Structure-based design of potent and selective cell-permeable inhibitors of human β-secretase (BACE-1)

Article abstract of DOI:10.1021/jm049379g

We describe the development of cell-permeable β-secretase inhibitors that demonstratively inhibit the production of the secreted amino terminal fragment of an artificial amyloid precursor protein in cell culture. In addition to potent inhibition in a cell

Full text of DOI:10.1021/jm049379g

Supporting Information
Structure-Based Design of a Series of Potent and Selective Cell-
Permeable Inhibitors of Human β-Secretase (BACE-1)
Shawn J. Stachel,† Craig A. Coburn,† Thomas G. Steele,† Kristen G. Jones,† Elizabeth F. Loutzenhiser,† Alison R.
Gregro,† Hemaka A. Rajapakse,† Ming-Tain Lai,‡ Ming-Chih Crouthamel,‡ Min Xu,‡ Katherine Tugusheva,‡
Janet E. Lineberger,‡ Beth L. Pietrak,‡ Amy S. Espeseth,‡ Xiao-Ping Shi,‡ Elizabeth Chen-Dodson,‡ M. Katharine
Holloway,¶ Sanjeev Munshi,§ Adam J. Simon,‡ Lawrence Kuo§ and Joseph P. Vacca†
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Experimental
General: 1H NMR spectra were recorded on a Varian Inova 400 (400 MHz)
spectrometer. Chemical shifts are reported in ppm from tetramethylsilane with
tetramethylsilane as the internal standard. Data are reported as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet),
coupling constants (Hz), integration. Low resolution mass spectra were recorded on a
Bruker Daltonics 3T Fourier transform ion cyclotron resonance mass spectrometer
(FT/ICR) with electrospray ionization. Analytical thin layer chromatography was
performed on EM Reagent 0.25 mm silica gel 60-F plates. Automated flash
chromatography was performed on an ISCO Combiflash Sg 100c with Biotage Flash 40
cartridges. Analytical HPLC was preformed on a HP 1100 with UV detection at 215 and
254 nm, Preparative reverse phase chromatography was performed using a Gilson 215
liquid handler and a YMC CombiPrep Pro C18 column (50 mm x 20 mm i.d.) with a
linear gradient over 20 minutes (95:5 to 5:95 water/acetonitrile, containing 0.1% TFA)
with collection triggered by UV detection at 220 or 254 nm. Solvents for extraction and
chromatography were HPLC grade. Unless otherwise noted, all reactions were
conducted in oven (80oC) or flame-dried glassware with magnetic stirring under an inert
atmosphere of dry nitrogen.
Dimethyl-5-[methyl(methylsulfonyl)amino]isophthalate (5). To a stirred slurry of
dimethyl 5-aminoisophthalate (5.0 g, 23.90 mmol) in 100 mL CH2Cl2/Pyridine (3:1) at 0
oC was added methanesulfonyl chloride (1.85 mL, 23.90 mmol). The resulting mixture
was stirred for 4 h at room temperature. The solvent was removed in vacuo and
ethylacetate (100 mL) was added resulting in precitate formation. The product was
collected by filtration to give 5.14 g (75%) of the sulfonamide as a white solid. 1H NMR
(400 MHz, DMSOd6) δ 8.15 (s, 1H), 8.02 (s, 2H), 3.89 (s, 6H), 3.02 (s, 3H) Electrospray
Mass Spectrum [M-OCH3]+ = 256.16
To a solution of sodium hydride (0.153 g, 3.83 mmol, 60 % oil dispersion) in 10 mL
DMF was added the above sulfonamide (1.0 g, 3.48 mmol) followed by methyl iodide
(0.43 mL, 6.97 mmol). After 1 hr the reaction was quenched with H2O (100 mL) and
extracted with EtOAc ( 3 x 50 mL). The organic extracts were dried over MgSO4 and
evaporated to give 1.03 g (98 %) of N-methylsulfonamide 5. 1H NMR (400 MHz,
DMSOd6) δ 8.40 (s, 1H), 8.19 (s, 2H), 3.91 (s, 6H), 3.34 (s, 3H), 3.01 (s, 3H).
