PRMT5 inhibitors

Abstract

A compound of formula (Ia), (Ib) or (Ic) 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; X is either N or CH; R.sup.7 is selected from H and C.sub.1-4 alkyl; (a) one of R.sup.8a, R.sup.8b, R.sup.8c and R.sup.8d is selected from H, halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, NHC.sub.1-4 alkyl; (b) another of R.sup.8a, R.sup.8b, R.sup.8c and R.sup.8d is selected from H, C.sub.1-4 alkyl, C.sub.1-4 fluoroalkyl, C.sub.3-6 cycloalkyl, C.sub.5-6 heteroaryl, C.sub.5-6 heteroaryl methyl, C.sub.4-6 heterocyclyl, C.sub.4-6 heterocyclyl methyl, phenyl, benzyl, halo, amido, amidomethyl, acylamido, acylamidomethyl, C.sub.1-4 alkyl ester, C.sub.1-4 alkyl ester methyl, C.sub.1-4 alkyl carbamoyl, C.sub.1-4 alkyl carbamoyl methyl, C.sub.1-4 alkylacyl, C.sub.1-4 alkylacyl methyl, phenylcarbonyl, carboxy, carboxymethyl, ether, amino, amino methyl, sulfonamido, sulfonamino, sulfone, sulfoxide, nitrile and nitrilemethyl; (c) the others of R.sup.8a, R.sup.8b, R.sup.8c and R.sup.8d are H. ##STR00001##

Claims

1. A compound of formula Ia, Ib or Ic: ##STR00034## 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; X is either N or CH; R.sup.7 is selected from H and C.sub.1-4 alkyl; (a) one of R.sup.8a, R.sup.8b, R.sup.8c and R.sup.8d is selected from H, halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, NHC.sub.1-4 alkyl; (b) another of R.sup.8a, R.sup.8b, R.sup.8c and R.sup.8d is selected from H, C.sub.1-4 alkyl, C.sub.1-4 fluoroalkyl, C.sub.3-6 cycloalkyl, C.sub.5-6 heteroaryl, C.sub.5-6 heteroaryl methyl, C.sub.4-6 heterocyclyl, C.sub.4-6 heterocyclyl methyl, phenyl, benzyl, halo, amido, amidomethyl, acylamido, acylamidomethyl, C.sub.1-4 alkyl ester, C.sub.1-4 alkyl ester methyl, C.sub.1-4 alkyl carbamoyl, C.sub.1-4 alkyl carbamoyl methyl, C.sub.1-4 alkylacyl, C.sub.1-4 alkylacyl methyl, phenylcarbonyl, carboxy, carboxymethyl, ether, amino, amino methyl, sulfonamido, sulfonamino, sulfone, sulfoxide, nitrile and nitrilemethyl; (c) the others of R.sup.8a, R.sup.8b, R.sup.8c and R.sup.8d are H.

2. A compound according to claim 1, wherein n is 1.

3. A compound according to claim 1, wherein R.sup.N is H.

4. A compound according to claim 1, wherein there are no R.sup.1 substituents.

5. A compound according to claim 1, wherein R.sup.2a, R.sup.2b, R.sup.2c and R.sup.2d (if present) are all H.

6. A compound according to claim 1, wherein R.sup.3a and R.sup.3b are both Me.

7. A compound according to claim 1, wherein R.sup.4a is OH.

8. A compound according to claim 1, wherein R.sup.4b is H.

9. A compound according to claim 1 which is of formula Ia1, Ib1 or Ic1: ##STR00035##

10. A compound according to claim 1, wherein the compound is a racemate at the carbon atom to which R.sup.4b and R.sup.4a are attached.

11. A compound according to claim 1, wherein the compound is the (S)-enantiomer at the carbon atom to which R.sup.4b and R.sup.4a are attached.

12. A compound according to claim 1, wherein X is N.

13. A compound according to claim 1, wherein R.sup.7 is methyl or ethyl.

14. A compound according to claim 13, wherein R.sup.7 is ethyl.

15. A compound according to claim 1, wherein R.sup.8a is selected from H, halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy and NHC.sub.1-4 alkyl.

