Supporting Information A second generation of hydroxyethylamine (HEA) BACE-1 inhibitors: optimizing potency and oral bioavailability. Nicolas Charrier, Brian Clarke, Leanne Cutler, Emmanuel Demont,* Colin Dingwall, Rachel Dunsdon, Philip East, Julie Hawkins, Colin Howes, Ishrut Hussain, Philip Jeffrey, Graham Maile, Rosalie Matico, Julie Mosley, Alan Naylor, Alistair O’Brien, Sally Redshaw, Paul Rowland, Virginie Soleil, Kathrine J. Smith, Sharon Sweitzer, Pam Theobald, David Vesey, Daryl S. Walter, Gareth Wayne. Neurology and Gastrointestinal Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Park, Harlow, Essex, CM19 5AW, U.K.
* To whom correspondence should be addressed. Current address: GlaxoSmithKline R&D, Medicines Research Centre, Stevenage, SG1 2NY, Hertfordshire, UK. Phone: 44 (0) 1438-764319. Fax: 44 (0) 1438-768322. E-mail:
[email protected].
Contents: Experimental Section: Synthetic Procedures of Compounds 1, 8a-f and 9b-f: S2-S20 Biology Experimental: S21 X-Ray crystallography: S22 Tabulated Analytical Data for Compounds 1, 8a-f and 9b-f: S23 LCMS traces of compounds 1, 8a-f and 9b-f.: S24-S34 1
H and 13C NMR of compound 8e: S35
S1
General: All solvents were purchased from Romil Ltd (Hy-Dry anhydrous solvents) and commercially available reagents were used as received. Melting points were recorded on Buchi B-545 apparatus and are uncorrected. All reactions were followed by TLC analysis (TLC plates GF254, Merck) or LCMS (liquid chromatography mass spectrometry) using a Waters ZQ instrument. NMR spectra were recorded on a Bruker AVANCE 400 spectrometer and are referenced as follows: 1H (400 MHZ), internal standard TMS at = 0.00; 13C (100.6 MHz), internal standard CDCl3 at = 77.23 or DMSO-D6 at = 39.70. Column chromatography was performed on pre-packed silica gel columns (30-90 mesh, IST). Mass spectra were recorded on Waters ZQ (ESI-MS) and Q-Tof 2 (HRMS) spectrometers. Mass Directed Auto Prep was performed on a Waters 2767 with a MicroMass ZQ Mass Spectrometer using Supelco LCABZ++ column.
LCMS analysis of all chemical intermediates was conducted on a Sunfire C18 column (30 mm x 4.6 mm i.d. 3.5µm packing diameter) at 30 degrees centigrade. The solvents employed were: A = 0.1% v/v solution of Formic Acid in Water. B = 0.1% v/v solution of Formic Acid in Acetonitrile. The gradient employed was: Time (min) 0 0.1 4.2 4.8 4.9 5.0
Flow Rate (ml/min) 3 3 3 3 3 3
%A
%B
97 97 0 0 97 97
3 3 100 100 3 3
The UV detection was an averaged signal from wavelength of 210 nm to 350 nm and mass spectra was recorded on a mass spectrometer using alternate-scan positive and negative electrospray ionisation. Compounds 1, 8a-f and 9b-f LCMS analysis was conducted on a Supelco ABZ+ column (33 mm x 4.6 mm i.d. 3.0µm packing diameter) at 22 degrees centigrade. The solvents employed were: A = 10mM aqueous Ammonium Acetate 0.1% v/v solution of Formic Acid. B = 95% CH3CN + 5% of a 0.1% v/v solution of Formic Acid in Water. The gradient employed was identical to the one used for intermediates.
Methyl 7-[(ethenylsulfonyl)amino]-3-ethyl-1H-indole-5-carboxylate (3)
S2
O S O
H N
O
O HN
To a solution of methyl 7-amino-3-ethyl-1H-indole-5-carboxylate (1.03 g, 4.74 mmol, 1 equiv) in CH2Cl2 (20 ml) at room temperature were added pyridine (575 µl, 7.11 mmol, 1.5 equiv), DMAP (66 mg, 0.47 mmol, 0.1 equiv), then 2-chloroethanesulfonyl chloride (545 µl, 5.22 mmol, 1.1 equiv). The resulting mixture was stirred for 5 min then diluted with AcOEt. The organic phase was washed with a 2N HCl aqueous solution then dried over MgSO4 and concentrated in vacuo. The residue was dissolved in CH2Cl2 (20 ml) and NEt3 (1 ml, excess) was added and the resulting solution stirred for 16 h then diluted with AcOEt. The organic phase was washed with a 2N HCl aqueous solution and brine, then dried over MgSO4 and concentrated in vacuo to give the title compound (1.7 g, 110%) which was used in the next step without further purification. A small sample was purified for analytical purpose by flash chromatography on silica gel (iso-hexane/AcOEt: 9/1 to 3/1). White solid; mp = 184-186 oC. MS (Electrospray): m/z = 309.0 [M+H]+. R. T. = 2.93 min. (> 99%). 1 H (400 MHz, CD6SO): = 1.26 (t, J = 7.6 Hz, 3H), 2.72 (q, J = 7.6 Hz, 2H), 3.85 (s, 3H), 5.98 (d, J = 10.0 Hz, 1H), 6.00 (d, J = 9.6 Hz, 1H), 6.84 (dd, J = 10.0, 9.6 Hz, 1H), 7.25 (s, 1H), 7.61 (s, 1H), 8.05 (s, 1H), 9.68 (bs, 1H), 10.91 (bs, 1H). 13 C (100.6 MHz, CD6SO): = 14.4, 17.5, 51.7, 115.9, 118.4, 118.7, 120.0, 121.1, 123.7, 127.7, 128.0, 133.1, 135.8, 166.8. HRMS (EI): m/z calcd for C14H17N2O4S: 309.0904; found: 309.0903. 7-Ethyl-1-methyl-3,4-dihydro-1H-[1,2,5]thiadiazepino[3,4,5-hi]indole-9-carboxylic acid 2,2-dioxide (4) O
O O S N
OH
N
Step 1: Methyl 7-ethyl-1-methyl-3,4-dihydro-1H-[1,2,5]thiadiazepino[3,4,5-hi]indole9-carboxylate 2,2-dioxide. O
O O S N
O
N
To a solution of methyl 7-amino-3-ethyl-1H-indole-5-carboxylate (124 mg, 0.4 mmol, 1 equiv) in DMF (10 ml) at room temperature was added NaH (60% in mineral oil, 19 mg, 0.45 mmol, 1.2 equiv). After 5 min, the mixture was heated to 100oC for 1 h and then cooled to room temperature. Ethanol (1 ml) was added and the solution dissolved in AcOEt. The organic phase was washed with a 2N HCl aqueous solution, dried over MgSO4 and concentrated in vacuo to give methyl 7-ethyl-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indole-9-carboxylate 2,2-dioxide (95 mg, 77%) which
S3
was used in the next stage without further purification. To a solution of methyl 7ethyl-3,4-dihydro-1H-[1,2,5]thiadiazepino[3,4,5-hi]indole-9-carboxylate 2,2-dioxide (191 mg, 0.62 mmol, 1equiv) in DMF (10 ml) at room temperature were added NaH (60% in mineral oil, 50 mg, 1.24 mmol, 2 equiv) and after 2 min, MeI (46 µl, 0.74 mmol, 1.2 equiv). The resulting mixture was stirred at room temperature for 30 min then EtOH (1 ml) was added and the solution concentrated in vacuo. The residue was dissolved in AcOEt and the organic phase was washed with H2O, dried over MgSO4 and concentrated in vacuo. Purification of the residue by flash chromatography on silica gel (iso-hexane/AcOEt: 3/1 to 1/1) gave the title compound (152 mg, 76%). White solid; mp = 197-198 oC. MS (Electrospray): m/z = 323.0 [M+H]+. R. T. = 2.78 min. (> 99%). 1 H (400 MHz, CD6SO): = 1.26 (t, J = 7.6 Hz, 3H), 2.73 (q, J = 7.6 Hz, 2H), 3.44 (s, 3H), 3.87 (s, 3H), 4.01-4.04 (m, 2H), 4.52-4.55 (m, 2H), 7.3 (s, 1H), 7.62 (d, J = 1.2 Hz, 1H), 8.10 (d, J = 1.2 Hz, 1H). 13 C (100.6 MHz, CD6SO): = 14.3, 17.4, 39.5, 42.6, 51.9, 56.1, 118.4, 118.8, 119.7, 121.4, 126.5, 129.3, 129.7, 134.4, 166.5. HRMS (EI): m/z calcd for C15H19N2O4S: 323.1060; found: 323.1058. Step 2: 7-Ethyl-1-methyl-3,4-dihydro-1H-[1,2,5]thiadiazepino[3,4,5-hi]indole-9carboxylic acid 2,2-dioxide (4). O
O O S N
OH
N
To a solution of methyl 7-ethyl-1-methyl-3,4-dihydro-1H-[1,2,5]thiadiazepino[3,4,5hi]indole-9-carboxylate 2,2-dioxide (135 mg, 0.42 mmol, 1 equiv) in EtOH (20 ml) was added 2N aqueous NaOH solution (20 ml, 40 mmol, 95 equiv). The resulting mixture was stirred for 14 h at room temperature then most of EtOH was removed in vacuo. The residue was extracted with Et2O. The aqueous layer was acidified using a 2N HCl aqueous solution and the white precipitate formed was extracted twice with AcOEt. The combined organic phases were dried over MgSO4 and concentrated in vacuo to give the title compound (74 mg, 57%) which was used in the next step without further purification. White solid; mp > 295 oC. MS (Electrospray): m/z = 308.9 [M+H]+. R. T. = 2.27 min. (> 99%). 1 H (400 MHz, CD6SO): = 1.26 (t, J = 7.6 Hz, 3H), 2.73 (q, J = 7.6 Hz, 2H), 3.43 (s, 3H), 4.00-4.03 (m, 2H), 4.51-4.54 (m, 2H), 7.3 (s, 1H), 7.62 (d, J = 1.6 Hz, 1H), 8.10 (d, J = 1.6 Hz, 1H). 12.5 (bs, 1H). 13 C (100.6 MHz, CD6SO): = 14.3, 17.3, 39.4, 42.5, 56.1, 118.2, 119.0, 119.7, 122.5, 126.2, 129.0, 129.6, 134.2, 167.5. HRMS (EI): m/z calcd for C14H17N2O4S: 309.0904; found: 309.0900.