Electrospray Mass Spectrum M + H =302.2
Methyl-3-[methyl(methylsulfonyl)amino]-5-(1R)-1-phenylethylaminocarbonyl) benzoate
(6). Compound 5 (1.03 g, 3.38 mmol) was dissolved in 50 mL THF:MeOH (1:1) and
cooled to 0oC. 1N NaOH (3.38 mL, 3.38 mmol) was added and the reaction was allowed
to warm to RT over 8 hours. The solution was acidified with 1N HCl (30 mL) and
extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with
brine and dried over MgSO4, filtered and concentrated in vacuo. Purification on silica gel
1
(5 % MeOH/CHCl3 containing 1% HOAc) gave mono acid 6 (0.795 g ,82%). H NMR
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(400 MHz, DMSO d6) δ 8.30 (s, 1H), 8.10 (s, 2H), 3.84 (s, 3H), 3.27 (s, 3H), 2.94 (s, 3H).
Electrospray Mass Spectrum M + H = 288.2
3-[methyl(methylsulfonyl)amino]-5-(1R)-1-phenylethylaminocarbonyl)benzoic acid (7).
A solution containing 0.133 g (0.46 mmol) of 6 in 5 mL CH2Cl2, BOP reagent (0.235 g,
0.55 mmol), (R)-(+)-α-methylbenylamine ( 0.071 mL, 0.55 mmol), and diisopropylamine
(0.24 mL, 1.39 mmol) was stirred at ambient temperature for 1 h. Evaporation of the
solvent and column chromatography on silica gel (90% EtOAc/Hexanes) afforded 0.071
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g, (95%) of the benzyl amide. H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 8.17 (s, 1H),
8.06 (s, 1H), 7.31 (m, 5H), 6.50 (d, J = 7.1 Hz, 1H), 5.33 (q, J = 7.1 Hz, 1H), 3.96 (s,
3H), 3.37 (s, 3H), 2.88 (s, 3H), 1.64 (d, J = 7.0 Hz, 3H). Electrospray Mass Spectrum M
+ H = 391.2. To 0.171 g (0.438 mmol) of the above benzyl amide in 10 mL THF:MeOH
(1:1) was added 2 N NaOH (0.66 mL, 1.32 mmol). The solution was heated to 50oC for 1
h. After cooling the solution was acidified by the addition of 1 N HCl (20 mL) and
extracted with EtOAc (3 x 30 mL). The combined organic extraction were dried over
1
MgSO4, filtered, and concentrated in vacuo to yield 7 (0.165 g, 100%). H NMR (400
MHz, CDCl3) δ 8.22 (m, 1H), 8.11 (m, 1H), 8.06 (m, 1H), 7.34 (m, 5H), 6.47 (d, J = 7.1
Hz, 1H), 5.33 (m, 1H), 3.37 (s, 3H), 2.87 (s, 3H), 1.64 (d, J = 7.0 Hz, 3H). Electrospray
Mass Spectrum M + H =377.2
(2R, 3S)-N-1-Cyclopropyl-2-hydroxy-4-1,3-diamine dihydrochloride (8). A solution
containing 2.63 g (10.0 mmol) of tert-butyl[S-(R*,R*)]-(-)-(1-oxiranyl-2-phenylethyl)-
carbamate in 30 mL of i-PrOH was treated with 6 mL of cyclopropyl amine and heated at
50oC in a sealed tube for 16 h. The reaction mixture was cooled and evaporated to give
3.2 g (100%) of amino alcohol a white solid that was pure by HPLC and used without
further purification. A 0oC solution containing 3.0 g (13.9 mmol) of the Boc protected
amino alcohol in 50 mL of 4:1 EtOAc/MeOH was subjected to a slow stream of HCl gas
for 15 minutes. After stirring for 3 h, the solvents were removed by rotory evaporation
and the resulting solid was triturated with ether and filtered leaving 8 (2.5 g, 97%) as a
crystalline white solid. 1H NMR (400 MHz, DMSOd6 ) δ 7.41-7.30 (m 5H), 4.26 (m, 1H),
3.71 (m, 1H), 3.38 (dd, J = 13, 2.3 Hz, 1H), 3.16 (t, J = 12 Hz, 1H), 3.08 (dd, J = 14, 6.5
Hz, 1H), 2.95 (dd, J = 14, 8.7 Hz, 1H), 2.78 (m, 1H), 0.98-0.89 (m, 4H). Electrospray
Mass Spectrum M + H == 186.3.
