.20 = +46.4° 9 0 racemic 9A [α].sub.D.sup.20 = −56.5° 9B [α].sub.D.sup.20 = +55.3°

(220) Antiviral Activity of the Compounds of the Invention

(221) DENV-2 Antiviral Assay

(222) The antiviral activity of all the compounds of the invention was tested against the 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 μL 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 μM-0.000064 μM or 2.5 μM-0.0000064 μM 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 μL of culture medium was added instead of Vero cells. Once the cells are 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 μL of virus suspension was added to all the wells containing test compound and to the wells assigned as virus control. In parallel, 15 μL 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 (EC50) was determined. Therefore, the percent inhibition (I) for every test concentration is calculated using the following formula: I=100*(S.sub.T−S.sub.CC)/(S.sub.VC−S.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).

(223) In parallel, the toxicity of the compounds was assessed on the same plates. Once the read-out for the eGFP signal was done, 40 μL 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.

(224) TABLE-US-00005 TABLE 1 EC.sub.50, CC.sub.50, and SI for the compounds of the invention in the DENV-2 antiviral assay EC.sub.50 CC.sub.50 compound# (μM) N (μM) N SI N 1 0.0032 4 14 4 4397 4 1A 2.4 3 12 3 4.8 3 1B 0.0014 3 3.4 3 >1980 3 2 0.0010 4 12 4 13700 4 2A 0.00093 3 9.6 4 16400 3 2B 0.17 3 18 3 105 3 3 0.00074 3 7.9 3 10700 3 3A 0.57 3 11 3 19 3 3B 0.00061 3 8.7 3 16300 3 4 0.00066 3 7.7 3 11600 3 4A 0.00041 7 6.0 7 15000 7 4B 0.016 6 11 7 693 6 5 0.00070 3 11 3 15700 3 5A 0.076 3 16 3 210 3 5B 0.00023 4 8.5 4 >16800 4 6 0.00043 4 3.6 4 7070 4 6A 0.00023 7 7.3 8 >12000 7 6B 0.020 5 10 6 492 5 7 0.00058 3 13 3 21800 3 7A 0.069 3 11 4 165 3 7B 0.00025 5 11 7 90600 5 8 0.0019 3 14 3 7460 3 8A 0.095 4 12 5 126 4 8B 0.0012 3 14 3 6780 3 9 0.00031 3 12 3 40200 3 9A 0.12 3 12 3 93 3 9B 0.00015 4 13 4 83000 4 N = the number of independent experiments in which the compounds were tested.

(225) Tetravalent Reverse Transcriptase Quantitative-PCR (RT-qPCR) Assay

(226) The antiviral activity of the compounds of the invention was tested against DENV-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 DENV-1, 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 3′UTR of DENV; Table 2) and a cellular reference gene (1-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 3′UTR gene. On the other hand, if a test compound is toxic to the cells, a similar effect on β-actin expression will be observed. The comparative ΔΔCp method is used to calculate EC.sub.50, which is based on the relative gene expression of the target gene (3′UTR) normalized with the cellular housekeeping gene (β-actin). In addition, CC.sub.50 values are determined based on the C.sub.p values acquired for the housekeeping gene β-actin.

(227) TABLE-US-00006 TABLE 2 Primers and probes used for the real-time,  quantitative RT-PCR. Primer/ probe Target Sequence.sup.a, b F3utr258 DENV 3'- 5′-CGGTTAGAGGAGACCCCTC-3′ UTR R3utr425 DENV 3'- 5′-GAGACAGCAGGATCTCTGGTC-3′ UTR P3utr343 DENV 3'- -5′-AAGGACTAG-ZEN- UTR AGGTTAGAGGAGACCCCCC-3′-/ Factin743 β-actin 5′-GGCCAGGTCATCACCATT-3′ Ractin876 β-actin 5′-ATGTCCACGTCACACTTCATG-3′ Pactin773 β-actin -5′-TTCCGCTGC- -CCTGAGG CTCTC-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 3′UTR 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).

(228) 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 μL/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 μM-0.000064 μM or 2.5 μM-0.0000064 μM 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 μL of virus suspension was added to all the wells containing test compound and to the wells assigned as virus control. In parallel, 25 μL 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 μL). 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.

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

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

(231) Using the LightCycler software and an in-house LIMS system, dose response curves for each compound were calculated and the half maximal effective concentration (EC50) and the half maximal cytotoxic concentration (CC.sub.50) were determined (Tables 5-8).

