TETRAHYDROISOQUINOLINE DERIVED PRMT5-INHIBITORS

Abstract

A compound of formula (I) wherein: 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 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.4 is either H or Me; R.sup.5 is either H or Me; A is either (i) optionally substituted phenyl; (ii) optionally substituted naphthyl; or (iii) optionally substituted C.sub.5-12 heteroaryl.

##STR00001##

Claims

1. A compound of formula I: ##STR00113## wherein: 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 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.4 is either H or Me; R.sup.5 is either H or Me; A is either (i) optionally substituted phenyl; (ii) optionally substituted naphthyl; or (iii) optionally substituted C.sub.5-12 heteroaryl.

2. (canceled)

3. A compound according to claim 1, wherein R.sup.1 represents one to four Me or halo groups.

4. A compound according to claim 1, wherein: R.sup.2a, R.sup.2b, R.sup.2c and R.sup.2d are all H; or (b) R.sup.2a, R.sup.2b, R.sup.2c and R.sup.2d are comprised of three H and one Me or CH.sub.2OH group; or (c) R.sup.2a, R.sup.2b, R.sup.2c and R.sup.2d are comprised of two H and two Me groups.

5-11. (canceled)

12. A compound according to claim 1, wherein: (a) R.sup.3a is H and R.sup.3b is Me; or (b) R.sup.3a and R.sup.3b are both H; or (c) R.sup.3a and R.sup.3b are both Me.

13-18. (canceled)

19. A compound according to claim 1 which is of formula Ia: ##STR00114##

20. A compound according to claim 1, which is a racemate at the carbon atom to which R.sup.4 is attached.

21. A compound according to claim 1, which is a single enantiomer at the carbon atom to which R.sup.4 is attached.

22-23. (canceled)

24. A compound according to claim 1, wherein the optional substituents on A are independently selected from the group consisting of: 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, aminomethyl, sulfonamido, sulfonamino, sulfone, sulfoxide, nitrile and nitrilemethyl and when A is phenyl, the optional substituent may also be a fused C.sub.5-6 N.sub.1-containing heterocyclic ring.

25. A compound according to claim 1, wherein A is optionally substituted phenyl, wherein the substituents are 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, —CFH, —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)N.sup.iPrH, —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, —O-tetrahydropyranyl, —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, -γ-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, dimethylpyrazolyl, pyrazinyl, pyridazinyl , methyloxadiazolyl, oxadiazolyl, dimethyloxadiazolyl, isoxazolyl, dimethyltriazolyl, imidazolyl, benzimidazolyl and thiadiazolyl.

26-31. (canceled)

32. A compound according to claim 25, wherein: (a) in the ortho position of the phenyl group there is a halo or methoxy susbtituent; or (b) in the para position of the phenyl group there is an amido or amidomethyl susbtituent; or (c) the phenyl group bears a halo or methoxy substituent in the ortho position, and an amido or amidomethyl substituent in the para position of the phenyl group; or (d) in the meta position of the phenyl group there is an amino susbtituent.

33-35. (canceled)

36. A compound according to claim 1, wherein: (a) A is optionally substituted naphthyl; or (b) optionally substituted C.sub.5-12 heteroaryl selected from the group consisting of: pyridinyl, pyrimidinyl, pyrazinyl, isoxazolyl, oxazolyl, thiophenyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridonyl, imidazolyl, benzimidazolyl, imidazopyridinyl and quinolinyl.

37-52. (canceled)

53. A compound according to claim 1, wherein A is selected from one of the following groups: ##STR00115##

54. A compound according to claim 1, wherein A is selected from one of the following groups: ##STR00116##

55. A compound according to claim 54, wherein A is selected from one of the following groups: ##STR00117##

56. A compound according to claim 1, wherein A is selected from one of the following groups: ##STR00118##

57. A compound according to claim 1, wherein A is selected from one of the following groups: ##STR00119##

58. (canceled)

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

60. A method of treatment of cancer, comprising administering to a patient in need of treatment, a compound according to a composition according to claim 59.

61-65. (canceled)

Description

EXAMPLES

[0349] 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.

[0350] Acronyms

[0351] 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), n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), biphenyl (biPh), benzyl (Bn), naphthyl (naph), methoxy (MeO), ethoxy (EtO),benzoyl (Bz), trimethylsilyl (TMS), and acetyl (Ac).

[0352] 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 (Et2O), ethyl acetate (EtOAc), acetic acid (AcOH), acetonitrile (MeCN), dichloromethane (methylene chloride, DCM), trifluoroacetic acid (TFA), N,N-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), 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (Ruphos), tert-butyloxycarbonyl (Boc, BOC), 2-(trimethylsilypethoxymethyl (SEM), triethylamine (Et.sub.3N), 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 4-dimethylaminopyridine (DMAP), N,N-diisopropylethylamine (DIPEA), lithium bis(trimethylsilyl)amide (LiHMDS) magnesium sulfate (MgSO.sub.4), 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), Propylphosphonic anhydride (T3P), tetra-n-butylammonium bromide (TBAB), and 1-hydroxybenzotriazole (HOBt).

[0353] General Experimental Details

[0354] Unless otherwise stated the following generalisations apply. .sup.1H NMR spectra were recorded on a Bruker Ultrashield plus (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.

[0355] LC/MS data was generated using either an Agilent 6100 Series Single Quad LC/MS (LCMS-A) or Agilent 1260 Infinity Series UPLC/MS (LCMS-B). Chlorine isotopes are reported as .sup.35Cl, Bromine isotopes are reported as either .sup.79Br or .sup.81Br or both .sup.79Br/.sup.81Br.

[0356] Preparative mass-directed LC was carried out using a Waters ZQ 3100.

[0357] LCMS Method A (LCMS-A)

[0358] Instrument: Agilent 6100 Series Single Quad LC/MS

[0359] Agilent 1200 Series HPLC

[0360] Pump: 1200 Series G1311A Quaternary pump

[0361] Autosampler: 1200 Series G1329A Thermostatted Autosampler

[0362] Detector: 1200 Series G1314B Variable Wavelength Detector

[0363] LC conditions:

[0364] Reverse Phase HPLC analysis

[0365] Column: Luna C8 (2) 5 μm 50×4.6 mm 100 Å

[0366] Column temperature: 30° C.

[0367] Injection Volume: 5 μL

[0368] Solvent A: Water 0.1% Formic Acid

[0369] Solvent B: MeCN 0.1% Formic Acid

[0370] Gradient: 5-100% B over 10 min

[0371] Detection: 254 nm or 214 nm

[0372] MS conditions:

[0373] Ion Source: Quadrupole

[0374] Ion Mode: Multimode-ES

[0375] Drying gas temp: 300° C.

