Second generation of hydroxyethylamine BACE-1 inhibitors: Optimizing potency and oral bioavailability

Article abstract of DOI:10.1021/jm800138h

BACE-1 inhibition has the potential to provide a disease-modifying therapy for the treatment of Alzheimer's disease. Optimization of a first generation of BACE-1 inhibitors led to the discovery of novel hydroxyethylamines (HEAs) bearing a tricyclic nonprime side. These derivatives have nanomolar cell potency and are orally bioavailable.

Full text of DOI:10.1021/jm800138h

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:
emmanuel.h.demont@gsk.com.
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
1H and 13C NMR of compound 8e: S35
S 1

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
1
AVANCE 400 spectrometer and are referenced as follows: H (400 MHZ), internal
13
standard TMS at δ = 0.00; C (100.6 MHz), internal standard CDCl3 at δ = 77.23 or
DMSO-D6 at δ = 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:
Flow Rate
(ml/min)
Time (min)
% A
% B
0
3
3
3
3
3
3
97
97
0
0
97
97
3
3
100
100
3
0.1
4.2
4.8
4.9
5.0
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)
S 2

O
H
N
S
O
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%).
1H (400 MHz, CD6SO): δ = 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).
13C (100.6 MHz, CD6SO): δ = 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
N
O
S
OH
N
Step 1: Methyl 7-ethyl-1-methyl-3,4-dihydro-1H-[1,2,5]thiadiazepino[3,4,5-hi]indole-
9-carboxylate 2,2-dioxide.
O
O
N
O
S
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
S 3

was used in the next stage without further purification. To a solution of methyl 7-
ethyl-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%).
1H (400 MHz, CD6SO): δ = 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).
13C (100.6 MHz, CD6SO): δ = 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-9-
carboxylic acid 2,2-dioxide (4).
O
O
N
O
S
OH
N
To a solution of methyl 7-ethyl-1-methyl-3,4-dihydro-1H-[1,2,5]thiadiazepino[3,4,5-
hi]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%).
1H (400 MHz, CD6SO): δ = 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).
13C (100.6 MHz, CD6SO): δ = 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.
S 4

3-(1,1-Dioxidotetrahydro-2H-1,2-thiazin-2-yl)-5-(ethylamino)benzoic acid.
O
O
S
N
O
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%)
1H (400 MHz, CD6SO): δ = 3.87 (s, 3H), 6.17 (s, 2H), 7.55 (s, 1H), 7.59 (s, 1H), 7.74
(s, 1H).
13C (100.6 MHz, CD6SO): δ = 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
O
SO2
N
O
SO2
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.
S 5

1H (400 MHz, CDCl3): δ = 2.01 (m, 2H), 2.25 (m, 2H), 3.61 (t, J = 6.0 Hz, 2H), 3.73
(t, J = 7.6 Hz, 2H).
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-1-
butanesulfonyl 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%).
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = 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
S
N
O
O
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%)
1H (400 MHz, CD6SO): δ = 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).
13C (100.6 MHz, CD6SO): δ =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
O
S
N
O
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
S 6

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%)
1H (400 MHz, CD6SO): δ = 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).
13C
(100.6
MHz,
CD6SO):
δ = 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
O
S
N
O
O
NH
To a solution of methyl 3-amino-5-(1,1-dioxidotetrahydro-2H-1,2-thiazin-2-
yl)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%)
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = 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
O
S
N
O
OH
NH
S 7

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%).
1H (400 MHz, CD6SO): δ = 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).
13C (100.6 MHz, CD6SO): δ = 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)
CF3
H2N
N
H
2
OH
SO3H
Step 1: 1,1-Dimethylethyl [(1S,2R)-2-hydroxy-1-(phenylmethyl)-3-({[3-
(trifluoromethyl)phenyl]methyl}amino)propyl]carbamate (6a).
O
CF3
O
N
N
H
H
OH
To a solution of 1,1-dimethylethyl {(1S)-1-[(2S)-2-oxiranyl]-2-phenylethyl}carbamate
(2.63 g, 10 mmol, equiv) in EtOH (40 ml) was added {[3-
1
(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)-2-
butanol bis(4-methylbenzenesulfonate) (7a).
S 8

