CHEMICAL COMPOUNDS

20190038607 · 2019-02-07

Assignee

Astrazeneca Ab (Sodertalje, SE)

Inventors

Cpc classification

International classification

Abstract

The invention concerns compounds of Formula (I)

##STR00001##

or pharmaceutically-acceptable salts thereof, wherein R.sup.1 to R.sup.5 have any of the meanings defined hereinbefore in the description; processes for their preparation, pharmaceutical compositions containing them and their use in the treatment of cell proliferative disorders.

Claims

1. A pharmaceutical composition comprising a compound and a pharmaceutically-acceptable diluent or carrier, wherein the compound is (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid or a pharmaceutically-acceptable salt thereof.

2. A pharmaceutical composition according to claim 1, wherein the compound is (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.

3. A pharmaceutical composition according to claim 1, wherein the compound is the maleic acid salt of (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.

4. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition contains less than 5% w/w of (R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate.

5. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises an anti-oxidant and optionally further comprises a metal-chelating agent.

6. A method for making a compound of Formula (I): ##STR00075## wherein: R.sup.1 and R.sup.2 are each independently H or F; R.sup.3 is H or methyl; and either: a) R.sup.4 is H and R.sup.5 is F; or b) R.sup.4 is F and R.sup.5 is H, comprising hydrolysis of a compound of Formula (II): ##STR00076## wherein R.sup.6 is (1-6C) alkyl.

7. A method according to claim 6, wherein the compound of Formula (I) is (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid.

8. A method according to claim 7, wherein R.sup.6 is methyl.

9. (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate.

10. A method for making a compound of Formula (II): ##STR00077## wherein: R.sup.1 and R.sup.2 are each independently H or F; R.sup.3 is H or methyl; either: a) R.sup.4 is H and R.sup.5 is F; or b) R.sup.4 is F and R.sup.5 is H; R.sup.6 is (1-6C) alkyl, comprising a reaction of a compound of Formula (III): ##STR00078## with a compound of Formula (IV): ##STR00079##

11. A method according to claim 10, wherein the compound of Formula (II) is (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0366] FIG. 1 shows an X-Ray Powder Diffraction Pattern of Example 7 Form A

[0367] FIG. 2 shows an X-Ray Powder Diffraction Pattern of Example 1 Form B

[0368] FIG. 3 shows DSC Thermogram of Example 1 Form B

[0369] FIG. 4 shows X-Ray Powder Diffraction Pattern of Example 1 Form A

[0370] FIG. 5 shows TGA Thermogram of Example 1 Form A

[0371] FIG. 6 shows X-Ray Powder Diffraction Pattern of Example 1 Form C

[0372] FIG. 7 shows TGA Thermogram of Example 1 Form C

[0373] FIG. 8 shows an X-Ray Powder Diffraction Pattern of Example 11

[0374] FIG. 9 shows a DSC trace of Example 11.

[0375] FIG. 10 shows the results of an MCF-7 xenograft study with Example 1 and AZD2014.

[0376] FIGS. 11 and 12 show the results of an HCC1428 long term estrogen deprived (LTED) xenograft efficacy study with Example 1.

EXAMPLES

[0377] The invention will now be illustrated in the following Examples in which, generally: [0378] (i) operations were carried out at ambient temperature, i.e. in the range 17 to 25? C. and under an atmosphere of an inert gas such as nitrogen unless otherwise stated; [0379] (ii) evaporations were carried out by rotary evaporation or utilising Genevac equipment or Biotage v10 evaporator in vacuo and work-up procedures were carried out after removal of residual solids by filtration; [0380] (iii) flash chromatography purifications were performed on an automated Teledyne Isco CombiFlash? Rf or Teledyne Isco CombiFlash? Companion? using prepacked RediSep Rf Gold? Silica Columns (20-40 ?m, spherical particles), GraceResolv? Cartridges (Davisil? silica) or Silicycle cartridges (40-63 ?m). [0381] (iv) preparative chromatography was performed on a Gilson prep HPLC instrument with UV collection; [0382] (v) chiral preparative chromatography was performed on a Gilson instrument with UV collection (233 injector/fraction collector, 333 & 334 pumps, 155 UV detector) or a Varian Prep Star instrument (2?SD1 pumps, 325 UV detector, 701 [0383] fraction collector) pump running with Gilson 305 injection; [0384] (vi) yields, where present, are not necessarily the maximum attainable; [0385] (vii) in general, the structures of end-products of the Formula I were confirmed by nuclear magnetic resonance (NMR) spectroscopy; NMR chemical shift values were measured on the delta scale [proton magnetic resonance spectra were determined using a Bruker Avance 500 (500 MHz) or Bruker Avance 400 (400 MHz) instrument]; measurements were taken at ambient temperature unless otherwise specified; the following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublet; dt, doublet of triplets; bs, broad signal [0386] (viii) in general, end-products of the Formula I were also characterised by mass spectroscopy following liquid chromatography (LCMS or UPLC); UPLC was carried out using a Waters UPLC fitted with Waters SQ mass spectrometer (Column temp 40, UV=220-300 nm, Mass Spec=ESI with positive/negative switching) at a flow rate of 1 ml/min using a solvent system of 97% A+3% B to 3% A to 97% B over 1.50 mins (total runtime with equilibration back to starting conditions etc 1.70 min), where A=0.1% formic acid in water (for acid work) or 0.1% ammonia in water (for base work) B=acetonitrile. For acid analysis the column used was Waters Acquity HSS T3 1.8 ?m 2.1?50 mm, for base analysis the column used was Waters Acquity BEH 1.7 ?m 2.1?50 mm; LCMS was carried out using a Waters Alliance HT (2795) fitted with a Waters ZQ ESCi mass spectrometer and a Phenomenex GeminiNX (50?2.1 mm 5 ?m) column at a flow rate of 1.1 ml/min 95% A to 95% B over 4 min with a 0.5 min hold. The modifier is kept at a constant 5% C (50:50 acetonitrile:water 0.1% formic acid) or D (50:50 acetonitrile:water 0.1% ammonium hydroxide (0.88 SG) depending on whether it is an acidic or basic method. [0387] (ix) ion exchange purification was generally preformed using a SCX-2 (Biotage, Propylsulfonic acid functionalized silica. Manufactured using a trifunctional silane. Non end-capped) cartridge. [0388] (x) intermediates purity was assessed by thin layer chromatographic, mass spectral, HPLC (high performance liquid chromatography) and/or NMR analysis; [0389] (xi) For XRPD analysis of Example 7, samples were mounted on zero background silicon wafers and analysed using the PANalytical CubiX Pro diffractometer (1=1.5418 ?). Samples were spun to improve counting statistics. Data was collected in reflection geometry in theta ?2theta configuration over the scan range 2? to 40? 2-theta with 25 second exposure per 0.025067? increment. X-rays were generated by a copper long-fine focus tube operated at 45 kV and 40 mA. Persons skilled in the art of X-ray powder diffraction will realise that the relative intensity of peaks can be affected by, for example, grains above 30 microns in size and non-unitary aspect ratios that may affect analysis of samples. The skilled person will also realise that the position of reflections can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer. The surface planarity of the sample may also have a small effect. Hence the diffraction pattern data presented are not to be taken as absolute values; [0390] (xii) For XRPD analysis of Example 1, The X-ray powder diffractogram was determined by mounting a sample of the crystalline material on a Panalytical single silicon crystal (SSC) wafer mount and spreading out the sample into a thin layer with the aid of a microscope slide. The sample was spun at 30 revolutions per minute (to improve counting statistics) and irradiated with X-rays generated by a copper long-fine focus tube operated at 45 kV and 40 mA with a wavelength of 1.5418 angstroms. The X-ray beam was passed through a 0.04 rad soller slit, then an automatic variable divergence slit set at 20 mm and finally a 20 mm beam mask. The reflected radiation was directed through a 20 mm antiscatter slit and a 0.04 rad soller slit. The sample was exposed for 1.905 seconds per 0.0025067? 2-theta increment (continuous scan mode) over the range 2 degrees to 40 degrees 2-theta in theta-theta mode. The running time was 3 minutes and 36 seconds. The instrument was equipped X-Celerator detector. Control and data capture was by means of a Dell Pentium 4HT Workstation operating with X'Pert Industry software. [0391] (xiii) Differential Scanning calorimetry: Analytical Instrument: TA Instruments Q1000 DSC. Typically less than 5 mg of material contained in a standard aluminium pan fitted with a lid was heated over the temperature range 25? C. to 300? C. at a constant heating rate of 10? C. per minute. A purge gas using nitrogen was usedflow rate 50 ml per minute. [0392] (xiv) Thermogravimetric Analysis Analytical Instrument: TA Instruments Q5000 TGA. Typically less than 10 mg of material is placed on a 100 ?l platinum pan and heated over the temperature range 30? C. to 150? C. at a constant heating rate of 10? C. per minute. [0393] (xv) the following abbreviations have been used: [0394] aq. aqueous [0395] CDCl.sub.3 deutero-chloroform [0396] Conc. concentrated [0397] DCM dichloromethane [0398] DMA N,N-dimethylacetamide [0399] DMSO dimethyl sulphoxide [0400] DSC differential scanning calorimetry, [0401] EtOH ethanol [0402] EtOAc ethyl acetate [0403] IPA/iPrOH isopropyl alcohol [0404] MeCN acetonitrile [0405] MTBE methyltertbutyl ether [0406] rt/RT room temperature [0407] sat. saturated [0408] sol. solution [0409] THF tetrahydrofuran [0410] TFA trifluoroacetic acid [0411] TGA Thermogravimetric analysis

Example 1: (E)-3-(3,5-Difluoro-44(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid

[0412] ##STR00019##

[0413] The following processes should be carried out under an atmosphere of nitrogen in the absence of light as a light degradation product [(R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate] may be formed.

[0414] (E)-Methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (350 g, 766.69 mmol) was charged to a 5 L fixed vessel. Isopropyl alcohol (2.80 L) was added to the vessel. Sodium hydroxide (5M, 460 ml, 2.30 mol) was added in one portion and the mixture was stirred at 21? C. for 16 hrs. The dark solution was screened through a filter to remove particulates. The filtrate was returned to the reactor vessel. The filter and filtrate collection vessel were washed with isopropanol (700 ml) and the washings were added to the reactor vessel. The reaction mixture was agitated and water (1.75 L) w added. Concentrated hydrochloric acid (37% w/w, 165 ml, 1.92 mol) was charged to the vessel. Further hydrochloric acid (21.5 ml) was added to the vessel to adjust the pH between 4.0 and 4.5. The solution was heated to 50? C. Water (1.92 L) added to the vessel over 1 hour maintaining the internal temperature between 50-53? C. The jacket temperature was raised to 70? C. to maintain the reactor temperature in this range during the addition. Within 10 minutes of completion of the water addition, the mixture self seeded and started to crystallise. The mixture was held at 50-52? C. for 1.5 hours (jacket set temperature 58? C.). The resulting yellow suspension was cooled to 5? C. (jacket temperature) over 6 hours. The slurry was held at 5? C. (jacket temperature) for 11 hrs. The resultant yellow solid was isolated by filtration. The cake was pasted with a spatula to prevent cracking of the cake. The vessel was washed with water (1.05 L). The washings were used to wash the cake. The cake was pulled dry in air then dried in vacuo to constant weight over 4 days (oven temperature=30? C.). (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid was thus isolated as a yellow crystalline solid, Form B (319.45 g, 94%). .sup.1H NMR (500 MHz, DMSO, 27? C.) 1.05 (3H, d), 1.08-1.28 (6H, m), 2.35 (1H, dd), 2.58 (1H, dd), 2.8-2.97 (2H, m), 3.47-3.57 (1H, m), 5.22 (1H, s), 6.67 (1H, d), 6.91-7.06 (2H, m), 7.19 (1H, d), 7.41 (1H, d), 7.46 (2H, d), 7.54 (1H, d), 10.58 (1H, s), 12.62 (1H, s).

[0415] An alternative method for synthesising Example 1, which results in formation of Form B crystalline material, is as follows:

[0416] The following processes should be carried out under an atmosphere of nitrogen in the absence of light as a light degradation product (as described above) may be formed. (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (50.0 g; 109.53 mmol) was stirred in isopropyl alcohol (450 ml). Sodium hydroxide (68.34 g, 65.72 ml, 328.58 mmol) was added in one portion and the mixture stirred at 20? C. for 16 hrs. The reaction mixture was diluted with water (250 ml), the pH adjusted to pH4 with conc. hydrochloric acid (27.28 ml, 317.63 mmol) and the mixture heated to 50? C. Further water (225 ml) was added over 30 minutes, maintaining the temperature above 45? C. During the addition, the material began to crystallise. The mixture was cooled from 50? C. to 5? C. over 5 hours then the suspension was held at 0? C. for a further 11 hours. The yellow solid was isolated by filtration. The filter cake was washed with water (100 ml), dried on the filter for a further 20 minutes then dried in a vacuum oven for 16 hours to constant weight (30? C., air bleed) to give the title compound (46.52 g) as a crystalline solid (Form B).

[0417] .sup.1H NMR (500 MHz, DMSO, 27? C.) 1.02-1.09 (3H, m), 1.17 (6H, dd), 2.37 (1H, dd), 2.59 (1H, dd), 2.8-2.98 (2H, m), 3.47-3.58 (1H, m), 5.24 (1H, s), 6.68 (1H, d), 6.9-7.06 (2H, m), 7.20 (1H, d), 7.38-7.51 (3H, m), 7.55 (1H, d), 10.59 (1H, s), 12.60 (1H, br). Crystalline form B may also be isolated from ethanol/water mixtures and ethanol/MTBE mixtures.

[0418] In a further aspect of the invention there is provided crystalline form B of Example 1, isolated from isopropanol/water mixture.

[0419] In a further aspect of the invention, there is provided a process for isolation of crystalline form B of Example 1 which comprises hydrolysis of (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate in isopropyl alcohol and base, followed by acidification and isolation of crystalline product from aqueous isopropanol.