[0376] Vaporizer temperature: 200° C.

[0377] Capillary voltage (V): 2000 (positive)

[0378] Capillary voltage (V): 4000 (negative)

[0379] Scan Range: 100-1000

[0380] Step size: 0.1 sec

[0381] Acquisition time: 10 min

[0382] LCMS Method B (LCMS-B:)

[0383] Instrument:

[0384] Pump: 1260 Infinity G1312B Binary pump

[0385] Autosampler: 1260 Infinity G1367E 1260 HiP ALS

[0386] Detector: 1290 Infinity G4212A 1290 DAD

[0387] LC conditions:

[0388] Reverse Phase HPLC analysis

[0389] Column: Poroshell 120 EC-C18 2.7 μm 50×3.0 mm

[0390] Column temperature: 35° C.

[0391] Injection Volume: 1 μL

[0392] Solvent A: Water 0.1% Formic Acid

[0393] Solvent B: MeCN 0.1% Formic Acid

[0394] Gradient: 5-100% B over 3.8 min

[0395] Detection: monitored at 254 nm and 214 nm

[0396] MS conditions:

[0397] Ion Source: Quadrupole

[0398] Ion Mode: API-ES

[0399] Drying gas temp: 350° C.

[0400] Capillary voltage (V): 3000 (positive)

[0401] Capillary voltage (V): 3000 (negative)

[0402] Scan Range: 100-1000

[0403] Step size: 0.1 sec

[0404] Acquisition time: 5 min

[0405] Additional Cartridges used are as follows

[0406] Phase Separator

[0407] Manufacturer: Biotage

[0408] Product: ISOLUTE® Phase Separator (3 mL unless otherwise stated)

[0409] SCX and SCX-2 Cartridges

[0410] Manufacturer: Biotage

[0411] Product: ISOLUTE ® SCX-2 1 g (6 mL Column)

[0412] Manufacturer: Silicycle

[0413] Product: SCX-2 500 mg or 5 g

[0414] Manufacturer: Agilent

[0415] Product: Bond elutO SCX 10 g

[0416] Sample Extraction Cartridge

[0417] Manufacturer: Waters

[0418] Product: Oasis® HLB 35 cc (6 g) LP extraction cartridge

Example 1

4-Chloro-N-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)benzamide (1)

[0419] ##STR00058##

[0420] (a) 2-(But-3-en-1-yl)isoindoline-1,3-dione (A1)

[0421] Potassium phthalimide (500 mg, 2.70 mmol), acetonitrile (5 mL) and 4-bromobut-1-ene (0.548 mL, 5.40 mmol) were stirred at 90° C. After 18 hours, the mixture was cooled to room temperature, filtered and the collected solids were washed with ethyl acetate (5 mL). The combined filtrates were concentrated and column chromatography (12 g SiO.sub.2 cartridge, 0-100% ethyl acetate in petroleum benzine 40-60° C.) gave the desired compound as a white solid (431 mg, 79%). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.87-7.80 (m, 2H), 7.73-7.67 (m, 2H), 5.86-5.72 (m, 1H), 5.11-4.99 (m, 2H), 3.77 (t, J=7.1 Hz, 2H), 2.49-2.40 (m, 2H). LCMS-B RT 3.71 min; m/z 202.1 [M+H].sup.+.

[0422] (b) 2-(2-(Oxiran-2-yl)ethyl)isoindoline-1,3-dione (A2)

[0423] 2-(But-3-en-1-yl)isoindoline-1,3-dione A1 (428 mg, 2.13 mmol), chloroform (5 mL) and 70-75% m-CPBA (629 mg, 2.55 mmol) were stirred at room temperature. After 18 hours, the mixture was quenched with a 10% w/v aqueous solution of sodium thiosulfate (2.5 mL) and was stirred vigorously for 5 minutes. The mixture was diluted with a saturated aqueous solution of sodium bicarbonate (5 mL), water (20 mL) and chloroform (10 mL). The separated aqueous phase was extracted with chloroform (2×10 mL). The combined organic extracts were dried over sodium sulfate and concentrated in vacuo to give the desired compound as a white solid (451 mg, 98%). The material was carried forward without further purification. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.88-7.82 (m, 2H), 7.74-7.69 (m, 2H), 3.97-3.80 (m, 2H), 3.03-2.94 (m, 1H), 2.74-2.68 (m, 1H), 2.47-2.41 (m, 1H), 2.04-1.93 (m, 1H), 1.90-1.79 (m, 1H); LCMS-B RT 3.43 min; m/z 218.1 [M+H].sup.+

[0424] (c) 2-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)isoindoline-1,3-dione (A3)

[0425] 2-(2-(Oxiran-2-yl)ethyl)isoindoline-1,3-dione A2 (100 mg, 0.46 mmol), dry acetonitrile (2 mL), tetrahydroisoquinoline (0.061 mL, 0.48 mmol) and calcium triflate (78 mg, 50 mol %) were stirred at room temperature. After two hours, the mixture was purified by column chromatography (12 g SiO.sub.2 cartridge, 0-10% methanol/DCM) to give the desired compound as a pale yellow syrup (44 mg, 27%): .sup.1H NMR (400 MHz, d.sub.4-methanol) δ 7.88-7.76 (m, 4H), 7.11-6.96 (m, 4H), 3.97-3.76 (m, 3H), 3.75-3.62 (m, 2H), 2.93-2.75 (m, 4H), 2.59-2.55 (m, 2H), 1.96-1.86 (m, 1H), 1.84-1.68 (m, 1H); LCMS-B m/z: 351.2 [M+H].sup.+.