S4
3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5-(ethylamino)benzoic acid. O S O
O
N
OH
NH
Step 1: Methyl 3-amino-5-nitrobenzoate. O
H2N
O
NO2
To a solution of 3-amino-5-nitrobenzoic acid (5.0 g, 27.5 mmol, 1 equiv) in MeOH (200 ml) was added thionyl chloride (2.1 ml, 28.8 mmol, 1.05 equiv) dropwise. The resulting mixture was refluxed for 16 h then cooled to room temperature and concentrated in vacuo. The residue was dissolved in AcOEt and the organic phase was washed with a saturated NaHCO3 aqueous solution then brine, dried over MgSO4 and concentrated in vacuo to give methyl 3-amino-5-nitrobenzoate (5.11 g, 95%) as an orange solid which was used in the next step without further purification. MS (Electrospray): m/z = 197.0 [M+H]+. R. T. = 2.25 min (> 99%) 1 H (400 MHz, CD6SO): = 3.87 (s, 3H), 6.17 (s, 2H), 7.55 (s, 1H), 7.59 (s, 1H), 7.74 (s, 1H). 13 C (100.6 MHz, CD6SO): = 52.5, 109.6, 110.7, 119.5, 131.3, 148.7, 150.5, 165.0. Step 2: Methyl 3-{bis[(4-chlorobutyl)sulfonyl]amino}-5-nitrobenzoate. Cl
Cl SO2
O
N SO2
O
NO2
Synthesis of 4-chloro-1-butanesulfonyl chloride: To a mixture of 1,2-oxathiane 2,2-dioxide (commercially available from Aldrich, 50 g, 367 mmol, 1 equiv) and SOCl2 (29 ml, 401 mmol, 1.1 equiv) was added DMF (4 ml, 51.6 mmol, 0.14 equiv). The resulting mixture was stirred under nitrogen at 70ºC for 3 days. A second portion of SOCl2 (10 ml, 137 mmol, 0.37 equiv) was added. The mixture was stirred at 70ºC for another 3 days and then cooled to room temperature and concentrated in vacuo. The residue was diluted with toluene then concentrated in vacuo. This procedure was repeated and the residue was dried under vacuum for 16 h to give crude 4-chloro-1-butanesulfonyl chloride (63 g, 90%) as black oil which was used in the next step without further purification.
S5
1
H (400 MHz, CDCl3): (t, J = 7.6 Hz, 2H).
= 2.01 (m, 2H), 2.25 (m, 2H), 3.61 (t, J = 6.0 Hz, 2H), 3.73
To a solution of methyl 3-amino-5-nitrobenzoate (1.96 g, 10 mmol, 1 equiv) and NEt3 (4.2 ml, 30 mmol, 3 equiv) at 0oC in CH2Cl2 (60 ml) was added 4-chloro-1butanesulfonyl chloride (4.8 g, 25 mmol, 2.5 equiv) in CH2Cl2 (20 ml) via canula. The resulting mixture was stirred at room temperature for 2 h then NEt3 (2.1 ml, 15 mmol, 1.5 equiv) and 4-chloro-1-butanesulfonyl chloride (2.4 g, 12.5 mmol, 1.25 equiv) in CH2Cl2 (5 ml) were added. After 1 h, the aqueous phase was washed with a 2N aqueous HCl solution then H2O, dried over MgSO4 and concentrated in vacuo. Purification of the residue by flash chromatography on silica gel (iso-hexane/AcOEt: 9/1 to 1/1) gave methyl 3-{bis[(4-chlorobutyl)sulfonyl]amino}-5-nitrobenzoate (4.27 g, 84%) as a pale brown solid. MS (Electrospray): m/z = 349.0 [M-H-SO2(CH2)4Cl]-. R. T. = 3.39 min (> 98%). 1 H (400 MHz, CDCl3): = 1.92-1.98 (m, 4H), 2.06 2.14 (m, 4H), 3.58 (t, J = 6.4 Hz, 4H), 3.66 (t, J = 7.6 Hz, 4H), 4.02 (s, 3H), 8.36 (dd, J = 2.4, 1.6 Hz, 1H), 8.43 (dd, J = 2.4, 2.0 Hz, 1H), 8.96 (dd, J = 2.0, 1.6 Hz, 1H). 13 C (100.6 MHz, CDCl3): = 20.7, 30.5, 43.6, 53.3, 55.1, 126.0, 129.9, 133.1, 135.2, 137.5, 148.6, 163.6. Step 3: Methyl 3-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5-nitrobenzoate. O O
S O N
O
NO2
To a solution of methyl 3-{bis[(4-chlorobutyl)sulfonyl]amino}-5-nitrobenzoate (1.51 g, 3 mmol, 1 equiv) in MeOH (30 ml) was added NEt3 (920 µl, 6.6 mmol, 2.2 equiv) and the resulting mixture was refluxed for 5 h. NEt3 (920 µl, 6.6 mmol, 2.2 equiv) was added and the mixture refluxed for 6 h then cooled to room temperature. The precipitate formed was filtered off and washed with MeOH to give the title compound (1.21 g, 80 %) as a white solid. MS (Electrospray): m/z = 315.0 [M+H]+. R. T. = 2.49 min (> 99%) 1 H (400 MHz, CD6SO): = 1.84-1.90 (m, 2H), 2.16-2.22 (m, 2H), 3.42-3.45 (m, 2H), 3.81-3.85 (m, 2H), 3.94 (s, 3H), 8.20 (s, 1H), 8.32 (s, 1H), 8.50 (s, 1H). 13 C (100.6 MHz, CD6SO): =23.3, 23.7, 50.0, 52.5, 53.0, 121.0, 124.4, 131.4, 131.9, 141.8, 148.0, 163.9. HRMS (EI): m/z calcd for C12H15N2O6S: 315.06453; found: 315.06396. Step 4: Methyl 3-amino-5-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2-yl)benzoate. O S O
O
N
O
NH2
A mixture of ethyl 3-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5-nitrobenzoate (500 mg, 1.59 mmol, 1 equiv), NH4COOH (1.0 g, 15.9 mmol, 10 equiv) and Pd on S6