N-[(1S, 2R)-1-benzyl-3-(cyclopropylamino)-2-hydroxypropyl]-5-
[methyl(methylsulfonyl)amino-N’-[(1R)-1-phenylethyl]isophthalamide (3).
A solution containing 7 (0.020 g, 0.054 mmol) in 2 mL of CH2Cl2 was treated
sequentially with 8 (0.019 g, 0.064 mmol), diisopropylethylamine (0.015 g, 0.117 mmol),
and BOP reagent (0.024 g, 0.054 mmol),. The reaction mixture was stirred at ambient
temperature for 30 minutes. Evaporation of the solvent and purification by reverse phase
HPLC afforded 3 (0.026 g, 84%).
1H NMR (400 MHz, CD3OD) δ 8.06 (s, 1H), 7.97 (s, 1H), 7.81 (s, 1H), 7.40 (d, J
= 7.3 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 7.28 (d, J = 7.2 Hz, 2H), 7.26-7.21 (m, 3H), 7.13
(t, J = 7.2 Hz, 1H), 5.22 (q, J = 7.1 Hz, 1H), 4.25 (m, 1H), 3.96 (m, 1H), 3.33 (s, 3H),
3.24 (m, 1H), 3.11 (m, 1H), 2.93 (s, 3H), 2.85 (m, 1H), 2.65 (m, 1H), 1.58 (d, J = 7.1 Hz,
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3H), 0.86-0.80 (m, 5H); Electrospray Mass Spectrum M + H = 579.1.; HPLC retention
time = 6.84 min; HRMS calcd for C31H39N4O5S [M+H]: 579.2636; found 579.2640.
Compounds 9-15 were prepared similarly to 3.
N-[(1S,2R)-1-benzyl-3-(cyclopropylamino)-2-hydroxypropyl]-N’-[(1R)-1-(4-
fluorophenyl)ethyl]-5-(methylsulfonyl)amino]isophthalamide (9).
1H NMR (400 MHz, CD3OD) δ 7.76 (s, 1H), 7.70 (s, 2H), 7.43-7.40 (m, 2H),
7.14 (m, 1H), 7.08-7.04 (m, 2H), 5.20 (m, 1H), 4.25 (m, 1H), 3.96 (m, 1H), 3.34-3.08 (m,
2H), 3.11 (m, 1H), 3.01 (s, 3H), 2.82 (m, 1H), 2.68 (m, 1H), 1.55 (d, J = 7.0 Hz, 3H),
0.86-0.82 (m, 5H). Electrospray Mass Spectrum M + H = 583.1; HPLC retention time =
5.61 min; HRMS calcd for C30H36FN4O5S [M+H]: 583.2385; found 583.2366.
N-[(1S,2R)-1-benzyl-3-(cyclopropylamino)-2-hydroxypropyl]-N’-[(1R)-1-(4-
fluorophenyl)ethyl]-5-[methyl(methylsulfonyl)amino]isophthalamide (10).
1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H), 7.87 (s, 1H), 7.72 (s, 1H), 7.60 (d, J =
7.8 Hz, 1H), 7.38-7.34 (m, 3H), 7.31 -7.20 (m, 3H), 5.25 (t, J = 7.1 Hz, 1H), 4.36 (m,
1H), 4.25 (m, 1H), 3.25 (s, 3H), 3.15 (m, 1H), 3.04-2.92 (m, 2H), 2.78 (s, 3H), 2.60 (m,
1H), 1.60 (d, J = 7.0 Hz, 3H), 1.24 (m, 1H), 1.01 (m, 2H), 0.83 (m, 2H). Electrospray
Mass Spectrum M + H = 597.1; HPLC retention time = 6.15 min; HRMS calcd for
C31H38FN4O5S [M+H]: 597.2542; found 597.2569.