[0426] (d) 4-Amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol (A4)

[0427] 2-(4-(3,4-Dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)isoindoline-1,3-dione A3 (44 mg, 0.13 mmol), ethanol (2 mL) and hydrazine hydrate (0.2 mL) were stirred at 80° C. for 3 hours. The mixture was cooled to room temperature, the resulting slurry was filtered and the collected solids were washed with cold ethanol (2 mL). The combined filtrates were concentrated to give the desired compound as a white semi-solid (29 mg, >100%). The material was carried forward without further purification

[0428] (e) 4-Chloro-N-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)benzamide (1)

[0429] 4-Amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol A4 (28 mg, 0.13 mmol), MeCN (2 mL), DIPEA (0.044 mL, 0.25 mmol) and 4-chlorobenozyl chloride (0.016 mL, 0.13 mmol) were stirred at room temperature. After 18 hours, methanol (0.1 mL) was added and the mixture loaded onto a 5 g SCX cartridge. The cartridge was washed with methanol (40 mL) then eluted with 3.5 M ammonia in methanol (40 mL). The basic eluate was concentrated in vacuo and the material was purified by preparative TLC (SiO.sub.2, 5% methanol/DCM) to give the desired compound as a white solid (21 mg, 46%): .sup.1H NMR (400 MHz, d.sub.4-methanol) δ 7.80-7.76 (m, 2H), 7.46-7.42 (m, 2H), 7.14-6.98 (m, 4H), 4.02-3.93 (m, 1H), 3.78-3.65 (m, 2H), 3.61-3.46 (m, 2H), 2.95-2.80 (m, 4H), 2.65-2.53 (m, 2H), 1.92-1.81 (m, 1H), 1.76-1.65 (m, 1H); LCMS-A RT 1.55 min; m/z 359.1 [M+H].sup.+.

[0430] Intermediate Preparation

[0431] (i) Alternate synthesis of 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol (A4)

##STR00059##

[0432] (a) 2-(but-3-en-1-yl)isoindoline-1,3-dione (A1)

[0433] Potassium phthlamide (5.00 g, 27.0 mmol), acetonitrile (50 mL) and 4-bromobut-1-ene (5.48 mL, 54.0 mmol) were stirred at 90° C. After 18 hours the mixture was cooled to room temperature, filtered and the collected solids washed with ethyl acetate (5 mL). The combined filtrates were evaporated, chromatography (40 g silica cartridge, 10-60% ethyl acetate/ petroleum benzine) gave the title compound as a white solid (4.31 g, 79% yield). LCMS-B: rt 3.71 min; m/z (positive ion) 202.1 [M+H].sup.+

[0434] (b) 2-(2-(oxiran-2-yl)ethyl)isoindoline-1,3-dione (A2)

[0435] 2-(But-3-en-1-yl)isoindoline-1,3-dione A1 (4.31 g, 21.4 mmol), chloroform (50 mL) and mCPBA (70-75%, 6.34 g, 26 mmol @70%) were stirred at room temperature. After 20 hours the mixture was diluted with 10% w/v aqueous sodium thiosulfate solution (100 mL) and stirred vigorously for five minutes. The mixture was diluted with saturated aqueous sodium bicarbonate (100 mL), the organic layer separated and the aqueous layer extracted with chloroform (2×100 mL). The pooled organic extracts were washed with saturated aqueous sodium bicarbonate (100 mL), brine (100 mL), dried over sodium sulfate and concentrated in vacua to give the title compound as a white solid (4.60 g, 99% yield). LCMS-B: rt 3.43 min; m/z (positive ion) 218.1 [M+H].sup.+

[0436] (c) 2-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)isoindoline-1,3-dione (A3)

[0437] 2-(2-(Oxiran-2-yl)ethyl)isoindoline-1,3-dione A2 (4.60 g, 21.2 mmol), ethanol (100 mL) and 1,2,3,4-tetrahydroisoquinoline (3.18 mL, 25.4 mmol) were stirred at 80° C. After 3.5 hours the mixture was concentrated in vacuo and the residue suspended in cold petroleum benzine (150 mL). The mixture was filtered and the collected solid washed with further petroleum benzine (3×50 mL) and air dried to give the title compound as a white solid (6.54 g, 88% yield). LCMS-B: rt 3.30 min; m/z (positive ion) 351.2 [M+H].sup.+; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.88-7.82 (m, 2H), 7.74-7.68 (m, 2H), 7.17-6.96 (m, 4H), 3.98-3.78 (m, 4H), 3.63 (d, J=14.9 Hz, 1H), 2.98-2.86 (m, 3H), 2.76-2.68 (m, 1H), 2.59-2.47 (m, 2H), 1.85-1.78 (m, 2H).

[0438] (d) 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol (A4)

[0439] 2-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutypisoindoline-1,3-dione (6.54 g, 18.7 mmol) and ethanol (200 mL) were brought to reflux and hydrazine hydrate (50-60%, 5.81 mL, 93.3 mmol @50%) added. The mixture was vigorously stirred at reflux for three hours then cooled to room temperature. The mixture was filtered and the collected solids washed with ethanol (2×25 mL). The pooled filtrates were concentrated in vacuo, the residue dissolved in ethanol (100 mL) and again concentrated in vacuo to give the title compound as a yellow oil (4.66 g). LCMS-B, m/z (positive ion) 221.2 [M+H].sup.+

[0440] (ii) Lithium 4-((4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)carbamoyl)benzoate (I2)

##STR00060##

[0441] (a) Methyl 4-((4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)carbamoyl)benzoate (I1)

[0442] To a solution of 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol A4 (0.300 g, 1.36 mmol, 1 equiv) in MeCN (20 mL) was added monomethyl terephthalic acid (0.245 g, 1.36 mmol, 1 equiv), DIPEA (712 μL, 4.09 mmol, 3 equiv) and HATU (0.777 g, 2.04 mmol, 1.5 equiv). The reaction was stirred at room temperature for 16 h. The mixture was quenched with a saturated aqueous solution of sodium carbonate (15 mL) and extracted with ethyl acetate (3×30 mL). The pooled organic extracts were washed with water (30 mL), brine (30 mL), dried over magnesium sulfate and evaporated. The crude residue was taken up in MeOH and purified by solid-phase extraction (10 g SCX-2 cartridge, 3 column volumes of methanol followed by 3 column volumes of 0.2 M methanolic ammonia) to give the title compound (215 mg) as a dark yellow solid, containing ˜20% of 1-(3,4-dihydroisoquinolin-2(1H)-yl)-4-(4-(methoxycarbonyl)benzamido)butan-2-ylmethyl terephthalate which was used without further purification. LCMS-B: rt=3.16 min, m/z=383 [m+H].sup.+

[0443] (b) Lithium 4-((4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)carbamoyl)benzoate (I2)

[0444] To a solution of methyl 4-((4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)carbamoyl)benzoate I1 (215 mg, 0.562 mmol, 1 equiv) in H.sub.2O:MeOH (1:2; 20 mL) was added LiOH.H.sub.2O (100 mg, 2.38 mmol, 4 equiv). The reaction was stirred at room temperature for 16 h. The solvent was removed in vacuo to give the title compound (310 mg) as a white solid. LCMS-B: rt 3.14 min, m/z 369 [M+H].sup.+.