CF3
H2N
N
H
2
OH
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
methylbenzenesulfonate) (4.3 g, 83%) as a white solid.
bis(4-
MS (Electrospray): m/z = 339.2 [M+H]+. R. T. = 0.92 min
1H (400 MHz, CD6SO): δ = 2.29 (s, 6H), 2.80-2.92 (m, 3H), 3.17-3.21 (m, 1H), 3.52-
3.57 (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).
13C (100.6 MHz, CD6SO): δ = = 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(4-
methylbenzenesulfonate) (7b)
2
H2N
N
H
OH
SO3H
(2R,3S)-3-Amino-4-phenyl-1-[(phenylmethyl)amino]-2-butanol
bis(4-
methylbenzenesulfonate) 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)-3-
amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol
methylbenzenesulfonate), using benzylamine instead
bis(4-
{[3-
of
(trifluoromethyl)phenyl]methyl}amine (65%, 2 steps).
MS (Electrospray): m/z = 271.1 [M+H]+. R. T. = 1.29 min.
1H (400 MHz, CD6SO): δ = 2.29 (s, 6H), 2.81-2.89 (m, 3H), 3.11-3.14 (m, 1H), 3.51-
3.55 (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).
13C (100.6 MHz, CD6SO): δ = 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.
S 9

(2R,3S)-3-amino-4-phenyl-1-[(1,1,5-trimethylhexyl)amino]-2-butanol bis(4-
methylbenzenesulfonate) (7c)
2
H2N
N
H
SO3H
OH
(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 2,2,5,5-
Tetramethylpyrrolidine, J. Chem. Soc. (C), 1971, 1653-1658.
(2R,3S)-3-amino-4-phenyl-1-[(1,1,5-trimethylhexyl)amino]-2-butanol
bis(4-
methylbenzenesulfonate) 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)-3-
Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol
bis(4-
methylbenzenesulfonate), using (1,1,5-trimethylhexyl)amine
(trifluoromethyl)phenyl]methyl}amine (59%, 2 steps).
instead of {[3-
MS (Electrospray): m/z = 307.3 [M+H]+. R. T. = 1.18 min.
1H (400 MHz, CD6SO): δ = 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), 3.58-
3.62 (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).
13C (100.6 MHz, CD6SO): δ = 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(4-
methylbenzenesulfonate) (7d)
2
H2N
N
H
OH
SO3H
((2R,3S)-3-Amino-1-(cyclohexylamino)-4-phenyl-2-butanol
bis(4-
methylbenzenesulfonate) 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)-3-
Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol
methylbenzenesulfonate), using cyclohexylamine instead
bis(4-
{[3-
of
(trifluoromethyl)phenyl]methyl}amine (61%, 2 steps).
MS (Electrospray): m/z = 263.3 [M+H]+. R. T. = 1.13 min.
1H (400 MHz, CD6SO): δ = 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

13C (100.6 MHz, CD6SO): δ = 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(4-
methylbenzenesulfonate) (7e)
2
H2N
N
H
OH
SO3H
(2R,3S)-3-Amino-1-(cyclopropylamino)-4-phenyl-2-butanol
bis(4-
methylbenzenesulfonate) 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)-3-
Amino-4-phenyl-1-({[3-(trifluoromethyl)phenyl]methyl}amino)-2-butanol
methylbenzenesulfonate), using cyclopropylamine instead
bis(4-
{[3-
of
(trifluoromethyl)phenyl]methyl}amine (53%, 2 steps).
MS (Electrospray): m/z = 221.1 [M+H]+. R. T. = 1.08 min.
1H (400 MHz, CD6SO): δ = 0.71−0.79 (m, 3Η), 0.83−0.87 (m, 1H), 2.29 (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).
13C (100.6 MHz, CD6SO): δ = 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
NH
H
OH
1,1-Dimethylethyl
[(1S,2R)-2-hydroxy-3-(methylamino)-1-
(phenylmethyl)propyl]carbamate was obtained as a white solid from 1,1-
dimethylethyl {(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