charcoal (10% w/w, 50% wet, 100 mg, 10% w/w) in MeOH/H2O 9/1 (20 ml) was stirred at 80oC for 2 h then cooled to room temperature. The catalyst was filtered off and most of the MeOH was removed in vacuo. The residue was partitioned between AcOEt and H2O and the two layers were separated. The organic phase was dried over MgSO4 and concentrated in vacuo. Trituration of the residue with Et2O gave the title compound (379 mg, 82%) as a white solid which was used in the next step without further purification. MS (Electrospray): m/z = 285.0 [M+H]+. R. T. = 1.92 min. (> 99%) 1 H (400 MHz, CD6SO): = 1.76-1.82 (m, 2H), 2.11-2.17 (m, 2H), 3.24-3.27 (m, 2H), 3.55-3.58 (m, 2H), 3.80 (s, 3H), 5.58 (bs, 2H), 6.75 (s, 1H), 6.98 (s, 1H), 7.11 (s, 1H). 13 C (100.6 MHz, CD6SO): = 23.7, 23.8, 50.1, 51.9, 53.1, 112.7, 114.3, 116.3, 130.5, 141.4, 149.6, 166.1. HRMS (EI): m/z calcd for C12H17N2O4S: 285.09035; found: 285.09003. Step 5: Methyl 3-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)benzoate. O S O
O
N
O
NH
To a solution of methyl 3-amino-5-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2yl)benzoate (1.0 g, 3.52 mmol, 1 equiv) in (CH2Cl)2 at room temperature were added acetaldehyde (240 µl, 4.22 mmol, 1.2 equiv), AcOH (200 µl, 3.53 mmol, 1 equiv) and NaHB(OAc)3 (1.04 g, 4.93 mmol, 1.4 equiv) and the resulting mixture was stirred for 16 h. Further acetaldehyde (120 µl, 2.11 mmo, 0.6 equiv), AcOH (100 µl, 1.76 mmol, 0.5 equiv) and NaHB(OAc)3 (500 mg, 2.46 mmol, 2 equiv) were added and the mixture stirred for a further 10 h. The solvant was removed in vacuo and the residue disolved in AcOEt. The organic phase was washed with a saturated NaHCO3 aqueous solution, dried over MgSO4 and concentrated in vacuo. Purification of the residue by flash chromatography on silica gel (iso-hexane/AcOEt: 3/1 to 2/3) gave the title compound (870 mg, 79%) as a white solid. MS (Electrospray): m/z = 313.1 [M+H]+. R. T. = 2.40 min (> 99%) 1 H (400 MHz, CDCl3): = 1.25 (t, J = 7.2 Hz, 3H), 1.87-1.93 (m, 2H), 2.30-2.36 (m, 2H), 3.18 (q, J = 7.2 Hz, 2H), 3.17-3.21 (m, 2H), 3.71-3.74 (m, 2H), 3.88 (s, 3H), 6.76 (dd, J = 2.4, 2.0 Hz, 1H), 7.17 (dd, J = 2.4, 1.6 Hz, 1H), 7.27 (dd, J = 2.0, 1.6 Hz, 1H). 13 C (100.6 MHz, CDCl3): = 14.6, 24.2, 24.5, 38.4, 50.7, 52.2, 53.5, 112.3, 115.7, 116.0, 131.8, 141.5, 149.0, 166.8. HRMS (EI): m/z calcd for C14H21N2O4S: 313.12165; found: 313.12126. Step 6: 3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5-(ethylamino)benzoic acid. O S O
O
N
OH
NH
S7
To a solution of methyl 3-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)benzoate (870 mg, 2.79 mmol, 1 equiv) in MeOH (10 ml) at room temperature was added NaOH (2N in H2O, 2.8 ml, 2 equiv) and the resulting mixture was stirred for 4 h. Most of the MeOH was removed in vacuo and the residue partitioned between AcOEt and a 2N HCl aqueous solution. The layers were separated and the organic phase was dried over MgSO4 and concentrated in vacuo. Trituration of the residue with Et2O gave the title compound (750 mg, 90%) as a white solid. MS (Electrospray): m/z = 299.1 [M+H]+. R. T. = 1.62 min (> 99%). 1 H (400 MHz, CD6SO): = 1.16 (t, J = 7.2 Hz, 3H), 1.77-1.83 (m, 2H), 2.11-2.17 (m, 2H), 3.04 (q, J = 7.2 Hz, 2H), 3.24-3.27 (m, 2H), 3.58-3.62 (m, 2H), 6.00 (bs, 1H), 6.68 (dd, J = 2.4, 2.0 Hz, 1H), 7.02 (dd, J = 2.0, 1.2 Hz, 1H), 7.06 (dd, J = 2.4, 1.2 Hz, 1H), 12.8 (bs, 1H). 13 C (100.6 MHz, CD6SO): = 14.1, 23.7, 23.8, 37.2, 50.1, 53.2, 110.8, 114.3, 114.4, 131.9, 141.4, 149.4, 167.3. HRMS (EI): m/z calcd for C13H20N2O4S: 299.1058; found: 299.1060.
(2R,3S)-3-Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol bis(4-methylbenzenesulfonate)(7a)
H2N
CF3
N H
OH
2
SO3H
Step 1: 1,1-Dimethylethyl [(1S,2R)-2-hydroxy-1-(phenylmethyl)-3-({[3(trifluoromethyl)phenyl]methyl}amino)propyl]carbamate (6a). O O
N H
OH
N H
CF3
To a solution of 1,1-dimethylethyl {(1S)-1-[(2S)-2-oxiranyl]-2-phenylethyl}carbamate (2.63 g, 10 mmol, 1 equiv) in EtOH (40 ml) was added {[3(trifluoromethyl)phenyl]methyl}amine (4.2 ml, 30 mmol, 3 equiv) and the resulting mixture was refluxed for 4 h then cooled to room temperature and concentrated in vacuo. The residue was dissolved in AcOEt and the organic phase was washed twice with a saturated NaHCO3 aqueous solution, dried over MgSO4 and concentrated in vacuo to give 1,1-dimethylethyl [(1S,2R)-2-hydroxy-1-(phenylmethyl)-3-({[3(trifluoromethyl)phenyl]methyl}amino)propyl]carbamate (3.32 g, 76%) as a white solid which was used in the next step without further purification. Step 2: (2R,3S)-3-Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2butanol bis(4-methylbenzenesulfonate) (7a).