[0420] Example 1 Form B is characterised in providing at least one of the following 20 values measured using CuK? radiation: 8.4 and 10.9. Example 1 Form B is characterised in providing an X-ray powder diffraction pattern, substantially as shown in FIG. 2. Ten X-Ray powder diffraction peaks are shown in Table A:

TABLE-US-00002 TABLE A Ten X-Ray Powder Diffraction peaks for Example 1 Form B Angle 2- Theta (2?) Intensity % 8.4 100 10.9 88.5 18.3 84.5 24.0 78.5 14.0 66.4 19.0 55.9 14.4 54.3 13.0 45 15.3 44.7 20.6 44.2

[0421] DSC analysis of Example 1 Form B shows it to be a high melting solid with an endotherm showing onset of melting at 188.6? C. (FIG. 3). Example 1 shows degradation through the melt which may lead to variation in melting onset, thus the value of 188.6? C. should not be taken as absolute.

[0422] Two solvated forms of Example 1 have also been observed.

[0423] Form A is a methyl tertiary butyl ether mono-solvate. The X-ray powder diffraction pattern is shown in FIG. 4. TGA shows an associated weight loss of 14.7% w/w between 55-150? C. (FIG. 5). A theoretical loss for a mono methyl tertiary butyl ether solvate is calculated to be 16.6%.

[0424] Form C is an Acetone mono-solvate, the X-ray powder diffraction pattern is shown in FIG. 6. TGA shows an associated weight loss of 10.0% w/w 50-150? C. %. (FIG. 7). The theoretical loss for a mono-acetone solvate is calculated to be 11.0%.

[0425] The (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as starting material was prepared as follows:

Preparation of 2-fluoro-2-methylpropyl trifluoromethanesulfonate

[0426] ##STR00020##

[0427] Under an atmosphere of nitrogen, 2-fluoro-2-methyl-propan-1-ol (1000 g, 10.86 moles) was charged to a 20 L vessel. DCM (8.5 L) was added to the vessel. The mixture was agitated and cooled to 1? C. 2,6-Lutidine (1395 g, 13.02 moles) was added to the mixture. A solution of trifluoromethanesulfonic anhydride (3220 g; 11.41 moles) in DCM (1 L) was added over 1 hour maintaining the temperature of the reaction mixture below 5? C. (the jacket set temperature was lowered to ?20? C. during the addition). The addition vessel was and lines were washed with DCM (0.5 L) and the washings were added to the vessel in one portion. The mixture was agitated at 0? C. for 1 hour affording a red solution.

[0428] A solution of concentrated hydrochloric acid (1.23 L, 37% w/w, 16.3 moles) was added to water (7 L). The dilute hydrochloric acid solution was added to the red solution and the stirred mixture was warmed to 25? C. The layers were allowed to separate and the upper aqueous layer was discarded. The organic layer was washed with water (2?5 L). The organic solution was concentrated under reduced pressure to afford a red oil. The red oil was purified by distillation using a wiped film evaporator (4.5 mbar, jacket temperature 50? C., condenser temperature 4? C.) to afford 2-fluoro-2-methylpropyl trifluoromethanesulfonate (1.69 Kg, 69%) as a pale red oil. .sup.1H NMR (500 MHz, DMSO-d6, 27? C.) ? 1.40 (6H, d), 4.79 (2H, d).

Preparation of (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine

[0429] ##STR00021##

[0430] (2R)-1-(1H-Indol-3-yl)propan-2-amine (3.81 kg, 21.21 moles) was added to a 100 L glass lined jacketed vessel under an atmosphere of nitrogen. 1,4-dioxane (23 L) was added, and the agitator was switched on. Diisopropylethylamine (5.55 L; 31.82 moles) was added to the stirred suspension followed by (2-fluoro-2-methyl-propyl)trifluoromethanesulfonate (5.55 kg, 23.77 moles). 1,4-Dioxane (4 L) was added to the vessel, and the mixture was heated to 75? C. Heating was continued for 24 hours before cooling the mixture to 25? C. Water (30.5 L) was added to the vessel, followed by toluene (30.5 L). After 40 minutes the agitator was switched off and the layers were allowed to separate. The aqueous layer was removed and water (30.5 L) was added to the organic solution. The mixture was agitated for 15 minutes before allowing the layers to separate. The aqueous layer was removed from the vessel. The organic solution was concentrated by vacuum distillation (jacket temperature 65? C., 110 mbar pressure) until approximately 27 L of distillate had been removed. The remaining solution in the vessel was cooled to afford (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine as a solution in toluene (33% w/w) (15.4 Kg, 97%). .sup.1H NMR (500 MHz, DMSO, 27? C.) 0.98 (3H, d), 1.26 (3H, d), 1.30 (3H, d), 2.57-2.75 (3H, m), 2.81 (1H, dd), 2.84-2.92 (1H, m), 6.97 (1H, t), 7.06 (1H, t), 7.11-7.22 (1H, multiplet obscured by toluene signals), 7.34 (1H, d), 7.52 (1H, d), 10.80 (1H, s).

Preparation of sodium {2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)methanesulfonate

[0431] ##STR00022##

[0432] 2,6-Difluoro-4-bromobenzaldehyde (1000 g, 4.39 mol) and 1,1-bis(di-tert-butylphosphino)ferrocene palladium dichloride (57.2 g; 87.76 mmol) were charged to a 20 L vessel. Tetra-n-butylammonium chloride (122 g, 438.97 mmol) was added followed by dimethylacetamide (5 L). The vessel was purged with a stream of nitrogen gas. Diisopropylethylamine (1.5 L, 8.78 mol) was added to the vessel followed by methyl acrylate (0.435 L, 4.82 mol). The mixture was agitated and heated to 60? C. The mixture was held at this temperature for 20 hours. Ethyl acetate (10 L) was added to the mixture and the heating was switched off. Water (5 L) was added to the vessel. The stirred mixture was cooled to 25? C. and stirring was continued for 10 minutes. Agitation was stopped and the layers were allowed to separate. The aqueous layer was removed and discarded. The organic layer was washed sequentially with hydrochloric acid (2.2M, 6 L) and water (5 L). Phosphonics SPM32 Scavenger (1050 g, 1050 mol) was added to the vessel and the mixture was stirred for 3 days at 25? C. The solid material was removed by filtration. The cake was washed with ethanol (5 L) and the combined filtrates were concentrated under reduced pressure to afford a solid. The solid was dissolved in ethanol (9 L) and the solution was agitated in a 20 L vessel. The solution was heated to 50? C. A solution of sodium bisulfite (460 g, 4.42 mol) in water (2.5 L) was added over 30 minutes. A thick slurry resulted which was stirred for 4 hours at 50? C. The slurry was cooled to 20? C. over 2 hours. The solid was isolated by filtration and the vessel and filter cake were washed with MTBE (2?3 L). The resulting solid was dried in vacuo to afford sodium {2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)methanesulfonate (1035 g, 71%) as alight brown solid. .sup.1H NMR (500 MHz, DMSO, 27? C.) ? 3.73 (3H, s), 5.32 (1H, d), 5.94 (1H, d), 6.76 (1H, d), 7.40 (2H, d), 7.60 (1H, d).

Preparation of (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate

[0433] ##STR00023##

[0434] Under an atmosphere of nitrogen, sodium {2,6-difluoro-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl}(hydroxy)methanesulfonate (1.211 kg, 3.52 mol) was to a 100 L vessel followed by potassium carbonate (0.974 kg, 7.05 mol). Water (9.1 L) was added and the agitator was started. Ethyl acetate (9.1 L) was added. The mixture was agitated at 25? C. for 5 hours. The agitator was stopped and the mixture was allowed to stand for 14 hours at 25? C. The lower aqueous phase was removed and discarded. The upper organic phase was concentrated under reduced pressure to afford a pale brown solid. The solid was dried in vacuo to afford (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate as a brown solid (608 g, 76%). .sup.1H NMR (500 MHz, DMSO, 27? C.) ? 3.77 (3H, s), 6.94 (1H, d), 7.66 (1H, d), 7.71 (2H, d), 10.20 (1H, s).

Preparation of (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0435] ##STR00024##

[0436] Under an atmosphere of nitrogen, (E)-Methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (0.606 kg, 2.65 mol) and (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine (33% w/w solution in toluene, 2.0 kg, 2.65 mol) were charged to a 20 L vessel followed by toluene (4.22 L). Acetic acid (304 ml, 5.31 mol) was added to the vessel. The mixture was agitated and heated to 80? C. The mixture was agitated at 80? C. overnight before being cooled to 20? C. A solution of potassium carbonate (0.916 kg, 6.63 mol) in water (3.3 L) was added to the mixture. The mixture was stirred for 10 minutes before the agitator was switched off and the layers were allowed to separate. The aqueous layer was removed and discarded. Water (3.3 L) was charged to the reactor. The mixture was agitated for 10 minutes then allowed to stand for 10 minutes. The lower aqueous phase was removed and the organic layer was allowed to stand at room temperature overnight. The batch was heated to 80? C. Heptane (4.61 L) was added to the hot solution over 35 minutes. The stirred mixture was held at approximately 80? C. for 1 hour. The mixture was cooled to 30? C. over 2 hours during which time the product crystallised. The slurry was stirred at 30? C. for 2.5 hours. The solid was isolated by filtration. The reactor vessel walls were washed with heptane and the washings were used to wash the filter cake. The solid was dried in vacuo to afford (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate as a pink solid (0.763 kg, 61%). .sup.1H NMR (400 MHz, DMSO, 27? C.) ? 1.06 (3H, d), 1.13 (3H, d), 1.21 (3H, d), 2.35 (1H, dd), 2.58 (1H, dd), 2.8-2.98 (2H, m), 3.44-3.61 (1H, m), 3.74 (3H, s), 5.24 (1H, s), 6.80 (1H, d), 6.9-7.05 (2H, m), 7.19 (1H, d), 7.41 (1H, d), 7.50 (2H, d), 7.63 (1H, d), 10.58 (1H, s). m/z: ES+ [M+H]+ 457.

Alternative Preparation of Example 1: (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid

[0437] ##STR00025##

[0438] 7.5M Sodium hydroxide (32.9 ml, 247.10 mmol) was added to a solution of (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (11.28 g, 24.71 mmol) in THF (143 ml) and methanol (71.4 ml). The reaction was stirred at room temperature for 4 h. The pH of the aqueous was adjusted to ?6.5 by addition of 2N HCl solution, then the solution was extracted with diethyl ether (3?150 ml). The combined organics were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% methanol in DCM which afforded a yellow solid. Attempted trituration with acetone/heptane failed due to higher than expected solubility. The solvents were removed to give a yellow solid which was triturated in isohexane (50 ml) with a few drops of diethyl ether, the resulting solid was filtered off and dried to give crude product (11.14 g) as a yellow powder. The solid was dissolved in ethanol (100 ml), under nitrogen and in the dark. The solution was then evaporated to 5mBar using a vacuum pump at 62? C. in the dark. This procedure was repeated twice and the resulting yellow glass scratched with a spatula into a fine powder and subjected to 5mBar using a vacuum pump at 62? C. for 60 min, to afford a yellow powder. The powder was then left in a vacuum oven over P.sub.2O.sub.5 at 62? C. at 300 mBar overnight to afford the title product (9.77 g, 89%) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO, 27? C.) ? 1.07-1.16 (3H, m), 1.18-1.29 (6H, m), 2.39 (1H, dd), 2.62 (1H, dd), 2.92 (2H, dd), 3.56 (1H, d), 5.26 (1H, s), 6.70 (1H, d), 7.02 (2H, dd), 7.22 (1H, d), 7.47 (3H, dd), 7.58 (1H, d), 10.60 (1H, s), 12.60 (1H, s). m/z: ES+ (ElectroSpray+) [M+H]+ 443.

[0439] The (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as starting material was prepared as follows:

Preparation of 2-fluoro-2-methylpropan-1-ol

[0440] ##STR00026##

[0441] Lithium aluminium hydride (3.37 g, 88.56 mmol) was added portionwise over 15 min to a cooled solution of ethyl 2-fluoro-2-methylpropanoate (9.9 g, 73.80 mmol) in diethyl ether (184 ml) at 0? C. The reaction was stirred for 1 hr, then water (3.3 ml), followed by 15% NaOH solution (3.3 ml) and water (6.7 ml) were added sequentially. The suspension was stirred for 15 min, then filtered and the solids washed with diethyl ether. The filtrate was evaporated to give 2-fluoro-2-methylpropan-1-ol (5.90 g, 87%) as a colourless oil.

[0442] .sup.1H NMR (400 MHz, CDCl.sub.3, 27? C.) ? 1.37 (6H, d), 3.56 (2H, d), OH not observed.

Alternative Preparation of 2-fluoro-2-methylpropyl trifluoromethanesulfonate

[0443] ##STR00027##

[0444] Trifluoromethanesulfonic anhydride (12.06 ml, 71.24 mmol), followed by 2,6-lutidine (11.42 ml, 81.42 mmol) were added to a solution of 2-fluoro-2-methylpropan-1-ol (6.25 g, 67.85 mmol) in DCM (146 ml) at ?10? C. The reaction was stirred for 1 hr, then washed with 2N HCl (2?100 ml) and saturated NaHCO.sub.3 solution (2?100 ml). The organic phase was then dried over Na.sub.2SO.sub.4 and concentrated to give 2-fluoro-2-methylpropyl trifluoromethanesulfonate (12.89 g, 85%) as a red oil.

[0445] .sup.1H NMR (400 MHz, CDCl.sub.3, 27? C.) ? 1.46 (6H, d), 4.41 (2H, d).

[0446] This intermediate may be purified by vacuum distillation. Analysis by DSC showed the material had the potential to self heat. For reasons of process safety a wiped film evaporator or similar may be preferable to a batch distillation.

