3-oxa-8-azabicyclo[3.2.1]octane derivatives and their use in the treatment of cancer and hemoglobinopathies

Abstract

A compound of formula Ia, Ib, Ic or Id: ##STR00001##
wherein: n is 1 or 2; R.sup.N is H or Me; R.sup.1 is optionally one or more halo or methyl groups; R.sup.2a and R.sup.2b are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH.sub.2OH; R.sup.2c and R.sup.2d (if present) are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH.sub.2OH; R.sup.3a and R.sup.3b are independently selected from H and Me; R.sup.4a is selected from OH, —NH.sub.2, —C(═O)NH.sub.2, and —CH.sub.2OH; R.sup.4b is either H or Me; R.sup.5 is either H or Me; m is 1 or 2; q is 0 or 1; R.sup.11a, R.sup.11b, R.sup.11c and R.sup.11d are independently selected from H, halo, C.sub.1-4 alkyl, C.sub.1-4 fluoroalkyl, C.sub.3-4 cycloalkyl, C.sub.1-4 alkyloxy, NH—C.sub.1-4 alkyl and cyano; R.sup.12a and R.sup.12b are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH.sub.2OH; R.sup.12c and R.sup.12d are independently selected from the group consisting of: (i) F; (ii) H; (iii) Me; and (iv) CH.sub.2OH; R.sup.12e is H or Me; R.sup.13a and R.sup.13b are independently selected from H and Me; R.sup.14 is either H or Me; R.sup.16a and R.sup.16b are independently selected from H and Me; R.sup.6 is selected from H, OMe, and OEt.

Claims

1. A compound of formula: ##STR00045##

2. A method for the treatment of cancer, comprising administering to a patient in need of treatment, a therapeutically effective PRMT5 activity inhibiting amount of a composition comprising a compound of the following formula: ##STR00046## or pharmaceutically acceptable salts thereof; and a pharmaceutical carrier; wherein the cancer is any one or more of the following: leukemia, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), non-Hodgkin's lymphoma, Hodgkin's disease, prostate cancer, lung cancer, melanoma, breast cancer, colon and rectal cancer, colon cancer, squamous cell carcinoma and gastric cancer.

Description

EXAMPLES

(1) The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.

(2) Acronyms

(3) For convenience, many chemical moieties are represented using well known abbreviations, including but not limited to, methyl (Me), ethyl (Et), n-propyl (nPr), isopropyl (iPr), n-butyl (nBu), tert-butyl (tBu), phenyl (Ph), benzyl (Bn), methoxy (MeO), ethoxy (EtO), trimethylsilyl (TMS), and acetyl (Ac).

(4) For convenience, many chemical compounds are represented using well known abbreviations, including but not limited to, methanol (MeOH), deuterated methanol (d.sub.4-MeOD) ethanol (EtOH), isopropanol (i-PrOH), ether or diethyl ether (Et.sub.2O), ethyl acetate (EtOAc), acetic acid (AcOH), acetonitrile (MeCN), dichloromethane (methylene chloride, DCM), trifluoroacetic acid (TFA), dimethylformamide (DMF), tetrahydrofuran (THF), dimethylsulfoxide (DMSO), deuterated chloroform (CDCl.sub.3), diethylamine (DEA), deuterated dimethylsulfoxide (d.sub.6-DMSO), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl.HCl, EDCl), meta-chloroperoxybenzoic acid (mCPBA), 1,1′-bis(diphenylphosphino)ferrocene (dppf), tert-butyloxycarbonyl (Boc, BOC), 2-(trimethylsilyl)ethoxymethyl (SEM), triethylamine (Et.sub.3N or TEA), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIPEA or DIEA), 1,1′-bis(diphenylphosphino)ferrocene dichloropalladium (II) (PdCl.sub.2(dppf)), trans-dichlorobis(triphenylphosphine)palladium(II) (PdCl.sub.2(PPh.sub.3).sub.2), tris(dibenzylideneacetone) dipalladium(0) (Pd.sub.2(dba).sub.3), tetrakis(triphenylphosphine)palladium(0) (Pd(PPh.sub.3).sub.4), propylphosphonic anhydride (T3P), hexamethylphosphoramide (HMPA), 1,2-dichloroethane (DCE), benzyl (Bn) and 1-hydroxybenzotriazole (HOBt).

(5) In addition, TLC refers to thin layer chromatography.

(6) General Experimental Details

(7) Unless otherwise stated the following generalisations apply. .sup.1H NMR spectra were recorded on a Bruker Ultrashield Plus (400 MHz) or a Bruker AVANCE (400 MHz). The multiplicity of a signal is designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; tt, triplet of triplets; br, broad; m, multiplet. All observed coupling constants, J, are reported in Hertz.

(8) LCMS data was generated using either an Agilent 6100 Series Single Quad LCMS (LCMS-A), an Agilent 1260 Infinity Series UPLC/MS (LCMS-B), an Agilent 1200 Series G6110A Quadrupole LCMS or Waters 2695 alliance (LCMS-C). Chlorine isotopes are reported as .sup.35Cl, Bromine isotopes are reported as either .sup.79Br or .sup.81Br or both .sup.79Br/.sup.81Br.

(9) LCMS Method A (LCMS-A):

(10) Instrument: Agilent 6100 Series Single Quad LC/MS

(11) Agilent 1200 Series HPLC

(12) Pump: 1200 Series G1311A Quaternary pump

(13) Autosampler: 1200 Series G1329A Thermostatted Autosampler

(14) Detector: 1200 Series G1314B Variable Wavelength Detector

(15) LC conditions:

(16) Reverse Phase HPLC analysis

(17) Column: Luna C.sub.8 (2) 5 μm 50×4.6 mm 100 Å

(18) Column temperature: 30° C.

(19) Injection Volume: 5 μL

(20) Solvent A: Water 0.1% Formic Acid

(21) Solvent B: MeCN 0.1% Formic Acid

(22) Gradient: 5-100% solvent B over 10 min

(23) Detection: 254 nm or 214 nm

(24) MS conditions:

(25) Ion Source: Quadrupole

(26) Ion Mode: Multimode-ES

(27) Drying gas temp: 300° C.

(28) Vaporizer temperature: 200° C.

(29) Capillary voltage (V): 2000 (positive)

(30) Capillary voltage (V): 4000 (negative)

(31) Scan Range: 100-1000

(32) Step size: 0.1 sec

(33) Acquisition time: 10 min

(34) LCMS Method B (LCMS-B):

(35) Instrument: Agilent 1260 Infinity Series UPLC/MS

(36) Pump: 1260 Infinity G1312B Binary pump

(37) Autosampler: 1260 Infinity G1367E 1260 HiP ALS

(38) Detector: 1290 Infinity G4212A 1290 DAD

(39) LC conditions:

(40) Reverse Phase HPLC analysis

(41) Column: Poroshell 120 EC-C18 2.7 μm 50×3.0 mm

(42) Column temperature: 35° C.