16. A compound according to claim 1, wherein the one of R.sup.8a, R.sup.8b, R.sup.8c and R.sup.8d that is selected from H, C.sub.1-4 alkyl, C.sub.1-4 fluoroalkyl, C.sub.3-6 cycloalkyl, C.sub.5-6 heteroaryl, C.sub.5-6 heteroaryl methyl, C.sub.4-6 heterocyclyl, C.sub.4-6 heterocyclyl methyl, phenyl, benzyl, halo, amido, amidomethyl, acylamido, C.sub.1-4 alkyl ester, C.sub.1-4 alkyl ester methyl, C.sub.1-4 alkyl carbamoyl, C.sub.1-4 alkyl carbamoyl methyl, C.sub.1-4 alkylacyl, C.sub.1-4 alkylacyl methyl, phenylcarbonyl, carboxy, carboxymethyl, ether, amino, amino methyl, sulfonamido, sulfonamino, sulfone, sulfoxide, nitrile and nitrilemethyl is selected from C.sub.1-4 alkyl, fluoro, chloro, bromo, acetyl, methoxy, ethoxy, C(O)Me, C(O)Et, CH.sub.2C(O)Me, phenyl, CF.sub.3, CF.sub.2H, CN, CH.sub.2CN, OBn, OPh, OCF.sub.3,OCF.sub.2H, O(C.sub.6H.sub.4)CN, COOH, CH.sub.2COOH, C(O)OMe, C(O)NH.sub.2, C(O)NMeH, C(O)NMe.sub.2, C(O)NPrH, C(O)-piperidinyl, C(O)-pyrrolidinyl, C(O)-morpholino (which may be bridged or substituted with one or two methyl groups), C(O)-azetidinyl, CH.sub.2C(O)NH.sub.2, CH.sub.2C(O)-azetidinyl, CH.sub.2C(O)NMeH, CH.sub.2C(O)NPrH, CH.sub.2C(O)-pyrrolidinyl, CH.sub.2C(O)-morpholino, CH.sub.2-morpholino, CH.sub.2-methylpiperazinyl, OCH.sub.2pyridinyl, OCH.sub.2-methyloxadiazolyl, CH.sub.2-imidazolyl, Otetrahydropyranyl, CH.sub.2-tetraydropyanyl, NHmethylpyrazinyl, CH.sub.2-triazolyl, NHSO.sub.2Ph, NHSO.sub.2Me, SO.sub.2NMePh, SO.sub.2NMe.sub.2, SO.sub.2NHEt, SO.sub.2CF.sub.3,- -lactam, CH.sub.2NHC(O)Me, CH.sub.2NHC()OMe, CH.sub.2NHC(O)CF.sub.3, morpholino, CH.sub.2NH.sub.2, C(O)Ph, OCH.sub.2-isoxazolyl, NH-pyrimidinyl, pyridizinyl, pyrimidinyl, pyridinyl, pyrazolyl, methylpyrazolyl, dimethylpyrazolyl, pyrazinyl, pyridazinyl , methyloxadiazolyl, oxadiazolyl, dimethyloxadiazolyl, isoxazolyl, dimethyltriazolyl, imidazolyl, benzimidazolyl and thiadiazolyl.

17. A compound according to claim 1, wherein one of R.sup.8b, R.sup.8c and R.sup.8d is selected from H, C.sub.1-4 alkyl, C.sub.1-4 fluoroalkyl, C.sub.3-6 cycloalkyl, C.sub.5-6 heteroaryl, C.sub.5-6 heteroaryl methyl, C.sub.4-6 heterocyclyl, C.sub.4-6 heterocyclyl methyl, phenyl, benzyl, halo, amido, amidomethyl, acylamido, C.sub.1-4 alkyl ester, C.sub.1-4 alkyl ester methyl, C.sub.1-4 alkyl carbamoyl, C.sub.1-4 alkyl carbamoyl methyl, C.sub.1-4 alkylacyl, C.sub.1-4 alkylacyl methyl, phenylcarbonyl, carboxy, carboxymethyl, ether, amino, amino methyl, sulfonamido, sulfonamino, sulfone, sulfoxide, nitrile and nitrilemethyl.

18. A compound according to claim 17, wherein one of R.sup.8b, R.sup.8c and R.sup.8d is selected from C.sub.1-4 alkyl, fluoro, chloro, bromo, acetyl, methoxy, ethoxy, C(O)Me, C(O)Et, CH.sub.2C(O)Me, phenyl, CF.sub.3, CF.sub.2H, CN, CH.sub.2CN, OBn, OPh, OCF.sub.3, OCF.sub.2H, O(C.sub.6H.sub.4) CN, COOH, CH.sub.2COOH, C(O)OMe, C(O)NH.sub.2, C(O)NMeH, C(O)NMe.sub.2, C(O)NPrH, C(O)-piperidinyl, C(O)-pyrrolidinyl, C(O)-morpholino (which may be bridged or substituted with one or two methyl groups), C(O)-azetidinyl, CH.sub.2C(O)NH.sub.2, CH.sub.2C(O)-azetidinyl, CH.sub.2C(O)NMeH, CH.sub.2C(O)NPrH, CH.sub.2C(O)-pyrrolidinyl, CH.sub.2C(O)-morpholino, CH.sub.2-morpholino, CH.sub.2-methylpiperazinyl, OCH.sub.2pyridinyl, OCH.sub.2-methyloxadiazolyl, CH.sub.2-imidazolyl, Otetrahydropyranyl, CH.sub.2-tetraydropyanyl, NH-methylpyrazinyl, CH.sub.2-triazolyl, NHSO.sub.2Ph, NHSO.sub.2Me, SO.sub.2NMePh, SO.sub.2NMe.sub.2, SO.sub.2NHEt, SO.sub.2CF.sub.3,--lactam, CH.sub.2NHC(O)Me, CH.sub.2NHC(O)OMe, CH.sub.2NHC(O)CF.sub.3, morpholino, CH.sub.2NH.sub.2, C(O)Ph, OCH.sub.2-isoxazolyl, NH-pyrimidinyl, pyridizinyl, pyrimidinyl, pyridinyl, pyrazolyl, methylpyrazolyl, di methylpyrazolyl, pyrazinyl, pyridazinyl , methyloxadiazolyl, oxadiazolyl, dimethyloxadiazolyl, isoxazolyl, dimethyltriazolyl, imidazolyl, benzimidazolyl and thiadiazolyl.