[0445] (iii) 4-Methoxy-3-(pyrimidin-5-yl)benzoic acid (I4)

##STR00061##

[0446] (a) Methyl 4-methoxy-3-(pyrimidin-5-yl)benzoate (I3)

[0447] A mixture of methyl 3-iodo-4-methoxybenzoate (500 mg, 1.71 mmol), pyrimidin-5-ylboronic acid (318 mg, 2.57 mmol), PdCl.sub.2(dppf).DCM complex (71 mg, 5 mol %), 1,4-dioxane (10 mL) and aqueous 1.0 M Cs.sub.2CO.sub.3 (3.42 mL, 3.42 mmol) was degassed with bubbling nitrogen and heated in the microwave (120° C130 min). The mixture was added to water (100 mL) and EtOAc (100 mL) and the mixture was filtered through Celite. The filtrate was separated and the aqueous phase was extracted with further EtOAc (100 mL). The pooled organic phases were washed with brine (100 mL), dried over Na.sub.2SO.sub.4 and concentrated. Chromatography (0-80% EtOAc in petroleum benzine 40-60° C.) gave the title product as a white solid (321 mg, 77%). .sup.1H NMR (400 MHz, d.sub.4-DMSO) δ 9.18 (s, 1H), 8.96 (s, 2H), 8.07 (dd, J=8.7, 2.2 Hz, 1H), 7.97 (d, J=2.2 Hz, 1H), 7.32 (d, J=8.7 Hz, 1 H), 3.89 (s, 3H), 3.84 (s, 3H). LCMS-B rt 3.45 min; m/z 245.1 [M+H].sup.+.

[0448] (b) 4-methoxy-3-(pyrimidin-5-yl)benzoic acid (I4)

[0449] A suspension of methyl 4-methoxy-3-(pyrimidin-5-yl)benzoate I3 (319 mg, 1.31 mmol) and lithium hydroxide monohydrate (164 mg, 3.92 mmol) in THF (10 mL), MeOH (10 mL) and water (5 mL) was stirred at room temperature. After 18 hours, the mixture was concentrated in vacuo and the aqueous residue diluted to 40 mL with water. The pH was adjusted to 1 with aqueous 6 M HCl, the precipitate collected by filtration, washed with water (10 mL) and dried in vacuo to give the title compound as a white solid (229 mg, 76%). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 9.17 (s, 1H), 8.96 (s, 2H), 8.05 (dd, J=8.7, 2.2 Hz, 1H), 7.94 (d, J=2.2 Hz, 1H), 7.29 (d, J=8.7 Hz, 1H), 3.89 (s, 3H). LCMS-B: rt 3.27 min; m/z 231.1 [M+H].sup.+; m/z 229.1 [M−H].sup.−.

[0450] (iv) 3-(Pyridazin-4-yl)benzoic acid (I6)

##STR00062##

[0451] (a) 4-bromopyridazine hydrobromide (I5)

[0452] Potassium acetate (1.84 g, 19 mmol) and 3-bromofuran (0.612 mL, 6.8 mmol) were stirred in acetic acid (5 mL) and a solution of bromine (0.349 mL, 6.8 mmol) in acetic acid (2 mL) was added dropwise. After one hour the mixture was filtered, the solids washed with acetic acid (3 mL) and the filtrate concentrated. The mixture was dissolved in EtOH (10 mL) and hydrazine hydrate (1 mL) added. After 3 hours the mixture was added to EtOAc (50 mL) and the EtOAc washed with brine (2×50 mL). The brine extracts were extracted with EtOAc (50 mL), and the pooled EtOAc extracts dried over Na.sub.2SO.sub.4 and evaporated. The residue was diluted with 1,4-dioxane (5 mL) and treated with 33% HBr in acetic acid (1 mL) dropwise. The dark suspension was filtered, the collected solids washed with 1,4-dioxane (5 mL), acetone (5 mL) and air dried to give the title compound as a brown solid (806 mg, 49% yield). .sup.1H NMR (400 MHz, d.sub.5-DMSO) δ 9.50 (dd, J=2.6, 1.1 Hz, 1H), 9.14 (dd, J=5.7, 1.0 Hz, 1H), 8.14 (dd, J=5.6, 2.5 Hz, 1H). LCMS-B: rt 2.68 min; m/z 161.0 [M+H].sup.+ for .sup.81Br.

[0453] (b) 3-(Pyridazin-4-yl)benzoic acid (I6)

[0454] 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (0.496 g, 2.0 mmol), 4-bromopyridazine hydrobromide I5 (576 mg, 2.4 mmol), PdCl.sub.2(dppf) (83 mg, 5 mol %) and dioxane (10 mL) were loaded into a microwave tube. A solution of potassium carbonate (829 mg, 6.0 mmol) in water (5 mL) was added, the mixture degassed with a stream of nitrogen bubbles then heated in the microwave (120° C./30 minutes). The mixture was cooled, and the volatile solvents removed in vacuo. The aqueous residue was diluted with water to 75 mL, and shaken with DCM (75 mL). The mixture was filtered through celite, the aqueous layer separated and washed with further DCM (75 mL). The DCM extracts were discarded. The aqueous phase was diluted with water (25 mL) and treated with 5% w/v aqueous citric acid solution until pH 3 to pH paper. The resulting precipitate was collected by filtration, washed with water and dried under vacuum to give the title compound as a pale brown solid (312 mg, 78% yield). LCMS-B rt 3.15 min, m/z (positive ion) 201.1 [M+H].sup.+; m/z (negative ion) 199.1 [M−H].sup.−; .sup.1H NMR (400 MHz, DMSO) δ 9.70-9.65 (m, 1H), 9.33-9.28 (m, 1H), 8.38 (s, 1H), 8.17 (d, J=7.8 Hz, 1H), 8.12-8.05 (m, 2H), 7.71 (t, J=7.8 Hz, 1H).

[0455] (v) 2((1-acetylpiperidin-4-yl)amino)isonicotinic acid (I11)

##STR00063##

[0456] (a) tert-butyl (1-acetylpiperidin-4-yl)carbamate (I7)

[0457] Acetic anhydride (4.71 mL, 49.9 mmol) was added to a solution of tert-butyl piperidin-4-ylcarbamate (10.0 g, 49.9 mmol) and triethylamine (10.4 mL, 74.9 mmol) in anhydrous DCM (100 mL) at 0° C. The reaction mixture was stirred at 0° C. for 2 hours before water (-100 mL) and DCM (˜50 mL) were added. The organic phase was separated, washed with a saturated aqueous NaHCO.sub.3 solution (˜100 mL) and dried (MgSO.sub.4). The solvent was removed in vacuo to give the title compound as a white solid (10.72 g, 89%). .sup.1H NMR (400 MHz, CDC.sub.3) δ 4.54-4.42 (m, 2H), 3.80-3.70 (m, 1H), 3.70-3.58 (m, 1H), 3.12 (ddd, J=14.2, 11.8, 2.9 Hz, 1H), 2.78-2.65 (m, 1H), 2.07 (s, 3H), 2.05-1.97 (m, 1H), 1.96-1.87 (m, 1H), 1.43 (s, 9H), 1.36-1.20 (m, 2H).