S8
H2N OH
CF3
N H
2
SO3H
To a solution of 1,1-dimethylethyl [(1S,2R)-2-hydroxy-1-(phenylmethyl)-3-({[3(trifluoromethyl)phenyl]methyl}amino)propyl]carbamate (3.32 g, 7.6 mmol, 1equiv) in CH3CN (40 ml) was added tosic acid monohydrate (4.33 g, 22.8 mmol, 3 equiv) and the resulting mixture was stirred at room temperature for 5 h. The precipitate formed was filtered off and washed with Et2O to give (2R,3S)-3-amino-4-phenyl-1({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol bis(4methylbenzenesulfonate) (4.3 g, 83%) as a white solid. MS (Electrospray): m/z = 339.2 [M+H]+. R. T. = 0.92 min 1 H (400 MHz, CD6SO): = 2.29 (s, 6H), 2.80-2.92 (m, 3H), 3.17-3.21 (m, 1H), 3.523.57 (m, 1H), 4.03-4.08 (m, 1H), 4.28 (bs, 2H), 6.14 (d, J = 5.6 Hz, 1H), 7.12 (d, J = 7.6 Hz, 4H), 7.28-7.36 (m, 5H), 7.49 (d, J= 7.6 Hz, 4H), 7.69 (dd, J = 8.4, 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H),7.93 (s, 1H), 7.96 (bs, 3H), 9.01 (bs, 1H), 9.08 (bs, 1H). 13 C (100.6 MHz, CD6SO): = = 20.7, 33.1, 47.6, 49.6, 54.8, 65.7, 124.0 (q, J = 272.5 Hz), 125.4, 125.7 (q, J = 3.7 Hz), 126.8 (q, J = 3.4 Hz), 126.9, 128.0, 128.6, 129.1 (q, J = 31.7 Hz), 129.2, 129.7, 132.8, 134.4, 135.9, 137.7, 145.2. (2R,3S)-3-Amino-4-phenyl-1-[(phenylmethyl)amino]-2-butanol bis(4methylbenzenesulfonate) (7b)
2
H2N OH
N H SO3H
(2R,3S)-3-Amino-4-phenyl-1-[(phenylmethyl)amino]-2-butanol bis(4methylbenzenesulfonate) was obtained as a white solid from 1,1-dimethylethyl {(1S)1-[(2S)-2-oxiranyl]-2-phenylethyl}carbamate in an analogous manner than (2R,3S)-3amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol bis(4methylbenzenesulfonate), using benzylamine instead of {[3(trifluoromethyl)phenyl]methyl}amine (65%, 2 steps). MS (Electrospray): m/z = 271.1 [M+H]+. R. T. = 1.29 min. 1 H (400 MHz, CD6SO): = 2.29 (s, 6H), 2.81-2.89 (m, 3H), 3.11-3.14 (m, 1H), 3.513.55 (m, 1H), 4.06 (d, J = 10.4 Hz, 1H), 4.14-4.18 (m, 2H), 6.11 (bs, 1H), 7.13 (d, J = 7.6 Hz, 4H), 7.27-7.36 (m, 5H), 7.42-7.47 (m, 5H), 7.49 (d, J= 7.6 Hz, 4H), 7.97 (bs, 3H), 8.89 (bs, 1H), 9.00 (bs, 1H). 13 C (100.6 MHz, CD6SO): = 20.8, 33.1, 47.3, 50.2, 54.9, 65.7, 125.5, 127.0, 128.1, 128.7, 128.9, 129.1, 129.3, 130.2, 131.3, 135.9, 137.9, 145.3.
S9
(2R,3S)-3-amino-4-phenyl-1-[(1,1,5-trimethylhexyl)amino]-2-butanol bis(4methylbenzenesulfonate) (7c) 2
H2N OH
N H
SO3H
(1,1,5-Trimethylhexyl)amine was obtained using the procedure described by Berg, S. S.; Cowling, D. T. Acylation of 2,2,6,6-Tetramethylpiperidine and 2,2,5,5Tetramethylpyrrolidine, J. Chem. Soc. (C), 1971, 1653-1658. (2R,3S)-3-amino-4-phenyl-1-[(1,1,5-trimethylhexyl)amino]-2-butanol bis(4methylbenzenesulfonate) was obtained as a white solid from 1,1-dimethylethyl {(1S)1-[(2S)-2-oxiranyl]-2-phenylethyl}carbamate in an analogous manner than (2R,3S)-3Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol bis(4methylbenzenesulfonate), using (1,1,5-trimethylhexyl)amine instead of {[3(trifluoromethyl)phenyl]methyl}amine (59%, 2 steps). MS (Electrospray): m/z = 307.3 [M+H]+. R. T. = 1.18 min. 1 H (400 MHz, CD6SO): = 0.87 (d, J = 6.8 Hz, 6H), 1,10-1.16 (m, 2H), 1.22 (s, 6H), 1.21-1.25 (m, 2H), 1.46-1.55 (m, 3H), 2.29 (s, 6H), 2.78-2.83 (m, 1H), 2.87 (dd, J = 19,2 and 7.2 Hz, 1H), 2.97 (dd, J = 19,2 and 7.2 Hz, 1H), 3.02-3.07 (m, 1H), 3.583.62 (m, 1H), 3.92-3.97 (m, 1H), 6.00 (d, J = 6.0 Hz, 1H), 7.12 (d, J = 7.6 Hz, 4H), 7.29-7.38 (m, 5H), 7.49 (d, J= 7.6 Hz, 4H), 7.98 (bs, 3H), 8.11 (bs, 1H), 8.39 (bs, 1H). 13 C (100.6 MHz, CD6SO): = 20.7, 22.3, 22.5, 22.6, 27.1, 33.1, 36.9, 38.5, 42.4, 54.9, 59.4, 66.2, 125.4, 126.9, 128.0, 128.6, 129.2, 135.9, 137.8, 145.1. (2R,3S)-3-Amino-1-(cyclohexylamino)-4-phenyl-2-butanol bis(4methylbenzenesulfonate) (7d)
H2N OH
2
N H
SO3H
((2R,3S)-3-Amino-1-(cyclohexylamino)-4-phenyl-2-butanol bis(4methylbenzenesulfonate) was obtained as a white solid from 1,1-dimethylethyl {(1S)1-[(2S)-2-oxiranyl]-2-phenylethyl}carbamate in an analogous manner than (2R,3S)-3Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol bis(4methylbenzenesulfonate), using cyclohexylamine instead of {[3(trifluoromethyl)phenyl]methyl}amine (61%, 2 steps). MS (Electrospray): m/z = 263.3 [M+H]+. R. T. = 1.13 min. 1 H (400 MHz, CD6SO): = 1.01-1.35 (m, 5H), 1.59 (d, J = 12.4 Hz, 1H), 1.75 (d, J = 12 Hz, 2H), 1.93-1.99 (m, 2H), 2.29 (s, 6H), 2.81-3.02 (m, 4H), 3.14-3.18 (m, 1H), 3.52-3.57 (m, 1H), 3.98-4.02 (m ,1H), 6.08 (d, J = 5.6 Hz, 1H), 7.12 (d, J = 7.6 Hz, 4H), 7.27-7.38 (m, 5H), 7.49 (d, J= 7.6 Hz, 4H), 7.97 (bs, 3H), 8.37 (bs, 1H), 8.46 (bs, 1H). S10
13
C (100.6 MHz, CD6SO): = 20.7, 23.9, 24.5, 28.5, 33.1, 44.7, 54.9, 56.3, 65.7, 125.4, 126.9, 128.0, 128.6, 129.2, 135.9, 137.8, 145.2. (2R,3S)-3-Amino-1-(cyclopropylamino)-4-phenyl-2-butanol bis(4methylbenzenesulfonate) (7e)
H2N
2
N H
OH
SO3H
(2R,3S)-3-Amino-1-(cyclopropylamino)-4-phenyl-2-butanol bis(4methylbenzenesulfonate) was obtained as a white solid from 1,1-dimethylethyl {(1S)1-[(2S)-2-oxiranyl]-2-phenylethyl}carbamate in an analogous manner than (2R,3S)-3Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol bis(4methylbenzenesulfonate), using cyclopropylamine instead of {[3(trifluoromethyl)phenyl]methyl}amine (53%, 2 steps). MS (Electrospray): m/z = 221.1 [M+H]+. R. T. = 1.08 min. 1 H (400 MHz, CD6SO): = 0.71 0.79 (m, 3 ), 0.83 0.87 (m, 1H), 2.29T (s, 6H), 2.69 (bs, 1H), 2.84 (dd, J = 14.0 and 7.6 Hz, 1H), 2.91 (dd, J = 14.0 and 7.6 Hz, 1H), 3.00 (t, J = 11.6 Hz, 1H), 3.29 (d, J = 13.2 Hz, 1H), 3.53 (bs, 1H), 4.03 (bs, 1H), 6.10 (d, J = 5.6 Hz, 1H), 7.13 (d, J = 7.6 Hz, 4H), 7.28-7.31 (m, 1H), 7.32-7.38 (m, 4H), 7.50 (d, J= 7.6 Hz, 4H), 7.97 (bs, 3H), 8.74 (bs, 2H). 13 C (100.6 MHz, CD6SO): = 3.0, 20.8, 29.8, 33.2, 48.6, 55.1, 65.3, 125.5, 127.0, 128.1, 128.7, 129.3, 136.0, 137.9, 145.3. Phenylmethyl [(2R,3S)-3-amino-2-hydroxy-4-phenylbutyl]methylcarbamate hydrochloride (7f) O
HCl H2N
N
O
OH
Step 1:1,1-Dimethylethyl [(1S,2R)-2-hydroxy-3-(methylamino)-1(phenylmethyl)propyl]carbamate.