19. A compound according to claim 18, wherein R.sup.8c is selected from amido or amidomethyl.

20. A compound according to claim 19, wherein R.sup.8c is selected from: ##STR00036##

21. A compound according to claim 1, which is selected from: 2-(3-((2,3-Dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one (1); 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-((S)-3-((2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one (2); and 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-((S)-3-((2,3-dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-4-ethylphthalazin-1(2H)-one (3).

22. A pharmaceutical composition comprising a compound according to claim 1, and a pharmaceutically acceptable excipient.

23. A method of treatment of a PRMT5-overexpressing cancer, comprising administering to a patient in need of treatment, a compound according to claim 1.

24. A method of treatment of hemoglobinopathies, comprising administering to a patient in need of treatment, an effective PRMT5-inhibiting amount a compound according to claim 1.

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.

Acronyms

(2) 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).

(3) 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 (EDCI.HCl, EDCI), 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).

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

(5) General Experimental Details

(6) 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.

(7) 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.

(8) LCMS Method A (LCMS-A):

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

(10) Agilent 1200 Series HPLC

(11) Pump: 1200 Series G1311A Quaternary pump

(12) Autosampler: 1200 Series G1329A Thermostatted Autosampler

(13) Detector: 1200 Series G1314B Variable Wavelength Detector

(14) LC Conditions:

(15) Reverse Phase HPLC Analysis

(16) Column: Luna C8 (2) 5 m 504.6 mm 100

(17) Column temperature: 30 C.

(18) Injection Volume: 5 L

(19) Solvent A: Water 0.1% Formic Acid

(20) Solvent B: MeCN 0.1% Formic Acid

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

(22) Detection: 254 nm or 214 nm

(23) MS Conditions:

(24) Ion Source: Quadrupole

(25) Ion Mode: Multimode-ES

(26) Drying gas temp: 300 C.

(27) Vaporizer temperature: 200 C.

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

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

(30) Scan Range: 100-1000

(31) Step size: 0.1 sec

(32) Acquisition time: 10 min

(33) LCMS Method B (LCMS-B):

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

(35) Pump: 1260 Infinity G1312B Binary pump

(36) Autosampler: 1260 Infinity G1367E 1260 HiP ALS

(37) Detector: 1290 Infinity G4212A 1290 DAD

(38) LC Conditions:

(39) Reverse Phase HPLC Analysis

(40) Column: Poroshell 120 EC-C18 2.7 m 503.0 mm

(41) Column temperature: 35 C.

(42) Injection Volume: 1 L

(43) Solvent A: Water 0.1% Formic Acid

(44) Solvent B: MeCN 0.1% Formic Acid

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

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

(47) MS Conditions:

(48) Ion Source: Quadrupole

(49) Ion Mode: API-ES

(50) Drying gas temp: 350 C.

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

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

(53) Scan Range: 100-1000

(54) Step size: 0.1 sec

(55) Acquisition time: 5 min

(56) LCMS Method C (LCMS-C):

(57) Instrument: Agilent 1200 Series G6110A Quadrupole

(58) Pump: Binary pump

(59) Detector: DAD

(60) LC Conditions:

(61) Reverse Phase HPLC Analysis

(62) Column: Xbridge-C18, 2.5 m, 2.130 mm

(63) Column temperature: 30 C.

(64) Injection Volume: 1-10 L

(65) Solvent A: Water 0.07% Formic acid

(66) Solvent B: Methanol

(67) 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)

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

(69) MS Conditions:

(70) Ion Source: Quadrupole

(71) Ion Mode: ES+

(72) Drying gas temp: 350 C.

(73) Drying gas flow: 10 L/min

(74) Nebulizer pressure: 35 psi

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

(76) Scan Range: 50-900

(77) Or

(78) Instrument: Waters 2695 alliance

(79) Pump: Quaternary Pump

(80) Detector: 2996 Photodiode Array Detector

(81) MS model: Micromass ZQ

(82) LC Conditions:

(83) Column: Xbridge-C18, 3.5 m, 2.150 mm

(84) Column temperature: 30 C.

(85) Injection volume: 1-10 L

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

(87) Solvent A: 0.07% HCOOH aqueous solution

(88) Solvent B: MeOH

(89) Run time: 8 min

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

(91) Detection: 254 nm and 214 nm

(92) MS Condition:

(93) Ion source: ES+(or ES) MS range: 50900 m/z

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

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

(96) Desolvation temperature: 300 C.

(97) Source temperature: 100 C.

(98) Sample Preparation:

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

(100) Preparative RP-HPLC:

(101) Agilent 1260 Infinity HPLC System

(102) UV detection at 210 nm and 254 nm

(103) Gradient or isocratic elution through a Phenomenex Luna C8 (2) column 100 Axia (25021.2 mm; particle size 5 m)

(104) Flow rate: 10 mL/min

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

(106) 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.

(107) Preparative thin-layer chromatography (prep TLC) was performed using Tklst (China), grand grade: (HPTLC): 82 m>80%; (TLC): 10-40 m. Type: GF254. Compounds were visualised by UV (254 nm).

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

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

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

(111) 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.