(43) Injection Volume: 1 μL

(44) Solvent A: Water 0.1% Formic Acid

(45) Solvent B: MeCN 0.1% Formic Acid

(46) Gradient: 5-100% solvent B over 3.8 min

(47) Detection: monitored at 254 nm and 214 nm

(48) MS conditions:

(49) Ion Source: Quadrupole

(50) Ion Mode: API-ES

(51) Drying gas temp: 350° C.

(52) Capillary voltage (V): 3000 (positive)

(53) Capillary voltage (V): 3000 (negative)

(54) Scan Range: 100-1000

(55) Step size: 0.1 sec

(56) Acquisition time: 5 min

(57) LCMS Method C (LCMS-C):

(58) Instrument: Agilent 1200 Series G6110A Quadrupole

(59) Pump: Binary pump

(60) Detector: DAD

(61) LC conditions:

(62) Reverse Phase HPLC analysis

(63) Column: Xbridge-C18, 2.5 μm, 2.1×30 mm

(64) Column temperature: 30° C.

(65) Injection Volume: 1-10 μL

(66) Solvent A: Water 0.07% Formic acid

(67) Solvent B: Methanol

(68) Gradient: 30-95% solvent B over 3.5 min (for medium polarity samples) or 10-95% solvent B over 3.7 min (for large polarity samples)

(69) Detection: monitored at 254 nm and 214 nm

(70) MS conditions:

(71) Ion Source: Quadrupole

(72) Ion Mode: ES+

(73) Drying gas temp: 350° C.

(74) Drying gas flow: 10 L/min

(75) Nebulizer pressure: 35 psi

(76) Capillary voltage (V): 3500 (positive)

(77) Scan Range: 50-900

(78) Or

(79) Instrument: Waters 2695 alliance

(80) Pump: Quaternary Pump

(81) Detector: 2996 Photodiode Array Detector

(82) MS model: Micromass ZQ

(83) LC conditions:

(84) Column: Xbridge-C18, 3.5 μm, 2.1×50 mm

(85) Column temperature: 30° C.

(86) Injection volume: 1-10 μL

(87) Acquisition of wavelength: 214 nm, 254 nm

(88) Solvent A: 0.07% HCOOH aqueous solution

(89) Solvent B: MeOH

(90) Run time: 8 min

(91) Gradient: 20-95% solvent B over 5 min

(92) Detection: 254 nm and 214 nm

(93) MS condition:

(94) Ion source: ES+(or ES−) MS range: 50-900 m/z

(95) Capillary: 3 kV Cone: 3 V Extractor: 3 V

(96) Drying gas flow: 600 L/hr cone: 50 L/hr

(97) Desolvation temperature: 300° C.

(98) Source temperature: 100° C.

(99) Sample Preparation:

(100) The sample was dissolved in methanol, the concentration about 0.1-1.0 mg/mL, then filtered through the syringes filter with 0.22 μm.

(101) Preparative RP-HPLC:

(102) Agilent 1260 Infinity HPLC system

(103) UV detection at 210 nm and 254 nm

(104) Gradient or isocratic elution through a Phenomenex Luna C8 (2) column 100 Å Axia (250×21.2 mm; particle size 5 μm)

(105) Flow rate: 10 mL/min

(106) Gradients are as specified in the individual examples.

(107) Analytical thin-layer chromatography was performed on Merck silica gel 60 F254 aluminium-backed plates which were visualised using fluorescence quenching under UV light or a basic KMnO.sub.4 dip or Ninhydrin dip.

(108) Preparative thin-layer chromatography (prep TLC) was performed using Tklst (China), grand grade: (HPTLC): 8±2 μm>80%; (TLC): 10-40 μm. Type: GF254. Compounds were visualised by UV (254 nm).

(109) Flash chromatography was performed using a Biotage Isolera purification system using either Grace or RediSep® silica cartridges.

(110) Column chromatography was performed using Tklst (China), grand grade, 100-200 meshes silica gel.

(111) Microwave irradiation was achieved using a CEM Explorer SP Microwave Reactor.

(112) Where necessary, anhydrous solvents were purchased from Sigma-Aldrich or dried using conventional methods. Solutions of inorganic acids or bases where made up as aqueous solutions unless stated otherwise.

(113) Additional Cartridges used are as follows:

(114) Phase Separator:

(115) Manufacturer: Biotage

(116) Product: ISOLUTE Phase Separator (3 mL unless otherwise stated)

(117) SCX and SCX-2 Cartridges:

(118) Manufacturer: Biotage

(119) Product: ISOLUTE SCX 1 g, (6 mL SPE Column unless otherwise stated)

(120) Manufacturer: Biotage

(121) Product: ISOLUTE SCX-2 1 g (6 mL Column)

(122) Manufacturer: Silicycle

(123) Product: SCX-2 500 mg or 5 g

(124) Manufacturer: Agilent

(125) Product: Bond Elute SCX 10 g

(126) Sample Extraction Cartridge:

(127) Manufacturer: Waters

(128) Product: Oasis HLB 35 cc (6 g) LP extraction cartridge

Intermediate Preparations

(i) (R)-tert-Butyl(3-amino-2-hydroxypropyl)(2,3-dihydro-1H-inden-2-yl)carbamate (I2)

(129) ##STR00036##

(a) (R)-tert-Butyl(2,3-dihydro-1H-inden-2-yl)(3-(1,3-dioxoisoindolin-2-yl)-2-hydroxypropyl)carbamate (I1)

(130) A mixture of 2,3-dihydro-1H-inden-2-amine (5.0 g, 24.61 mmol) and (R)-2-(oxiran-2-ylmethyl)isoindoline-1,3-dione (4.91 g, 36.91 mmol) in EtOH (100 mL) was heated at reflux overnight. After cooling to room temperature, triethylamine (9.94 g, 98.44 mmol) and (Boc).sub.2O (10.74 g, 49.22 mmol) were added and the reaction mixture was stirred at room temperature overnight. The mixture was concentrated and purified by column (EtOAc/petroleum ether=¼, v/v) to give the title compound (5.4 g, 50%) as an oil.

(b) (R)-tert-Butyl(3-amino-2-hydroxypropyl)(2,3-dihydro-1H-inden-2-yl)carbamate (I2)

(131) A mixture of (R)-tert-butyl(2,3-dihydro-1H-inden-2-yl)(3-(1,3-dioxoisoindolin-2-yl)-2-hydroxypropyl)carbamate I1 (4.4 g, 10.08 mmol) and NH.sub.2NH.sub.2.H.sub.2O (80%, 1.89 g, 30.24 mmol) in EtOH (100 mL) was heated at reflux for 4 hours. After cooling to room temperature, the mixture was filtered and the filtercake was washed with EtOH (50 mL). The filtrate was concentrated and then dissolved in CH.sub.2Cl.sub.2, washed with saturated aqueous NaHCO.sub.3 solution (80 mL×3), brine (20 mL×2), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound (3.1 g, crude) as a yellow oil, which was used for the next step without further purification. LCMS: RT 2.17 min; m/z 307.2 [M+H].sup.+.