O O
N H
NH OH
1,1-Dimethylethyl [(1S,2R)-2-hydroxy-3-(methylamino)-1(phenylmethyl)propyl]carbamate was obtained as a white solid from 1,1dimethylethyl {(1S)-1-[(2S)-2-oxiranyl]-2-phenylethyl}carbamate in an analogous manner than 1,1-dimethylethyl [(1S,2R)-2-hydroxy-1-(phenylmethyl)-3-({[3(trifluoromethyl)phenyl]methyl}amino)propyl]carbamate, using methylamine (2N in MeOH, 10 equiv) instead of {[3-(trifluoromethyl)phenyl]methyl}amine (74%). MS (Electrospray): m/z = 295.1 [M+H]+. R. T. = 2.12 min.
S11
1
H (400 MHz, CD6SO): = 1.14 and 1.26 (s, 9 ), 2.27 (s, 3 ), 2.43 (dd, J = 14.0, 7.6 Hz, 1H), 2.49-2.56 (m, 3H), 2.98 (dd, J = 14.0 Hz, 3.6 Hz, 1H), 3.40-3.45 (m, 1H), 3.51-3.53 (m, 1H), 4.62-4.95 (bs, 1H), 6.65 (d, J = 8.8 Hz, 1H), 7.12-7.25 (m, 5H). 13 C (100.6 MHz, CD6SO): =28.1, 36.0, 36.2, 54.7, 55.1, 71.5, 77.2, 125.5, 127.7, 129.0, 139.7, 155.2. Step 2: 1,1-Dimethylethyl [(1S,2R)-2-hydroxy-3(methyl{[(phenylmethyl)oxy]carbonyl}amino)-1-(phenylmethyl)propyl]carbamate.
O
O
N H
O
N
O
OH
To a solution of 1,1-dimethylethyl [(1S,2R)-2-hydroxy-3-(methylamino)-1(phenylmethyl)propyl]carbamate (950 mg, 3.23 mmol, 1 equiv) in CH2Cl2 (10 ml) was added pyridine (390 µl, 4.84 mmol, 1.5 equiv) then phenylmethyl chloridocarbonate (500 µl, 3.55 mmol, 1.1 equiv) and the resulting mixture was stirred at room temperature for 4h. The organic solution was washed with a 2N HCl aqueous solution, a saturated NaHCO3 aqueous solution then brine, dried over MgSO4 and concentrated in vacuo. Purification of the residue by flash chromatography on silica gel (iso-hexane/AcOEt: 9/1 to 3/1) gave 1,1-dimethylethyl [(1S,2R)-2-hydroxy3-(methyl{[(phenylmethyl)oxy]carbonyl}amino)-1-(phenylmethyl)propyl]carbamate (1.3 g, 94%) as a white foam. MS (Electrospray): m/z = 429.2 [M+H]+. R. T. = 2.83 min. 1 H (400 MHz, CDCl3): = 1.35 (s, 9 ), 2.70 2.87 (m, 1 ), 2.98 (s, 3H), 3.33-3.41 (m, 2H), 3.72-3.88 (m, 2H), 4.18-4.24 (m, 1H), 4.62-4.72 (m, 1H), 5.14 (s, 2H), 7.12 (bs, 1H), 7.17-7.35 (m, 10H). 13 C (100.6 MHz, CDCl3): =28.3, 35.7, 36.0, 53.1, 55.0, 67.5, 73.0, 79.6, 126.3, 127.8, 128.1, 128.4, 128.5, 129.5, 136.5, 137.9, 156.2, 158.1. Step 3: Phenylmethyl [(2R,3S)-3-amino-2-hydroxy-4-phenylbutyl]methylcarbamate hydrochloride (7f). O
H2N
N
O
. HCl
OH
To a solution of 1,1-dimethylethyl [(1S,2R)-2-hydroxy-3(methyl{[(phenylmethyl)oxy]carbonyl}amino)-1-(phenylmethyl)propyl]carbamate (1.3 g, 3 mmol, 1 equiv) in Dioxan (10 ml) was added HCl (4N in Dioxan, 7.5 ml, 10 equiv) and the resulting mixture was stirred at room temperature for 12 h then concentrated in vacuo. The residue was co-evaporated with toluene then triturated with Et2O to give phenylmethyl [(2R,3S)-3-amino-2-hydroxy-4phenylbutyl]methylcarbamate hydrochloride (950 mg, 87%) as a white solid. MS (Electrospray): m/z = 329.3 [M+H]+. R. T. = 1.26 min.
S12
1
H (400 MHz, CDCl3): = 2.85 (s, 3 ), 2.95 3.27 (m, 3H), 3.70-3.76 (m, 2H), 4.25 (bs, 1H), 4.89-4.99 (m, 2H), 5.23 (bs, 1H), 7.12-7.36 (m, 10 H), 8.09 (bs, 3H). 13 C (100.6 MHz, CDCl3): =33.5, 35.4, 51.0, 56.3, 67.5, 68.0, 127.2, 127.6, 128.0, 128.5, 129.0, 129.4, 135.8, 136.3, 157.3.
General procedure for amide coupling: To a solution of acid (0.3 mmol, 1 equiv) in CH2Cl2 or DMF (4 ml) were added EDAC.HCl (69 mg, 0.36 mmol, 1.2 equiv), HOBT (49 mg, 0.36 mmol, 1.2 equiv) then N-Ethyl morpholine (150 µl, 1.2 mmol, 4 equiv) followed by the appropriate amine di-tosylate (0.3 mmol, 1 equiv). The resulting mixture was stirred at room temperature for 16 h, and then diluted with CH2Cl2. The organic phase was washed with a saturated NaHCO3 aqueous solution, H2O then dried over MgSO4 and concentrated in vacuo. Purification of the residue by Mass-Directed Auto-Prep gave the corresponding formate salt amide (> 99 % purity) as foam. (2R,3S)-3-{[(7-Ethyl-1-methyl-2,2-dioxido-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indol-9-yl)carbonyl]amino}-2-hydroxy-4-phenyl-N-{[3(trifluoromethyl)phenyl]methyl}-1-butanaminium formate (8a) O
O O S N
N H
+
OH H
N H
CF3
N
O H
O
76% Yield. MS (Electrospray): m/z = 629.2 [M+H]+. R. T. = 3.09 min. 1 H (400 MHz, CDCl3): = 1.29 (t, J = 7.2 Hz, 3H), 2.69 (q, J = 7.2 Hz, 2H), 2.83 (dd, J = 12.4, 5.2 Hz, 1H), 2.93 (dd, J = 12.4, 3.2 Hz, 1H), 3.03 (dd, J = 8.0, 6.0 Hz, 1H), 3.14 (dd, J = 8.0, 5.2 Hz, 1H), 3.46 (s, 3H), 3.70-3.77 (m, 1H), 3.81-3.86 (m, 2H), 3.90-4.30 (b, 3H), 3.92 (d, J = 13.2 Hz, 1H), 4.02 (d, J = 13.2 Hz, 1H), 4.30-4.39 (m, 1H), 4.40-4.46 (m, 2H), 6.58 (d, J = 8.4 Hz, 1H), 6.83 (s, 1H), 7.18-7.22 (m, 1H), 7.23-7.30 (m, 4H), 7.35 (s, 1H), 7.46 (dd, J = 8.0, 8.0 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.67 (s, 1H), 7.70 (s, 1H), 8.49 (s, 1H). 13 C (100.6 MHz, CDCl3): = = 14.3, 17.9, 36.4, 39.4, 43.6, 50.7, 52.6, 53.4, 56.9, 70.4, 117.5, 117.8, 120.2, 124.0 (q, J = 272.9 Hz), 124.9 (q, J = 3.9 Hz), 125.8 (q, J = 3.9 Hz), 126.0, 126.4, 126.8, 127.8, 128.7, 129.3, 129.5, 130.6, 131.0 (q, J = 32.4 Hz), 132.4, 133.9, 137.2, 137.4, 168.4, 169.3. HRMS (EI): m/z calcd for C32H36F3N4O4S: 629.2404; found: 629.2395. (2R,3S)-3-{[(7-Ethyl-1-methyl-2,2-dioxido-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indol-9-yl)carbonyl]amino}-2-hydroxy-4-phenyl-N(phenylmethyl)-1-butanaminium formate (8b)