(112) Additional Cartridges used are as follows:

(113) Phase Separator

(114) Manufacturer: Biotage

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

(116) SCX and SCX-2 Cartridges:

(117) Manufacturer: Biotage

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

(119) Manufacturer: Biotage

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

(121) Manufacturer: Silicycle

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

(123) Manufacturer: Agilent

(124) Product: Bond Elut SCX 10 g

(125) Sample Extraction Cartridge:

(126) Manufacturer: Waters

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

(128) Intermediate Preparations

(i) N-(2,4-Dimethoxybenzyl)-2,3-dihydro-1H-inden-2-amine (I1)

(129) ##STR00027##

(130) To a solution of 2,3-dihydro-1H-inden-2-amine (20.0 g, 0.150 mol) in ethanol (300 mL) was added 2,4-dimethoxybenzaldehyde (25.0 g, 0.150 mol). The reaction mixture was stirred at 70 C. for 2 hours, then cooled to 0 C. before NaBH.sub.4 (11.4 g, 0.300 mol) was added. The resulting mixture was stirred at 0 C. for 30 minutes and poured into water (150 mL). Chloroform (150 mL) was added and the resulting mixture filtered. The filtrate was concentrated under reduced pressure and the aqueous mixture extracted with EtOAc (350 mL). The organic layer was washed with brine (280 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired compound as a yellow oil (41 g, 96%). LCMS: RT 1.45 min; m/z 284.2 [M+H].sup.+.

(ii) 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethylphthalazin-1(2H)-one (I5)

(131) ##STR00028##

(a) Dimethyl 2-propionylterephthalate (I2)

(132) A mixture of zinc powder (0.720 g, 11.0 mmol), CoBr.sub.2 (150 mg, 0.69 mmol), allyl bromide (60 uL, 0.7 mmol) and TFA (1 drop) was stirred under nitrogen in acetonitrile (7 mL) for 10 minutes at room temperature. Dimethyl 2-bromoterephthalate (2.0 g, 7.0 mmol) and propionic anhydride (1.05 g, 8.07 mmol) were then added and the mixture stirred for 5 hours. The mixture was quenched with aqueous sodium bicarbonate and water and the aqueous phase was then extracted with diethyl ether. The combined organic fractions were dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue obtained was purified by column chromatography (5-10% EtOAc in hexanes) to give the desired product (1.1 g, 61%) as a yellow solid. LCMS RT 2.37 min; m/z 251.1 [M+H].sup.+.

(b) Methyl 4-ethyl-1-oxo-1,2-dihydrophthalazine-6-carboxylate (I3)

(133) To a solution of dimethyl 2-propionylterephthalate 12 (1.0 g, 4.0 mmol) in DCM (10 mL) was added N.sub.2H.sub.4.H.sub.2O (80% aqueous solution, 250 mg, 4.0 mmol), and the resulting mixture was stirred at room temperature overnight. DCM (100 mL) and water (100 mL) were added. The organic layer was washed with 1 M aqueous HCl (550 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired product (300 mg, 32%) as an off-white solid. LCMS RT 2.23 min; m/z 233.1 [M+H].sup.+.

(c) 4-Ethyl-1-oxo-1,2-dihydrophthalazine-6-carboxylic Acid (I4)

(134) To a solution of methyl 4-ethyl-1-oxo-1,2-dihydrophthalazine-6-carboxylate I3 (0.300 g, 1.29 mmol) in THF (6 mL), MeOH (2 mL) and H.sub.2O (1 mL) was added LiOH.H.sub.2O (164 mg, 3.91 mmol). The reaction mixture was stirred at room temperature overnight, then concentrated before the resultant residue was taken up in water (15 mL). The aqueous solution was acidified with 1M aqueous HCl to pH 4 and the aqueous phase extracted with DCM (330 mL). The pooled organic fractions were dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired product (260 mg, 92%) as an off-white solid. LCMS RT 1.18 min; m/z 219.1 [M+H].sup.+.

(d) 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethylphthalazin-1(2H)-one (I5)

(135) To a solution of 4-ethyl-1-oxo-1,2-dihydrophthalazine-6-carboxylic acid I4 (120 mg, 0.56 mmol) and 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (91 mg, 0.61 mmol) in DCM (10 mL) was added DIPEA (399 L, 2.24 mmol), EDCI.HCl (215 mg, 1.12 mmol) and HOBt (8 mg, 0.06 mmol). The resulting mixture was stirred at room temperature overnight. Saturated aqueous NaHCO.sub.3 (30 mL) was added and the aqueous extracted with DCM (350 mL). The combined organic fractions were dried (Na.sub.2SO.sub.4), filtered and concentrated to give the desired product (80 mg, 46%) as a grey solid. LCMS RT 1.35 min; m/z 314.1 [M+H].sup.+.

EXAMPLES

Example 1: 2-(3-((2,3-Dihydro-1H-inden-2-yl)amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one (1)

(136) ##STR00029##

(a) 4-Methyl-2-(oxiran-2-ylmethyl)phthalazin-1(2H)-one (A1)

(137) To a suspension of Na.sub.2SO.sub.4 (0.400 g, 2.82 mmol) in DMF (2.5 mL) was added 4-methylphthalazin-1(2H)-one (0.200 g, 1.25 mmol) and NaH (60% in mineral oil, 0.070 g, 1.7 mmol). A solution of 2-(chloromethyl)oxirane (0.185 g, 2.00 mmol) in DMF (1.5 mL) was added dropwise and the mixture stirred at room temperature for 1 hour then at 60 C. for 4 hours. The mixture was filtered and the filtrate diluted with EtOAc (50 mL), washed with water (350 mL), brine (50 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (10% EtOAc in petroleum ether) to give the title compound as a white solid (0.100 g, 37%). .sup.1H NMR (400 MHz, CDCl.sub.3) 8.48-8.46 (m, 1H), 7.85-7.75 (m, 3H), 4.47-4.42 (m, 1H), 4.32-4.27 (m, 1H), 3.46-3.42 (m, 1H), 2.87-2.85 (m, 1H), 2.77-2.75 (m, 1H), 2.60 (s, 3H); LCMS: RT 1.64 min; m/z 217.1 [M+H].sup.+.