(ii) (R)-4((3-((tert-Butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoic acid

(132) ##STR00037##

(a) (R)-Methyl 4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoate (I3)

(133) To a solution of (R)-tert-butyl (3-amino-2-hydroxypropyl)(2,3-dihydro-1H-inden-2-yl) carbamate I2 (300 mg, 0.98 mmol) and 2-ethoxy-4-(methoxycarbonyl)benzoic acid (220 mg, 0.98 mmol) in DCM (10 mL) was added DIPEA (697 μL, 3.92 mmol), EDCl (387 mg, 1.96 mmol) and HOBt (14 mg, 0.10 mmol). The reaction was stirred at room temperature overnight, saturated aqueous NaHCO.sub.3 solution (50 mL) was added, and the aqueous phase was extracted with DCM (3×50 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (petroleum ether:EtOAc=3:1) to give the desired product (370 mg, 74%) as a white solid. LCMS RT 3.23 min; m/z 513.3 [M+H].sup.+

(b) (R)-4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoic acid (I4)

(134) To a solution of (R)-methyl 4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoate I3 (370 mg, 0.72 mmol) in a mixture of THF (10 mL), MeOH (3 mL) and H.sub.2O (1 mL) was added LiOH.H.sub.2O (91 mg, 2.16 mmol). The reaction was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and the residue obtained suspended in water (15 mL). The mixture was acidified to pH 4 with 1 M aqueous HCl and the aqueous phase extracted with DCM (3×30 mL). The pooled organic extracts were dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired product (370 mg, 100%) as an off-white solid. LCMS RT 3.09 min; m/z 499.3 [M+H]+

(iii) tert-Butyl (S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I9)

(135) ##STR00038##

(136) (a) (S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (I5) (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (5.00 g, 28.2 mmol) was vigorously stirred in 1,4-dioxane (100 mL) and water (50 mL). Sodium bicarbonate (4.74 mg, 56.4 mmol) and Boc anhydride (6.77 g, 31.0 mmol) were added and the mixture was stirred vigorously at room temperature. After 17 hours the mixture was concentrated in vacuo and the residue dissolved in water (200 mL). A 30% w/v aqueous solution of sodium hydrogen sulfate monohydrate (30 mL) was added and the mixture extracted with chloroform (3×200 mL). The pooled organic extracts were washed with brine, dried over sodium sulfate and concentrated in vacuo to give the desired compound (7.50 g, 96% yield) as a thick syrup. LCMS-B: RT 3.64 min; m/z 178.1 [M-Boc+2H].sup.+; m/z 276.1 [M−H].sup.−

(b) tert-Butyl (S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6)

(137) (S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (I5) (7.50 g, 27.0 mmol) was dissolved in THF (150 mL) and CDI (8.77 g, 54.1 mmol) was added. The mixture was stirred for 30 minutes at room temperature then cooled to 0° C. A solution of sodium borohydride (1.16 g, 30.5 mmol) in water (15 mL) was added dropwise. After 40 minutes the mixture was quenched with acetone (25 mL) and concentrated in vacuo. The residue was partitioned between water (250 mL) and ethyl acetate (200 mL). The separated aqueous phase was extracted with ethyl acetate (2×250 mL), the combined organic extracts washed with 5% w/v aqueous NaHSO.sub.4 (250 mL), brine (200 mL), dried over sodium sulfate and concentrated in vacuo. The residue was loaded in diethyl ether (50 mL) onto a plug of basic alumina and silica (50 mL each). The plug was eluted with diethyl ether (250 mL) and the eluate evaporated to give the desired compound (5.93 g, 83% yield) as a colourless syrup. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.25-7.06 (m, 4H), 4.82-4.59 (m, 1H), 4.57-4.38 (m, 1H), 4.37-4.19 (m, 1H), 3.57-3.40 (m, overlaps with trace solvent), 3.03 (dd, J=16.1, 5.7 Hz, 1H), 2.80 (d, J=16.1 Hz, 1H), 1.50 (s, 9H). LCMS-B: RT 3.66 min; m/z 164.2 [M-Boc+2H].sup.+, 286.2 [M+Na].sup.+

(c) tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7)

(138) tert-Butyl (S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6) (1.50 g, 5.70 mmol), DCM (25 mL) and DMSO (5 mL) were cooled to 0° C. Triethylamine (2.38 mL, 17.1 mmol) was added, followed by pyridine-sulfur trioxide complex (2.72 g, 17.1 mmol). The mixture was stirred at 0° C. for 10 minutes then allowed to come to room temperature. After 2 hours, saturated sodium bicarbonate (75 mL) and water (75 mL) were added, and the mixture extracted with diethyl ether (3×150 mL). The pooled ether extracts were washed with 1:1 water: saturated aqueous NH.sub.4Cl (200 mL), brine (200 mL), dried over sodium sulfate, filtered and concentrated in vacuo to give the desired compound as a colourless oil which was used without further purification.

(d) tert-Butyl (S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I8A) and tert-butyl (S)-3-((S)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I8B)

(139) A solution of tert-butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7) (5.70 mmol @ 100% conversion) in i-propanol (50 mL) was cooled to 0° C. Nitromethane (1.22 mL, 22.8 mmol) and potassium fluoride (331 mg, 5.70 mmol) were added and the mixture stirred for 18 hours, allowing the temperature to come to room temperature as the ice bath thawed. The mixture was diluted with water (200 mL) and extracted with DCM (3×200 mL). The pooled DCM extracts were washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography (40 g silica cartridge, 0-20% ethyl acetate/hexanes) gave two partly overlapping peaks, which were split into early (8A major, colourless syrup, 697 mg, 37% yield) and late (8B minor, colourless syrup, 170 mg, 9% yield) fractions. Overall: 867 mg, 47% yield.

(140) Data for major isomer tert-butyl (S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate I8A:

(141) .sup.1H NMR (400 MHz, d.sub.4-MeOD) δ 7.25-7.14 (m, 4H), 4.85-4.49 (m, 5H), 4.44 (dd, J=12.6, 9.3 Hz, 1H), 4.37-3.99 (m, overlaps with solvent), 3.19 (dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd, J=15.8, 5.6 Hz, 1H), 1.51 (s, 9H). LCMS-B: RT 3.71 min; m/z 223.2 [M-Boc+2H].sup.+, 345.2 [M+Na].sup.+; m/z 321.2 [M−H].sup.−

(142) Data for minor isomer tert-butyl (S)-3-((S)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate I8B:

(143) .sup.1H NMR (400 MHz, d.sub.4-MeOD) δ 7.25-7.11 (m, 4H), 4.75 (d, J=16.5 Hz, 1H), 4.68-4.48 (m, 4H), 4.42-4.23 (m, overlaps with residual nitromethane), 3.06 (dd, J=16.3, 6.1 Hz, 1H), 2.91 (d, J=16.1 Hz, 1H), 1.50 (s, 9H). LCMS-B: RT 3.70 min; m/z 223.2 [M-Boc+2H].sup.+, 345.2 [M+Na].sup.+; m/z 321.2 [M−H].sup.−

(e) tert-Butyl (S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I8A)

(144) Copper catalyst used:

(145) ##STR00039##

(146) tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7) (1.9 mmol @100% conversion), absolute ethanol (5 mL), nitromethane (1.02 mL, 19.0 mmol) and the copper catalyst (91 mg, 10 mol %) (see above FIGURE, prepared according to Tetrahedron:

(147) Asymmetry (2008) 2310-2315) were stirred at room temperature. After 90 hours the mixture was concentrated in vacuo, chromatography (40 g silica cartridge, 0-15% ethyl acetate/hexanes) gave the desired compound (352 mg, 58% yield over two steps). .sup.1H NMR (400 MHz, d.sub.4-MeOD) δ 7.25-7.13 (m, 4H), 4.85-4.68 (m, 1H), 4.65-4.49 (m, 1H), 4.49-4.39 (m, 1H), 4.36-3.96 (m, overlaps with trace solvent), 3.19 (dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd, J=15.9, 5.6 Hz, 1H), 1.51 (s, 9H). LCMS-B: RT 3.25 min; m/z 321.1 [M−H].sup.−

(f) tert-Butyl (S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I9)

(148) tert-Butyl (S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I8A) (1.54 g, 4.78 mmol), absolute ethanol (75 mL) and 10% Pd/C (53% wetted with water, 1.5 g) were stirred under hydrogen (balloon). After 3 hours the mixture was filtered through celite, the celite was washed with absolute ethanol (100 mL) and the combined filtrates concentrated in vacuo to give the desired compound (1.34 g, 96% yield) as a pale grey-green syrup. LCMS-B: RT 3.27 min, m/z 293.2 [M+H].sup.+

(149) Alternate Synthesis Method

(a) (S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (I5)

(150) (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (50.0 g, 282 mmol) was vigorously stirred in a mixture of 1,4-dioxane (1000 mL) and water (500 mL). Sodium bicarbonate (47.4 g, 564 mmol) and Boc anhydride (67.7 g, 310 mmol) were added and the reaction was stirred vigorously at room temperature for 6 days. The mixture was concentrated in vacuo and the residue dissolved in water (2000 mL). A 30% w/v aqueous solution of sodium hydrogen sulfate monohydrate (300 mL) was added and the mixture extracted with chloroform (3×1000 mL). The pooled organic extracts were washed with brine, dried over sodium sulfate and concentrated in vacuo to give the desired compound (90.0 g, quantitative) as a thick syrup. LCMS-B: RT 3.64 min; m/z 178.1 [M-Boc+2H].sup.+; m/z 276.1 [M−H].sup.−

(b) tert-Butyl (S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6)

(151) (S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (I5) (54.0 g, 195 mmol) was dissolved in THF (1000 mL) and CDI (63.2 g, 390 mmol) was added. The mixture was stirred for 2 hours at 30° C. then cooled to 0° C. A solution of sodium borohydride (14.7 g, 390 mmol) in water (120 mL) was added dropwise. After 3 hours the mixture was quenched with acetone (300 mL) and concentrated in vacuo. The residue was partitioned between water (1000 mL) and ethyl acetate (1000 mL). The separated aqueous phase was extracted with ethyl acetate (4×500 mL) and the combined organic extracts washed with 5% w/v aqueous NaHSO.sub.4 (1000 mL), brine (500 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by chromatography (5-20% ethyl acetate/petroleum ether) to give the desired compound (30.4 g, 59% yield) as a yellow syrup. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.25-7.06 (m, 4H), 4.82-4.59 (m, 1H), 4.57-4.38 (m, 1H), 4.37-4.19 (m, 1H), 3.57-3.40 (m, overlaps with trace solvent), 3.03 (dd, J=16.1, 5.7 Hz, 1H), 2.80 (d, J=16.1 Hz, 1H), 1.50 (s, 9H). LCMS-B: RT 3.66 min; m/z 164.2 [M-Boc+2H].sup.+, 286.2 [M+Na].sup.+

(c) tert-Butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7)

(152) tert-Butyl (S)-3-(hydroxymethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I6) (16 g, 0.06 mol), DCM (250 mL) and DMSO (75 mL) were cooled to 0° C. Triethylamine (25.1 mL, 0.18 mol) was added, followed by pyridine-sulfur trioxide complex (28.6 g, 0.18 mol). The mixture was stirred at 0° C. for 30 minutes then allowed to come to room temperature and stirred at room temperature overnight. Saturated sodium bicarbonate (200 mL) and water (200 mL) were added, and the mixture extracted with diethyl ether (3×300 mL). The pooled ether extracts were washed with 1:1 water: saturated aqueous NH.sub.4Cl (200 mL), dried over sodium sulfate and concentrated in vacuo to give the desired compound (16.0 g) as an orange oil which was used without further purification.

(e) tert-Butyl (S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I8A)

(153) To a solution of tert-butyl (S)-3-formyl-3,4-dihydroisoquinoline-2(1H)-carboxylate (I7) (0.06 mol @100% conversion) in absolute ethanol (50 mL) was added a solution of the copper catalyst (6.8 g, 20 mol %) (see above FIGURE, prepared according to Tetrahedron: Asymmetry (2008) 2310-2315) in absolute ethanol (10 mL). The mixture was cooled to 0° C. and nitromethane (36.0 g, 0.6 mol) was added. The reaction was stirred at 0° C. for 3 days, the mixture was concentrated in vacuo and purified by chromatography (5% ethyl acetate/petroleum ether) to give the desired compound (7.5 g, 39% yield over two steps). .sup.1H NMR (400 MHz, d.sub.4-MeOD) δ 7.25-7.13 (m, 4H), 4.85-4.68 (m, 1H), 4.65-4.49 (m, 1H), 4.49-4.39 (m, 1H), 4.36-3.96 (m, overlaps with trace solvent), 3.19 (dd, J=15.9, 3.2 Hz, 1H), 2.92 (dd, J=15.9, 5.6 Hz, 1H), 1.51 (s, 9H). LCMS-B: RT 3.25 min; m/z 321.1 [M−H].sup.−

(f) tert-Butyl (S)-3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I9)

(154) To a solution of tert-butyl (S)-3-((R)-1-hydroxy-2-nitroethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I8A) (7.5 g, 23.3 mmol) in absolute ethanol (100 mL) was added 10% Pd/C (7.5 g) and the reaction was stirred under an atmosphere of hydrogen. After 3 hours, the mixture was filtered through Celite, the Celite was washed with absolute ethanol (200 mL) and the combined filtrates concentrated in vacuo to give the desired compound (5.3 g, 78% yield) as a pale grey solid. LCMS-B: RT 3.27 min, m/z 293.2 [M+H].sup.+

(iv) 4-(((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl)carbamoyl)-3-ethoxybenzoic acid (I16)

(155) ##STR00040##

(a) Ethyl-4-bromo-2-ethoxybenzoate (I10)

(156) To a mixture of 4-bromo-2-hydroxybenzoic acid (20.0 g, 92.6 mmol) and K.sub.2CO.sub.3 (38.4 g, 278 mmol) in dimethylsulfoxide (50 mL) at 40° C. was added ethyl bromide (15.2 g, 139 mmol) dropwise over 30 minutes, the reaction mixture was stirred for 2 hours then further ethyl bromide (15.2 g, 139 mmol) was added over 30 minutes. The reaction was stirred a further 8 hours then diluted with CH.sub.2Cl.sub.2 (150 mL) and filtered. The filtrate was washed with water (200 mL×10), brine (200 mL×3), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound as a brown solid (24.3 g, 96%): LCMS: RT 2.93 min; m/z 273.0 [M+H].sup.+.