S13
O
O O S N
N
N H
+
OH H H
N O H
O
70% Yield. MS (Electrospray): m/z = 561.3 [M+H]+. R. T. = 2.88 min. 1 H (400 MHz, CDCl3): = 1.30 (t, J = 7.6 Hz, 3H), 2.70 (q, J = 7.6 Hz, 2H), 2.84 (dd, J = 12.4, 5.6 Hz, 1H), 3.01 (dd, J = 12.4, 3.6 Hz, 1H), 3.05 (dd, J = 14.4, 8.0 Hz, 1H), 3.14 (dd, J = 14.4, 4.8 Hz, 1H), 3.45 (s, 3H), 3.5-4.0 (bs, 3H), 3.72-3.78 (m, 1H), 3.82-3.87 (m, 2H), 3.91 (d, J = 13.2 Hz, 1H), 4.03 (d, J = 13.2 Hz, 1H), 4.35-4.43 (m, 1H), 4.42-4.48 (m, 2H), 6.63 (d, J = 8.4 Hz, 1H), 6.83 (s, 1H), 7.19-7.24 (m, 1H), 7.28-7.41 (m, 10 H), 7.41 (s, 1H), 8.56 (s, 1H). 13 C (100.6 MHz, CDCl3): = 14.3, 18.0, 36.4, 39.5, 43.6, 50.4, 52.8, 53.1, 56.9, 70.0, 110.0, 117.6, 117.8, 120.2, 126.0, 126.4, 126.8, 127.8, 128.5, 128.7, 128.9, 129.2, 129.5, 130.6, 133.9, 137.4, 168.4, 169.7. HRMS (EI): m/z calcd for C31H37N4O4S: 561.2530; found: 561.2528. N-((2R,3S)-3-{[(7-Ethyl-1-methyl-2,2-dioxido-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indol-9-yl)carbonyl]amino}-2-hydroxy-4phenylbutyl)cyclohexanaminium formate (8c) O
O
O S N
N H
N
+
OH H H
N
O H
O
64% Yield. MS (Electrospray): m/z = 553.3 [M+H]+. R. T. = 3.18 min. 1 H (400 MHz, CDCl3): = 1.08 1.30 (m, 3H), 1.26 (t, J = 7.6 Hz, 3H), 1.32-1.45 (m, 2H), 1.61 (d, J = 11.2 Hz, 2H), 1.77 (d, J = 11.2 Hz, 2H), 2.06 (t, J = 9.6 Hz, 2H), 2.68 (q, J = 7.6 Hz, 2H), 2.81-3.02 (m, 3H), 3.20-3.26 (m, 2H), 3.41 (s, 3H), 3.80-3.84 (m, 2H), 4.05-4.12 (m, 1H), 4.37-4.45 (m, 2H), 6.80 (s, 1H), 7.10-7.26 (m, 5H), 7.47 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 8.62 (s, 1H). 13 C (100.6 MHz, CDCl3): =14.2, 17.9, 24.5, 24.9, 29.4, 36.6, 39.4, 43.6, 48.6, 53.2, 56.8, 58.0, 69.7, 117.7, 118.3, 120.4, 125.8, 126.3, 126.5, 127.6, 128.5, 129.4, 130.6, 133.9, 137.7, 168.3, 169.9. HRMS (EI): m/z calcd for C30H41N4O4S: 553.2843; found: 553.2842. N-((2R,3S)-3-{[(7-Ethyl-1-methyl-2,2-dioxido-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indol-9-yl)carbonyl]amino}-2-hydroxy-4-phenylbutyl)2,6-dimethyl-2-heptanaminium formate (8d)
S14
O
O O S N
N
N H
OH H
+
H
O
N O
H
68% Yield. MS (Electrospray): m/z = 597.3 [M+H]+. R. T. = 2.91 min. 1 H (400 MHz, CDCl3): = 0.81 (d, J = 6.8 Hz, 6 ), 1.07 1.14 (m, 2 ), 1.24 1.32 (m, 12H), 1.49 (sp, J = 6.8 Hz, 1H), 1.52-1.60 (m, 2H), 2.73 (q, J = 7.6 Hz, 2H), 2.84-3.00 (m, 2H), 3.15 (dd, J = 12.4, 2.0 Hz, 1H), 3.25 (dd, J = 12.4, 4.4 Hz, 1H), 3.41 (s, 3H), 3.78-3.83 (m, 2H), 4.10-4.14 (m, 1H), 4.38-4.43 (m, 2H), 4.41-4.51 (m, 1H), 6.80 (s, 1H), 7.04-7.25 (m, 5H), 7.45 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 8.61 (s, 1H). 13 C (100.6 MHz, CDCl3): = 14.2, 17.9, 21.3, 22.5, 23.6, 27.7, 36.3, 38.5, 38.9, 39.4, 43.6, 45.2, 53.1, 56.8, 59.2, 69.6, 117.7, 118.2, 120.4, 125.8, 126.4, 126.5, 127.6, 128.5, 129.5, 130.6, 13.9, 137.7, 168.2, 169.6. HRMS (EI): m/z calcd for C33H49N4O4S: 597.3469; found: 597.3461. N-[(1S,2R)-1-Benzyl-3-(cyclopropylamino)-2-hydroxypropyl]-7-ethyl-1-methyl-3,4dihydro-1H-[1,2,5]thiadiazepino[3,4,5-hi]indole-9-carboxamide 2,2-dioxide (8e) O
O
O S N
N H
N H
OH
N
60% Yield. MS (Electrospray): m/z = 511.2 [M+H]+. R. T. = 2.67 min. 1 H (400 MHz, CD6SO): = 0.20 0.23 (m, 2H), 0.32-0.35 (m, 2H), 1.27 (t, J = 7.6 Hz, 3H), 2.07-2.10 (m, 1H), 2.30 (bs, 1H), 2.62-2.79 (m, 4H), 2.83 (dd, J = 13.6, 10.4 Hz, 1H), 3.11 (dd, J = 13.6, 3.6 Hz, 1H), 3.41 (s, 3H), 3.57-3.64 (m, 1H), 3.97-4.01 (m, 2H), 4.11-4.18 (m, 1H), 4.45-4.49 (m, 2H), 4.91 (d, J = 5.6 Hz, 1H), 7.12 (t, J = 6.8 Hz, 1H), 7.18-7.25 (m, 5H), 7.49 (s, 1H), 7.92 (s, 1H), 8.26 (d, J = 8.8 Hz, 1H). 13 C (100.6 MHz, CD6SO) : = 6.2, 14.4, 17.5, 30.2, 35.7, 38.8, 42.5, 52.3, 54.4, 55.8, 71.5, 117.1, 117.5, 117.9, 1 25.6, 125.9, 126.5, 127.8, 128.7, 129.1, 129.5, 133.3, 139.7, 165.8. HRMS (EI): m/z calcd for C27H35N4O4S: 511.2373; found: 511.2371. (2R,3S)-3-{[(7-Ethyl-1-methyl-2,2-dioxido-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indol-9-yl)carbonyl]amino}-2-hydroxy-N-methyl-4phenyl-1-butanaminium formate (8f) O
O O S N
N H
N
+
OH H H
O
N
H
S15
O
Step 1: Phenylmethyl ((2R,3S)-3-{[(7-ethyl-1-methyl-2,2-dioxido-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indol-9-yl)carbonyl]amino}-2-hydroxy-4phenylbutyl)methylcarbamate. O
O
O
O S N
N
N H
O
OH
N