1H (400 MHz, CD6SO): δ = 1.14 and 1.26 (s, 9Η), 2.27 (s, 3Η), 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).
13C (100.6 MHz, CD6SO): δ =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
O
N
N
O
H
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-hydroxy-
3-(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.
1H (400 MHz, CDCl3): δ = 1.35 (s, 9Η), 2.70−2.87 (m, 1Η), 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).
13C (100.6 MHz, CDCl3): δ =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
. HCl
H2N
N
O
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-4-
phenylbutyl]methylcarbamate hydrochloride (950 mg, 87%) as a white solid.
MS (Electrospray): m/z = 329.3 [M+H]+. R. T. = 1.26 min.
S12

1H (400 MHz, CDCl3): δ = 2.85 (s, 3Η), 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).
13C (100.6 MHz, CDCl3): δ =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
N
CF3
N+
O
S
N
H
H
H
OH
N
O
H
O
76% Yield. MS (Electrospray): m/z = 629.2 [M+H]+. R. T. = 3.09 min.
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = = 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
N
N+
O
S
N
H
H
H
OH
N
O
H
O
70% Yield. MS (Electrospray): m/z = 561.3 [M+H]+. R. T. = 2.88 min.
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = 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-4-
phenylbutyl)cyclohexanaminium formate (8c)
O
O
N
N+
O
S
N
H
H
H
OH
N
O
H
O
64% Yield. MS (Electrospray): m/z = 553.3 [M+H]+. R. T. = 3.18 min.
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ =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
N
N+
O
S
N
H
H
H
O
OH
N
H
O
68% Yield. MS (Electrospray): m/z = 597.3 [M+H]+. R. T. = 2.91 min.
1H
(400 MHz, CDCl3): δ = 0.81 (d,
J
=
6.8
Hz, 6Η), 1.07−1.14 (m, 2Η), 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).
13C (100.6 MHz, CDCl3): δ = 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-3,4-
dihydro-1H-[1,2,5]thiadiazepino[3,4,5-hi]indole-9-carboxamide 2,2-dioxide (8e)
O
O
N
O
S
N
N
H
H
OH
N
60% Yield. MS (Electrospray): m/z = 511.2 [M+H]+. R. T. = 2.67 min.
1H (400 MHz, CD6SO): δ = 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).
13C (100.6 MHz, CD6SO) :
δ = 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-4-
phenyl-1-butanaminium formate (8f)
O
O
N
N+
O
S
N
H
H
H
OH
O
N
H
O
S15

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-4-
phenylbutyl)methylcarbamate.
O
O
O
N
O
S
N
N
O
H
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-4-
phenyl-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.
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = 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
O
S
N
O
CF3
N+
N
H
H
H
OH
O
NH
H
O
S16

77% Yield. MS (Electrospray): m/z = 619.2 [M+H]+. R. T. = 2.94 min.
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = 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)-1-
butanaminium formate (9b)
O
O
S
N
O
N+
N
H
H
H
OH
NH
O
H
O
73% Yield. (Electrospray): m/z = 551.2 [M+H]+. R. T. = 2.75 min.
1H (400 MHz, CDCl3): δ = 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), 4.30-
4.38 (m, 2H), 6.60 (s, 1H), 6.72 (s, 1H), 6.82 (s, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.12-
7.42 (m , 10H), 8.49 (s, 1H).
13C (100.6 MHz, CDCl3): δ = 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
N
O
O
N+
N
H
H
H
OH
NH
O
H
O
67% Yield. S (Electrospray): m/z = 543.2 [M+H]+. R. T. = 3.11 min.
1H (400 MHz, CDCl3): δ = 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), 2.82-
2.95 (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).
13C (100.6 MHz, CDCl3): δ = 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-2-
heptanaminium formate (9d)
O
O
S
N
O
N+
N
H
H
H
OH
NH
O
H
O
71% Yield. MS (Electrospray): m/z = 587.4 [M+H]+. R. T. = 2.75 min.
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = 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
N
O
O
N+
N
H
H
H
OH
O
NH
H
O
62% Yield. MS (Electrospray): m/z = 501.1 [M+H]+. R. T. = 2.54 min.
1H (400 MHz, CDCl3): δ = 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).
13C (100.6 MHz, CDCl3): δ = 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
O
S
N
O
N+
N
H
H
H
OH
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 Auto-
Prep.
O
O
S
N
O
O
N
N
O
H
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
N
O
O
N+
H
N
H
H
OH
Cl
NH
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.
1H (400 MHz, CD6SO): δ = 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), 5.88-
5.94 (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).
13C (100.6 MHz, CDCl3): δ = 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 β-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 β-Secretase
Protein Asp-2 Expressed in a Variety of Cell Lines. J. Biol. Chem. 276, 16739-
16748). 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 Aβ 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 <0.5%. A DMSO solvent control was included in each
experiment. 24hr later, medium was harvested for Aβ analysis. The effect of
compounds on cellular viability was assessed using the cell proliferation reagent
WST-1 (Roche).
Medium from cells was assayed for Aβ40 or Aβ42 using a sensitive BioVerisTM
immunoassay employing Aβ C-terminal specific antibodies. Briefly, Aβ peptides
were captured using biotinylated 6E10 (Signet Laboratories). C-terminal Aβ40
(G210; The Genetics Company) and Aβ42 (5G5; raised to C-terminal Aβ residues
MVGGVVIA and shown to lack cross-reactivity with Aβ40) antibodies labelled with
a BVTM Ruthenium NHS ester tag were used to detect the specific Aβ species.
Antibody-Aβ complexes were captured with streptavidin coated Dynabeads® (Dynal)
and assayed in a BioVerisTM M384 analyser.
S21