(b) 2-(3-((2,3-Dihydro-1H-inden-2-yl)(2,4-dimethoxybenzyl)amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one (A2)

(138) To a solution of 4-methyl-2-(oxiran-2-ylmethyl)phthalazin-1(2H)-one A1 (0.090 g, 0.42 mmol) in EtOH (5 mL) was added a solution of N-(2,4-dimethoxybenzyl)-2,3-dihydro-1H-inden-2-amine 11 (238 mg, 0.840 mmol) in EtOH (5 mL). The resulting mixture was heated at 80 C. overnight. The solvent was removed under reduced pressure and the residue was purified by column chromatography (33% EtOAc in petroleum ether) to give the title compound as yellow oil (170 mg, 81%). LCMS: RT 2.29 min; m/z 500.2[M+H].sup.+.

(c) 2-(3-((2,3-Dihydro-H-inden-2-yl)amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one (1)

(139) A solution of 2-(3-((2,3-dihydro-1H-inden-2-yl)(2,4-dimethoxybenzyl)amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one A2 (170 mg, 0.34 mmol) in TFA (5 mL) was heated at reflux for 4 hours. The solvent was removed under reduced pressure and the residue obtained was suspended in saturated aqueous NaHCO.sub.3 (20 mL). The aqueous was extracted with DCM (210 mL) and the combined organic layers dried (Na.sub.2SO.sub.4), filtered and concentrated. The crude product was purified by preparative TLC 5% MeOH in DCM to give the title compound as a white solid (33 mg, 28%). .sup.1H NMR (400 MHz, CD.sub.3OD) 8.39 (d, J=8 Hz, 1H), 8.01-7.87 (m, 3H), 7.26-7.19 (m, 4H), 4.45-4.27 (m, 3H), 4.14-4.06 (m, 1H), 3.45-3.37 (m, 2H), 3.28-3.24 (m, 1H), 3.19-3.09 (m, 3H), 2.63 (s, 3H); LCMS: RT 1.90 min; m/z 350.1 [M+H].sup.+.

Example 2: 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-((S)-3-((2,3-dihydro-1H-Inden-2-yl)amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one (2)

(140) ##STR00030## ##STR00031##

(a) Dimethyl 2-bromoterephthalate (A3)

(141) To a solution of 2-bromoterephthalic acid (2.0 g, 8.2 mmol) in MeOH (20 mL) at 0 C. under N.sub.2 was added thionyl chloride (5.8 g, 49 mmol) dropwise. The resulting mixture was stirred at room temperature overnight. The mixture was concentrated and the residue partitioned between DCM (20 mL) and saturated aqueous NaHCO.sub.3 (15 mL). The aqueous phase was extracted with DCM (310 mL). The combined organic fractions were washed with saturated aqueous NaHCO.sub.3 (310 mL), brine (310 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound (1.85 g, 83%) as a white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.30 (d, J=1.6 Hz, 1H), 7.99 (dd, J=8.0, 1.2 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 3.95 (s, 3H), 3.94 (s, 3H); LCMS RT 2.63 min; m/z 273,275 [M+H].sup.+.

(b) Dimethyl 2-acetylterephthalate (A4)

(142) To a solution of dimethyl 2-bromoterephthalate A3 (2.65 g, 9.70 mmol) in CH.sub.3CN (30 mL) was added PPh.sub.3 (918 mg, 3.50 mmol), TEA (1.96 g, 19.4 mmol), 1-(vinyloxy)butane (4.9 g, 49 mmol) and Pd(OAc).sub.2 (392 mg, 1.75 mmol) at room temperature under N.sub.2. The resulting mixture was heated at reflux overnight under N.sub.2. The mixture was filtered, the filtrate was concentrated and the residue obtained was diluted with THF (30 mL) and 3 M HCl (30 mL). The mixture was stirred at room temperature for 1 hour. The organics were removed under reduced pressure and the aqueous phase was extracted with DCM (320 mL). The combined organic fractions were washed with saturated aqueous NaHCO.sub.3 aqueous solution (310 mL), brine (310 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (EtOAc/PetroleumEther=1/10) to give the title compound (1.1 g, 48%) as a yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.15 (dd, J=8.0, 1.6 Hz, 1H), 8.12 (d, J=1.6 Hz, 1H), 7.86 (d, J=8.0 Hz, 1H), 3.95 (s, 3H), 3.91 (s, 3H), 2.58 (s, 3H); LCMS RT 2.12 min; m/z 237.1 [M+H].sup.+

(c) Lithium 2-acetylterephthalate (A5)

(143) To a solution of dimethyl 2-acetylterephthalate A4 (1.1 g, 4.7 mmol) in THF (10 mL), MeOH (6 mL) and H.sub.2O (3 mL) was added LiOH.H.sub.2O (978 mg, 23.3 mmol). The resulting mixture was stirred at room temperature overnight, then concentrated in vacuo to dryness. The compound was dried by azeotroping with methanol to give the crude title compound (2.6 g, >95%) which was used in the next step without purification. LCMS RT 0.61 min; m/z 209 [M-Li+H.sub.2].sup.+.