This compound was obtained using the general procedure for amide coupling using phenylmethyl [(2R,3S)-3-amino-2-hydroxy-4-phenylbutyl]methylcarbamate hydrochloride as amine. The crude material was used in the next step without purification. Step 2: (2R,3S)-3-{[(7-Ethyl-1-methyl-2,2-dioxido-3,4-dihydro-1H[1,2,5]thiadiazepino[3,4,5-hi]indol-9-yl)carbonyl]amino}-2-hydroxy-N-methyl-4phenyl-1-butanaminium formate (8f). To a solution of the crude material from Step 1 in MeOH (5 ml) was added Pd on charcoal (10% w/w, 50 mg) and the resulting mixture was stirred under an atmosphere of Hydrogen (105 Pa) for 12 h. The catalyst was filtered off and the solution concentrated in vacuo. The residue was purified by Mass-Directed Auto-Prep to give the title compound as white foam (40%, 2 steps). MS (Electrospray): m/z =485.3 [M+H]+. R. T. = 2.59 min. 1 H (400 MHz, CDCl3): = 1.28 (t, J = 7.6 H, 3H), 2.61 (s, 3H), 2.68 (q, J = 7.6 Hz, 2H), 2.87-2.95 (m, 2H), 3.12 (d, J = 11.6 Hz, 1H), 3.21 (dd, J = 13.2 Hz, 3.6 Hz, 1H), 3.42 (s, 3H), 3.77-3.84 (m, 2H), 3.87-3.93 (m, 1H), 4.31-4.38 (m, 2H), 4.37-4.43 (m, 2H), 4.50-5.00 (bs, 2H), 6.81 (s, 1H), 7.15-7.26 (m, 6H), 7.44 (s, 1H), 7.79 (s, 1H), 8.57 (bs, 1H). 13 C (100.6 MHz, CDCl3): = 14.3, 17.9, 33.8, 36.2, 39.4, 43.5, 53.0, 53.2, 56.9, 69.6, 117.6, 118.3, 120.3, 125.9, 126.2, 126.7, 127.8, 128.6, 129.4, 130.6, 133.9, 137.6, 168.5, 169.9. HRMS (EI): m/z calcd for C25H33N4O4S: 485.2217; found: 485.2216. (2R,3S)-3-({[3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-4-phenyl-N-{[3(trifluoromethyl)phenyl]methyl}-1-butanaminium formate (1) O S O
O
N
N
N H
+
OH H H O
NH H
S16
O
CF3
77% Yield. MS (Electrospray): m/z = 619.2 [M+H]+. R. T. = 2.94 min. 1 H (400 MHz, CDCl3): = 1.20 (t, J = 8.8 Hz, 3H), 1.81-1.87 (m, 2H), 2.25-2.34 (m, 2H), 2.79 (dd, J = 12.4, 6.0 Hz, 1H), 2.87-2.96 (m, 2H), 3.04 (q, J = 8.8 Hz, 2H), 3.03-3.08 (m, 1H), 3.10-3.17 (m, 2H), 3.59-3.65 (m, 2H), 3.76-3.80 (m, 1H), 3.90 (d, J = 13.2 Hz, 1H), 3.97 (d, J = 13.2 Hz, 1H), 4.26-4.33 (m, 1H), 4.0-4.4 (bs, 4H), 6.59 (s, 1H), 6.71 (s, 1H), 6.82 (s, 1H), 6.98 (d, J = 8.8 Hz, 1H), 7.14-7.30 (m, 5H), 7.43 (dd, J = 7.6, 7.6 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.65 (s, 1H), 8.46 (s, 1H). 13 C (100.6 MHz, CDCl3): = 14.5, 24.2, 24.5, 36.2, 38.2, 50.6, 50.7, 52.5, 53.6, 53.9, 70.2, 110.4, 113.2, 114.2, 123.8 (q, J = 275.2 Hz), 124.8 (q, J = 4.0 Hz), 125.8 (q, J = 4.0 Hz), 126.6, 128.6, 129.2, 129.4, 130.9 (q, J = 30.2 Hz), 132.4, 135.7, 137.4, 137.7, 141.5, 149.2, 168.0, 169.5. HRMS (EI): m/z calcd for C31H39F3N4O4S: 619.2560; found: 619.2551. (2R,3S)-3-({[3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-4-phenyl-N-(phenylmethyl)-1butanaminium formate (9b) O S O
O
N
N H
NH
+
N
OH H H
O H
O
73% Yield. (Electrospray): m/z = 551.2 [M+H]+. R. T. = 2.75 min. 1 H (400 MHz, CDCl3): = 1.20 (t, J = 7.2 Hz, 3H), 1.81-1.87 (m, 2H), 2.26-2.33 (m, 2H), 2.80 (dd, J = 12.4, 6.8 Hz, 1H), 2.86-2.98 (m, 2H), 3.01-3.06 (m, 1H), 3.06 (q, J = 7.2 Hz, 2H), 3.12-3.19 (m, 2H), 3.58-3.63 (m, 2H), 3.68-3.72 (m, 1H), 3.89 (d, J = 13.2 Hz, 1H), 3.96 (d, J = 13.2 Hz, 1H), 4.26-4.30 (m, 1H), 4.0-4.7 (bs, 2H), 4.304.38 (m, 2H), 6.60 (s, 1H), 6.72 (s, 1H), 6.82 (s, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.127.42 (m , 10H), 8.49 (s, 1H). 13 C (100.6 MHz, CDCl3): = 14.6, 24.2, 24.5, 36.1, 38.3, 50.5, 50.7, 52.6, 53.6, 53.7, 69.8, 110.4, 113.2, 114.2, 125.0, 126.5, 128.6, 128.8, 129.2, 129.4, 134.8, 135.7, 137.8, 141.5, 149.2, 167.9, 169.6. HRMS (EI): m/z calcd for C30H39N4O4S: 551.2686; found: 551.2682. N-[(2R,3S)-3-({[3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-4-phenylbutyl]cyclohexanaminium formate (9c)
S17
O S O
O
N
N H
NH
+
N
OH H H
O H
O
67% Yield. S (Electrospray): m/z = 543.2 [M+H]+. R. T. = 3.11 min. 1 H (400 MHz, CDCl3): = 1.15 1.28 (m, 6H), 1.34-1.39 (m, 2H), 1.59-1.64 (m, 1H), 1.68-1.89 (m, 5H), 1.98-2.06 (m, 2H), 2.20-2.37 (m, 3H), 2.73-2.82 (m, 1H), 2.822.95 (m, 2H), 3.01-3.11 (m, 4H), 3.10-3.19 (m, 3H), 3.59-3.63 (m, 2H), 3.88-3.95 (m, 1H), 4.29-4.40 (m, 1H), 6.62 (s, 1H), 6.77 (s, 1H), 6.83 (s, 1H), 7.10-7.19 (m, 2H), 7.21-7.28 (m, 5H), 8.59 (s, 1H). 13 C (100.6 MHz, CDCl3): = 14.6, 24.2, 24.5, 24.6, 25.1, 29.9, 36.3, 38.3, 48.5, 50.7, 53.1, 53.6, 57.8, 69.5, 110.5, 113.2, 114.4, 126.5, 128.6, 129.5, 135.5, 137.7, 141.6, 149.2, 176.8, 169.8. HRMS (EI): m/z calcd for C29H43N4O4S: 543.2999; found: 543.2996. N-[(2R,3S)-3-({[3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-4-phenylbutyl]-2,6-dimethyl-2heptanaminium formate (9d) O S O
O
N
N H
NH
+
N
OH H H
O H
O
71% Yield. MS (Electrospray): m/z = 587.4 [M+H]+. R. T. = 2.75 min. 1 H (400 MHz, CDCl3): = 0.84 (d, J = 6.8 Hz, 6H), 1.08-1.14 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H), 1.23 (s, 3H), 1.25 (s, 3H), 1.27-1.31 (m, 2H), 1.47-1.53 (m, 3H), 1.81-1.85 (m, 2H), 2.25-2.30 (m, 2H), 2.80 (dd, J = 12.4, 7.6 Hz, 1H), 2.89 (dd, J = 12.4, 8.8 Hz, 1H), 3.01-3.07 (m, 3H), 3.13-3.20 (m, 3H), 3.61-3.64 (m, 2H), 3.80-4.10 (bs, 1H), 3.97-4.04 (m, 1H), 4.28-4.41 (m, 1H), 6.61 (s, 1H), 4.50-5.50 (bs, 2H), 6.83 (s, 1H), 6.85 (s, 1H), 7.12-7.18 (m, 2H), 7.21-7.26 (m, 4H), 7.67 (d, J = 8.0 Hz, 1H), 8.60 (s, 1H). 13 C (100.6 MHz, CDCl3): = 14,5, 21.4, 22.5, 24.0, 24.2, 24.4, 27.8, 36.4, 38.2, 38.8, 39.0, 45.0, 50.6, 53.4, 53.5, 58.4, 69.6, 110.7, 113.1, 114.1, 126.4, 128.5, 129.5, 135.6, 137.9, 14.6, 149.2, 167.8, 169.7. HRMS (EI): m/z calcd for C32H52N4O4S: 587.3625; found: 587.3619. N-[(2R,3S)-3-({[3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-4-phenylbutyl]cyclopropanaminium formate (9e)
S18
O S O
O
N
N
N H
+
OH H H O
NH H
O