(b) 4-Bromo-2-ethoxybenzoic acid (I11)

(157) To a solution of ethyl 4-bromo-2-ethoxybenzoate I10 (24.1 g, 88.6 mmol) in a mixture of THF (150 mL), methanol (15 mL) and water (15 mL) was added LiOH H.sub.2O (18.6 g, 44.3 mmol). The resulting mixture was stirred at room temperature for 24 hours. The solvent was removed, and the residue diluted with water (200 mL). The pH of the aqueous mixture was adjusted to 6 by addition of 2 M aqueous HCl. The aqueous mixture was extracted with CH.sub.2Cl.sub.2 (150 mL×3) and the combined organic layers washed with brine (100 mL×3), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound as a yellow solid (19.8 g, 92%): LCMS: RT 2.47 min; m/z, 244.9/246.9 [M+H].sup.+.

(c) Benzyl-4-bromo-2-ethoxybenzoate (I12)

(158) To a solution of 4-bromo-2-ethoxybenzoic acid I11 (16.4 g, 67.2 mmol) in acetonitrile (75 mL) was added benzyl bromide (13.8 g, 80.7 mmol) and K.sub.2CO.sub.3 (18.6 g, 134.4 mmol). The resulting mixture was stirred at 40° C. overnight. The reaction mixture was filtered and concentrated. The residue was diluted with CH.sub.2Cl.sub.2 (50 mL) and washed with water (100 mL×2), brine (70 mL×3), dried (Na.sub.2SO.sub.4), filtered and concentrated. The crude product was purified by column chromatography (1% EtOAc in petroleum ether) to give the title compound as a light-yellow oil (20.9 g, 93%): LCMS: RT 3.31 min; m/z 334.9 [M+H].sup.+.

(d) 1-Benzyl 4-methyl 2-ethoxyterephthalate (I13)

(159) To a solution of benzyl 4-bromo-2-ethoxybenzoate I12 (20.0 g, 59.9 mmol) in methanol (100 mL) was added Pd(dppf)Cl.sub.2 (2.2 g, 3 mmol) and triethylamine (13.3 g, 131.7 mmol). The resulting mixture was heated at reflux overnight under an atmosphere of carbon monoxide. The reaction mixture was concentrated and the residue obtained was diluted with water and was extracted with CH.sub.2Cl.sub.2 (50 mL×3). The combined organic layers were washed with brine (60 mL×2), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (2% EtOAc in petroleum ether) to give the title compound as a white solid (15.1 g, 81%): LCMS: RT 3.03 min; m/z 315.1 [M+H].sup.+.

(e) 2-Ethoxy-4-(methoxycarbonyl)benzoic acid (I14)

(160) To a solution of 1-benzyl 4-methyl 2-ethoxyterephthalate I13 (14.9 g, 47.4 mmol) in THF (100 mL) was added 10% Pd/C (1.5 g). The resulting mixture was stirred at room temperature overnight under H2. The catalyst was removed by filtration through Celite and the filter cake washed with THF (50 mL). The filtrate was concentrated and the residue was diluted with water. The pH of the aqueous solution was adjusted to 6 by addition of 2 M aqueous HCl and the resultant mixture extracted with CH.sub.2Cl.sub.2 (50 mL×3). The combined organic layers were washed with brine (50 mL×2), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound as a white solid (10.2 g, 96%): LCMS: RT 2.13 min; m/z 225.0 [M+H].sup.+.

(f) (S)-tert-Butyl-3-((R)-2-(2-ethoxy-4-(methoxycarbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (I15)

(161) To a solution of 2-ethoxy-4-(methoxycarbonyl)benzoic acid I14 (1.7 g, 7.5 mmol) in CH.sub.2Cl.sub.2 (50 mL) were added (S)-tert-butyl 3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate I9 (2.0 g, 6.8 mmol), HOBt (100 mg, 0.7 mmol), DIPEA (3.9 g, 20.4 mmol) and EDCl (2.6 g, 13.6 mmol). The resulting mixture was stirred at room temperature overnight. The mixture was diluted with water (80 mL) and extracted with CH.sub.2Cl.sub.2 (80 mL×3). The combined organic layers were washed with water (150 mL), brine (150 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (33% EtOAc in petroleum ether) to give the title compound as a yellow solid (2.2 g, 65%):

(162) LCMS: RT 3.12 min; m/z 499.2 [M+H].sup.+.

(g) 4-(((R)-2-((S)-2-(tert-Butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyhcarbamoyl)-3-ethoxybenzoic acid (I16)

(163) To a solution of (S)-tert-butyl-3-((R)-2-(2-ethoxy-4-(methoxycarbonyhbenzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate 15 (2.1 g, 4.2 mmol) in methanol (4 mL) was added a solution of NaOH (340 mg, 8.4 mmol) in water (6 mL). The resulting mixture was stirred at room temperature overnight. The solvent was removed, and the residue diluted with water (30 mL). The pH of the aqueous mixture was adjusted to 5 by addition of 2 M aqueous HCl solution. The aqueous layer was then extracted with CH.sub.2Cl.sub.2 (50 mL×4) and the combined organic layers washed with brine, dried (Na.sub.2SO.sub.4) and concentrated to give the title compound as a yellow solid (1.9 g, 95%): LCMS: RT 2.95 min; m/z 485.2 [M+H].sup.+.

(v) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acid I20

(164) ##STR00041##

(a) Ethyl-2-ethoxy-4-methylbenzoate I17

(165) To a mixture of 2-hydroxy-4-methylbenzoic acid (8.2 g, 53.9 mmol) and K.sub.2CO.sub.3 (22.4 g, 161.7 mmol) in dimethylsulfoxide (70 mL) at 40° C. was added ethyl iodide (12.6 g, 80.8 mmol) dropwise over a period of 30 min. The reaction was stirred for 2 hours then further ethyl iodide (12.6 g, 80.8 mmol) was added over 30 min. The resulting mixture was stirred a further 8 hours at 40° C., diluted with CH.sub.2Cl.sub.2 (150 mL) and filtered. The filtrate was washed with water (200 mL×10) and brine (200 mL×2), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired compound as a yellow liquid (10.1 g, 90%): LCMS: RT 2.70 min; m/z 209.1 [M+H].sup.+.

(b) 3-Ethoxy-4-(ethoxycarbonyl)-benzoic acid I18

(166) To a solution of ethyl 2-ethoxy-4-methylbenzoate I17 (10.0 g, 48.1 mmol) in a mixture of pyridine (25 mL) and water (75 mL) was added KMnO.sub.4 (22.8 g, 144.2 mmol). The resulting mixture was heated at 50° C. for 48 hours, then cooled and allowed to stir at room temperature for 24 hours. The mixture was filtered and the filter cake washed with hot water. The combined aqueous filtrates were washed with EtOAc (75 mL×3) and acidified with 2M aqueous HCl solution. The mixture was extracted with CH.sub.2Cl.sub.2 (150 mL×3). The combined organic layers were washed with brine, dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired compound as a white solid (5.0 g, 44%): LCMS: RT 0.25 min; m/z 239.0 [M+H].sup.+.