(232) TABLE-US-00007 TABLE 3 cDNA synthesis using Mix A, denaturation, Mix B and reverse transcription. A Mix A Plates 8 Samples 828 Reaction Vol. (μl) 20 Concentration Volume for (μl) Mix Item Unit Stock Final 1 sample x samples Milli-Q H.sub.2O 7.27 6019.56 R3utr425 μM 20 0.27 0.15 12420 Ractin876 μM 20 0.27 0.15 124.20 Volume 7.57 mix/well (μl) 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 (μl) Mix Item Unit Stock Final 1 sample x samples Expand HIFI buffer 2 X 10.00 1.00 2.00 1728.0 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/μl 40.00 1.00 0.50 432.0 Expand RT U/μl 50.00 0.33 0.13 112.3 Total Volume 7.43 Mix (μl) D Protocol cDNA synthesis Step Temp Time Rev transc 42° C. 30′ Denaturation 99° C.  5′ Hold  4° C. hold

(233) TABLE-US-00008 TABLE 4 qPCR mix and protocol. A Mix c Samples 833 Reaction Vol. 25 (μl) Concentration Volume for (μl) Mix Item Unit Stock Final 1 sample x samples H.sub.2O PCR grade Roche 7.74 6447.42 Roche 2xMM mix X 2 1 12.50 10412.50 F3utr258 μM 20 0.3 0.38 316.54 R3utr425 μM 20 0.3 0.38 316.54 P3utr343 μM 20 0.1 0.13 108.29 Factin743 μM 20 0.3 0.38 316.54 Ractin876 μM 20 0.3 0.38 316.54 Pactin773 μM 20 0.1 0.13 108.29 Volume 22.02 Mix/Tube (μl) 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

(234) TABLE-US-00009 TABLE 5 EC.sub.50, CC.sub.50, and SI for the compounds against serotype 1 in the RT-qPCR assays RT-qPCR serotype 1 TC974#666 EC.sub.50 CC.sub.50 compound# (μM) N (μM) N SI N 1B 0.0015 3 >2.5 3 >2860 3 2A 0.0046 5 >2.5 4 >981 4 3B 0.0011 3 >2.5 3 >3640 3 4A 0.00075 3 >2.5 3 >5470 3 5B 0.00094 4 5.1 4 8640 4 6A 0.00016 3 >2.5 3 >41300 3 7B 0.00013 3 >2.5 3 >19200 3 8B 0.0012 3 14 3 10500 3 9B 0.00010 3 >2.5 3 >45500 3 N = the number of independent experiments in which the compounds were tested.

(235) TABLE-US-00010 TABLE 6 EC.sub.50, CC.sub.50, and SI for the compounds against serotype 2 in the RT-qPCR assays RT-qPCR serotype 2 16681 EC.sub.50 CC.sub.50 compound# (μM) N (μM) N SI N 1B 0.0011 3 4.1 3 4670 3 2A 0.0013 4 12 4 4910 4 3B 0.00090 3 3.6 3 4760 3 4A 0.00045 3 2.7 3 11700 3 5B 0.00024 5 4.2 5 >17100 5 6A 0.00016 3 4.2 3 >12600 3 7B 0.00019 3 >2.5 2 >13500 2 8B 0.00030 3 16 2 54300 2 9B 0.000068 3 >2.5 3 >56500 3 N = the number of independent experiments in which the compounds were tested.

(236) TABLE-US-00011 TABLE 7 EC.sub.50, CC.sub.50, and SI for the compounds against serotype 3 in the RT-qPCR assays RT-qPCR serotype 3 H87 EC.sub.50 CC.sub.50 compound# (μM) N (μM) N SI N 1B 0.020 3 >2.5 3 >127 3 2A 0.020 3 >2.5 3 >157 3 3B 0.013 3 >2.5 3 >444 3 4A 0.013 3 >2.5 3 >234 3 5B 0.0067 4 >2.5 4 >752 4 6A 0.0026 3 >2.5 3 >1480 3 7B 0.0052 3 >2.5 3 >473 3 8B 0.019 3 12 3 796 3 9B 0.0017 3 >2.5 3 >1840 3 N = the number of independent experiments in which the compounds were tested.

(237) TABLE-US-00012 TABLE 8 EC.sub.50, CC.sub.50, and SI for the compounds against serotype 4 in the RT-qPCR assays RT-qPCR serotype 4 H241 EC.sub.50 CC.sub.50 compound# (μM) N (μM) N SI N 1B 0.13 3 >2.4 3 23 3 2A 0.10 3 2.8 3 35 3 3B 0.072 3 >2.3 3 >32 3 4A 0.044 4 2.2 4 69 4 5B 0.026 4 2.5 2 86 2 6A 0.026 4 2.3 4 119 4 7B 0.024 3 >2.5 3 >186 3 8B 0.084 3 7.4 3 88 3 9B 0.0072 3 5.0 2 1390 2 N = the number of independent experiments in which the compounds were tested.