[0458] (b) 1-(4-aminopiperidin-1-yl)ethan-1-one hydrochloride (I8)

[0459] A solution of tert-butyl (1-acetylpiperidin-4-yl)carbamate I7 (10.72 g, 44.24 mmol) in 1,4-dioxane (100 mL) was cooled to 0° C. and treated with 4.0 M HCl in 1,4-dioxane (12.2 mL, 48.7 mmol). A white precipitate formed following addition of the acid which was isolated by filtration. The precipitate was dissolved in MeOH (100 mL) and treated with 4.0 M HCl in 1,4-dioxane (12.2 mL, 48.7 mmol) and the mixture was stirred at room temperature for 16 hours. Another aliquot of 4.0 M HCl in 1,4-dioxane (6.10 mL, 24.4 mmol) was added and the reaction mixture was stirred for 1.5 hours at 40° C. The volatiles were removed in vacuo and the white solid was dried under high vacuum to give the title compound (8.60 g, ˜90% purity, >95% yield). .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.52-8.23 (m, 3H), 4.39-4.26 (m, 1H), 3.89-3.77 (m, 1H), 3.28-3.14 (m, 1H), 3.11-3.00 (m, 1H), 2.65-2.54 (m, 1H), 1.99 (s, 3H), 1.97-1.86 (m, 2H), 1.54-1.41 (m, 1H), 1.41-1.27 (m, 1H).

[0460] (c) Methyl 2-bromoisonicotinate (I9)

[0461] A solution of 2-bromoisonicotinic acid (5.00 g, 24.8 mmol) in MeOH (50 mL) was treated with sulfuric acid (0.50 mL, 9.4 mmol) and the reaction mixture was stirred at 80° C. for 1 hour. The mixture was returned to room temperature and stirred for a further 96 hours before heating to 80° C. and stirring for 24 hours. The reaction mixture was cooled to room temperature, and the volatiles were removed in vacuo. An aqueous NaOH solution (2 M, ˜50 mL) was added to the residue and the aqueous was extracted with EtOAc (3×50 mL). The organic layers were combined, washed with brine, dried (MgSO.sub.4) and the solvent removed in vacua to give the title compound as a yellow oil (4.14 g, 77%). LCMS-B: rt 3.55 min; m/z 216 [M+H].sup.+ for .sup.79Br, 218 [M+H].sup.+ for .sup.81Br; .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.52 (dd, J=5.0, 0.8 Hz, 1H), 8.04 (t, J=1.2 Hz, 1H), 7.80 (dd, J=5.0, 1.4 Hz, 1H), 3.96 (s, 3H).

[0462] (d) Methyl 2-((1-acetylpiperidin-4-amino)isonicotinate (I10)

[0463] A mixture of 1-(4-aminopiperidin-1-yl)ethan-1-one hydrochloride I8 (2.25 g, 12.6 mmol), methyl 2-bromoisonicotinate I9 (1.81 g, 8.38 mmol), Cs.sub.2CO.sub.3 (10.92 g, 33.51 mmol), xantphos (0.242 g, 0.419 mmol) and Pd.sub.2(dba).sub.3 (0.384 g, 0.419 mmol) in 1,4-dioxane (40 mL) was bubbled with nitrogen for 10 min. The mixture was then stirred under an atmosphere of nitrogen at 80° C. for 24 hours. Further Cs.sub.2CO.sub.3 (5.46 g, 16.8 mmol), xantphos (0.121 g, 0.209 mmol) and Pd.sub.2(dba).sub.3 (0.192 g, 0.210 mmol) were added and the mixture was stirred under an atmosphere of nitrogen at 80° C. for 5 days. The reaction mixture was returned to room temperature and diluted with EtOAc (˜150 mL). Solid impurities were removed by filtration and the filtrate solvent was removed in vacuo. The resultant solid was purified by column chromatography (Biotage Isolera, 40 g SiO.sub.2 cartridge, 0-100% EtOAc in petroleum benzine 40-60° C., then 0-20% MeOH (containing 1% v/v TEA) in EtOAc) to give the title compound as a yellow solid (0.558 g, 24%). LCMS-B: rt 3.05 min; m/z 278.2 [M+H].sup.+.

[0464] (e) 2-((1-acetylpiperidin-4-yl)amino)isonicotinic acid (I11)

[0465] A mixture of methyl 2-((1-acetylpiperidin-4-yl)amino)isonicotinate I10 (0.558 g, 2.01 mmol), LiOH.H.sub.2O (1.69 g, 40.2 mmol), THF (7 mL), MeOH (7 mL) and water (1.5 mL) was stirred at 40° C. for 2 hours. The mixture was returned to room temperature and the volatiles were removed in vacuo. Water (˜30 mL) was added and the pH was adjusted to ˜6 with an aqueous solution of HCl (2 M). The aqueous phase was passed through an Oasis HLB 35 cc LP extraction cartridge (6 g) which was washed with 4 column volumes of water. The lipophilic component was then eluted with 4 column volumes of MeOH. Evaporation of the MeOH in vacuo gave the title compound as a yellow solid (0.197 g, 37%). The aqueous phase from the first iteration of cartridge purification was passed through another Oasis HLB 35 cc LP extraction cartridge (6 g). The column was washed with 4 column volumes of water and the product was eluted with 4 column volumes of MeOH. Evaporation of the MeOH in vacuo gave further title compound as a white solid (0.128 g, 24%), with NMR data in agreement. Overall yield: 0.325 g, 61%. LCMS-B: rt 1.17 min; m/z 262.1 [M−H].sup.−. .sup.1H NMR (400 MHz, d.sub.6-DMSO) δ 8.08 (d, J=5.2 Hz, 1H), 6.98 (s, 1H), 6.86-6.79 (m, 2H), 4.26-4.16 (m, 1H), 4.03-3.90 (m, 1H), 3.81-3.72 (m, 1H), 3.21-3.12 (m, 1H), 2.85-2.74 (m, 1H), 2.00 (s, 3H), 1.97-1.83 (m, 2H), 1.41-1.16 (m, 2H).