(c) Ethyl 4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoate I19

(167) To a solution of 3-ethoxy-4-(ethoxycarbonyl)benzoic acid I18 (2.5 g, 10.4 mmol) in CH.sub.2Cl.sub.2 (20 mL) was added 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (1.42 g, 9.5 mmol), HOBt (135.1 mg, 1.0 mmol), DIPEA (2.5 g, 19.0 mmol) and EDCl (2.2 g, 11.4 mmol). The resulting mixture was stirred at room temperature overnight. The mixture was partitioned with saturated aqueous NaHCO.sub.3, and extracted with CH.sub.2Cl.sub.2 (20 mL×2). The combined organic layers were washed with brine, dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (1% methanol in dichloromethane) to give the desired compound as a yellow oil (2.5 g, 80%): LCMS: RT 2.40 min; m/z 334.1 [M+H].sup.+.

(d) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acid I20

(168) To a solution of ethyl-4-(3-oxa-8-azabicyclo-[3.2.1]-octane-8-carbonyl)-2-ethoxybenzoate I19 (2.4 g, 7.2 mmol) in a mixture of THF (20 mL), methanol (2 mL) and water (2 mL) was added LiOH H.sub.2O (1.5 g, 36 mmol). The resulting mixture was stirred at room temperature for 24 h. The solvent was removed, and the residue obtained diluted with water (20 mL). The pH of the aqueous mixture was adjusted to 6 by addition of a 2 M aqueous HCl solution. The mixture was extracted with CH.sub.2Cl.sub.2 (20 mL×3) and the combined organic layers washed with brine (10 mL×2), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired compound as a yellow oil (1.7 g, 79%): .sup.1H NMR (400 MHz, d.sub.4-MeOD) δ 7.83 (d, J=7.8 Hz, 1H), 7.19 (d, J=1.0 Hz, 1H), 7.10 (dd, J=7.8, 1.3 Hz, 1H), 4.65 (br s, 1H), 4.20 (q, J=7.0 Hz, 2H), 3.97 (br s, 1H), 3.82 (d, J=10.8 Hz, 1H), 3.72 (d, J=11.0 Hz, 2H), 3.59 (d, J=10.9 Hz, 1H), 2.13-1.94 (m, 4H), 1.45 (t, J=7.0 Hz, 3H). LCMS: RT 1.20 min; m/z 306.1 [M+H].sup.+.

EXAMPLES

Example 1: 2-Ethoxy-N—((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamide hydrochloride (1)

(169) ##STR00042##

(a) 2-(2,2-Diethoxyethoxy)-1,1-diethoxyethane (A1)

(170) To a suspension of NaH (60% dispersion in mineral oil, 3.8 g, 95.0 mmol) in xylene (100 mL) was added 2,2-diethoxyethanol (11.6 g, 86.4 mmol) under N.sub.2. The mixture heated at 110° C. for 2 hours, then cooled to room temperature and 2-bromo-1,1-diethoxyethane (25.6 g, 130.0 mmol) added. The reaction was heated at 110° C. overnight, then cooled to room temperature and the organic layer washed with water, dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue obtained was purified by distillation to give the title compound (7 g, 33%) as colorless oil.

(b) 9-Benzyl-3-oxa-9-azabicyclo[3.3.1]nonan-7-one (A2)

(171) 2-(2,2-Diethoxyethoxy)-1,1-diethoxyethane A1 (5 g, 20.0 mmol) in a mixture of acetic acid (1.2 mL) and water (5 mL) was heated at reflux for 1 hour then cooled to room temperature. Benzylamine hydrochloride (2.1 g, 20.0 mmoL) and 3-oxopentanedioic acid (2.5 g, 16.6 mmoL), NaOAc (0.69 g, 83.8 mmoL) and water (10 mL) were added and the reaction heated at 50° C. for 5 hours. The reaction was cooled to room temperature and aqueous NaOH solution (42 mL, 50% w/v) was added into the mixture. The aqueous phase was extracted with EtOAc (3×30 mL) and the combined organic fractions concentrated and purified by column chromatography (100% petroleum ether to 10% EtOAc in petroleum ether) to give the title compound as a yellow solid. LCMS: RT 0.50 min, m/z 232.1 [M+H].sup.+

(c) tert-Butyl 7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate (A3)

(172) A mixture of 9-benzyl-3-oxa-9-azabicyclo[3.3.1]nonan-7-one A2 (425 mg, 2.2 mmol), (Boc).sub.2O (1.3 g, 6.5 mmol) and 10% Pd/C (50 mg) in MeOH (10 mL) was stirred vigorously under an atmosphere of H2 gas overnight. The catalyst was removed by filtration through celite and the filtrate was concentrated. The residue obtained was purified by column chromatography (100% petroleum ether to 10% EtOAc in petroleum ether) to give the title compound as a yellow solid (250 mg, 57%). LCMS: RT 0.53 min, m/z 264.1 [M+Na].sup.+.

(d) 3-Oxa-9-azabicyclo[3.3.1]nonan-7-one hydrochloride (A4)

(173) A solution of tert-butyl 7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carboxylate A3 (160 mg, 0.66 mmol) in HCl in Et.sub.2O (3 M solution, 3 mL) was stirred at room temperature for 2-3 hours. The mixture was concentrated and the crude product obtained washed with ether to give the desired product as a yellow solid (120 mg, 100%). LCMS: RT 0.24 min, m/z 142.1 [M+H].sup.+.

(e) (S)-tert-Butyl-3-((R)-2-(2-ethoxy-4-(7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (A5)

(174) To a solution of -oxa-9-azabicyclo[3.3.1]nonan-7-one hydrochloride A4 (80 mg, 0.37 mmol) and 4-(((R)-2-((S)-2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinolin-3-yl)-2-hydroxyethyl) carbamoyl)-3-ethoxybenzoic acid I16 (151 mg, 0.31 mmol) in DCM (10 mL) was added EDCl (107 mg, 0.56 mmol), HOBt (76 mg, 0.56 mmol) and DIPEA (145 mg, 1.12 mmol). The reaction was stirred at room temperature for 3 hours, then concentrated. The residue obtained was purified by column chromatography (100% DCM to 10% MeOH in DCM) to give the title compound as a yellow solid (150 mg, 79%). LCMS: RT 2.74 min, m/z 608.3 [M+H].sup.+.