Crystallography
Asp2N153, 172, 223, 354Q/Fc was expressed in CHO secretion system with thrombin
cleavage sites engineered after residues 45 and 460 (pro/thr/Asp2QQQQ/thr/Fc).
Pro/thr/Asp2QQQQ/thr/Fc was captured by ProSep-A High Capacity resin
(Bioprocessing Limited) and neutralized eluate dialyzed into 25mM Hepes, 0.25M
NaCl, pH 7.4. Soluble Asp2QQQQ was released by cleavage with bovine alpha
thrombin (Haematologic Technologies Inc). ProSep A unbound of the thrombin digest
was further truncated by pepsin at pH 4.6. V61-E452 form of Asp2QQQQ was
purified from the neutralized pepsin digest by MonoQ (GE Healthcare) at pH=7.4.
Crystals of apo Asp2 were grown at 20ºC using the hanging drop vapour diffusion
method combined with streak seeding. Protein at 10mg/mL was mixed in a 1:1 ratio
with the reservoir solution consisting of 10% PEG8000 and 0.1M glycine pH 3.2.
Crystals were soaked overnight in the mother liquor to which approximately 0.1mg of
solid compound was added. The crystals were cryoprotected by serial transfer into a
solution of mother liquor with 30% glycerol and then flash frozen in liquid nitrogen
prior to data collection. Crystals grew in the spacegroup P212121 with 1 molecule per
asymmetric unit. Data was collected at the ESRF beamline ID23-1. For both
inhibitors data was collected to 1.8Å (with Rmerge=0.085 for inhibitor 1 and
R
merge=0.073 for inhibitor 8a). After cycles of building and refinement using the
programs COOT and REFMAC the final models were refined to 1.8Å resolution with
Rwork=0.191, Rfree=0.231 for inhibitor 1 and Rwork=0.173, Rfree=0.211 for inhibitor 8a.
Coordinates have been deposited in the PDB with codes 2vnm (inhibitor 1) and 2vnn
(inhibitor 8a).
S22

Tabulated Analytical Data for 1, 8a-f and 9b-f
Compound
Yield
77%
76%
70%
64%
70%
60%
40%
73%
67%
71%
61%
45%
Purity
>98%
100%
100%
100%
100%
>98%
>98%
100%
100%
100%
>99%
100%
MS
HRMS
619.2
629.2
561.3
553.3
597.3
511.2
485.3
551.2
543.2
587.4
501.1
475.3
619.2551
629.2395
561.2528
553.2842
597.3461
511.2371
485.2216
551.2682
543.2996
587.3619
501.2529
475.2373
1
8a
8b
8c
8d
8e
8f
9b
9c
9d
9e
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