62% Yield. MS (Electrospray): m/z = 501.1 [M+H]+. R. T. = 2.54 min. 1 H (400 MHz, CDCl3): = 0.66 (d, J = 7.2 Hz, 2H), 0.79 (s, 2H), 1.21 (t, J = 6.8 Hz, 3H), 1.83-1.89 (m, 2H), 2.27-2.32 (m, 2H), 2.35-2.39 (m, 1H), 2.90-3.00 (m, 2H), 3.06-3.27 (m, 5H), 3.62-3.64 (m, 2H), 3.82-3.86 (m, 1H), 4.0-4.3 (bs, 4H), 4.27-4.35 (m, 2H), 6.61 (s, 1H), 6.76 (s, 1H), 6.82 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.16-7.20 (m, 1H), 7.25-7.29 (m, 4H), 8.49 (s, 1H). 13 C (100.6 MHz, CDCl3): = 4.7, 14.6, 24.2, 24.5, 30.8, 36.2, 38.3, 50.7, 51.9, 53.5, 53.6, 69.6, 110.4, 113.2, 114.4, 126.6, 128.6, 129.5, 135.6, 137.7, 141.5, 149.2, 167.9, 169.2. HRMS (EI): m/z calcd for C26H37N4O4S: 501.2530; found: 501.2529. (2R,3S)-3-({[3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-N-methyl-4-phenyl-1-butanaminium chloride (9f) O S O
O
N
N H
+
N
OH H H
Cl NH
Step 1: Phenylmethyl [(2R,3S)-3-({[3-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-4-phenylbutyl]methylcarbamate. The title compound was obtained using the procedure described in Step 1 for the synthesis of inhibitor 8e. The crude material was purified by Mass-Directed AutoPrep. O S O
O
O
N
N H
N
O
OH
NH
Step 2: (2R,3S)-3-({[3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5(ethylamino)phenyl]carbonyl}amino)-2-hydroxy-N-methyl-4-phenyl-1-butanaminium chloride (9f).
S19
O S O
O
N
N H
NH
+
N
OH H H
Cl
The product from Step 1 was hydrogenated according to the procedure described for inhibitor 8e (Step 2). The crude material was dissolved in dioxan and treated with HCl (2N in dioxan, 1 ml, excess) to give the title compound as a white solid (> 99%). 45%, 2 Steps. MS (Electrospray): m/z = 475.3 [M+H]+. R. T. = 2.41 min. 1 H (400 MHz, CD6SO): = 1.15 (t, J = 7.2 Hz, 3H), 1.78-1.82 (m, 2H), 2.12-2.18 (m, 2H), 2.57 (s, 3H), 2.81-2.92 (m, 2H), 2.98-3.07 (m, 3H), 3.14 (dd, J = 14, 2.8 Hz, 1H), 3.23-3.27 (m, 2H), 3.53-3.57 (m, 2H), 3.78-3.89 (m, 1H), 4.00-4.11 (m, 1H), 5.885.94 (m, 2H), 6.62 (s, 1H), 6.77 (s, 1H), 6.80 (s, 1H), 7.11-7.16 (m, 1H), 7.20-7.28 (m, 4H), 8.23 (d, J = 8.8 Hz, 1H)), 8.37 (bs, 1H), 8.56 (bs, 1H). 13 C (100.6 MHz, CDCl3): = 14.5, 24.2, 33.9, 35.6, 38.3, 48.8, 50.6, 52.7, 53.5, 54.2, 69.7, 111.3, 113.6, 114.3, 126.4, 128.4, 129.4, 135.3, 138.1, 141.5, 149.1, 167.7. HRMS (EI): m/z calcd for C24H35N4O4S: 475.2374; found: 475.2373.
S20
Enzyme Assays
BACE-1, BACE-2 and Cathepsin D activity and inhibition were assayed using a fluorescent FAM-[SEVNLDAEFK]-TAMRA substrate. Briefly, increasing concentrations of compound were incubated with 5mM FAM-[SEVNLDAEFK]TAMRA substrate and either 1nM BACE-1, 25nM BACE-2 or 0.036 units/ml Cathepsin D (Calbiochem) at pH 4.5 for 4hr (BACE-1) or 1hr (BACE-2, Cathepsin D) at room temperature. The BACE-1 and BACE-2 preparations used were recombinant Fc fusion proteins of the ectodomains of these proteins stably expressed in CHO cells and purified from the medium of these cells using immobilised protein A, as described previously ((a) Hussain, I.; Powell, D. J.; Howlett, D. R.; Chapman, G. A.; Gilmour, L.; Murdock, P. R.; Tew, D. G.; Meek, T. D.; Chapman, C.; Schneider, K.; Ratcliffe, S. J.; Tattersall, D.; Testa, T. T.; Southan, C.; Ryan, D. M.; Simmons, D. L.; Walsh, F. S.; Dingwall, C.; Christie, G. (2000). ASP1 (BACE2) Cleaves the Amyloid Precursor Protein at the -Secretase Site. Mol. Cell. Neurosci. 16: 409-419. (b) Charlwood, J.; Dingwall, C.; Matico, R.; Hussain, I.; Johanson, K.; Moore, S.; Powell, D. J.; Skehel, J. M.; Ratcliffe, S.; Clarke, B.; Trill, J.; Sweitzer, S.; Camilleri, P. (2001). Characterization of the Glycosylation Profiles of Alzheimer’s -Secretase Protein Asp-2 Expressed in a Variety of Cell Lines. J. Biol. Chem. 276, 1673916748). Control reactions with no enzyme were included in each assay. Quantification of substrate cleavage was assessed using an LJL analyst spectrophotometer (485nm excitation, 535nm emission). Cell treatments and A measurements
Human neuroblastoma cells expressing human Amyloid Precursor Protein (APP) wild type sequence (SHSY5Y-APPwt) were cultured in DMEM Hams F12 (Invitrogen) containing 10% foetal bovine serum, 2mM glutamine and 400µg/ml geneticin (Invitrogen) at 37oC with 5% CO2. For treatment with inhibitor, cells were cultured to confluency in 96-well plates in medium lacking geneticin. Compounds were dissolved in DMSO and added to cells over a range of concentrations, ensuring that the final DMSO concentration was 98% 619.2 619.2551 1 76% 100% 629.2 629.2395 8a 70% 100% 561.3 561.2528 8b 64% 100% 553.3 553.2842 8c 70% 100% 597.3 597.3461 8d 60% >98% 511.2 511.2371 8e 40% >98% 485.3 485.2216 8f 73% 100% 551.2 551.2682 9b 67% 100% 543.2 543.2996 9c 71% 100% 587.4 587.3619 9d 61% >99% 501.1 501.2529 9e 45% 100% 475.3 475.2373 9f Table 1. Analytical data for 1, 8a-f and 9b-f: Overall yield, purity, MS (electrospray, [M+H]+), HRMS (EI, [M+H]+).
S23
LCMS traces LCMS trace Compound 1:
S24
LCMS trace Compound 8a:
S25
LCMS trace Compound 8b:
S26
LCMS trace Compound 9b:
S27
LCMS trace Compound 8c:
S28
LCMS trace Compound 9c:
S29
LCMS trace Compound 8d:
S30
LCMS trace Compound 9d:
S31
LCMS trace Compound 8e:
S32
LCMS trace Compound 9e:
S33
LCMS trace Compound 8f:
S34
1H and 13C NMR of compound 8e:
S35