(f) (3S)-tert-Butyl 3-((1R)-2-(2-ethoxy-4-(7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (A6)

(175) A mixture of (S)-tert-butyl 3-((R)-2-(2-ethoxy-4-(7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate A5 (110 mg, 0.18 mmol) in THF (5 mL) was added NaBH.sub.4(34 mg, 0.90 mmol) and the reaction stirred at room temperature for 3 hours. The mixture was concentrated and the residue obtained purified by column chromatography (100% DCM to 10% MeOH in DCM) to give the title compound as a white solid (70 mg, 63%). LCMS: RT 2.78 min, m/z 632.3 [M+Na].sup.+. The relative stereochemistry of the bicyclic alcohol was not determined.

(g) 2-Ethoxy-N—((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-4-(7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamide hydrochloride (1)

(176) A solution of (3S)-tert-butyl 3-((1R)-2-(2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1] nonane-9-carbonyl)benzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate A6 (50 mg, 0.082 mmol) in a solution of HCl in Et.sub.2O (3M, 5 mL) was stirred at room temperature for 2-3 h. The mixture was concentrated and the crude product washed with diethyl ether to give the desired product as a white solid (30 mg, 90%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.97 (d, J=8.0 Hz, 1H), 7.31-7.22 (m, 4H), 7.19 (d, J=0.8 Hz, 1H), 7.11 (dd, J=7.6 Hz, 1.2 Hz, 1H), 4.68 (br s, 1H), 4.43 (q, J=7.2 Hz, 2H), 4.27-4.22 (m, 3H), 4.0-4.97 (m, 2H), 3.89-3.85 (m, 3H), 3.76-3.70 (m, 2H), 3.66-3.59 (m, 2H), 3.27 (br s, 1H), 3.22-3.17 (m, 1H), 2.39-2.33 (m, 1H), 2.23-2.18 (m, 1H), 1.96 (m, 1H), 1.82-1.78 (m, 1H), 1.43 (t, J=7.2 Hz, 3H); LCMS: RT 3.25 min, m/z 510.3 [M+H].sup.+.

Example 2: N—((S)-3-((2,3-Dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamide (2)

(177) ##STR00043##

(a) tert-Butyl (2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamate (A7)

(178) To a solution of (R)-4-((3-((tert-butoxycarbonyl)(2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)carbamoyl)-3-ethoxybenzoic acid I4 (200 mg, 0.40 mmol) and 3-oxa-9-azabicyclo[3.3.1]nonan-7-one hydrochloride A4 (76 mg, 0.44 mmol) in DCM (5 mL) was added DIPEA (0.356 mL, 2.00 mmol), EDCl (153 mg, 0.81 mmol) and HOBt (5 mg, 0.07 mmol). The resultant mixture was stirred at room temperature overnight. Saturated aqueous NaHCO.sub.3 (30 mL) was added, and the mixture extracted with DCM (3×30 mL). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue obtained was purified by preparative TLC (DCM:MeOH=10:1) to give the desired product (60 mg, 24%) as a grey solid. LCMS RT 2.85 min; m/z 622.3 [M+H].sup.+

(b) tert-Butyl (2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamate (A8)

(179) To a solution of tert-butyl (2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-(7-oxo-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamate A7 (60 mg, 0.10 mmol) in MeOH (5 mL) was added NaBH.sub.4(11 mg, 0.30 mmol) and the reaction stirred at room temperature for 3 hours. The solvent was removed and the residue purified by preparative TLC (DCM:MeOH=10:1) to give the title compound (50 mg, 81%) as a white solid. LCMS: RT 1.31 min; m/z 524.3 [M-Boc+2H].sup.+.

(c) N—((S)-3-((2,3-Dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-2-ethoxy-4-(7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamide (2)

(180) tert-Butyl (2,3-dihydro-1H-inden-2-yl)((R)-3-(2-ethoxy-4-(−7-hydroxy-3-oxa-9-azabicyclo[3.3.1]nonane-9-carbonyl)benzamido)-2-hydroxypropyl)carbamate A8 (50 mg, 0.08 mmol) was stirred in a solution of 2.0 M solution of HCl in Et.sub.2O at room temperature for 2-3 hours. Saturated aqueous NaHCO.sub.3 (30 mL) was added, and the mixture was extracted with DCM (3×30 mL). The combined organic layers were dried (Na.sub.2SO.sub.4), filtered and concentrated. The reaction was repeated on another 60 mg of starting material and the combined crude products were purified by preparative TLC (DCM:MeOH=10:1) provide the desired product (30 mg, 30%) as a white solid. .sup.1H NMR (400 MHz, MeOD) δ 8.01 (d, J=8.0 Hz, 1H), 7.24-7.11 (m, 6H), 4.66 (m, 2H), 4.29 (q, J=6.8 Hz, 2H), 4.06-3.76 (m, 7H), 3.63-3.59 (m, 1H), 3.54-3.49 (m, 1H), 3.37-3.33 (m, 2H), 3.09-2.90 (m, 4H), 2.40-2.34 (m, 1H), 2.25-2.18 (m, 1H), 2.04-1.95 (m, 1H), 1.83-1.79 (m, 1H), 1.53 (t, J=6.8 Hz, 3H); LCMS: RT 1.26 min; m/z 524.3 [M+H].sup.+.

Example 3: 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methylbenzamide hydrochloride (3)

(181) ##STR00044##

(a) (S)-tert-Butyl 3-((R)-2-(benzylamino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (A9)

(182) To a solution of (S)-tert-butyl 3-((R)-2-amino-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate I9 (200 mg, 0.68 mmol) in MeOH (2 mL) was added benzaldehyde (80 mg, 0.75 mmol). The mixture was stirred at room temperature for 2 hours then NaBH.sub.4 (85 mg, 1.36 mmol) was added in portions. The mixture was stirred at room temperature for 2 hours then the solvent was removed and the residue dissolved in water (20 mL). The solution was adjusted to pH 10 by addition of 1.0 M aqueous NaOH solution and the aqueous layer extracted with DCM (3×20 mL). The combined organic extracts were washed with brine (30 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (2% MeOH in DCM) to give the title compound as a yellow oil (120 mg, 46%). LCMS: RT 2.33 min; m/z 383.2 [M+H].sup.+.

(b) (S)-tert-Butyl 3-((R)-2-(benzyl(methyl)amino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (A10)

(183) To a solution of (S)-tert-butyl 3-((R)-2-(benzylamino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate A9 (120 mg, 0.31 mmol) in CH.sub.3OH (2 mL) was added 37% aqueous formaldehyde (150 μL, 1.55 mmol), NaBH.sub.3CN (40 mg, 0.62 mmol) and AcOH (1 drop) and the reaction stirred at room temperature overnight. The solvent was removed and the residue dissolved in water (10 mL). The pH was adjusted to 10 by addition of 0.5 M aqueous NaOH and the aqueous extracted with EtOAc (3×20 mL). The combined organic fractions were dried (Na.sub.2SO.sub.4) and concentrated. The residue was purified by preparative TLC (5% MeOH in DCM) to give the title compound as yellow oil (90 mg, 73%). LCMS: RT 2.23 min; m/z 397.2 [M+H].sup.+.