(d) Lithium 4-methyl-1-oxo-1,2-dihydrophthalazine-6-carboxylate (A6)

(144) To a solution of lithium 2-acetylterephthalate A5 (2.6 g crude product, 4.7 mmol) in isopropanol (10 mL) was added 50% hydrazine hydrate (932 mg, 9.32 mmol). The resulting mixture was heated at reflux overnight. The mixture was concentrated to dryness and azeotroped with methanol to give the crude title compound (2.1 g, >95%) as a grey solid, which was used in the next step without purification. LCMS RT 2.59 min; m/z 205.1 [M+H].sup.+.

(e) 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one A7

(145) A solution of lithium 4-methyl-1-oxo-1,2-dihydrophthalazine-6-carboxylate A6 (2.1 g crude product, 4.7 mmol) in thionyl chloride (10 mL) was heated at reflux for 2 hours. The mixture was concentrated to dryness to give the crude acid chloride. The residue was dissolved in DCM (20 mL), then TEA (2.35 g, 23.3 mmol) and 3-oxa-8-azabicyclo[3.2.1]octane hydrochloride (1.05 g, 6.99 mmol) were added. The resulting mixture was stirred at room temperature overnight then diluted with water (10 mL) and the aqueous phase extracted with DCM (310 mL). The combined organic extracts were washed with saturated aqueous NaHCO.sub.3 (310 mL), brine (310 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue obtained was purified by column chromatography (MeOH/DCM=1/50) to give the title compound (600 mg, 45% yield over three steps) as a yellow solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 10.57 (s, 1H), 8.50 (d, J=8.0 Hz, 1H), 7.95 (d, J=1.2 Hz, 1H), 7.81 (dd, J=8.0, 1.2 Hz, 1H), 4.78 (s, 1H), 3.92-3.90 (m, 2H), 3.76-3.59 (m, 3H), 2.61 (s, 3H), 2.09-1.99 (m, 4H); LCMS RT 0.83 min; m/z 300.1 [M+H].sup.+.

(f) 2-(((2S)-3-(6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzoic Acid (A8)

(146) To a solution of 6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one A7 (0.300 g, 1.00 mmol) in DMF (10 mL) under N.sub.2 was added (S)-2-(oxiran-2-ylmethyl) isoindoline-1,3-dione (0.610 g, 3.00 mmol) and K.sub.2CO.sub.3 (553 mg, 4.00 mmol). The resulting mixture was stirred at 85 C. overnight then the mixture filtered and the filtrate acidified to pH 3-4 with 4 M aqueous HCl. The aqueous was extracted with DCM (55 mL) and the combined organic layers were dried (Na.sub.2SO.sub.4), filtered and concentrated in vacuo to give the crude product as a yellow oil (contains some DMF). LCMS RT 1.94 min; m/z 521.2 [M+H].sup.+.

(g) 2-((2S)-3-(6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)isoindoline-1,3-dione (A9)

(147) A solution of 2-(((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzoic acid A8 (crude product from above step, 1.0 mmol) in EtOH (10 mL) was heated at reflux overnight. The mixture was concentrated and the residue diluted with EtOAc (10 mL) and water (10 mL). The separated aqueous phase was extracted with EtOAc (310 mL) and the combined organic extracts washed with saturated aqueous NaHCO.sub.3 (35 mL), brine (35 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by preparative TLC (MeOH/DCM=1/20) to give the title compound (260 mg, 51% yield over two steps) as an off-white solid. .sup.1H NMR (400 MHz, MeOD) 8.40 (d, J=8.4 Hz, 1H), 7.99-7.97 (m, 1H), 7.91 (dd, J=8.0, 1.6 Hz, 1H), 7.82-7.76 (m, 4H), 4.69 (s, 1H), 4.55-4.48 (m, 1H), 4.37-4.24 (m, 2H), 3.93-3.83 (m, 3H), 3.81-3.73 (m, 3H), 3.60-3.57 (m, 1H), 2.57 (s, 3H), 2.10-2.03 (m, 4H); LCMS RT 2.29 min; m/z 503.2 [M+H].sup.+.

(h) 2-((S)-3-Amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one (A10)

(148) To a solution of 2-((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)isoindoline-1,3-dione A9 (250 mg, 0.50 mmol) in EtOH (5 mL) was added 75% hydrazine hydrate (100 mg, 1.5 mmol). The resulting mixture was heated at reflux for 4 hours. The mixture was filtered and the filtrate was concentrated in vacuo to give the crude product (210 mg, >95%) as a yellow solid. LCMS RT 2.22 min; m/z 373.2 [M+H].sup.+.