Example 2

[0466] General Procedure A

##STR00064##

[0467] To lithium 4-((4-(3,4-dihydroisoquinolin-2(1 H)-yl)-3-hydroxybutyl)carbamoyl)benzoate I2 (70 mg, 0.19 mmol, 1 equiv) in CH.sub.3CN (2 mL) was added DIPEA (130 μL, 0.75 mmol, 4 equiv) and HATU (107 mg, 0.281 mmol, 1.5 equiv). The desired amine (0.561 mmol, 3 equiv) in DMF (1 mL) was added and the reaction stirred at room temperature for 16 h. The reactions were quenched by the addition of a 1M aqueous solution of NaOH (2 mL) and stirred for 1 h. DCM (3 mL) was added, the layers mixed thoroughly and then passed through a phase separation cartridge (3mL). The aqueous layer was further extracted with DCM utilising the phase separation cartridge (2×3 mL). The combined organic layers were concentrated by a stream of air. An equivalent volume of MeOH was added and the solution purified by solid-phase extraction (1 g SCX-2 cartridge, 3 column volumes of methanol followed by 3 column volumes of 0.2 M methanolic ammonia). The organic solvent was removed in vacuo to give the title compound. Where specified, the compound was further purified by column chromatography (12 g, 50-100% EtOAc (modified by the addition of 1% v/v of 3.5 M methanolic ammonia) in petroleum benzine followed by 0-20% MeOH in EtOAc modified by the addition of 1% v/v of 2.0 M methanolic ammonia) to give the title compound.

[0468] General Procedure B

##STR00065##

[0469] To the acid (0.23 mmol, 1 equiv) in CH.sub.3CN (2 mL) was added DIPEA (120 μL, 0.69 mmol, 3 equiv) and HATU (131 mg, 0.345 mmol, 1.5 equiv). 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol A4 (51 mg, 0.23 mmol, 1 equiv) in DMF (1 mL) was added and the reaction stirred at room temperature for 16 h. The reactions were quenched by the addition of a 1M solution of NaOH (2 mL) and the stirred for 3 h. DCM (3 mL) was added, the layers mixed thoroughly and then passed through a phase separation cartridge (3 mL). The aqueous layers were further extracted with DCM (3 mL) and the organic layers collected by passage through a phase separation cartridge (2 repeats). The DCM layers were concentrated by a stream of air. An equivalent volume of MeOH was added and the solution purified by solid-phase extraction (1 g SCX-2 cartridge, 3 column volumes of methanol followed by 3 column volumes of 0.2 M methanolic ammonia). The organic solvent was removed in vacuo to give the title compounds.

[0470] General Procedure C

##STR00066##

[0471] The acid (0.20 mmol) and triethylamine (0.084 mL, 0.60 mmol) were dissolved in DCM. The mixture was cooled to 0° C. and a 1.0 M solution of isopropyl chloroformate in toluene (0.20 mL, 0.20 mmol) added. The mixture was stood for thirty minutes then a 0.40 M solution of 4-amino-1-(3,4-dihydroisoquinolin-2(1 H)-yl)butan-2-ol A4 in DCM (0.50 mL, 0.20 mmol) added. The mixtures were stood at room temperature for 17 hours then diluted with methanol (1 mL). The mixtures were loaded onto 2 g SCX cartridges, washed with methanol (15 mL) and eluted with 3.5 M ammonia in methanol (15 mL). The basic eluates were concentrated to give the amide products.

[0472] General Procedure D:

##STR00067##

[0473] The acyl chlorides (0.20 mmol each) and triethylamine (0.084 mL, 0.60 mmol) were dissolved in DCM (1 mL). A 0.40 M solution of 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol A4 in DCM (0.50 mL, 0.20 mmol) was added. The mixtures were stood at room temperature for 17 hours then diluted with methanol (1 mL). The mixtures were loaded onto 2 g SCX cartridges, washed with methanol (15 mL) and eluted with 3.5 M ammonia in methanol (15 mL). The basic eluates were concentrated to give the amide products.

TABLE-US-00003 Cpd Structure Analytical Data Method 2 [00068] LCMS-B: rt 3.54 min; m/z 457.2 [M + H].sup.+ C 3 [00069] LCMS-B: rt 3.30 min; m/z 441.2 [M + H].sup.+ C 4 [00070] LCMS-B rt 3.10 min; m/z 433.3 [M + H].sup.+ C 5 [00071] LCMS-B: rt 3.39 min; m/z 393.2 [M + H].sup.+ C 6 [00072] LCMS-B: rt 3.18 min; m/z 422.3 [M + H].sup.+ C 7 [00073] LCMS-B: rt 3.32 min; m/z 421.3 [M + H].sup.+ C 8 [00074] LCMS-B: rt 3.49 min; m/z 422.3 [M + H].sup.+ C 9 [00075] LCMS-B: rt 3.16 min; m/z 382.3 [M + H].sup.+ C 10 [00076] LCMS-B: rt 3.34 min; m/z 437.3 [M + H].sup.+ C 11 [00077] LCMS-B: rt 3.31 min; molecular ion not detected C 12 [00078] LCMS-B: 3.23 min; m/z 432.3 [M + H].sup.+ C 13 [00079] LCMS-B: 3.10 min; m/z 406.3 [M + H].sup.+ C 14 [00080] LCMS-B: 3.02 min; m/z 411.3 [M + H].sup.+ C 15 [00081] LCMS-B: 3.19 min; m/z 305.2 [M + H].sup.+ C 16 [00082] LCMS-B: 3.22 min; m/z 350.3 [M + H].sup.+ C 17 [00083] LCMS-B: 3.05 min; m/z 326.2 [M + H].sup.+ C 18 [00084] LCMS-B: 3.26 min; m/z 359.2 [M + H].sup.+ C 19 [00085] LCMS-B: 3.46 min; m/z 409.3 [M + H].sup.+ D 20 [00086] LCMS-B: 3.48 min; m/z 451.2 [M + H].sup.+ D 21 [00087] LCMS-B: 3.36 min; m/z 391.3 [M + H].sup.+ C 22 [00088] LCMS-B: 3.21 min; m/z 403.3 [M + H].sup.+ C 23 [00089] LCMS-B: 3.58 min; m/z 405.2 [M + H].sup.+ C 24 [00090] LCMS-B: 3.27 min; m/z 340.2 [M + H].sup.+ C 25 [00091] LCMS-B: 3.19 min; m/z 403.3 [M + H].sup.+ C, From I6 26 [00092] LCMS-B: 3.13 min; m/z 387.2 [M + H].sup.+ C 27 [00093] LCMS-B: 3.21, 3.26 min; m/z 433.3 [M + H].sup.+ C, From I4 28 [00094] LCMS-B: 3.20 min; m/z 392.3 [M + H].sup.+ C 29 [00095] LCMS-B: 3.09 min; m/z 356.3 [M + H].sup.+ C, except on a 0.25 mmol scale 30 [00096] LCMS-B: rt 3.20 min, m/z 422.3 [M + H].sup.+. A Compound was subjected to column chromatography 31 [00097] LCMS-B: rt 3.14 min, m/z 408.3 [M + H].sup.+. A 32 [00098] LCMS-B: rt 3.13 min, m/z 396.3 [M + H].sup.+. A Compoun was subjected to column chromatography 33 [00099] LCMS-B: rt 3.21 min, m/z 406 [M + H].sup.+. B 34 [00100] LCMS-B: rt 3.34 min, m/z 393 [M + H].sup.+. B 35 [00101] LCMS-B: rt 3.31 min, m/z 392 [M + H].sup.+. B 36 [00102] LCMS-B: rt 3.17 min, m/z 410 [M + H].sup.+. B 37 [00103] LCMS-B: rt 3.16 min, m/z 396 [M + H].sup.+. B 38 [00104] LCMS-B: rt 3.13 min, m/z 392 [M + H].sup.+. B 39 [00105] LCMS-B: rt 3.22 min, m/z 407 [M + H].sup.+. B 40 [00106] LCMS-B: rt 3.47 min, m/z 401 [M + H].sup.+. B 41 [00107] LCMS-B: rt 3.48 min, m/z 401 [M + H].sup.+. B 42 [00108] LCMS-B: rt 3.18 min, m/z 407 [M + H].sup.+. B 43 [00109] LCMS-B: rt 3.18 min, m/z 361 [M + H].sup.+. B