(c) (S)-tert-Butyl 3-((R)-1-hydroxy-2-(methylamino)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (A11)

(184) A mixture of (S)-tert-butyl 3-((R)-2-(benzyl(methyl)amino)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate A10 (80 mg, 0.20 mmol) and Pd(OH).sub.2 (14 mg, 0.1 mmol) in EtOAc (2 mL) was stirred at 45° C. under a hydrogen atmosphere overnight. The catalyst was removed by filtration through Celite and the filtrate was concentrated. The residue obtained was purified by preparative TLC (7% MeOH/DCM) to give the title compound as a yellow oil (40 mg, 65%). LCMS: RT 2.19 min; m/z 307.2 [M+H].sup.+

(d) (3S)-tert-Butyl 3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N-methylbenzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (A12)

(185) To a solution of 4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxybenzoic acid I20 (40 mg, 0.13 mmol) and (S)-tert-butyl3-((R)-1-hydroxy-2-(methylamino)ethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate A11 (40 mg, 0.13 mmol) in DCM was added DIPEA (75 mg, 0.59 mmol), HOBt (2 mg, 0.013 mmol), and EDCl.HCl (50 mg, 0.26 mmol). The resulting mixture was stirred at room temperature for two days. The mixture was diluted with DCM (20 mL) and the organic layer washed with saturated aqueous NaHCO.sub.3 (20 mL), 0.5 M aqueous HCl (20 mL), water (20 mL) and brine (20 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue obtained was purified by preparative TLC (5% MeOH in DCM) to give the title compound as a yellow solid (35 mg, 45%). LCMS: RT 2.80 min; m/z 594.3 [M+H]

(e) 4-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N—((R)-2-hydroxy-2-((S)-1,2,3,4-tetrahydroisoquinolin-3-yl)ethyl)-N-methylbenzamide hydrochloride (3)

(186) To a solution of (3S)-tert-butyl 3-((1R)-2-(4-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-ethoxy-N-methylbenzamido)-1-hydroxyethyl)-3,4-dihydroisoquinoline-2(1H)-carboxylate A12 (30 mg, 0.05 mmol) in Et.sub.2O (1 mL) was added HCl in Et.sub.2O (1.8 M, 1 mL). The mixture was stirred at room temperature overnight. The solvent was removed and the solid washed with Et.sub.2O (3×3 mL) to give the title compound as a yellow solid (23 mg, 85%). .sup.1H NMR (400 MHz, MeOD) δ 7.34-7.32 (m, 5H), 7.14-7.06 (m, 2H), 4.62 (br s, 1H), 4.42-4.29 (m, 3H), 4.09-3.59 (m, 11H), 3.22-3.19 (m, 1H), 3.04 (br s, 3H), 1.99 (br s, 4H), 1.22 (br s, 3H), LCMS: RT 3.39 min; m/z 494.3 [M+H].sup.+.

(187) Assays

(188) PRMT5 Biochemical Assay

(189) Compounds of the invention may be tested for in vitro activity in the following assay: A histone H4 derived peptide is used as substrate (amino acid sequence: Ser-Gly-Arg-Gly-Lys-Gly-Gly-Lys-Gly-Leu-Gly-Lys-Gly-Gly-Ala-Lys-Arg-His-Arg-Lys-Val-NH.sub.2). Full-length PRMT5 enzyme (NCBI Reference sequence NP_006100.2) was co-expressed with His.sub.6-MEP50 in insect cells and purified via Nickel immobilized metal affinity and gel filtration chromatography (“the enzyme”).

(190) The 6 μL reactions are run in Greiner brand black 384-well low volume assay plates. All reactions contained assay buffer (phosphate buffered saline, 0.01% (v/v) Tween-20, 0.01% (w/v) albumin from chicken egg white, 1 mM Dithiothreitol, 1 μM peptide substrate, 1 μM S-Adenosyl methionine, and 15 ng/reaction enzyme, with the enzyme being omitted from negative control reactions. Compounds were added in a volume of 100 nL from dilution series prepared in DMSO, positive and negative control reactions receiving the same volume DMSO without compound. The plates were sealed with adhesive seals and incubated for 4 hours at 37 degree Celsius. Reaction progress was measured using the Transcreener™ EPIGEN methyltransferase assay (BellBrook Labs, Madison, Wis.) as recommended by the manufacturer. To each reaction 2 μL detection mix were added, containing coupling enzymes, fluorescence polarisation tracer, and AMP antibody. Plates were incubated for 90 minutes before being read on a PerkinElmer EnVision™ plate reader in fluorescence polarisation mode. IC.sub.50 values were obtained from the raw readings by calculating percent inhibition (% I) for each reaction relative to controls on the same plate (% I=(I−CN)/(CP−CN) where CN/CP are the averages of the negative/positive reactions, respectively), then fitting the % I data vs. compound concentration [I] to % I=(A+((B−A)/(1+((C/[I]{circumflex over ( )}D)))) where A is the lower asymptote, B is the upper asymptote, C is the IC.sub.50 value, and D is the slope.

(191) TABLE-US-00001 Compound Number IC.sub.50 (μM) 1 0.019 2 0.019 3 0.036
PRMT5 Biomarker Assay

(192) Compounds of the invention may be tested for potency to inhibit symmetrical dimethylation of arginine in the following assay:

(193) The cell line TE11 was seeded at a density of 6,000 cells per well in 96 well optical quality tissue culture plates in DME medium and 10% foetal bovine serum, and allowed to adhere for 5 hours under standard culture conditions (37 degree Celsius, 5% CO.sub.2). Compound dilutions prepared in DMSO were added to the medium, with negative control wells reserved for treatment with DMSO only and maximum inhibition controls receiving a potent PRMT5 inhibitor compound at 1 μM concentration. After incubation for 72 hours, the cells were fixed with 3.7% formaldehyde in PBS for 30 minutes at room temperature, washed with phosphate buffer saline and blocked with Odyssey blocking buffer (LI-COR, Lincoln, Nebr.). Rabbit anti-Di-Methyl Histone H4 Arginine 3 specific antibody (Epigentek) in Odyssey blocking buffer was added and incubated for 14 hours at 4 degree Celsius. After washing, anti-rabbit secondary antibody labelled with Alexa647 dye (LifeTechnologies) and Hoechst 33342 (1 μg/mL, SigmaAldrich) were added for 1 hour incubation. Plates were washed and read on a PerkinElmer Envision 2103 in fluorescence intensity scanning mode (24 scans across the well area). The plates were imaged on a PerkinElmer Phenix high content imaging platform. Using a Columbus image analysis pipeline, individual nuclei were located by Hoechst 33342 stain and the methylation level was calculated from the Alexa647-related intensity in the same area. IC.sub.50 values were obtained from the mean Alexa647-related intensity per cell by calculating percent inhibition (% I) for each well relative to controls on the same plate (% I=(I−CN)/(CP−CN) where CN/CP are the averages of the negative/maximum inhibition controls, respectively), then fitting the % I data vs. compound concentration [I] to % I=(A+((B−A)/(1+((C/[I]){circumflex over ( )}D)))) where A is the lower asymptote, B is the upper asymptote, C is the IC.sub.50 value, and D is the slope.

(194) TABLE-US-00002 Compound Number IC.sub.50 (μM) 1 0.0039 2 0.0006 3 0.0117