(i) tert-Butyl ((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamate (A11)

(149) To a solution of 2-((S)-3-amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one A10 (190 mg, 0.51 mmol) in DCM (6 mL) was added TEA (155 mg, 1.53 mmol) and Boc.sub.2O (223 mg, 1.02 mmol). The mixture was stirred at room temperature for 2 hours then partitioned between water (5 mL) and DCM (10 mL). The aqueous phase was extracted with DCM (35 mL) and the combined organic fractions washed with saturated aqueous NaHCO.sub.3 (35 mL), brine (35 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue was purified by column chromatography (MeOH/DCM=1/50) to give the title compound (150 mg, 62%) as a white solid. .sup.1H NMR (400 MHz, CD.sub.3OD) 8.46 (d, J=8.0 Hz, 1H), 8.05 (d, J=1.2 Hz, 1H), 7.94 (dd, J=8.0, 1.6 Hz, 1H), 4.70 (s, 1H), 4.33-4.27 (m, 1H), 4.21-4.15 (m, 2H), 3.93-3.86 (m, 2H), 3.77-3.75 (m, 2H), 3.60-3.57 (m, 1H), 3.28-3.24 (m, 1H), 3.16-3.11 (m, 1H), 2.64 (s, 3H), 2.11-2.04 (m, 4H), 1.42 (s, 9H); LCMS RT 2.39 min; m/z 473.2[M+H].sup.+, 495.2 [M+Na].sup.+.

(j) 2-((S)-3-Amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one Dihydrochloride (A 12)

(150) A solution of tert-butyl ((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamate A11 (150 mg, 0.32 mmol) in 2 M HCl in Et.sub.2O (10 mL) was stirred at room temperature for 1 hour. The mixture was concentrated in vacuo to give the title compound (118 mg, 84%) as a off-white solid. .sup.1H NMR (400 MHz, CD.sub.3OD) 8.47 (d, J=8.0 Hz, 1H), 8.07 (d, J=0.8 Hz, 1H), 7.96 (dd, J=8.0, 1.6 Hz, 1H), 4.71 (s, 1H), 4.36-4.24 (m, 3H), 3.92-3.86 (m, 2H), 3.77-3.74 (m, 2H), 3.60-3.57 (m, 1H), 3.15-3.12 (m, 1H), 2.98-2.93 (m, 1H), 2.65 (s, 3H), 2.11-2.04 (m, 4H); LCMS RT 2.45 min; m/z 373.2 [M+H].sup.+.

(k) 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2((S)-3-((2,3-dihydro-1H-inden-2-yl) amino)-2-hydroxypropyl)-4-methylphthalazin-1(2H)-one (2)

(151) To a solution of 2-((S)-3-amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one dihydrochloride A12 (40 mg, 0.09 mmol) in 1,2-dichloroethane (5 mL) at room temperature under N2 was added TEA (46 mg, 0.45 mmol), AcOH (36 mg), 1H-inden-2(3H)-one (24 mg, 0.18 mmol) and NaBH(OAc).sub.3 (58 mg, 0.27 mmol). The mixture was stirred at room temperature overnight under N.sub.2, then concentrated and the residue diluted with water (10 mL) and DCM (10 mL). The aqueous phase was extracted with DCM (35 mL) and the combined organic extracts washed with saturated aqueous NaHCO.sub.3 (35 mL), brine (35 mL), dried (Na.sub.2SO.sub.4) filtered and concentrated. The residue was purified by preparative TLC (MeOH/DCM=1/20) to give the crude product (15 mg) as a black solid.

(152) The reaction was repeated twice more on the same scale and the three batches of crude products (batch 1 (16 mg), batch 2 (16 mg), batch 3 (15 mg)) combined and purified by preparative-HPLC to give the title compound (13 mg, 8%) as a white solid. .sup.1H NMR (400 MHz, CD.sub.3OD) 8.47 (d, J=8.0 Hz, 1H), 8.07 (d, J=1.2 Hz, 1H), 7.96 (dd, J=8.0, 1.6 Hz, 1H), 7.27-7.19 (m, 4H), 4.71 (s, 1H), 4.45-4.38 (m, 1H), 4.37-4.28 (m, 2H), 4.16-4.08 (m, 1H), 3.93-3.86 (m, 2H), 3.77-3.74 (m, 2H), 3.60-3.57 (m, 1H), 3.46-3.38 (m, 2H), 3.28-3.27 (m, 1H), 3.18-3.11 (m, 3H), 2.65 (s, 3H), 2.11-2.04 (m, 4H); LCMS RT 1.81 min; m/z 489.3 [M+H].sup.+.

Example 3: 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-((S)-3-((2,3-dihydro-1H-Inden-2-yl)amino)-2-hydroxypropyl)-4-ethylphthalazin-1(2H)-one (3)

(153) ##STR00032## ##STR00033##

(a) 2-(((2S)-3(6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzoic Acid (A13)

(154) To a solution of 6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethylphthalazin-1(2H)-one I5 (0.200 g, 0.638 mmol) in DMF (10 mL) under N.sub.2 was added (S)-2-(oxiran-2-ylmethyl) isoindoline-1,3-dione (130 mg, 0.64 mmol) and K.sub.2CO.sub.3 (133 mg, 0.962 mmol). The resulting mixture was heated at 85 C. overnight. The reaction was cooled and the solids removed by filtration. The filtrate was acidified to pH 3-4 with 4 M aqueous HCl then extracted with DCM (55 mL). The combined organic fractions were dried (Na.sub.2SO.sub.4) filtered and concentrated to give the crude product as yellow oil which was used in the next step without purification (contained some DMF). LCMS RT 2.13 min; m/z 535.2 [M+H].sup.+.