Example 3

2-Chloro-N-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)isonicotinamide 44

[0474] ##STR00110##

[0475] To 2-chloroisonicotinic acid (0.23 mmol, 1 equiv) in CH.sub.3CN (2 mL) was added DIPEA (120 μL, 0.69 mmol, 3 equiv) and HATU (131 mg, 0.345 mmol, 1.5 equiv). 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol A4 (51 mg, 0.23 mmol, 1 equiv) in DMF (1 mL) was added and the reaction stirred at room temperature for 16 h. The reaction was quenched by the addition of a 1M aqueous solution of NaOH (2 mL) and then stirred for 3 h. The reaction was extracted with DCM (3×3 mL) utilising a phase separation cartridge and the combined organic layers reduced under a stream of air. An equivalent volume of MeOH was added and the solution purified by solid-phase extraction (1 g SCX-2 cartridge, 3 column volumes of methanol followed by 3 column volumes of 0.2 M methanolic ammonia). The organic solvent was removed in vacuo and the residue was purified by column chromatography (12 g, 50-100% EtOAc (modified by the addition of 1% v/v of 3.5 M methanolic ammonia) in petroleum benzine followed by 0-50% MeOH in EtOAc modified by the addition of 1% v/v of 2.0 M methanolic ammonia) to give the title compound as a pale yellow oil (8 mg, 10% yield). .sup.1H NMR (400 MHz, MeOD) δ 8.47 (dd, J=5.2, 0.7 Hz, 1H), 7.80 (dd, J=1.5, 0.7 Hz, 1H), 7.68 (dd, J=5.1, 1.5 Hz, 1H), 7.15-7.06 (m, 3H), 7.05-6.95 (m, 1H), 4.03-3.91 (m, 1H), 3.81-3.66 (m, 2H), 3.62-3.48 (m, 2H), 3.00-2.82 (m, 4H), 2.70-2.54 (m, 2H), 1.96-1.82 (m, 1H), 1.78-1.63 (m, 1H).

Example 4

N-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)-3-(morpholine-4-carbonyl)benzamide 45

[0476] ##STR00111##

[0477] (a) Methyl 3-(morpholine-4-carbonyl)benzoate A5

[0478] Morpholine (958 SIL, 11.1 mmol, 1 equiv), mono-methyl isophthalate (2.00 g, 11.1 mmol, 1 equiv), MeCN (50 mL), DIPEA (5.80 mL, 33.3 mmol, 3 equiv) and HATU (4.64 g, 12.2 mmol, 1.1 equiv) were stirred at room temperature. After two hours the mixture was quenched with 5% w/v aqueous sodium carbonate (50 mL) and the organic solvents removed in vacuo. The aqueous residue was extracted with ethyl acetate (3×50 mL), and the pooled organic extracts washed with water (2×50 mL), dried over sodium sulfate and evaporated. Chromatography (40 g silica cartridge, 0-50% ethyl acetate in petroleum benzine) and collection of the suspected product fractions gave the title compound (2.718 g, 98% yield) as a pale brown oil. LCMS-B: rt 3.32 min, m/z 250 [M+H].sup.+.

[0479] (b) 3-(Morpholine-4-carbonyl)benzoic acid A6

[0480] LiOH.H.sub.2O (1.37 g, 32.7 mmol, 3 equiv) was added to a solution of Methyl 3-(morpholine-4-carbonyl)benzoate A5 (2.72 g, 10.9 mmol) in MeOH (20 mL) and water (10 mL) and the resulting suspension was stirred for 16 hours at room temperature. The volatiles were removed in vacuo to give a white solid. Water was added, followed by a 0.5 M aqueous solution of citric acid until the solution was at pH 4. The mixture was stirred for 30 minutes before it was extracted with EtOAc (3×70 mL). The combined organic layers were washed with brine (100 mL), dried (MgSO.sub.4) and concentrated in vacuo to give the title compound (1.74 g, 68% yield) as a white solid. LCMS-B: rt 3.19 min, m/z 236 [m+H].sup.+, 234 [M−H].sup.−.