Alternative Preparation of (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine

[0447] ##STR00028##

[0448] 2-Fluoro-2-methylpropyl trifluoromethanesulfonate (8.04 g, 35.87 mmol) was added to a solution of (R)-1-(1H-indol-3-yl)propan-2-amine (5.00 g, 28.70 mmol) and N-ethyl-N-isopropylpropan-2-amine (7.44 ml, 43.04 mmol) in dioxane (50 ml). The reaction was heated to 90? C. for 3 h. After cooling to room temperature, the reaction was diluted with EtOAc (200 ml) and washed with saturated. NaHCO.sub.3 solution (2?100 ml). The aqueous phase was extracted with EtOAc (150 ml), then the combined organics were dried over MgSO.sub.4 and concentrated. The crude product was purified by flash silica chromatography, elution gradient 100% EtOAc. Pure fractions were evaporated to dryness to afford (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine (6.49 g, 91%) as a brown oil. .sup.1H NMR (400 MHz, CDCl.sub.3, 27? C.) ? 1.14 (3H, d), 1.31 (3H, d), 1.37 (3H, d), 1.94 (1H, s), 2.63-2.87 (3H, m), 2.92 (1H, dd), 3.07 (1H, h), 7.07 (1H, d), 7.08-7.15 (1H, m), 7.16-7.24 (1H, m), 7.37 (1H, d), 7.62 (1H, d), 8.04 (1H, s). m/z: ES+ [M+H]+ 249

Alternative Preparation of (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate

[0449] ##STR00029##

[0450] 4-Bromo-2,6-difluorobenzaldehyde (9.99 g, 45.20 mmol) and methyl acrylate (6.14 ml, 67.81 mmol) were taken up in thoroughly degassed DMA (100 ml) and tri-o-tolylphosphine (1.376 g, 4.52 mmol), palladium(II) acetate (0.507 g, 2.26 mmol) and triethylamine (12.60 ml, 90.41 mmol) added. The reaction was stirred and heated to 80? C. for 6 hours. The reaction mixture was cooled and filtered through a layer of celite, and washed with methanol (50 ml). The crude product was pre-absorbed onto silica and purified by suction chromatography eluting with 0-10% diethyl ether/dichloromethane. Fractions containing the desired product were evaporated and triturated with diethyl ether (50 ml) to afford a yellow solid which was triturated with water (50 ml) and dried under high vacuum at 50? C. to afford (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (8.85 g, 87%) as a yellow solid. .sup.1H NMR (400 MHz, DMSO, 27? C.) ? 3.75 (3H, s), 6.93 (1H, d), 7.52-7.81 (3H, m), 10.18 (1H, s). No mass ion observed in LCMS.

Alternative Preparation of (E)-methyl 3-(3,5-difluoro-44(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0451] ##STR00030##

[0452] (E)-Methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (6.58 g, 29.09 mmol) was added to a suspension of (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine (6.02 g, 24.24 mmol) in toluene (51.1 ml) and acetic acid (2.78 ml, 48.48 mmol). The reaction was heated to 80? C. for 5 hours. The reaction mixture was purified by ion exchange chromatography, using an SCX-2 column. The desired product was eluted from the column using 7M NH.sub.3/methanol and pure fractions were evaporated to dryness to afford a brown solid. The crude product was purified by flash silica chromatography, elution gradient 0 to 30% EtOAc in heptane. Pure fractions were evaporated to dryness to afford the title product (7.52 g, 68.0%) as a yellow solid. .sup.1H NMR (400 MHz, DMSO, 100? C.) ? 1.10 (3H, d), 1.12-1.31 (6H, m), 2.28-2.72 (2H, m), 2.84-3.09 (2H, m), 3.52-3.69 (1H, m), 3.76 (3H, s), 5.30 (1H, s), 6.64 (1H, d), 6.9-7.11 (2H, m), 7.21 (1H, d), 7.32 (2H, d), 7.42 (1H, d), 7.58 (1H, d), 10.14 (1H, s). m/z: ES+ [M+H]+ 457

Example 2: (E)-3-(4-((1R,3R)-2-(2-Fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid

[0453] ##STR00031##

[0454] 7.5M Sodium hydroxide solution (0.983 ml, 7.37 mmol) was added to a solution of (E)-methyl 3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (310 mg, 0.74 mmol) in methanol (5 ml). The mixture was stirred at 20? C. for 2 hours. The reaction mixture was purified by ion exchange chromatography, using an SCX-2 column. Fractions containing the desired product were eluted from the column using 7M NH.sub.3/methanol and pure fractions were evaporated to dryness to afford a yellow solid. The crude product was purified by preparative HPLC (Waters SunFire column, 5? silica, 50 mm diameter, 100 mm length), using decreasingly polar mixtures of water (containing 0.1% formic acid) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness and then loaded onto SCX-2 column and eluted with 7N ammonia in methanol to afford the title product (63.0 mg, 21.02%) as a yellow solid. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 1.06 (3H, d), 1.30 (3H, d), 1.47 (3H, d), 2.53-2.64 (2H, m), 2.79 (2H, s), 3.10 (1H, d), 5.08 (1H, s), 6.47 (1H, d), 6.98 (1H, t), 7.06 (1H, t), 7.19-7.37 (3H, m), 7.44 (1H, d), 7.56 (1H, d), 7.63 (2H, d), 10.81 (1H, s), 12.30 (1H, s). m/z: ES+ [M+H]+ 407.

[0455] The (E)-methyl 3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as starting material was prepared as follows:

Preparation of (E)-methyl 3-(4-formylphenyl)acrylate

[0456] ##STR00032##

[0457] 4-Bromobenzaldehyde (30 g, 162.15 mmol) and methyl acrylate (20.94 g, 243.22 mmol) were taken up in thoroughly degassed DMA (300 ml) and treated with tri-o-tolylphosphine (4.94 g, 16.21 mmol), palladium(II) acetate (1.820 g, 8.11 mmol) and triethylamine (45.2 ml, 324.29 mmol) and heated to 110? C. for 16 hours. The reaction appeared complete after this time. The reaction mixture was poured into water (4 L) and the resulting precipitate was filtered and dried. The solid was chromatographed on silica, eluting with 100% heptane to 30% EtOAc in heptane. Relevant fractions were combined and evaporated to dryness to afford a yellow solid product which was triturated with heptane, filtered and washed with cold heptane. The solid was dried to afford (E)-methyl 3-(4-formylphenyl)acrylate (25.6 g, 83%) as a yellow crystalline product. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 3.75 (3H, s), 6.79 (1H, d), 7.72 (1H, d), 7.93 (4H, s), 10.03 (1H, s). No mass ion observed in LCMS.

Preparation of (E)-methyl 3-(44(1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0458] ##STR00033##

[0459] (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine (obtained as described in Example 1, preparation of starting materials) (450 mg, 1.81 mmol) and (E)-methyl 3-(4-formylphenyl)acrylate (345 mg, 1.81 mmol) were dissolved in toluene (15 ml), acetic acid (5 ml) and molecular sieves were added. The reaction was stirred at 110? C. for 16 hours under nitrogen then cooled to room temperature. The crude product was purified by ion exchange chromatography, using an SCX-2 column. The desired product was eluted from the column using 7M NH.sub.3/methanol and pure fractions were evaporated to dryness to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 30% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (317 mg, 41.6%) as a yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 1.09 (3H, d), 1.30 (3H, d), 1.47 (3H, d), 2.48-2.78 (4H, m), 3.30 (1H, m), 3.79 (3H, s), 5.09 (1H, s), 6.40 (1H, d), 7.12 (1H, td), 7.17 (1H, td), 7.29 (1H, d), 7.34 (2H, d), 7.43 (2H, d), 7.54 (1H, d), 7.66 (2H, m). m/z: ES+ [M+H]+ 421.

Example 3: (E)-3-(3,5-Difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid

[0460] ##STR00034##

[0461] 2M Sodium hydroxide (3.0 ml, 6.00 mmol) was added to a solution of (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (275 mg, 0.60 mmol) in THF (1.5 ml)/methanol (1.5 ml). The reaction was stirred at room temperature for 3 h. EtOAc (15 ml) and water (15 ml) were added, then the pH of the aqueous was adjusted to ?7 by addition of 2N HCl. The layers were separated and the aqueous was extracted with EtOAc (15 ml). The combined organics were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% methanol in DCM. Pure fractions were evaporated to dryness to afford the title product (250 mg, 94%) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 0.77 (3H, d), 1.06 (3H, d), 1.93 (1H, m), 2.18 (1H, dd), 2.58 (1H, dd), 2.65 (1H, dd), 2.84 (1H, dd), 3.35 (1H, dd), 4.32 (1H, d), 4.44 (1H, d), 5.16 (1H, s), 6.67 (1H, d), 6.93-7.04 (2H, m), 7.21 (1H, d), 7.42 (1H, d), 7.46 (2H, m), 7.54 (1H, d), 10.57 (1H, s), 12.51 (1H, s). m/z: ES+ [M+H]+ 443.

[0462] The (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as starting material was prepared as follows:

Preparation of (S)-3-fluoro-2-methylpropan-1-ol

[0463] ##STR00035##

[0464] N,N-Diethyl-1,1,2,3,3,3-hexafluoropropan-1-amine (18.25 ml, 100.57 mmol) was added dropwise to a solution of (R)-methyl 3-hydroxy-2-methylpropanoate (9.25 ml, 83.81 mmol) in DCM (77 ml) (reaction warms to ?40? C.). The reaction was stirred for 1 h at ambient temperature, then warmed to reflux for 4 h, before cooling to room temperature overnight. The reaction mixture was poured onto ice, and the layers separated. The aqueous was extracted with DCM (2?150 ml), then the combined organics were dried and carefully concentrated. The residue was dissolved in THF (200 ml) and cooled in an ice-bath. Lithium aluminium hydride (6.45 g, 167.61 mmol) was added in portions over 15 min. The reaction was stirred at 0? C. for 1 h and warmed to room temperature for a further 1 h. After cooling in an ice-bath, the reaction was quenched by addition of water (7 ml), followed by 15% NaOH (7 ml), and finally water (21 ml). MgSO.sub.4 was added until a granular solid was formed. The solid was filtered through celite and the solids washed with diethyl ether (50 ml). The filtrate was washed with 2N HCl (2?100 ml), then the organic phase was dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% EtOAc in DCM. Pure fractions were evaporated to dryness to afford (S)-3-fluoro-2-methylpropan-1-ol (6.42 g, 83%) as a straw coloured oil. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 0.97 (3H, dd), 1.96-2.14 (1H, m), 3.64 (2H, d), 4.3-4.42 (1H, m), 4.42-4.54 (1H, m), OH not observed.

Preparation of (S)-3-fluoro-2-methylpropyl trifluoromethanesulfonate

[0465] ##STR00036##

[0466] To a stirred solution of (S)-3-fluoro-2-methylpropan-1-ol (7.9 g, 85.77 mmol) in DCM (140 ml) at 0? C. was added trifluoromethanesulfonic anhydride (17.31 ml, 102.92 mmol) dropwise followed by dropwise addition of 2,6-dimethylpyridine (11.95 ml, 102.92 mmol). The reaction mixture was stirred at 0? C. for 45 min and room temperature for 30 min. The reaction mixture was diluted with DCM (60 ml), and washed sequentially with 1M HCl (3?100 ml), saturated sodium bicarbonate solution (100 ml) and saturated brine (50 ml). The organic layer was dried over Na.sub.2SO.sub.4, filtered and evaporated to near dryness. The solution was filtered through a plug of silica and washed through with DCM (50 ml) and evaporated to give (S)-3-fluoro-2-methylpropyl trifluoromethanesulfonate (14.38 g, 74.8%) as a brown oil. .sup.1H NMR (400 MHz, CDCl.sub.3, 27? C.) ? 1.09 (3H, dd), 2.24-2.44 (1H, m), 4.30 (0.5H, dd), 4.37-4.46 (1H, m), 4.52 (2.5H, tt).

Preparation of (S)N((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amine

[0467] ##STR00037##

[0468] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (666 mg, 2.97 mmol) was added to a solution of (R)-1-(1H-indol-3-yl)propan-2-amine (470 mg, 2.7 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.700 ml, 4.05 mmol) in 1,4-dioxane (6.05 ml). The reaction was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc (20 ml) and washed with water (20 ml). The aqueous was extracted with EtOAc (2?20 ml), then the combined organics were dried (MgSO.sub.4) and concentrated. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% methanol in DCM. Pure fractions were evaporated to dryness to afford (S)N((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amine (590 mg, 88%) as a brown oil. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 0.86 (3H, dd), 1.20 (3H, d), 1.94-2.11 (1H, m), 2.64-2.74 (2H, m), 2.85-2.98 (2H, m), 3.05-3.15 (1H, m), 4.13-4.39 (2H, m), 7.09 (1H, d), 7.12 (2H, ddd), 7.20 (1H, ddd), 7.33-7.41 (1H, m), 7.56-7.65 (1H, m), 8.10 (1H, s). m/z: ES+ [M+H]+ 249

Preparation of (E)-methyl 3-(3,5-difluoro-44(1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0469] ##STR00038##

[0470] Acetic acid (2.0 ml) was added to a solution of (S)N((R)-1-(1H-indol-3-yl)propan-2-yl)-3-fluoro-2-methylpropan-1-amine (273 mg, 1.10 mmol) and (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (obtained as described in Example 1, preparation of starting materials) (226 mg, 1 mmol) in toluene (8.0 ml). The reaction was warmed to 95? C. for 2.5 h. The volatiles were removed under vacuum, then the residue was passed through an SCX-2 column. The column was then eluted with 7M NH.sub.3/methanol to liberate the product. The filtrate was concentrated and the crude product was purified by flash silica chromatography, elution gradient 0 to 10% methanol in DCM. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (282 mg, 61.8%) as a pale yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 0.82 (3H, d), 1.12 (3H, d), 1.81-1.99 (1H, m), 2.24 (1H, ddd), 2.64 (1H, ddd), 2.71 (1H, dd), 2.93-3.01 (1H, ddd), 3.42 (1H, dq), 3.81 (3H, s), 4.25-4.39 (1H, m), 4.38-4.53 (1H, m), 5.20 (1H, s), 6.39 (1H, d), 6.99 (2H, m), 7.06-7.16 (2H, m), 7.21-7.25 (1H, m), 7.52 (3H, m). m/z: ES+ [M+H]+ 457.

Example 4: (E)-3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid

[0471] ##STR00039##

[0472] 7.5M Sodium hydroxide solution (0.904 ml, 6.78 mmol) was added to a solution of (E)-methyl 3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (285 mg, 0.68 mmol) in methanol (3 ml). The mixture was stirred at 20? C. for 2 hours. The reaction mixture was purified by ion exchange chromatography, using an SCX-2 column. Fractions containing the desired product were eluted from the column using 7M NH.sub.3/methanol and pure fractions were evaporated to dryness to afford a yellow solid. The crude product was purified by preparative LCMS (Phenomenex Gemini-NX axia Prep C18 OBD column, 5? silica, 50 mm diameter, 100 mm length), using decreasingly polar mixtures of water (containing 1% NH.sub.3) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford the title product (80 mg, 29.0%) as a yellow solid. .sup.1H NMR (500 MHz, DMSO, 33? C.) ? 0.87 (3H, d), 1.04 (3H, d), 1.91-2.27 (2H, m), 2.50 (1H, p), 2.57-2.75 (2H, m), 3.13 (1H, s), 4.51 (2H, dd), 4.86 (1H, s), 6.47 (1H, d), 6.92-6.99 (1H, m), 7-7.09 (1H, m), 7.15-7.35 (3H, m), 7.35-7.5 (2H, m), 7.57 (2H, d), 10.64 (1H, d), CO.sub.2H not observed. m/z: ES+ [M+H]+ 407.