N-[(1S,2R)-3-amino-1-benzyl-2-hydroxypropyl]-N’-[(1R)-1-(4-fluorophenyl)ethyl]-5-
[methyl(methylsulfonyl)amino]isophthalamide (11).
1H NMR (400 MHz, CD3OD) δ 8.85 (d, J = 1.9 Hz, 1H), 8.45 (d, J = 9.0 Hz, 1H),
7.98 (m, 1H), 7.95 (m, 1H), 7.76 (m, 1H), 7.42-7.38 (m, 2H), 7.27-7.20 (m, 4H), 7.11 (m,
1H), 7.08-7.04 (m, 2H), 5.21 (q, J = 7.1 Hz, 1H), 4.21 (m, 1H), 4.08 (dd, J = 7.1, 7.2 Hz,
1H), 3.84 (m, 1H), 3.31 (m, 1H), 3.28 (s, 3H), 3.13 (dd, J = 9.9, 3.1 Hz, 1H), 2.92 (s,
3H), 2.80 (dd, J = 11, 9.9 Hz, 1H), 1.55 (d, J = 7.1 Hz, 3H). Electrospray Mass Spectrum
M + H = 557.6; ; HPLC retention time = 7.18 min; HRMS calcd for C28H34FN4O5S
[M+H]: 557.2229; found 557.2224.
N-[(1S,2R)-1-benzyl-3-(ethylamino)-2-hydroxypropyl]-N’-[(1R)-1-(4-
fluorophenyl)ethyl]-5-[methyl(methylsulfonyl)amino]isophthalamide (12).
1H NMR (400 MHz, CD3OD) δ 8.86 (d, J = 7.4 Hz, 1H), 8.01 (s, 1H), 7.97 (s,
1H), 7.78 (s, 1H), 7.41 (m, 2H), 7.29-7.21 (m, 4H), 7.14 (t, J = 7.3 Hz, 1H), 7.06 (d, J =
8.8 Hz, 2H), 5.22 (t, J = 7.1 Hz, 1H), 4.25 (m, 1H), 3.94 (m, 1H), 3.38-3.26 (m, 2H), 3.33
(s, 3H), 3.19 (m, 1H), 3.09 (m, 2H), 2.94 (s, 3H), 2.83 (m, 1H), 1.56 (d, J = 7.1 Hz, 3H),
1.33 (t, J = 7.3 Hz, 3H). Electrospray Mass Spectrum M + H = 585.2; HPLC retention
time = 7.31 min; HRMS calcd for C30H38FN4O5S [M+H]: 585.2547; found 585.2533.
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N-[(1S, 2R)-1-benzyl-3-(tert-butylamino)-3-hydroxypropyl]-N’-[(1R)-1-(4-
fluorophenyl)ethyl]-5-[methyl(methylsulfonyl)amino]isophthalamide (13).
1H NMR (400 MHz, CD3OD) δ 8.89 (d, J = 7.24 Hz, 1H), 8.02 (s, 1H), 7.98 (s,
1H), 7.79 (s, 1H), 7.43-7.38 (m, 2H), 7.30-7.22 (m, 4H), 7.15 (t, J = 7.2 Hz, 1H), 7.06 (t,
J = 8.8 Hz, 2H), 5.22 (t, J = 7.1 Hz, 1H), 4.27 (m, 1H), 3.97 (m, 1H), 3.33 (s, 3H), 3.38-
3.27 (m 2H), 3.18 (m, 1H), 3.02 (m, 1H), 2.93 (s, 3H), 2.84 (m, 1H), 1.56 (d, J = 7.0 Hz,
3H), 1.37 (s, 9H). Electrospray Mass Spectrum M + H = 613.2; HPLC retention time =
7.31 min; HRMS calcd for C31H38FN4O5S [M+H]: 613.2840; found 613.2854.