[0481] (c) N-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)-3-(morpholine-4-carbonyl)benzamide 45

[0482] 3-(Morpholine-4-carbonyl)benzoic acid A6 (54 mg, 0.23 mmol, 1 equiv) in CH.sub.3CN (2 mL) was added DIPEA (120 μL, 0.69 mmol, 3 equiv) and HATU (131 mg, 0.345 mmol, 1.5 equiv). 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol A4 (51 mg, 0.23 mmol, 1 equiv) in DMF (1 mL) was added and the reaction stirred at room temperature for 16 h. The reaction was quenched by the addition of a 1M aqueous solution of NaOH (2 mL) and then stirred for 3 h. DCM (3 mL) was added, the layers mixed thoroughly and then passed through a phase separation cartridge (3 mL). The aqueous layer was further extracted with DCM (3 mL) and the organic layers collected by passage through a phase separation cartridge (2 repeats). The combined DCM layers were concentrated by a stream of air. An equivalent volume of MeOH was added and the solution purified by solid-phase extraction (1 g SCX-2 cartridge, 3 column volumes of methanol followed by 3 column volumes of 0.2 M methanolic ammonia). The crude material was purified by column chromatography (12 g SiO.sub.2 cartridge, 60-100% EtOAc (modified by the addition of 1% v/v 3.5 M methanolic ammonia) in petroleum benzine followed by 0-20% methanol in EtOAc (modified by the addition of 1% v/v 3.5 M methanolic ammonia) to give the title compound. LCMS-B: rt 3.16 min, m/z 438.3 [M+H].sup.+.

Example 5

24(1-acetylpiperidin-4-yl)amino)-N-(4-(3,4-dihydroisoquinolin-2(1H)-yl)-3-hydroxybutyl)isonicotinamide 46

[0483] ##STR00112##

[0484] 2-((1-Acetylpiperidin-4-yl)amino)isonicotinic acid I11 (30 mg, 0.115 mmol, 1 equiv) and triethylamine (0.096 mL, 0.69 mmol) were dissolved in DCM. The mixture was cooled to 0° C. and a 1.0 M solution of isopropyl chloroformate in toluene (0.23 mL, 0.23 mmol) added. The mixture was stood for thirty minutes then a 0.40 M solution of 4-amino-1-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol A4 in DCM (0.50 mL, 0.20 mmol) added. The mixture stood at room temperature for 17 hours and was then diluted with methanol (1 mL), loaded onto a 2 g SCX cartridge, washed with methanol (15 mL) and eluted with 3.5 M ammonia in methanol (15 mL). The basic eluent was concentrated and purified by column chromatography (12 g SiO.sub.2 cartridge, 60-100% EtOAc (modified by the addition of 1% v/v 3.5 M methanolic ammonia) in petroleum benzine followed by 0-45% methanol in EtOAc (modified by the addition of 1% v/v 3.5 M methanolic ammonia) to give the title compound as a colourless oil. LCMS-B: rt 3.03 min, m/z 466.3 [M+H].sup.+.

[0485] Assays

[0486] PRMT5 Biochemical Assay

[0487] Compounds of the invention may be tested for in vitro activity in the following assay:

[0488] 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 Hiss-MEP50 in insect cells and purified via Nickel immobilized metal affinity and gel filtration chromatography (“the enzyme”).

[0489] The 6 μL assay reactions are run in Greiner brand black 384-well low volume 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, 200 nM 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° C. 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 min 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 % l data vs. compound concentration [I] to % I=(A+((B−A)/(1+((C/[I])̂D)))) where A is the lower asymptote, B is the upper asymptote, C is the IC.sub.50 value, and D is the slope.

TABLE-US-00004 Example Number IC.sub.50 (μM) 1 0.359

[0490] PRMT5 Biomarker Assay

[0491] Compounds of the invention may be tested for potency to inhibit symmetrical demethylation of arginine in the following assay:

[0492] The cell line TE11 was seeded at a density of 12,000 cells per well in 96 well tissue culture plates in DME medium and 10% foetal bovine serum, and allowed to adhere overnight under standard culture conditions (37° C., 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 positive controls receiving a potent PRMT5 inhibitor. The concentration of the inhibitor had been previously determined to give maximum inhibition of the methylation. After incubation for 72 h, cells were washed twice in ice-cold PBS, lysed in lysis buffer (20 mM Tris pH 7.4, 135 mM NaCl, 1.5 mM MgCl.sub.2, 1mM EGTA, 10% glycerol and 1% Triton-X100), centrifuged at 15,000×g and the supernatants collected for subsequent analysis. The methylation level was determined using the EpiQuik™ Global Di-Methyl Histone H4R3 Quantification ELISA Kit (Epigentek, Farmingdale, N.Y.) as per the manufacturer's recommendations; in parallel the total protein amount in the lysate was quantified using a Lowry protein assay. The methylation level was corrected for the total protein amount of each sample, normalised to the controls, and the data fitted against a four-parameter logistic model to determine the 50% inhibitory concentration (IC.sub.50).

TABLE-US-00005 Compound Number IC.sub.50 (μM) 1 0.0404

[0493] Revised PRMT5 Biochemical Assay

[0494] Compounds of the invention may be tested for in vitro activity in the following assay:

[0495] 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 Hiss-MEP50 in insect cells and purified via Nickel immobilized metal affinity and gel filtration chromatography (“the enzyme”).

[0496] 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])̂D)))) where A is the lower asymptote, B is the upper asymptote, C is the IC.sub.50 value, and D is the slope.

TABLE-US-00006 Compound Number IC.sub.50 (μM) 2 1.132 3 0.925 4 51.680 5 1.331 6 13.103 7 1.941 8 3.089 9 4.351 10 3.982 11 39.185 12 1.595 13 7.089 14 0.910 15 3.977 16 13.024 17 15.026 18 9.794 19 0.319 20 0.214 21 0.415 22 7.019 23 1.689 24 25.379 25 6.350 26 3.746 27 5.621 28 12.294 29 10.792 30 3.121 31 3.164 32 3.542 33 7.452 34 56.096 35 1.214 36 1.198 37 1.186 38 2.883 39 1.220 40 0.769 41 5.286 42 2.834 43 105.667 44 13.686 45 5.228 46 0.186

[0497] Revised PRMT5 Biomarker Assay

[0498] Compounds of the invention may be tested for potency to inhibit symmetrical dimethylation of arginine in the following assay:

[0499] 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 positive 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 methylation level information was corrected for the number of cells as expressed by the Hoechst 33342 stain, converted to percent inhibition relative to controls on the same plate and the data fitted against a four-parameter logistic model to determine the 50% inhibitory concentration (IC.sub.50 plate-reader based). Alternatively, 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. The resulting mean intensity per cell was directly converted to percent inhibition as outlined above (IC.sub.50, imager based)).

TABLE-US-00007 IC.sub.50 (μM) plate- Compound Number reader based 1 0.318 19 0.089 20 0.505 36 0.023 40 0.048 46 0.007