[0473] The (E)-methyl 3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate used as starting material was prepared as follows:

Preparation of (E)-methyl 3-(44(1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0474] ##STR00040##

[0475] (E)-Methyl 3-(4-formylphenyl)acrylate (obtained as described in Example 2, preparation of starting materials) (18.56 g, 97.57 mmol) was added to a stirred solution of (R)-1-(1H-indol-3-yl)propan-2-amine (17 g, 97.57 mmol) in acetic acid (250 ml) at 23? C. under nitrogen. The resulting solution was stirred at 80? C. for 2 hours. The reaction mixture was evaporated to dryness and redissolved in DCM (500 ml), and washed sequentially with saturated NaHCO.sub.3 (300 ml?2), 2M NaOH (aq) (300 ml), water (300 ml), and saturated brine (300 ml). The organic layer was dried over MgSO.sub.4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 1 to 7% methanol in DCM. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (25.1 g, 74.3%) as a beige foam. The product was mostly cis isomer, containing about 12% trans isomer which was inseparable. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 1.25 (3H, d), 2.37-2.48 (1H, m), 2.74 (1H, d), 3.12 (1H, s), 3.73 (3H, s), 5.18 (1H, s), 6.64 (1H, d), 6.97 (2H, dd), 7.19 (1H, d), 7.36-7.46 (3H, m), 7.64-7.75 (3H, m), 10.19 (1H, s), no NH observed. m/z: ES+ [M+H]+ 347.

Preparation of (E)-methyl 3-(44(1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0476] ##STR00041##

[0477] (E)-Methyl 3-(4-((1S,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (35 g, 101.03 mmol), 3-bromoprop-1-ene (9.62 ml, 111.14 mmol) and N-ethyl-N-isopropylpropan-2-amine (19.36 ml, 111.14 mmol) were suspended in acetonitrile (160 ml), nitrogen was bubbled through for 5 min and then the mixture was sealed into a microwave tube. The reaction was heated to 140? C. for 3.5 hours in the microwave reactor and cooled to room temperature.

[0478] The reaction mixture was evaporated to dryness and redissolved in DCM (100 ml), and washed sequentially with 1M citric acid (100 ml), water (100 ml), and saturated brine (100 ml). The organic layer was dried over MgSO.sub.4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 20% EtOAc in heptane. Pure fractions were evaporated to dryness to afford a 50:50 mixture of (E)-methyl 3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate: (E)-methyl 3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (10.00 g, 25.6%) as a pale yellow solid. m/z: ES+ [M+H]+ 387.

Preparation of (E)-methyl 3-(44(1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0479] ##STR00042##

[0480] Trifluoroacetic acid (5.59 ml, 75.29 mmol) was added to (E)-methyl 3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (9.7 g, 25.10 mmol) in DCM (100 ml) at 20? C. under nitrogen. The resulting solution was stirred at 20? C. for 3 days. The reaction mixture was diluted cautiously with saturated NaHCO.sub.3 solution (250 ml), and the DCM layer washed sequentially with water (250 ml) and saturated brine (250 ml). The organic layer was dried over MgSO.sub.4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 10 to 20% EtOAc in heptane. Fractions were evaporated to dryness to afford a 65:35 mixture of (E)-methyl 3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate: (E)-methyl 3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (7.99 g, 82%) as a pale yellow solid. m/z: ES+ [M+H]+ 387

Preparation of (E)-methyl 3-(44(1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0481] ##STR00043##

[0482] 7 Separate batches of 65:35 (E)-methyl 3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate: (E)-methyl 3-(4-((1S,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (2.00 g, 5.17 mmol) were reacted as follows.

[0483] (E)-methyl 3-(4-((1R,3R)-2-allyl-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (2.00 g, 5.17 mmol) and chlorotris(triphenylphosphine)rhodium(I) (Wilkinson's catalyst) (2.346 g, 2.54 mmol) were suspended in acetonitrile (12 ml) and water (2.4 ml) and nitrogen was bubbled through for 5 min before being sealed into a microwave tube. The reaction was heated to 100? C. for 60 min in the microwave reactor and cooled to room temperature. The reaction mixtures were combined and evaporated to dryness and redissolved in DCM (200 ml) and saturated NaHCO.sub.3 solution (200 ml) added. The organic layer was washed sequentially with water (200 ml) and saturated brine (200 ml) before being dried over MgSO.sub.4, filtered and evaporated to afford crude product. The crude product was purified by flash silica chromatography, elution gradient 0 to 5% methanol in DCM. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (7.02 g, 55.9%) as a pale yellow solid. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 1.14 (3H, d), 2.27-2.4 (1H, m), 2.81 (1H, dd), 2.94-3.05 (1H, m), 3.72 (3H, s), 5.19 (1H, s), 6.60 (1H, d), 6.94-7 (1H, m), 7.01-7.09 (1H, m), 7.26 (3H, d), 7.43 (1H, d), 7.59-7.68 (3H, m), 10.70 (1H, s), NH not observed. m/z: ES+ [M+H]+ 347

Preparation of (E)-methyl 3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate

[0484] ##STR00044##

[0485] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as described in Example 3, preparation of starting materials) (291 mg, 1.30 mmol) was added to a solution of (E)-methyl 3-(4-((1R,3R)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (300 mg, 0.87 mmol) and N,N-diisopropylethylamine (0.226 ml, 1.30 mmol) in 1,4-dioxane (5 ml). The mixture was stirred at 90? C. for 1 hour then the mixture was evaporated to dryness and the residue was partitioned between DCM (30 ml) and water (30 ml). The aqueous layer was extracted with DCM (30 ml) and the extracts combined with the organic layer. The combined extracts were filtered through a phase-separating paper and evaporated. The residue was purified by flash silica chromatography, elution solvent 15% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(4-((1R,3R)-2-((S)-3-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (314 mg, 86%) as an off-white solid. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 0.87 (3H, d), 1.06 (3H, d), 1.9-2.28 (2H, m), 2.55-2.8 (3H, m), 2.97-3.21 (1H, m), 3.72 (3H, s), 4.31-4.69 (2H, m), 4.88 (1H, s), 6.60 (1H, dd), 6.98 (1H, t), 7.04 (1H, t), 7.17-7.35 (3H, m), 7.44 (1H, d), 7.55-7.76 (3H, m), 10.65 (1H, s). m/z: ES+ [M+H]+ 421.

Example 5: (E)-3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Isomer 1)*

[0486] ##STR00045##

[0487] Sodium hydroxide (184 mg, 4.61 mmol) was added to (E)-methyl 3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (217 mg, 0.46 mmol) in THF (1 ml)/methanol (1 ml). The resulting solution was stirred at 20? C. for 16 hours. The reaction was diluted with water (10 ml) and the pH was adjusted to 7 by the addition of 2N HCl. The solution was extracted with EtOAc (2?20 ml). The combined organics were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in heptane. The pure fractions were evaporated to give a crude material. The crude product was triturated using a diethyl ether/isohexane mixture to give the title product (53.0 mg, 25.2%) as a yellow solid. .sup.1H NMR (400 MHz, DMSO, 27? C.) ? 0.54 (3H, d), 1.06 (3H, s), 1.34 (3H, s), 2.14 (1H, dd), 2.66 (1H, d), 2.83 (1H, d), 3.03 (1H, dd), 4.21 (1H, t), 4.33 (1H, t), 5.12 (1H, s), 6.73 (1H, d), 7.01 (2H, dtd), 7.14-7.28 (1H, m), 7.43 (1H, d), 7.54 (2H, s), 7.59 (1H, d), 10.50 (1H, s), 12.61 (1H, s), CO.sub.2H not observed. m/z: ES+ [M+H]+ 457

[0488] Stereochemistry inferred to be (R) at the undefined centre by analogy with other examples, ie compound inferred to be: (E)-3-(3,5-difluoro-4-(1R)-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid

[0489] The 3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) used as starting material was prepared as follows:

Preparation of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate

[0490] ##STR00046##

[0491] 1-(1H-Indol-3-yl)-2-methylpropan-2-amine (807 mg, 4.29 mmol) and (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (obtained as described in Example 1, preparation of starting materials) (970 mg, 4.29 mmol) were combined in acetic acid (15 ml) and the mixture heated to 80? C. for 2 hours. The reaction mixture was purified by ion exchange chromatography, using an SCX-2 column. The desired product was eluted from the column using 2M NH.sub.3 in methanol and product-containing fractions were evaporated to dryness to afford (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate (1610 mg, 95%) as a yellow foam. .sup.1H NMR (500 MHz, DMSO, 20? C.) ? 1.15 (3H, s), 1.27 (3H, s), 2.23 (1H, s), 2.61 (2H, s), 3.75 (3H, s), 5.46 (1H, s), 6.84 (1H, d), 6.97 (2H, dtd), 7.18 (1H, d), 7.39 (1H, d), 7.58 (2H, s), 7.67 (1H, d), 10.60 (1H, s). m/z: ES? [M?H]? 395.

Preparation of (E)-methyl 3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)

[0492] ##STR00047##

[0493] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as described in Example 3, preparation of starting materials) (0.339 g, 1.51 mmol) was added to a solution of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate (0.3 g, 0.76 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.458 ml, 2.65 mmol) in 1,4-dioxane (2 ml). The stirring was continued for 24 hours then the volatiles were removed under vacuum and the crude product was purified by flash silica chromatography, elution gradient 0 to 25% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (0.202 g, 36%) as a white solid. The material was combined with another batch (0.36 g) and purified by preparative HPLC (Chiralpak IA column, 20 ?m silica, 20 mm diameter, 250 mm length), Heptane:IPA 70:30 at 80 ml/min (4 injections). Fractions containing the desired compounds were evaporated to yield (E)-methyl 3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1, first eluted, 217 mg) and (E)-methyl 3-(3,5-difluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 2, second eluted, 165 mg). Analysis was done on Chiralpak IA column, 5 ?m silica, 4.6 mm diameter, 50 mm length, Heptane:IPA 70:30 at 2 ml/min. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 0.50 (3H, d), 1.02 (3H, s), 1.30 (3H, s), 2.11 (1H, dd), 2.62 (2H, d), 2.80 (1H, d), 2.99 (1H, dd), 3.74 (3H, s), 4.09-4.23 (1H, m), 4.29 (1H, d), 5.09 (1H, s), 6.81 (1H, d), 6.97 (2H, dt), 7.16 (1H, d), 7.39 (1H, d), 7.53 (2H, d), 7.64 (1H, d), 10.44 (1H, s). m/z: ES+ [M+H]+ 471.

Example 6: (E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Isomer 1)*

[0494] ##STR00048##

[0495] 2M Sodium hydroxide (1.6 ml, 3.20 mmol) was added to a solution of (E)-methyl 3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (148 mg, 0.31 mmol) in THF (0.8 ml)/methanol (0.8 ml). The reaction was stirred at room temperature for 3 h. EtOAc (15 ml) and water (15 ml) were added, and the pH of the aqueous was adjusted to ?7 by addition of 2N HCl. The layers were separated, and the aqueous was extracted with EtOAc (15 ml). The combined organics were dried over Na.sub.2SO.sub.4 and concentrated. The crude product was purified by flash silica chromatography, elution gradient 25 to 100% EtOAc in heptane. Pure fractions were evaporated to dryness to afford the title product (isomer 1) (124 mg, 86%) as a pale yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 1.03 (3H, d), 1.08 (3H, s), 1.16 (3H, d), 1.35 (3H, s), 2.56 (1H, dd), 2.66 (1H, d), 3.02 (1H, d), 3.17 (1H, dd), 5.24 (1H, s), 6.39 (1H, d), 7.00 (2H, d), 7.05-7.16 (2H, m), 7.20 (1H, dd), 7.28 (1H, s), 7.49 (1H, dd), 7.61 (1H, d), CO.sub.2H not observed. m/z: ES+ [M+H]+ 457.

[0496] Stereochemistry inferred to be (R) at the undefined centre by analogy with other examples.

[0497] The (E)-methyl 3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) used as starting material was prepared as follows:

Preparation of (E)-methyl 3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)

[0498] ##STR00049##

[0499] 2-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as described in Example 1, preparation of starting materials) (679 mg, 3.03 mmol) was added to a solution of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3,5-difluorophenyl)acrylate (obtained as described in Example 5, preparation of starting materials) (600 mg, 1.51 mmol) and N-ethyl-N-isopropylpropan-2-amine (0.915 ml, 5.30 mmol) in 1,4-dioxane (2.5 ml). The reaction was stirred at room temperature for 1 h. The reaction was then heated to 105? C. for 88 hours. The volatiles were removed under vacuum and the crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (430 mg, 60.4%) as a white solid. The racemic product was purified by preparative HPLC (Chiralpak AD column, 20 ?m silica, 50 mm diameter, 250 mm length), Heptane:Ethanol 90:10 90 ml/min. Fractions containing the desired compounds were evaporated to dryness to afford (E)-methyl 3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1, first eluted, 149 mg, 34.6%) and (E)-methyl 3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 2, second eluted, 143 mg, 33.3%) as cream coloured solids. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 1.01 (3H, d), 1.08 (3H, s), 1.15 (3H, d), 1.34 (3H, s), 2.55 (1H, dd), 2.66 (1H, d), 2.98-3.06 (1H, m), 3.17 (1H, dd), 3.80 (3H, s), 5.23 (1H, s), 6.38 (1H, d), 6.97 (2H, d), 7.07-7.12 (2H, m), 7.17-7.22 (1H, m), 7.32 (1H, s), 7.46-7.5 (1H, m), 7.52 (1H, d). m/z ES? [M?H]? 469.