(b) 2-((2S)-3-(6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)isoindoline-1,3-dione (A14)

(155) A solution of 2-(((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)benzoic acid A13 (crude product obtained above, 0.64 mmol) in EtOH (10 mL) was heated at reflux overnight. The mixture was concentrated and the residue partitioned between EtOAc (10 mL) and water (10 mL). The aqueous phase was extracted with EtOAc (310 mL) and the combined organic extracts washed with saturated aqueous NaHCO.sub.3 (35 mL), brine (35 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated to give the title compound (300 mg, 91% yield over two steps) as an off-white solid. LCMS RT 2.49 min; m/z 517.2 [M+H].sup.+.

(c) 2-((S)-3-Amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethylphthalazin-1(2H)-one (A15)

(156) To a solution of 2-((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)isoindoline-1,3-dione A14 (0.300 g, 0.581 mmol) in a mixture of EtOH (5 mL) and DCM (5 mL) was added 80% hydrazine hydrate (44 mg, 0.7 mmol). The resulting mixture was heated at reflux for 4 hours under N.sub.2. The solids were removed by filtration and the filtrate concentrated to afford the crude product (280 mg, >95%) as yellow oil. LCMS RT 2.78 min; m/z 387.2 [M+H].sup.+.

(d) tert-Butyl ((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamate (A 16)

(157) To a solution of 2-((S)-3-amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.]octane-8-carbonyl)-4-ethylphthalazin-1(2H)-one A15 (crude, 280 mg, 0.58 mmol@100%) in THF (6 mL) was added TEA (117 mg, 1.16 mmol) and Boc.sub.2O (164 mg, 0.751 mmol). The mixture was stirred at room temperature overnight. The reaction was diluted with water (5 mL) and DCM (5 mL) and the aqueous phase was extracted with DCM (315 mL). The combined organic extracts were washed with saturated aqueous NaHCO.sub.3 (315 mL), brine (15 mL), dried (Na.sub.2SO.sub.4), filtered and concentrated. The residue obtained was purified by preparative TLC (MeOH/DCM=1/10) to give the title compound (170 mg, 60%) as a yellow solid. LCMS RT 2.51 min; m/z 487.3 [M+H].sup.+.

(e) 2-((S)-3-Amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one Dihydrochloride (A 17)

(158) A mixture of tert-butyl ((2S)-3-(6-(3-oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-4-ethyl-1-oxophthalazin-2(1H)-yl)-2-hydroxypropyl)carbamate A16 (170 mg, 0.35 mmol) in 2 M HCl in Et.sub.2O (15 mL) was stirred at room temperature for 2 hours. The mixture was concentrated to give the title compound (161 mg, 99%) as an off-white solid.

(f) 6-(3-Oxa-8-azabicyclo[3.2.1]octane-8-carbonyl)-2-((S)-3-((2,3-dihydro-1H-inden-2-yl) amino)-2-hydroxypropyl)-4-ethylphthalazin-1(2H)-one (3)

(159) To a solution of 2-((S)-3-amino-2-hydroxypropyl)-6-(3-oxa-8-azabicyclo[3.2.]octane-8-carbonyl)-4-methylphthalazin-1(2H)-one dihydrochloride A17 (0.080 g, 0.17 mmol) in DCM (5 mL) was added TEA (26 mg, 0.26 mmol), 1H-inden-2(3H)-one (28 mg, 0.21 mmol) and NaBH.sub.4 (13 mg, 0.34 mmol). The mixture was stirred at room temperature overnight, concentrated and the residue was purified by preparative TLC (MeOH/DCM=1/10) to give the product (15 mg) as a black solid.

(160) The reaction was repeated on the same scale and the two batches of product combined and purified again by preparative TLC to give the title compound (15 mg, 9%) as a grey solid. .sup.1H NMR (400 MHz, CD.sub.3OD) 8.48 (d, J=8.0 Hz, 1H), 8.12 (s, 1H), 7.96-7.94 (d, J=8.0 Hz, 1H), 7.25-7.21 (m, 4H), 4.71 (br s, 1H), 4.50-4.30 (m, 3H), 4.15-4.11 (m, 1H), 3.92-3.75 (m, 5H), 3.64-3.58 (m, 1H), 3.48-3.39 (m, 2H), 3.18-3.06 (m, 5H), 2.14-1.98 (m, 4H), 1.38-1.35 (m, 3H); LCMS RT 2.10 min; m/z 503.3 [M+H].sup.+.

(161) Assays

(162) PRMT5 Biochemical Assay

(163) 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).

(164) The 6 L 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 M peptide substrate, 1 M 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 L 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 (%1) for each reaction relative to controls on the same plate (% I=(ICN)/(CPCN) where CN/CP are the averages of the negative/positive reactions, respectively), then fitting the % I data vs. compound concentration [I] to % l=(A+((BA)/(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.

(165) TABLE-US-00001 Compound Number IC.sub.50 (M) 1 4.53 2 0.17 3 0.03
PRMT5 Biomarker Assay

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

(167) 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 M 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 g/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=(ICN)/(CPCN) 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+((BA)/(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.

(168) TABLE-US-00002 Compound Number IC.sub.50 (M) 2 0.0180 3 0.0019