N-[(1S, 2R)-1-benzyl-3-(cyclopropylamino)-2-hydroxypropyl]-5-
[(isopropylsulfonyl)(methyl)amino]-N’-[(1R)-1-phenylethyl]isophthalamide (14).
1H NMR (400 MHz, CD3OD) δ 8.85 (d, J = 7.7 Hz, 1H), 7.98 (s, 1H), 7.97 (s,
1H), 7.80 (s, 1H), 7.39 (d, J = 7.3 Hz, 2H), 7.34 (t, J = 7.3 Hz, 2H), 7.29-7.21 (m, 4H),
7.14 (t, J = 7.0 Hz, 1H), 5.23 (t, J = 7.1 Hz, 1H), 4.24 (m, 1H), 3.98 (m, 1H), 3.47 (m,
1H), 3.37 (s, 3H), 3.36-3.21 (m, 1H), 3.16 (m 1H), 2.86-2.73 (m, 2H), 1.57 (d, J = 7.1
Hz, 3H), 1.32 (d, J = 6.8 Hz, 6H), 0.95-0.88 (m, 5H). Electrospray Mass Spectrum M + H
= 607.1; HPLC retention time = 7.62 min; HRMS calcd for C33H43N4O5S [M+H]:
607.2949; found 607.2935.
N-[(1S, 2R)-1-benzyl-3-(cyclopropylamino)-2-hydroxypropyl]-5-
[[(dimethylamino)sulfonyl](methyl)amino]-N’-[(1R)-1-phenylethyl]isophthalamide (15).
1H NMR (400 MHz, CD3OD) δ 8.80 (d, J = 7.0 Hz, 1H), 7.97 (s, 2H), 7.88 (s,
1H), 7.41-7.39 (m, 2H), 7.38-7.32 (m, 2H), 7.28 -7.22 (m, 5H), 7.156 (m ,1H), 5.23 (m,
1H), 4.25 (m, 1H), 4.01 (m, 1H), 3.25 (s, 3H), 3.20-3.15 (m, 2H), 2.82 (s, 6H), 2.80-2.73
(m, 2H), 1.57 (d, J = 7.1 Hz, 3H), 0.95-0.87 (m, 5H). Electrospray Mass Spectrum M + H
= 608.2; HPLC retention time = 7.02 min; HRMS calcd for C32H42N5O5S [M+H]:
608.2901; found 608.2875.
Biochemical assay conditions
The biochemical assay employed to determine the IC50 of compounds consists of the
following. Briefly, the assay was carried out with BACE enzyme at pH 4.5 with a
coumarin labeled proprietary substrate (EVNFEVEF) discovered from peptide screening
[1]. The compounds were preincubated with BACE (2 nM) for 30 min before the
addition of substrate to initiate the reaction. Tris buffer (pH8.0) was introduced into the
reaction mixture to stop the reaction after 30 min incubation. The formation of product
was monitored by HPLC with excitation at 340 nm and emission at 440 nm. Data were fit
to a 4 parameter logistic regression using standard algorithms.
Cell based assay
HEK293T cells were stably transfected with APP695 encoding the NFEV BACE
substrate (replacing the endogenous APP) including the alpha-secretase mutant site K612V.
After being seeded for one day, the cells were treated for 20-24 hours with a medium
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containing a compound or DMSO (negative control). The conditioned medium was then
removed from the cultured cells and analyzed for the secreted n-terminal fragment
sAPPβ_NF. A standard Western blot protocol was conducted on the conditioned
medium. The sAPPβ_NF band was probed with a specific antibody recognizing the neo-
epitope of C-terminal sAPPβ_NF. Bands were quantitated in a scanner and integrated
intensities were fit to a 4 parameter logistic regression using standard algorithms.
1) Brady, S.; Bruce, J.; Singh, S.; Crouthamel, M.-C.; Holloway, K. M.; Coburn, C.;
Vacca, J. P.; Shafer, J. A.; Hazuda, D. "Development of BACE 1 Inhibitors" poster,
presented at 9th International conference on Alzheimer's Disease and Related Disorders,
Philadelphia, PA, July 17-22, (2004).
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