Example 7: (E)-3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Isomer 1)*

[0500] ##STR00050##

[0501] 2M Sodium hydroxide solution (20.42 ml, 40.85 mmol) was added to a solution of (E)-methyl 3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (3.55 g, 8.17 mmol) in methanol (100 ml), THF (100 ml) and water (75 ml). The mixture was heated at 40? C. for 16 hours. The mixture was diluted with water (100 ml) and concentrated to a volume such that the organic solvents had been removed. The resulting aqueous solution was acidified to pH 6 with 2M HCl. The resulting aqueous suspension was extracted with DCM (500 ml) (adding brine to help separate the emulsion which formed), filtered through a phase-separating paper, dried over MgSO.sub.4 then filtered through celite and evaporated to afford ?3.5 g of a pale yellow solid. The crude product was treated with diethyl ether/DCM (1:1, 150 ml) and sonicated. The fine suspension which formed was passed through a pad of silica (?100 g) and the silica was eluted with diethyl ether (?2 L). Product-containing fractions were combined, evaporated and dried under vacuum at 50? C. to afford the title product (2.305 g, 64.5%) as a beige solid. .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 0.52 (3H, d), 1.03 (3H, s), 1.05-1.16 (1H, m), 1.23 (3H, s), 2.21 (1H, dd), 2.65 (1H, d), 2.84 (1H, d), 2.89 (1H, dd), 4.19 (1H, ddd), 4.31 (1H, ddd), 4.66 (1H, s), 6.50 (1H, d), 6.93 (1H, ddd), 6.98 (1H, ddd), 7.18 (1H, d), 7.38 (2H, d), 7.40 (1H, d), 7.58 (1H, d), 7.63 (2H, d), 10.18 (1H, s), 12.30 (1H, s). m/z: ES+ [M+H]+ 421.

[0502] Stereochemistry inferred to be (R) at the undefined centre by analogy with other examples.

[0503] The product (9.0 g, 21.40 mmol) was slurried in acetonitrile (150 ml) under nitrogen in the dark for 1 hour in a stoppered 250 ml round bottomed flask. The mixture was stirred over the weekend at room temperature then filtered and washed with cold acetonitrile (60 ml) to afford a white solid which was dried under high vacuum at 40? C. for 5 hours to yield crystalline form A of the title product (7.81 g, 87%).

[0504] An XRPD trace of Crystalline form A includes the following peaks and is shown in FIG. 1.

TABLE-US-00003 2-Theta ? % 4.48 100 10.76 42.2 9.88 21.4 6.13 20.8 13.41 18.9 14.01 18.2 14.31 14.7 18.46 13.2 7.92 12.2 4.76 9.3

[0505] The (E)-methyl 3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) used as starting material was prepared as follows:

Preparation of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (racemate)

[0506] ##STR00051##

[0507] (E)-Methyl 3-(4-formylphenyl)acrylate (41.8 g, 219.62 mmol) (obtained as described in Example 2, preparation of starting materials) was added in one portion to 1-(1H-indol-3-yl)-2-methylpropan-2-amine (43.8 g, 219.62 mmol) in acetic acid (314 ml) under nitrogen. The resulting solution was stirred at 80? C. for 5 hours. The reaction mixture was concentrated in vacuo. Toluene (200 ml) was added and the residue evaporated to dryness. The azeotrope treatment was repeated twice more to give a brown solid. This was stirred in 1:1 EtOAc/heptane (500 ml) for 30 min before filtering and washing with 1:1 EtOAc/heptane. The compound was air dried to give a white solid. The crude material was suspended in 2-methyl tetrahydrofuran (750 ml), and saturated sodium bicarbonate solution was added over 10 min to the stirred mixture (effervescence), the mixture was stirred until the material dissolved and the aqueous phase remained basic. The phases were separated and the organic phase washed with brine, dried over MgSO.sub.4, filtered and concentrated in vacuo to give a pale yellow foam (?78 g). The material was dissolved in diethyl ether (200 ml) and concentrated to dryness (repeated twice). On the second addition a proper solid was obtained. This was stirred in diethyl ether and evaporated to dryness to give (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (racemate) (73.6 g, 89%). .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 1.26 (3H, s), 1.35 (3H, s), 1.42 (1H, br s), 2.69-2.82 (2H, m), 3.80 (3H, s), 5.12 (1H, s), 6.41 (1H, d), 7.06-7.16 (2H, m), 7.21 (1H, dd), 7.37 (2H, d), 7.46-7.54 (4H, m), 7.67 (1H, d). m/z: ES+ [M+H]+ 361.

Preparation of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)

[0508] ##STR00052##

[0509] (E)-Methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (racemate) (65 g) was purified in seven injections as follows.

[0510] The racemic material was purified by preparative HPLC (Chiralpak OD column, 20 ?m silica, 100 mm diameter, 250 mm length), Heptane:IPA 50:50. Fractions containing the desired compounds were evaporated to dryness to afford (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1, first eluted, 30.3 g, 93%) and (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 2, second eluted, 28.2 g, 86%). .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 1.27 (3H, s), 1.36 (3H, s), 2.69-2.82 (2H, m), 3.80 (3H, s), 5.14 (1H, s), 6.43 (1H, d), 7.12 (2H, pd), 7.2-7.24 (1H, m), 7.39 (3H, d), 7.51 (3H, d), 7.68 (1H, d), NH not observed. m/z: ES+ [M+H]+ 361.

Preparation of (E)-methyl 3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)

[0511] ##STR00053##

[0512] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as described in Example 3, preparation of starting materials) (5.32 g, 21.36 mmol) was added to a solution of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (3.5 g, 9.71 mmol) and N-ethyl-N-isopropylpropan-2-amine (6.34 ml, 36.41 mmol) in 1,4-dioxane (17.5 ml). The mixture was stirred at 22? C. for 3 days. The mixture was evaporated and the residue was partitioned between DCM (150 ml) and water (150 ml). The aqueous layer was extracted with DCM (50 ml) and the extracts combined with the organic layer. The combined extracts were filtered through a phase-separating paper and evaporated. The residue was purified by flash silica chromatography, elution solvent 15% EtOAc in heptane. Fractions containing significant amounts of product began to form crystals; the tubes were agitated to encourage further crystallisation. The crystals were collected by filtration and washed with a small amount of 15% EtOAc in heptane to afford (E)-methyl 3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (2.91 g, 69.0%) as a white crystalline solid. Liquors from the crystallisation and other product-containing fractions were combined and evaporated. The residue was recrystallised from EtOAc/heptane to afford more 3-(4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) as a white crystalline solid (635 mg, 15.1%). 1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 0.53 (3H, d), 0.95-1.07 (1H, m), 1.09 (3H, s), 1.32 (3H, s), 2.16 (1H, dd), 2.66 (1H, d), 2.94 (1H, d), 2.97 (1H, d), 3.80 (3H, s), 4.14 (1H, ddd), 4.31 (1H, ddd), 4.59 (1H, s), 6.42 (1H, d), 7.05-7.11 (2H, m), 7.13 (1H, s), 7.17 (1H, dd), 7.35 (2H, d), 7.45 (2H, d), 7.50 (1H, dd), 7.67 (1H, d). m/z: ES+ [M+H]+ 435.

Example 8: (E)-3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Isomer 1)*

[0513] ##STR00054##

[0514] 2M Sodium hydroxide solution (0.782 ml, 1.56 mmol) was added to a solution of (E)-methyl 3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) (68 mg, 0.16 mmol) in methanol (5 ml), THF (5.00 ml) and water (5.00 ml) and the mixture stirred for 40 hours at 22? C. The mixture was concentrated to a volume such that all of the organic solvent had been removed and was acidified to pH6 with 2M HCl. Concentrated aqueous ammonia (2 drops) was added, followed by methanol (?2 ml), giving a pale yellow solution. The crude product was purified by preparative HPLC (Waters XBridge Prep C18 OBD column, 5? silica, 50 mm diameter, 100 mm length), using decreasingly polar mixtures of water (containing 1% NH.sub.3) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford the title product (isomer 1) (50.0 mg, 76%) as a yellow solid. .sup.1H NMR (500 MHz, DMSO, 30? C.) ? 0.93 (3H, s), 1.19 (3H, d), 1.23 (3H, s), 1.51 (3H, d), 2.56-2.64 (2H, m), 2.75 (1H, d), 3.04 (1H, dd), 5.06 (1H, s), 6.40 (1H, d), 7.03 (1H, ddd), 7.09-7.15 (2H, m), 7.35 (1H, dd), 7.44-7.51 (5H, m), 10.87 (1H, s), CO.sub.2H not observed. m/z: ES+ [M+H]+ 421.

[0515] Stereochemistry inferred to be (R) at the undefined centre by analogy with other examples.

[0516] The (E)-methyl 3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1) used as starting material was prepared as follows:

Preparation of (E)-methyl 3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1)

[0517] ##STR00055##

[0518] 2-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as described in Example 1, preparation of starting materials) (389 mg, 1.73 mmol) was added to a solution of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (racemate) (obtained as described in Example 7, preparation of starting materials) (250 mg, 0.69 mmol) and N,N-diisopropylethylamine (0.453 ml, 2.60 mmol) in 1,4-dioxane (1.25 ml). The mixture was stirred at 95? C. for 64 hours and then partitioned between DCM (30 ml) and water (30 ml). The aqueous layer was extracted with DCM (20 ml) and the extracts combined with the organic layer. The combined extracts were filtered through a phase-separating paper and evaporated. The residue was purified by flash silica chromatography, elution solvent 15% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (racemate) (178 mg, 59.1%) as a beige solid. The racemic product was purified by preparative HPLC (Chiralpak AD column, 20 ?m silica, 50 mm diameter, 250 mm length), Heptane:Ethanol: 80:20. Fractions containing the desired compounds were evaporated to dryness to afford (E)-methyl 3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 1, first eluted, 70 mg) and (E)-methyl 3-(4-(2-(2-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (isomer 2, second eluted, 66 mg). .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 1.00 (3H, s), 1.13 (3H, d), 1.20 (3H, s), 1.44 (3H, d), 2.57-2.78 (3H, m), 2.93 (1H, dd), 3.80 (3H, s), 5.05 (1H, s), 6.41 (1H, d), 7.13 (1H, ddd), 7.18 (1H, ddd), 7.32 (1H, d), 7.43 (2H, d), 7.51 (2H, d), 7.54 (1H, d), 7.64 (1H, s), 7.67 (1H, d). m/z: ES+ [M+H]+ 435.

Example 9: (E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Isomer 1)*

[0519] ##STR00056##

[0520] 2M Sodium hydroxide (3.31 ml, 6.63 mmol) was added to a solution of (E)-methyl 3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (600 mg, 1.33 mmol) (mixture of diastereoisomers) in methanol (5 ml), and THF (20 ml) and the mixture stirred at ambient temperature for 4 hours. The mixture was concentrated to a volume such that all of the organic solvent had been removed, diluted with water (50 ml), acidified with dilute HCl to pH 6 and extracted with ethyl acetate (2?50 ml). The extracts were combined and evaporated under reduced pressure. The residue was purified by preparative HPLC (Chiralpak IA column, 20 ?m silica, 20 mm diameter, 250 mm length), Heptane:IPA 90:10, 0.2% acetic acid at 80 ml/min. Fractions containing the desired compounds were combined and analysed to yield (E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (isomer 1, first eluted, 130 mg, 22.36%) and (E)-3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (isomer 2, second eluted, 30.0 mg, 5.16%). .sup.1H NMR (500 MHz, DMSO, 30? C.) ? 0.49 (3H, d), 1.03 (3H, s), 1.14-1.21 (1H, m), 1.28 (3H, s), 2.17 (1H, dd), 2.59-2.71 (1H, m), 2.8-2.99 (2H, m), 4.09-4.34 (2H, m), 4.99 (1H, s), 6.59 (1H, d), 6.86-7.07 (2H, m), 7.1-7.19 (1H, m), 7.18-7.29 (1H, m), 7.36-7.49 (2H, m), 7.49-7.66 (2H, m), 10.31 (1H, s), CO.sub.2H not observed. m/z: ES+ [M+H]+ 439.

[0521] Stereochemistry inferred to be (R) at the undefined centre by analogy with other examples.

[0522] The (E)-methyl 3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (mixture of diastereomers) used as starting material was prepared as follows:

Preparation of (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate

[0523] ##STR00057##

[0524] 4-Bromo-2-fluorobenzaldehyde (20.88 g, 102.87 mmol) and methyl acrylate (13.98 ml, 154.30 mmol) were taken up in thoroughly degassed DMA (150 ml) and tri-o-tolylphosphine (3.13 g, 10.29 mmol), palladium(II) acetate (1.155 g, 5.14 mmol) and triethylamine (28.7 ml, 205.74 mmol) added. The reaction was stirred and heated to 100? C. for 16 hours. More tri-o-tolylphosphine (3.13 g, 10.29 mmol) and palladium(II) acetate (1.155 g, 5.14 mmol) were added and the reaction mixture was heated to 110? C. for a further 2 hours. Water (1 L) was added and the reaction mixture extracted with DCM (2?500 ml). Combined organics were dried (MgSO.sub.4), filtered and evaporated to give a brown solid. The crude product was purified by flash silica chromatography, elution gradient 0 to 25% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate (15.20 g, 71.0%) as a yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) ? 3.83 (3H, s), 6.53 (1H, d), 7.31 (1H, dd), 7.41 (1H, d), 7.65 (1H, d), 7.79-8 (1H, m), 10.25-10.41 (1H, m). No mass ion observed in LCMS.

Preparation of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluorophenyl)acrylate (racemate)

[0525] ##STR00058##

[0526] 1-(1H-Indol-3-yl)-2-methylpropan-2-amine (1 g, 5.31 mmol) and (E)-methyl 3-(3-fluoro-4-formylphenyl)acrylate (1.106 g, 5.31 mmol) in acetic acid (15 ml) were stirred at 80? C. for 2 hours under nitrogen. The crude product was purified by ion exchange chromatography, using an SCX-2 column. The desired product was eluted from the column using 7M NH.sub.3/methanol and pure fractions were evaporated to dryness to afford (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluorophenyl)acrylate (racemate) (2.000 g, 99%). .sup.1H NMR (400 MHz, DMSO, 30? C.) ? 1.14 (3H, s), 1.27 (3H, s), 2.52-2.74 (2H, m), 3.74 (3H, s), 5.12 (1H, s), 6.69 (1H, d), 6.86-7.05 (2H, m), 7.20 (1H, d), 7.25-7.33 (2H, m), 7.39 (1H, d), 7.73 (1H, d), 7.85 (1H, t), 10.29 (1H, s), NH not observed. m/z: ES+ [M+H]+ 379.

Preparation of (E)-methyl 3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (mixture of diastereomers)

[0527] ##STR00059##

[0528] (S)-3-Fluoro-2-methylpropyl trifluoromethanesulfonate (obtained as described in Example 3, preparation of starting materials) (1.259 g, 5.62 mmol) was added to a solution of (E)-methyl 3-(4-(3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)-3-fluorophenyl)acrylate (racemate) (850 mg, 2.25 mmol) and N-ethyl-N-isopropylpropan-2-amine (1.467 ml, 8.42 mmol) in 1,4-dioxane (5 ml). The mixture was heated at 60? C. for 4 hours, then stirred at ambient temperature for 12 hours. The mixture was partitioned between ethyl acetate (25 ml) and water (25 ml). The aqueous layer was extracted with ethyl acetate (2?25 ml) and the extracts combined with the organic layer. The combined extracts were evaporated under vacuum. The residue was purified by flash silica chromatography, elution solvent 10% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(3-fluoro-4-(2-((S)-3-fluoro-2-methylpropyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (mixture of diastereoisomers) (600 mg, 59.0%). m/z: ES+ [M+H]+ 453.

Example 10: (E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoic acid

[0529] ##STR00060##

[0530] Methyl (E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate (4.70 g, 10.27 mmol) was dissolved in iPrOH (42.8 ml) and 5M sodium hydroxide solution (6.16 ml, 30.82 mmol) was added in one portion, the reaction was then stirred at room temperature for 4 hours. Water was added (100 ml) and the pH was brought to ?5 by addition of 2N HCl. The solution was extracted with EtOAc (?2) and the combined organics were dried (MgSO.sub.4) and concentrated in vacuo. The residue was passed through a silica plug, eluting first with DCM, then up to 5% MeOH in DCM. Fractions containing product were evaporated to a yellow solid (?4.2 g). The residue (4.2 g) was dissolved in EtOH (20 ml) and warmed to 35? C. Water (30 ml) was added slowly over ?40 mins. The mixture was then stirred for another 30 minutes, then slowly cooled to room temperature. Additional water (30 ml) was added, and the reaction was then cooled to 0? C. The mixture was filtered and the solids were washed with water before being dried under vacuum at 35? C. overnight to afford (E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoic acid (3.34 g, 73.3%) as a pale yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) 1.12 (3H, d), 1.19 (3H, d), 1.26 (3H, d), 2.43 (1H, dd), 2.63 (1H, dd), 2.87 (1H, dd), 3.07 (1H, dd), 3.65 (1H, q), 6.41 (1H, d), 7.02 (2H, d), 7.06-7.16 (2H, m), 7.19-7.25 (1H, m), 7.41 (1H, s), 7.48-7.57 (1H, m), 7.63 (1H, d), CO.sub.2H not observed. m/z: ES+ [M+H]+ 444.

[0531] This compound could alternatively be named (E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]acrylic acid.

Preparation of (4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol

[0532] ##STR00061##

[0533] Lithium borodeuteride (0.497 g, 17.25 mmol) was added portionwise to a solution of methyl 4-bromo-2,6-difluorobenzoate (2.89 g, 11.5 mmol) in THF (46.0 ml). The reaction was heated to 50? C. for 2 hours. After cooling, (30 ml) 2N HCl was carefully added. The layers were separated, and the aqueous was extracted with EtOAc (2?50 ml). The combined organics were washed with brine, dried (MgSO.sub.4) and concentrated to afford (4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol (2.250 g, 87%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) 1.96 (1H, s), 7.07-7.13 (2H, m).

Preparation of 4-bromo-2,6-difluoro-1-deuterobenzaldehyde

[0534] ##STR00062##

[0535] Dess-Martin reagent (4.98 g, 11.73 mmol) was added to (4-bromo-2,6-difluoro-phenyl)-dideuterio-methanol (2.20 g, 9.78 mmol) in DCM (39.1 ml) at room temperature. The reaction was stirred for 1 hour, then was quenched by addition of (50 ml) sat. NaHCO.sub.3 containing 10% sodium thiosulfate. The layers were separated and the aqueous phase was extracted with DCM (2?50 ml). The organics were dried (MgSO.sub.4) and concentrated, then the crude product was purified by flash silica chromatography, elution gradient 0 to 25% EtOAc in heptane. Pure fractions were evaporated to dryness to afford 4-bromo-2,6-difluoro-1-deuterobenzaldehyde (2.040 g, 94%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) 7.18-7.25 (2H, m). No mass ion observed.

Preparation of methyl (E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate

[0536] ##STR00063##

[0537] 4-Bromo-2,6-difluoro-1-deuterobenzaldehyde (3.33 g, 15.0 mmol), triethylamine (4.18 ml, 30.00 mmol), palladium (II) acetate (0.168 g, 0.75 mmol) and tritolylphosphine (0.457 g, 1.50 mmol) were dissolved in DMF (36.6 ml), which was degassed. Methyl acrylate (2.026 ml, 22.50 mmol) was then added and the reaction was heated to 80? C. for 4 hours. After cooling, the mixture was added to water (150 ml) and extracted with EtOAc (2?150 ml). The combined organics were washed with 2N HCl (100 ml) then brine (100 ml), then dried (MgSO4) and concentrated. The crude product was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in heptane. Pure fractions were evaporated to dryness to afford methyl (E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate (2.93 g, 86%) as a yellow solid. 1H NMR (400 MHz, CDCl3, 30? C.) 3.83 (3H, d), 6.51 (1H, d), 7.12 (2H, m), 7.57 (1H, d). m/z (ES+), [M+H]+=228.

(R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine

[0538] ##STR00064##

[0539] (2R)-1-(1H-indol-3-yl)propan-2-amine (3.81 kg, 21.21 moles) was added to a 100 L glass lined jacketed vessel under an atmosphere of nitrogen. 1,4-dioxane (23 L) was added, and the agitator was switched on. Diisopropylethylamine (5.55 L; 31.82 moles) was added to the stirred suspension followed by (2-fluoro-2-methyl-propyl)trifluoromethanesulfonate (5.55 kg, 23.77 moles). 1,4-Dioxane (4 L) was added to the vessel, and the mixture was heated to 75? C. Heating was continued for 24 hours before cooling the mixture to 25? C. Water (30.5 L) was added to the vessel, followed by toluene (30.5 L). After 40 minutes the agitator was switched off and the layers were allowed to separate. The aqueous layer was removed and water (30.5 L) was added to the organic solution. The mixture was agitated for 15 minutes before allowing the layers to separate. The aqueous layer was removed from the vessel. The organic solution was concentrated by vacuum distillation (jacket temperature 65? C., 110 mbar pressure) until approximately 27 L of distillate had been removed. The remaining solution in the vessel was cooled to afford (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine as a solution in toluene (33% w/w) (15.4 Kg, 97%). 1H NMR (500 MHz, DMSO, 27? C.) 0.98 (3H, d), 1.26 (3H, d), 1.30 (3H, d), 2.57-2.75 (3H, m), 2.81 (1H, dd), 2.84-2.92 (1H, m), 6.97 (1H, t), 7.06 (1H, t), 7.11-7.22 (1H, multiplet obscured by toluene signals), 7.34 (1H, d), 7.52 (1H, d), 10.80 (1H, s).

Preparation of methyl (E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate

[0540] ##STR00065##

[0541] (R)N-(1-(1H-indol-3-yl)propan-2-yl)-2-fluoro-2-methylpropan-1-amine [33% w/w in toluene] (11.26 g, 14.97 mmol) and (E)-3-(4-deuteriocarbonyl-3,5-difluoro-phenyl)prop-2-enoate (3.40 g, 14.97 mmol) were heated in toluene (55.6 ml)/acetic acid (4.28 ml, 74.83 mmol) at 80? C. for 5 hr. After cooling, the volatiles were removed under vacuum. The residue was taken-up in DCM (200 ml) and washed with sat. NaHCO.sub.3 solution (200 ml). The aqueous phase was extracted with DCM (100 ml) then the combined organics were washed with brine, dried and concentrated in vacuo. The crude material was loaded to an SCX-2 column, eluting with methanol to remove unreacted aldehyde. The column was then eluted with 7M NH.sub.3-MeOH to liberate the product. The basic filtrate was evaporated and the crude product was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in heptane. Pure fractions were evaporated to dryness to afford methyl (E)-3-[4-[(1R,3R)-1-deuterio-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-1-yl]-3,5-difluoro-phenyl]prop-2-enoate (4.70 g, 68.6%) as a pale yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3, 30? C.) 1.11 (3H, d), 1.19 (3H, d), 1.25 (3H, d), 2.42 (1H, dd), 2.62 (1H, dd), 2.87 (1H, dd), 3.07 (1H, dd), 3.65 (1H, q), 3.81 (3H, s), 6.39 (1H, d), 6.99 (2H, d), 7.06-7.17 (2H, m), 7.23 (1H, dd), 7.45 (1H, s), 7.49-7.6 (2H, m). m/z: ES+ [M+H]+ 458.

Example 11: Preparation of (1R,3R)-1-{4-[(E)-2-carboxyethenyl]-2,6-difluorophenyl}-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-beta-carbolin-2-ium maleate

[0542] ##STR00066##

[0543] A solution of maleic acid (1.31 g, 11.29 mmol) in acetone (15 ml) was stirred under nitrogen. A solution of (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (Example 1) (5.00 g, 11.3 mmol) in acetone (25 ml) was added to the maleic acid solution to give a yellow solution. The reaction vessel was covered in foil to protect from light and purged with a stream of nitrogen gas overnight until the solvent evaporated. A solid was obtained which was dried in vacuo for 2 hours to give the title compound as a cream solid (6.23 g, 98%). .sup.1H NMR (500 MHz, DMSO, 27? C.) 0.95-1.34 (9H, m), 2.24-2.45 (1H, m), 2.54-2.66 (1H, m), 2.8-2.99 (2H, m), 3.52 (1H, s), 5.22 (1H, s), 6.26 (2H, s), 6.67 (1H, d), 6.89-7.07 (2H, m), 7.19 (1H, d), 7.39-7.51 (3H, m), 7.55 (1H, d), 10.59 (1H, s).

[0544] An XRPD trace of this maleate salt is shown in FIG. 8 and a DSC trace shown in FIG. 9.

Example 12

[0545] Exemplary compositions of Example 1 were manufactured at the 75 g scale using a wet granulation process. The active ingredient, mannitol, microcrystalline cellulose and sodium starch glycolate were weighed in the quantities tabulated below and transferred to a Diosna P1-6mixer-granulator and mixed (with chopping) at 600 rpm for 6 minutes. For Composition A, mixing was continued while 30 mL water was added in two aliquots at a rate of approximately 1 mL per second, pausing mixing in between aliquots, while for Composition B a solution prepared by stirring the required amounts of EDTA and ascorbic acid with 20 mL water at 50? C. for 20 minutes (protected from light) was added using an analogous process, the second aliquot in this case comprising approximately 10 mL of rinse liquor. Wet mixing was continued for a total of 1.5 minutes. The wet granules were passed through a 1.5 mm screen then dried under vacuum at 50-60? C. to a moisture content of <2% w/w. The resulting granules were milled using a 1 mm screen then mixed with the lubricant for 5 minutes at 32 rpm using a Turbula blender. Tablets containing 10 mg of the active ingredient were formed by compressing the granules to a nominal 100 mg compression weight using a Riva Mini-press equipped with 6 mm normal concave tooling.

TABLE-US-00004 Composition A Composition B Component Function % w/w Amount (g) % w/w Amount (g) Example 1 Form B Active ingredient 10.0 7.50 10.0 7.50 EDTA Chelating agent Not present 0.1 0.075 Ascorbic acid Anti-oxidant Not present 0.5 0.375 Mannitol Diluent 68.0 51.00 67.4 50.55 Microcrystalline Diluent 15.0 11.25 15.0 11.25 cellulose Sodium starch Disintegrant 5.0 3.75 5.0 3.75 glycolate Stearic acid Lubricant 2.0 1.50 2.0 1.50 Total 100 75.00 100 75.00

[0546] The stability of Compositions A and B with regard to degradant formation (wherein degradant means (R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate) was evaluated for tablets packed in induction sealed 75 cc HDPE bottles or exposed to the atmosphere in petri dishes, stored in the dark, under controlled temperature and humidity as tabulated below. It is apparent that Composition B, which contains both a chelating agent and anti-oxidant, is more stable to chemical degradation than Composition A which is a standard tablet formulation.

[0547] Stability of Exemplary Compositions with Regard to Degradant Formation (%)

TABLE-US-00005 4 Week Time Point (storage condition) Initial (25? C./ (25? C./ (40? C./ Time (5? C., 60% RH, 60% RH, 75% RH, Formulation Point packed) packed) exposed) exposed) Composition A 0.05 0.06 0.08 0.27 0.28 Composition B ND <0.05 <0.05 0.05 0.11 ND Not detected (<0.02% w/w)

Example 13: (E)-3-[3,5-difluoro-4-[(3R)-2-(2-fluoro-2-methyl-propyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate

[0548] ##STR00067##

[0549] (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (200 mg, 0.45 mmol) was added to a solution of cerium ammonium nitrate (248 mg, 0.45 mmol) in acetonitrile (6 ml)/water (1.500 ml) at room temperature. The reaction was stirred for 2 hr and further cerium ammonium nitrate (248 mg, 0.45 mmol) was added. The solution was stirred at 25? C. for a further 15 minutes. The reaction mixture was acidified with 2M HCl (3 ml) and extracted with DCM (2?10 ml). The organics were then concentrated in vacuo and the crude product was purified by preparative HPLC (Waters SunFire column, 5? silica, 50 mm diameter, 100 mm length), using decreasingly polar mixtures of water (containing 0.1% formic acid) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford (R,E)-3-(3,5-difluoro-4-(2-(2-fluoro-2-methylpropyl)-3-methyl-4,9-dihydro-3H-pyrido[3,4-b]indol-2-ium-1-yl)phenyl)acrylate (35.0 mg, 17.58%) as an orange glass. 1H NMR (500 MHz, DMSO, 30? C.) 1.27 (3H, d), 1.43-1.55 (6H, m), 3.51 (1H, d), 3.72 (1H, dd), 3.96 (1H, dd), 4.18-4.32 (1H, m), 4.67 (1H, s), 6.59 (1H, s), 7.09-7.3 (2H, m), 7.42 (1H, t), 7.52-7.73 (3H, m), 7.78 (1H, d), NH not observed. HRMS (ESI): [M+H].sup.+, found 441.17831, C.sub.23H.sub.24F.sub.2N.sub.2O.sub.2 requires 441.17844.

Preparation of (E)-3-[3,5-difluoro-4-[2-(2-fluoro-2-methyl-propyl)-3-methyl-9H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate

[0550] ##STR00068##

[0551] (E)-3-(3,5-Difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (0.500 g, 1.13 mmol) was dissolved in DMSO (10 mL) and heated to 120? C. in air and light for 16 h. The reaction was then heated to 180? C. for 2.5 h. The reaction mixture was cooled and purified by preparative HPLC (Waters XBridge Prep C18 OBD column, 5? silica, 50 mm diameter, 100 mm length), using decreasingly polar mixtures of water (containing 1% NH.sub.3) and MeCN as eluents. Fractions containing the desired compound were evaporated to dryness to afford (E)-3-[3,5-difluoro-4-[2-(2-fluoro-2-methyl-propyl)-3-methyl-9H-pyrido[3,4-b]indol-2-ium-1-yl]phenyl]prop-2-enoate (0.047 g, 9.40%) as an orange solid. 1H NMR (400 MHz, DMSO, 27? C.) 1.27 (3H, s), 1.33 (3H, d), 3.09 (3H, s), 4.59-4.8 (1H, m), 5.14-5.37 (1H, m), 6.54 (1H, d), 7.19 (1H, d), 7.39 (1H, t), 7.63 (2H, d), 7.74 (1H, t), 7.91 (1H, d), 8.45 (1H, d), 8.88 (1H, s), 10.79 (1H, s). m/z: ES+ [M+H]+ 439.

Example 14A: Preparation of (E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-3-hydroxy-2-methyl-propyl)-3-methyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoic acid (Isomer 1)

[0552] ##STR00069##

[0553] 2M Sodium hydroxide (1.27 mL, 2.54 mmol) was added to a solution of (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateIsomer 1 (120 mg, 0.25 mmol) in THF (0.635 mL)/methanol (0.635 mL). The reaction was stirred at room temperature for 1 h, then diluted with EtOAc and water. The aqueous was adjusted to pH 6 by addition of 2M HCl, and the layers were separated. The aqueous layer was extracted with EtOAc, then the combined organics were dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acidIsomer 1 (85 mg, 72.9%) as a beige solid. 1H NMR (400 MHz, DMSO, 27? C.) 0.97 (3H, d), 1.05 (3H, d), 2.32-2.4 (1H, m), 2.44-2.54 (1H, m), 2.73-2.93 (3H, m), 3-3.14 (2H, m), 3.32-3.49 (2H, m), 4.73 (1H, s), 5.14 (1H, s), 6.58 (1H, d), 6.82-6.96 (2H, m), 7.10 (1H, d), 7.33 (1H, d), 7.36 (1H, d), 7.45 (1H, d), 10.49 (1H, s). m/z (ES+), [M+H]+=459.

Example 14B: Preparation of (E)-3-[3,5-difluoro-4-[(1R,3R)-2-(2-fluoro-3-hydroxy-2-methyl-propyl)-3-methyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-1-yl]phenyl]prop-2-enoic acid (Isomer 2)

[0554] ##STR00070##

[0555] 2M Sodium hydroxide (1.27 mL, 2.54 mmol) was added to a solution of (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylateIsomer 2 (110 mg, 0.23 mmol) in THF (0.529 mL)/methanol (0.529 mL). The reaction was stirred at room temperature for 1 h, then diluted with EtOAc and water. The aqueous was adjusted to pH 6 by addition of 2M HCl, and the layers were separated. The aqueous layer was extracted with EtOAc, then the combined organics were dried (MgSO.sub.4) and concentrated in vacuo. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acidIsomer 2 (81 mg, 76%) as a beige solid. 1H NMR (400 MHz, DMSO, 27? C.) 1.02 (2H, s), 1.05 (3H, d), 1.23 (1H, s), 1.90 (3H, s), 2.28-2.46 (1H, m), 2.53-2.7 (1H, m), 2.86-3.03 (2H, m), 3.56 (1H, d), 4.83 (1H, s), 5.17 (1H, s), 6.66 (1H, d), 6.86-7.08 (2H, m), 7.17 (1H, d), 7.40 (1H, d), 7.44 (2H, d), 7.53 (1H, d), 10.54 (1H, s). m/z (ES+), [M+H]+=459.

[0556] The (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1 and 2) used as starting materials were prepared as follows:

2-Fluoro-2-methylpropane-1,3-diol

[0557] ##STR00071##

[0558] LiAlH.sub.4 (0.741 g, 19.25 mmol) was added portionwise to a cooled solution of diethyl 2-fluoro-2-methylmalonate (1.345 g, 7.00 mmol) in THF (35.0 ml). The reaction was allowed to warm to room temperature over 1 h. After cooling to 0? C., the reaction was quenched by addition of water (0.75 mL), 15% NaOH (0.75 mL), then water (1.5 mL). The suspension was stirred for 30 min, then filtered and the solids were washed with THF. The filtrate was evaporated to afford 2-fluoro-2-methylpropane-1,3-diol (0.745 g, 98%) as a colourless oil. 1H NMR (400 MHz, CDCl3, 27? C.) 1.34 (3H, d), 2.12-2.27 (2H, m), 3.75 (4H, d).

3-(((R)-1-(1H-Indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-ol

[0559] ##STR00072##

[0560] Trifluoromethanesulfonic anhydride (1.151 ml, 6.80 mmol) was added to a solution of 2-fluoro-2-methylpropane-1,3-diol (0.70 g, 6.47 mmol) in DCM (17.85 ml) at 0? C., followed by 2,6-lutidine (0.908 ml, 7.77 mmol). The reaction was allowed to warm to room temperature over 30 min, then was washed with 2M HCl. The organic phase was passed through a phase separator cartridge and concentrated in vacuo. The residue was dissolved in dioxane (12 mL), then (R)-1-(1H-indol-3-yl)propan-2-amine (1.128 g, 6.47 mmol) and DIPEA (1.678 ml, 9.71 mmol) were added and the reaction was heated to 90? C. for 2 h. After cooling, the reaction was diluted with DCM and washed with water. The aqueous was extracted with DCM, then the organics were concentrated in vacuo. The crude product was purified by flash silica chromatography, elution gradient 0 to 10% MeOH in DCM. Pure fractions were evaporated to dryness to afford 3-(((R)-1-(1H-indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-ol (0.815 g, 47.6%) as a brown gum. m/z (ES+), [M+H]+=265.

(E)-methyl 3-(3,5-difluoro-44(1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1 and

[0561] ##STR00073##

[0562] (E)-methyl 3-(3,5-difluoro-4-formylphenyl)acrylate (565 mg, 2.50 mmol) was added to a suspension of 3-(((R)-1-(1H-indol-3-yl)propan-2-yl)amino)-2-fluoro-2-methylpropan-1-01 (661 mg, 2.50 mmol) in toluene (11.3 ml)/acetic acid (1.25 ml). The reaction was heated to 90? C. for 5 h. After cooling, the volatiles were removed under vacuum, then the residue was passed through an SCX-2 column, eluting with methanol to remove unreacted aldehyde. The column was then eluted with NH.sub.3/MeOH to liberate the products. The basic filtrate was evaporated then the crude product was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in heptane. Pure fractions were evaporated to dryness to afford (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 1-122 mg, 10.3%) as a yellow solid and (E)-methyl 3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-3-hydroxy-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylate (Isomer 2-129 mg, 11% as a yellow/orange solid. Isomer 1-1H NMR (400 MHz, CDCl3, 27? C.) 1.10 (3H, d), 1.14 (3H, d), 2.54 (1H, dd), 2.62-2.73 (1H, m), 3.06-3.3 (2H, m), 3.40 (1H, dd), 3.56 (1H, t), 3.80 (3H, s), 3.87-4.08 (1H, m), 4.27 (1H, s), 5.16 (1H, s), 6.37 (1H, d), 7.01 (2H, d), 7.07-7.15 (2H, m), 7.14-7.24 (1H, m), 7.42-7.56 (2H, m), 7.71 (1H, s). m/z (ES+), [M+H]+=473. Isomer 2-1H NMR (400 MHz, CDCl3, 27? C.) 1.15 (3H, d), 1.20 (3H, d), 2.65 (1H, dd), 2.79 (1H, t), 2.93-3.09 (2H, m), 3.57 (1H, dt), 3.70 (1H, dd), 3.78 (3H, s), 4.2-4.67 (1H, m), 5.42 (1H, s), 6.32 (1H, d), 6.94 (2H, d), 7.06-7.15 (2H, m), 7.19-7.27 (2H, m), 7.42 (1H, s), 7.51 (1H, dd), 8.02 (1H, s). m/z (ES+), [M+H]+=473.

Reference Example 1: 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one

[0563] ##STR00074##

[0564] Pyridine 4-methylbenzenesulfonate (11.62 g, 46.24 mmol) was added to a suspension of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one (128 g, 231.19 mmol) in methanol (1 L) under nitrogen. The mixture was stirred at 60? C. for 1.5 hours. The mixture was soluble after 5 minutes. The mixture was held at 50? C. overnight during which time a precipitate formed. The solid material was isolated by filtration and washed with water and acetonitrile. This material still contained minor impurities from the previous stage and required further purification. The material was dissolved in dichloromethane and purified by flash chromatography on silica gel (0% methanol/DCM to 10% methanol/DCM). The desired product, 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (Reference Example 1) (92 g, 85%), was thus isolated as a cream solid (Form A): .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.4-1.51 (12H, m), 1.51-1.78 (2H, m), 1.89-2.05 (2H, m), 2.72-2.86 (1H, m), 2.91-3.05 (1H, m), 3.12-3.24 (1H, m), 3.64 (2H, q), 3.83-4.01 (1H, m), 4.29-4.41 (1H, m), 4.47 (1H, t), 4.58 (2H, q), 8.26 (2H, s), 8.85 (1H, s); Mass Spectrum [M+H].sup.+=470.

[0565] 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yL)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one was prepared as follows:

[0566] 1,8-Diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) was added to a suspension of tert-butyl 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (150 g, 319.46 mmol) in 2-methylTHF (1.2 L). Iodoethane (46 mL, 575.03 mmol) was added and the mixture was stirred for 16 hours at 35? C. Further iodoethane (46 mL, 575.03 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (76 mL, 511.14 mmol) were added and stirring was continued for 24 hours at 35? C. The mixture was poured into water and the insoluble material was isolated by filtration, washed with water and MTBE and dried in vacuo to afford tert-butyl 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (116 g, 73.0%) as a yellow solid. The filtrate was extracted with DCM and the organic solution was dried with magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica using gradient elution (30% MTBE/heptane to 100% MTBE). A second crop of the desired product (12 g, 24.12 mmol, 7.55%), was thus isolated as a yellow solid which was later combined with the first crop: .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.41 (9H, s), 1.44 (9H, s), 1.48 (3H, t), 1.52-1.69 (2H, m), 1.87-2.04 (2H, m), 2.79-3.03 (3H, m), 3.86-4.03 (2H, m), 4.59 (2H, q), 7.89 (2H, s), 8.85 (1H, s); Mass Spectrum [M+H]+=498.

[0567] A suspension of tert-butyl 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (3009.5 g, 6.05 mol) in DCM (9 L) was cooled to 5-10? C. under N.sub.2. TFA (9 L) was added portionwise to the suspension whilst maintaining the temperature <30? C. The reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated, the resulting residue was dissolved in water (30 L) and added slowly to a 35% aqueous ammonia solution (12 L) at 0-5? C. The suspension was stirred for 30 min then the product was filtered off and washed with water (2?6 L). The product was dried at 50? C. in vacuo for 2 days. to afford 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine ((2496 g): .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.4-1.52 (12H, m), 1.57-1.73 (2H, m), 1.83-1.93 (2H, m), 2.57-2.7 (2H, m), 2.71-2.84 (1H, m), 2.96-3.09 (2H, m), 4.58 (2H, q), 8.06 (2H, s), 8.84 (1H, s); Mass Spectrum [M+H].sup.+=398.

[0568] To a solution of 3-(tetrahydro-2H-pyran-2-yloxy) propanoic acid (HATU, 48.80 g 0.2774 mol) and N-ethyl-N-isopropylpropan-2-amine (86.96 mL, 0.4993 mol) in THF (552 mL) was added O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (115.73 g, 0.3051 mol) portionwise at RT under nitrogen. The resulting mixture was stirred for 20 min then 3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)-5-(1-ethyl-3-(piperidin-4-yl)-1H-1,2,4-triazol-5-yl)pyrazin-2-amine (122.5 g (110.25 g active), 0.2774 mol) was added portionwise over 1 h. After 3.5 h, the mixture was concentrated and the residue was slurried in MeCN (275 mL) for 15 min at room temperature. The product was filtered off, washed with MeCN (3?110 mL) and dried overnight at 50? C. in vacuo. This gave 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one (131.9 g, 96%). .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.29-1.48 (16H, m), 1.48-1.75 (4H, m), 1.83-1.99 (2H, m), 2.48-2.68 (2H, m), 2.68-2.79 (1H, m), 2.87-2.99 (1H, m), 3.07-3.19 (1H, m), 3.32-3.42 (1H, m), 3.47-3.6 (1H, m), 3.64-3.75 (1H, m), 3.75-3.84 (1H, m), 3.84-3.95 (1H, m), 4.24-4.39 (1H, m), 4.47-4.6 (3H, m), 7.84 (2H, s), 8.79 (1H, s): Mass Spectrum [M+Na].sup.+=577.

Alternative Preparation of Reference Example 1

[0569] To a suspension of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-(tetrahydro-2H-pyran-2-yloxy)propan-1-one (131.9 g, 0.2382 mol) in methanol (1045 mL) was added pyridinium p-toluenesulfonate (11.97 g, 47.7 mmol) under N2. The reaction mixture was stirred at 60? C. for 5.5 h then at 50? C. overnight. The reaction mixture was cooled to 0? C. and the solid was filtered off. The product was slurried in water (250 mL) for 20 min at room temperature, filtered off, washed with water (3?40 mL) and dried at 50? C. in vacuo. This gave 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (21.4 g) as Form A (see below).

[0570] The methanol liquors were concentrated and the resulting solid was slurried in water (0.6 L) for 20 min at room temperature. The solid was isolated by filtration and washed with water (3?100 mL). The filter cake was slurried for a second time in water (0.5 L) for a further 20 minutes. The product was isolated by filtration, washed with water (100 mL) and dried at 50? C. in vacuo. This gave 81.9 g 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (81.9 g) as Form A.

[0571] Both crops were combined (103.3 g), seeded with Form B (16.68 g) and slurried in MeCN (826 mL) at room temperature overnight. This gave 117.4 g of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one as a pale yellow solid (117.4 g), Form B (see below). This material was further purified by slurrying in heptane (7.5 rel vols) for 1 hour. The mixture was filtered, pulled dry on the filter, and dried at 50? C. in a vacuum oven overnight to afford 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (102.5 g) as Form B.

[0572] Form B may also be made by slurrying Form A in MeCN without seeding.

[0573] Form A or B may also be converted to Form C as follows:

[0574] A suspension of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (eg Form B made by the processes outlined above) in IPA (12 vol) was heated at reflux until the solid dissolved. The solution was hot filtered then cooled to room temperature. This gave 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1H-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one as a pale yellow solid (99.3 g, 97%) as Form C.

[0575] Form C may also be converted to Form B as follows:

[0576] In a 10 L flange flask, Form C (377.8 g portion 1) in MIBK (7900 mL) was heated to 110-115? C. to give a solution. The solution was allowed to cool to 97-103? C. and immediately polish filtered into a SOL vessel containing a seed of Form B (0.8 g) in acetonitrile (8220 mL) stirring at ?15? C. During the addition the temperature in the 50 L vessel was maintained between ?15 and 25? C. by means of jacket cooling. Three further portions of the compound dissolved in MIBK were added by a similar method. To the resulting slurry was added a seed of form B (0.8 g) and the mixture was then stirred at 10-20? C. overnight. In-process analysis confirmed the desired form (Form B) with no Form C or amorphous visible. The mixture was filtered and washed with acetonitrile (3340 mL). The solid was oven dried for 2 days (solid was broken up during the drying to a powder and a mixture of small lumps ?1 mm to ?3-4 mm size) until constant weight was obtained. Yield=1532.8 g (93.5%)

[0577] 3-(Tetrahydro-2H-pyran-2-yloxy)propanoic acid was prepared as follows:

[0578] To a stirred solution of methanol (2.4 L) and concentrated sulfuric acid (44.4 mL, 832.61 mmol) at 0? C. under nitrogen was added, dropwise, beta-propiolactone (175 mL, 2.78 mol). This solution was allowed to stir at room temperature for 2 days. The reaction mixture was cooled to 10? C. before adding, portionwise, sodium bicarbonate (145 g, 1.72 mol), the resulting suspension was left to stir at room temperature for 75 minutes. This solution was filtered, the filter-cake was washed with methanol (800 mL). The filtrate was evaporated to an oil which was redissolved in dichloromethane (1.2 L) and stirred for 60 minutes before refiltering. This solution was filtered before evaporating to give methyl 3-hydroxypropanoate (219 g, 76%) as an oil. .sup.1H NMR Spectrum: (CDCl.sub.3) 2.50 (2H, t), 3.63 (3H, s), 3.78 (2H, t).

[0579] Pyridinium p-toluenesulfonate (7.65 g, 30.45 mmol) was added to a clear solution of methyl 3-hydroxypropanoate (63.4 g, 609.00 mmol) and 3,4-dihydro-2H-pyran (78 mL, 852.60 mmol) in dichloromethane (650 mL) at room temperature under nitrogen to give a cloudy solution. This was allowed to stir at room temperature overnight. The reaction mixture was washed with water (250 mL) and brine (250 mL) before drying (MgSO.sub.4) and evaporating to an oil. This crude product was purified by flash silica chromatography, elution gradient 15 to 30% EtOAc in heptane. Pure fractions were evaporated to dryness to afford methyl 3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.7 g, 59.0%) as a colourless oil: .sup.1H NMR Spectrum: (CDCl.sub.3) 1.47 (4H, dddd), 1.55-1.84 (2H, m), 2.55 (2H, t), 3.33-3.53 (1H, m), 3.53-3.7 (4H, m), 3.78 (1H, ddd), 3.93 (1H, dt), 4.42-4.72 (1H, m); Mass Spectrum [MH].sup.+=189.

[0580] Sodium hydroxide (2M, 349 mL, 697.58 mmol) was added to a solution of methyl 3-(tetrahydro-2H-pyran-2-yloxy)propanoate (67.68 g, 359.58 mmol) in THF (680 mL) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. The THF was removed in vacuo, the aqueous layer was then washed with ethyl acetate (260 mL), before cooling to 0? C. and careful acidification to pH 5 by the addition of hydrochloric acid (2M). The product was extracted with ethyl acetate (3?250 mL) before drying (MgSO.sub.4) and evaporation to give 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (57.0 g, 91%) as a clear oil. This material was dissolved in ethyl acetate (750 mL) then washed with water (3?250 mL) and brine (250 mL) to remove remaining acetic acid. The organic solution was dried (MgSO.sub.4) and evaporated to give 3-(tetrahydro-2H-pyran-2-yloxy)propanoic acid (45.67 g, 72.9%) as a colourless oil: .sup.1H NMR Spectrum: .sup.1H NMR (CDCl.sub.3) 1.43-1.67 (4H, m), 1.65-1.95 (2H, m), 2.68 (2H, t), 3.48-3.58 (1H, m), 3.73 (1H, dt), 3.88 (1H, ddd), 4.02 (1H, dt), 4.59-4.7 (1H, m); Mass Spectrum [M?H].sup.?=173.

[0581] The tert-butyl 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate was prepared as follows:

[0582] Hydrazine hydrate (23.59 mL, 480.75 mmol) was added dropwise to a stirred mixture of methyl 3-amino-6-bromopyrazine-2-carboxylate (100 g, 418.04 mmol) in EtOH (2 L). The mixture was heated at 50? C. under nitrogen. The resulting thick suspension was stirred at 50? C. for 16 hours. Further hydrazine (2.5 mL) was added in one portion and the suspension was stirred at 50? C. for a further 24 hours. Ethanol (500 mL) was charged to the thick reaction mixture and the mixture was allowed to cool to room temperature. The resulting suspension was filtered and the solid washed with ethanol (1 L) and dried in vacuo to give 3-amino-6-bromopyrazine-2-carbohydrazide (98 g, quantitative) as a cream solid: .sup.1H NMR Spectrum (DMSO-d.sub.6) 4.52 (2H, s), 7.59 (2H, s), 8.30 (1H, s), 9.74 (1H, s); Mass Spectrum [M+H].sup.+=232.

[0583] Pivalic anhydride (165 mL, 815.38 mmol) was added to a stirred mixture of 3-amino-6-bromopyrazine-2-carbohydrazide (172 g, 741.26 mmol) in acetonitrile (1.8 L) and the mixture was heated at 80? C. for 1 hour. The reaction was left to stir for 16 hours. The required yellow solid material was isolated by filtration. The filtrate was partitioned between EtOAc (2 L) and aqueous sodium bicarbonate (2 L). The organic layer was washed with saturated brine and dried over MgSO.sub.4. The solution was filtered and concentrated to give an orange sticky solid which was triturated with MTBE (250 mL). The insoluble yellow solid was isolated by filtration and this material was shown to be identical to the first solid. The combined solids were dried in the vacuum oven at 50? C. for 3 days to afford 3-amino-6-bromo-N-pivaloylpyrazine-2-carbohydrazide (224 g, 96%) as a yellow solid: .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.17 (9H, s), 7.62 (2H, s), 8.37 (1H, s), 9.42-9.56 (1H, m), 10.09-10.23 (1H, m); Mass Spectrum [M+H].sup.+=318.

[0584] To 3-amino-6-bromo-N-pivaloylpyrazine-2-carbohydrazide (2301 g, 7.28 mol) in MeCN (10.8 L) was added DIPEA (3.044 L, 17.48 mol) and p-toluenesulfonyl chloride (1665 g, 8.73 mol) portion-wise (?280 g?6) at 50? C. over a period of 30 mins. The reaction temperature was maintained between 65-70? C. by controlling the rate of addition. After the addition was complete, the reaction mixture was stirred at 70? C. for 1 h. The mixture was cooled to room temperature and quenched with 5% NaHCO.sub.3 (aqueous, 24.2 L). The resulting suspension was stirred for 30 min then filtered. The product was washed with water (14.8 L), pulled dry and dried at 50? C. for 16 h. The product was dissolved in DCM (12 L) and the phases separated. The organic phase was loaded onto a silica pad (6 kg) and the product was eluted with 20% EtOAc/DCM (8?10 L). Concentration of the product containing fractions gave 1987 g (92% yield) with a purity of 99.8% by HPLC of 5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (36 g, 17%): .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.43 (9H, s), 7.70 (2H, s), 8.39 (1H, s); Mass Spectrum [M+H].sup.+=298.

[0585] A stream of nitrogen gas was passed through a solution of 5-bromo-3-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-amine (89.35 g, 239.75 mmol) in DMA (1.2 L) for 20 minutes. Dicyclohexyl(2,4,6-triisopropylbiphenyl-2-yl)phosphine (11.43 g, 23.98 mmol), tris(dibenzylideneacetone)dipalladium(0) (5.49 g, 5.99 mmol), zinc (1.568 g, 23.98 mmol) and dicyanozinc (16.89 g, 143.85 mmol) were added sequentially to the stirred mixture. The mixture was heated to 100? C. and stirred for 1 hour. The mixture was cooled and partitioned between DCM (3 L) and water (1 L). The black mixture was filtered through celite and the organic layer was separated. The solution was washed with water then brine. The solution was dried with magnesium sulfate and concentrated under reduced pressure. The residue was triturated with MTBE and isolated by filtration, washing with MTBE. The filter cake was dried in vacuo to afford 5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile (55.7 g, 95%) as a pale orange solid: .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.46 (9H, s), 6.02 (1H, s), 8.38 (2H, s); Mass Spectrum [M?H].sup.?=242.

[0586] Hydrazine hydrate (82 mL, 1.69 mol) was added to 5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbonitrile (55 g, 225.18 mmol) in IPA (200 mL) and the mixture was heated at 50? C. under nitrogen for 16 hours. The mixture was cooled in an ice bath. The resulting precipitate was collected by filtration, washed with IPA and diethyl ether and dried to a constant weight to afford (Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide (49.2 g, 79%) as a yellow solid: .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.45 (9H, s), 5.26 (2H, s), 5.58 (2H, s), 7.56 (2H, s), 8.75 (1H, s); Mass

[0587] Spectrum [M+H].sup.+=277.

[0588] O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (74.3 g, 195.44 mmol) was added to a solution of N-Boc-isonipecotic acid (41.1 g, 179.15 mmol) and 4-methylmorpholine (35.9 mL, 325.74 mmol) in DMA (800 mL). The mixture was stirred for 10 minutes then (Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide (45 g, 162.87 mmol) was added to the solution in one portion (exotherm observed from 22? C. to 27? C.). After a few minutes the product crystallised from the reaction mixture. The reaction mixture was removed from the vessel and filtered through a sinter. Additional DMA was added to wash product from the sides of the vessel (150 mL) and this was poured onto the filter cake. Isopropanol (600 mL) was added to the vessel and the remainder of the product in the vessel was suspended in this solvent using vigorous agitation. The isopropanol suspension was used to wash the filter cake once the DMA had been removed by suction. The filter cake was sucked dry then washed with MTBE and sucked dry once again. The filter cake was dried in vacuo to afford (Z)-tert-butyl 4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)methylene)hydrazinecarbonyl)piperidine-1-carboxylate (76 g, 95%) as a yellow solid:

[0589] .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.40 (9H, s), 1.46 (9H, s), 1.63-1.9 (2H, m), 2.33-2.6 (2H, m, obscured by DMSO signal), 2.63-3.03 (2H, m), 3.18-3.48 (4H, m, obscured by water signal), 3.88-4.11 (2H, m), 6.43 (2H, s), 7.76 (2H, br), 8.84 (0.5H, s), 8.87 (0.5H, s), 9.85 (1H, s); Mass Spectrum [M+H].sup.+=488

[0590] In an alternative preparation, the N-Boc-isonipecotic acid may be made in situ as follows: Isonipecotic acid (858 g, 3.74 mol) was dissolved in DMA (25.3 L) and 4-methylmorpholine (393 mL, 3.74 mol) added. Stirred for 5 mins and isobutyl chloroformate (489 mL, 3.74 mol) added. The reaction mixture was stirred at 25? C. for 2 h and cooled to 15? C. before (Z)-5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazine-2-carbohydrazonamide (940 g, 3.4 mol) was added portionwise over 10 mins. The reaction mixture was stirred for 1-2 h at 15? C. Water (20.5 L) was added portionwise over 1 h and stirred for a further 1 h before being filtered. The filtercake was then washed with water (4?4 L) and pulled dry on the filter before being dried in a vacuum oven at 50? C. until dry to give the desired product.

[0591] Acetic acid (200 mL) was added to dioxane (500 mL) in a 3 L fixed double jacketed vessel and the solution was heated to 70? C. under nitrogen. (Z)-tert-butyl 4-(2-(amino(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)methylene)hydrazinecarbonyl)-piperidine-1-carboxylate (74.5 g, 152.80 mmol) was added portionwise to the warm mixture. After 10 minutes the temperature was increased to 100? C. (slight reflux). The reaction mixture was stirred at 100? C. for 1.5 hours (suspension) then held at 80? C. overnight (solution formed after overnight hold). The resulting solution was concentrated under reduced pressure, then diluted with toluene, evaporated to dryness, taken up with toluene and concentrated again. The residual oil was mixed with some ethyl acetate and concentrated to dryness. A solid crystallised from solution which was triturated with MTBE (200 mL) and isolated by filtration. The filter cake was washed with water and MTBE to afford tert-butyl 4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1H-1,2,4-triazol-3-yl)piperidine-1-carboxylate (50 g, 70%) as a grey solid.

[0592] The filtrate was concentrated under reduced pressure to give a yellow solid. This material was triturated with MTBE and filtered. The filter cake was washed with ethyl acetate and then MTBE to give a second crop as a pale yellow solid (4.93 g, 7%). This material was identical to the first crop: .sup.1H NMR Spectrum: (DMSO-d.sub.6) 1.17 (9H, s), 1.22 (9H, s), 1.29-1.47 (2H, m), 1.67-1.78 (2H, m), 2.57-2.87 (3H, m), 3.57-3.92 (2H, m), 7.56 (2H, br), 8.56 (1H, s), 13.47 (2H, br s); Mass Spectrum [M+H]+=470.

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